Note: Descriptions are shown in the official language in which they were submitted.
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CLONING OF CYT"OCHROME P450 GENES FROM NICOTIANA
The present invention relates to nucleic acid sequences
encoding cytochrome P450 enzymes (hereinafter referred to as
P450 and P450 enzymes) in Nicotiana plants and methods for
using those nucleic acid sequences to alter plant phenotypes.
BACKGROUND
Cytochrome P450s catalyze enzymatic reactions for a
diverse range of chemically dissimilar substrates that include
the oxidative, peroxidative and reductive metabolism of
endogenous and xenobiotic substrates. In plants, P450s
I participate in biochemical pathways that include the synthesis
of plant products such as phenylpropanoids, alkaloids,
terpenoids, lipids, cyanogenic glycosides, and glucosinolates
(Chappel, .Annu. Rev. Plant Physiol. Plant Mol. Biol. 198,
49:311-343). Cytochrome P450s, also known as P450 heme-
thiolate proteins, usually act as terminal oxidases in multi-
component electron transfer chains, called P450- containing
monooxygenase systems. Specific reactions catalyzed include
demethylation, hydroxylation, epoxidation, N-oxidation,
sulfooxidation, N-, S-, and 0- dealkylations, desulfation,
deamination, and reduction of azo, vitro, and N-oxide groups.
The diverse role of Nicotiana plant P450 enzymes has been
implicated in effecting a variety of plant metabolites such as
phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic
glycosides, glucosinolates and a host of other chemical
entities. During recent years, it is becoming apparent that
some P450 enzymes can impact the composition of plant
metabolites in plants . For example, it has been long desired
to improve the flavor and aroma of certain plants by altering
its profile of selected fatty acids through breeding; however
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very little is known about mechanisms involved in controlling
the levels of these leaf constituents. The down regulation of
P450 enzymes associated with the modification of fatty acids
may facilitate accumulation of desired fatty acids that provide
more preferred leaf phenotypic qualities. The function of P450
enzymes and their broadening roles in plant constituents is
still being discovered. For instance, a special class of P450
enzymes was found to catalyze the breakdown of fatty acid into
volatile C6- and C9-aldehydes and -alcohols that are major
contributors of "fresh green" odor of fruits and vegetables.
The level of other novel targeted P450s may be altered to
enhance the qualities of leaf constituents by modifying lipid
composition and related break down metabolites in Nicotiana
leaf. Several of these constituents in leaf are affected by
senescence that stimulates the maturation of leaf quality
properties. Still other reports have shown that P450s enzymes
are play a functional role in altering fatty acids that are
involved in plant-pathogen interactions and disease resistance.
In other instances, P450 enzymes have been suggested to be
involved in alkaloid biosynthesis. Nornicotine is a minor
alkaloid found in Nicotiana tabaccum. It is has been
postulated that it is produced by the P450 mediated
demethylation of nicotine followed by acylation and nitrosation
at the N position thereby producing a series of N-
acylnonicotines and N-nitrosonornicotines. N-demethylation,
catalyzed by a putative P450 demethylase, is thought to be a
primary source of nornicotine biosyntheses in Nicot3ana. while
the enzyme is believed to be microsomal, thus far a nicotine
demethylase enzyme has not been successfully purified, nor have
the genes involved been isolated.
Furthermore, it is hypothesized but not proven that the
activity of P450 enzymes is genetically controlled and also
strongly influenced by~environment factors. For example, the
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demethylation of nicotine in Nicotiana is thought to increase
substantially when the plants reach a mature stage.
Furthermore, it is thought that the demethylase gene contains a
transposable element that can inhibit translation of RNA when
present.
The large multiplicity of P450 enzyme forms, their
differing structure and function have made their research on
Nicotiana P450 enzymes very difficult before the enclosed
invention. In addition, cloning of P450 enzymes has been
hampered at least in part because these membrane-localized
proteins are typically present in low abundance and often
unstable to purification. Hence, a need exists for the
identification of P450 enzymes in plants and the nucleic acid
sequences associated with those P450 enzymes. In particular,
only a few cytochrome Nicotiana P450 proteins have been
reported in tobacco. The inventions described herein entail
the discovery of a substantial number of cytochrome P450
fragments that correspond to several groups of P450 species
based on their sequence identity.
SUMMARY
The present invention is directed to Jplant P450 enzymes.
The present invention is further directed to plant P450 enzymes
from Nicotiana. The present invention is also directed to P450
enzymes in plants whose expression is induced by ethylene
and/or plant senescence. The present invention is yet further
directed to nucleic acid sequences in plants having enzymatic
activities, for example, oxygenase, demethylase and the like,
or other and the use of those sequences to reduce or silence
the expression of these enzymes. The invention also relates to
P450 enzymes found in plants containing higher nornicotine
levels than plants exhibiting lower nornicotine levels.
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In one aspect, the invention is directed to nucleic acid
sequences as set forth in SEQ. ID. Nos. l, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, ,
109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,
133, 135, 137, 139, 141, 143, 145 and 147.
In a second related aspect, those fragments containing
greater than 75% identity in nucleic acid sequence were placed
into groups dependent upon their identity in a region
corresponding to the first nucleic acid following the
cytochrome P450 motif GXRXCX(G/A) to the 'stop codon. The
representative nucleic acid groups and respective species are
shown in Table I.
In a third aspect, the invention is directed to amino acid
sequences as set forth in SEQ. TD. Nos. 2, 4, 6, 8, 10, 12, 14,
1&, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,
108, 110, 112, 114~ 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, 136, 138, 140, 142, 144, 146 and 148.
In a fourth related aspect, those fragments containing
greater than 71a identity in amino acid sequence were placed
into groups dependent upon their identity to each other in a
region corresponding to the first amino acid following the
cytochrome P450 motif GXRXCX(G/A) to the stop codon. The
representative amino acid groups and respective species are
shown in Table IT.
In a fifth aspect of the invention is the use of nucleic
acids sequences as set forth in SEQ. ID. Nos. 1, 3, 5, 7,
9,
11, 13, 15, 17, 19, 23, 25, 27, 29, 31, 33, 35, 37, 39,
21, 41,
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43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,
75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,
105, 107,1~109,~111, 113, 115, 117, 119, 121, 123, 125, 127,
129, 131, 133, 135, 137, 139, 141, 143, 145 and 147.
In a sixth related aspect, the reduction or elimination of
P450 enzymes in Nicotiana plants may be accomplished
transiently using RNA viral systems. Resulting transformed
or infected plants are assessed for phenotypic changes
including, but not limited to, analysis of endogenous P450 RNA
transcripts, P450 expressed peptides, and concentrations of
plant metabolites using techniques commonly available to one
having ordinary skill in the art.
In a seventh important aspect, the present invention is
also directed to generation of trangenic Nicotiana lines that
have altered P450 enzyme activity levels. In accordance with
the invention, these transgenic lines include nucleic acid
sequences that are effective for reducing or silencing the
expression of certain enzyme thus resulting in phenotypic
effects within Nicotiana. Such nucleic acid sequences include
SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,. 52, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, '79, 81, 83, 85, 87, 89,
91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 112, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,
141, 143, 145 and 147.
In a very important eight aspect of the invention, plant
cultivars including nucleic acids of the present invention in a
down regulation capacity will have altered metabolite profiles
relative to control plants.
In a ninth aspect, the present invention is directed to
the screening of plants, more preferably Nicotiana, that
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contain genes that have substantial nucleic acid identity to
the taught nucleic acid sequence. The use of the invention
would be advantageous to identify and select plants that
contain a nucleic acid sequence with exact or substantial
identity where such plants are part of a breeding program for
traditional or transgenic varieties, a mutagenesis program, or
naturally occurring diverse plant populations. The screening
of plants for substantial nucleic acid identity may be
accomplished by evaluating plant nucleic acid materials using a
nucleic acid probe in conjunction with nucleic acid detection
protocols including, but not limited to, nucleic acid
hybridization and PCR analysis. The nucleic acid probe may
consist of the taught nucleic acid sequence or fragment thereof
corresponding to SEQ ID 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, 91, 93', 95, 97, 99, 101, 103, 105, 107, 109, 111~ 113,
W
115, 117, 119, 121, 123, 125, 127, 129, 131., 133, 235~ 137,
139, 141, 143, 145 and 147.
In a tenth aspect, the present invention is directed to
the identification of plant genes, more preferably Nicotiana,
that share substantial amino acid identity corresponding to the
taught nucleic acid sequence. The identification of plant
genes including both cDNA and genomic clans of those cDNAs and
genomic clones, preferably from Nicotiana may be accomplished
by screening plant cDNA libraries using a nucleic acid probe in
conjunction with nucleic acid detection protocols including,
but not limited to, nucleic acid hybridization and PCR
analysis. The nucleic acid probe may be comprised of nucleic
acid sequence or fragment thereof corresponding to SEQ ID 1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
_
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101, 103, 105, 107, 109, 111~ 113, 115, 117, 119, 121, 123,
125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 and 147
In an alterative eleventh aspect, CDNA expression
libraries that express peptides may be screened using
antibodies directed to part or all of the taught amino acid
sequence. Such amino acid sequences include SEQ ID 2, 4, 8, 9,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,
104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,
128, 130, 132, 134, 136, 138, 140, 142, 144, 146 and 148.
BRIE' DESCRIPTIOI~1 ~E' DRAWINGS
Figure 1 shows nucleic acid SEQ. ID. No.:1 and amino acid
SEQ. ID. No.:2.
Figure 2 shows nucleic acid SEQ. ID. No.:3 and amino acid
SEQ. ID. No.:4.
Figure 3 shows nucleic acid SEQ. ID. No.:5 and amino acid
SEQ. ID. No.:6.
Figure 4 shows nucleic acid SEQ. ID. No.:7 and amino acid
SEQ. ID. No.:8.
Figure 5 shows nucleic acid SEQ. ID. No.:9 and amino acid
SEQ. ID. No.:lO.
Figure 6 shows nucleic acid SEQ. ID. No.:l1 and amino acid
SEQ. ID. No.:l2.
Figure 7 shows nucleic acid SEQ. ID. No.:l3 and amino acid
SEQ. ID. No.:l4.
Figure 8 shows nucleic acid SEQ. ID. No.:l5 and amino acid
SEQ. ID. No.:l6.
Figure~9 shows nucleic acid SEQ. ID. No.:l7 and amino acid
SEQ. ID. No.:l8.
Figure 10 shows nucleic acid SEQ. ID. No.:l9 and amino
acid SEQ. ID. No.:20.
__~ 7
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Figure 11 shows nucleic acid SEQ. ID. No .:21 and amino
acid SEQ. ID. No.:22.
Figure 12 shows nucleic acid SEQ. ID. No .:23 and amino
acid SEQ. ID. No.:24.
Figure 13 shows nucleic acid SEQ. ID. No .:25 and amino
acid SEQ. ID. No.:26.
Figure 14 shows nucleic acid SEQ, ID. No .:27 and amino
acid SEQ. ID. No.:28.
Figure 15 shows nucleic acid SEQ. ID. No .:29 and amino
acid SEQ. ID. No.:30.
Figure 16 shows nucleic acid SEQ. ID. No .:31 and amino
acid SEQ. ID. No.:32.
Figure 17 shows nucleic acid SEQ. ID. No .:33 and amino
acid SEQ. ID. No.:34.
Figure 18 shows nucleic acid SEQ. ID. No .:35 and amino
acid SEQ. ID. No.:36.
Figure 29 shows nucleic acid SEQ. ID. No .:37 and amino
acid SEQ. ID. No.:38.
Figure 20 shows nucleic acid SEQ. ID. No .:39 and amino
acid SEQ. ID. No.:40.
Figure 21 shows nucleic acid SEQ. ID. No .:41 and amino
acid SEQ. ID. No.:42.
Figure 22 shows nucleic acid SEQ.~ ID. No .:43 and amino
acid SEQ. ID. No.:44.
Figure 23 shows nucleic acid SEQ. ID. No .:45 and amino
acid SEQ. ID. No.:46.
Figure 24 shows nucleic acid SEQ. ID. No .:47 and amino
acid SEQ. ID. No.:48.
Figure 25 shows nucleic acid SEQ. ID. No .:49 and amino
acid SEQ. ID. No.:50.
Figure 26 shows nucleic acid SEQ. ID. No .:51 and amino
acid SEQ. ID. No.:52.
Figure 27 shows nucleic acid SEQ. ID. No .:53 and amino
acid SEQ. ID. No.:54.
_ _ -
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Figure 28 shows nucleic acid SEQ. ID. No .:55 and amino
acid -SEQ. ID. No.:56.
Figure 29 shows nucleic acid SEQ. ID. No .:57 and amino
acid SEQ. ID. No.:58.
Figure 30 shows nucleic acid SEQ. ID. No .:59 and amino
acid SEQ. ID. No.:60.
Figure 31 shows nucleic acid SEQ. ID. No .:61 and amino
acid SEQ. ID. No.:62.
Figure 32 shows nucleic acid SEQ. ID. No .:63 and amino
acid SEQ. ID. No.:64.
Figure 33 shows nucleic acid SEQ. ID. No .:65 and amino
acid SEQ. ID. No.:66.
Figure 34 shows nucleic acid SEQ. ID. No .:67 and amino
acid SEQ. ID. No.:68.
Figure 35 shows nucleic acid SEQ. ID. No .:69 and amino
acid SEQ. ID. No.:70.
Figure 36 shows nucleic acid SEQ. ID. No .:71 and amino
acid SEQ. ID. No.:72.
Figure 37 shows nucleic acid SEQ. ID. No .:73 and amino
acid SEQ. ID. No.:74.
Figure 38 shows nucleic acid SEQ. ID. No .:75 and amino
,
acid SEQ. ID. No.:76.
Figure 39 shows nucleic acid SEQ. ID. No .:77 and amino
acid SEQ. ID. No.:78.
Figure 40 shows nucleic acid SEQ. ID. No .:79 and amino
acid SEQ. ID. No.:80.
Figure 41 shows nucleic acid SEQ. ID. No .:81 and amino
acid SEQ. ID. No.:82.
Figure 42 shows nucleic acid SEQ. ID. No .:83 and amino
acid SEQ. ID. No.:84. _
Figure 43 shows nucleic acid SEQ. ID. No .:85 and amino
acid SEQ. ID. No.:86.
Figure 44 shows nucleic acid ,SEQ. ID. No .:87 and amino
acid SEQ. ID. No.:88.
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Figure 45 shows nucleic acid SEQ. ID. No.:89 and amino
acid SEQ. ID. No.:90.
Figure 46 shows nucleic acid SEQ. ID. No.:91 and amino
acid SEQ. TD. No.:92.
Figure 47 shows nucleic acid SEQ. ID. No.:93 and amino
acid SEQ. ID. No.:94.
Figure 48 shows nucleic acid SEQ. ID. No.:95 and amino
acid SEQ. ID. No.:96.
Figure 49 shows nucleic acid SEQ. ID. No.:97 and amino
acid SEQ. ID. No.:98.
Figure 50 shows nucleic acid 5EQ. ID. No.:99 and amino
acid SEQ. ID. No.:100.
Figure 51 shows nucleic acid SEQ. ID. No.:101 and amino
acid SEQ. ID. No.:102.
Figure 52 shows nucleic acid SEQ. ID. No.:103 and amino
acid SEQ. ID. No.:104.
Figure 53 shows nucleic acid SEQ. ID. No.:105 and amino
acid SEQ. ID. No.:106.
Figure 54 shows nucleic acid SEQ. ID. No.:107 and amino
acid SEQ. ID. No.:108.
Figure 55 shows nucleic acid SEQ. ID. No.:109 and amino
acid SEQ. ID. No.:110.
Figure 56 shows nucleic acid SEQ. ID. No.:111 and amino
acid SEQ. ID. No.:112.
Figure 57 shows nucleic acid SEQ. ID. No.:113 and amino
acid SEQ. ID. No.:114.
Figure 58 shows nucleic acid SEQ. ID. No.:115 and amino
acid SEQ. TD. No.:116.
Figure 59 shows nucleic acid SEQ. ID. No.:117 and amino
acid SEQ. ID. No.:118. ,
Figure 60 shows nucleic acid SEQ. ID. No.:119 and amino
acid SEQ. ID. No.:120.
Figure 61 shows nucleic acid SEQ. ID. No.:121 and amino
acid SEQ. TD. No.:122.
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Figure 62 shows nucleic acid SEQ. ID. No .:123 and amino
acid SEQ. ID. No.:124.
Figure 63 shows nucleic acid SEQ. TD. No .:125 and amino
acid SEQ. ID. No.:126.
Figure 64 shows nucleic acid SEQ. ID. No .:127 and amino
acid SEQ. ID. No.:128.
Figure 65 shows nucleic acid SEQ. ID. No .:129 and amino
acid SEQ. ID. No.:130.
Figure 66 shows nucleic acid SEQ. TD. No .:131 and amino
10acid SEQ. ID. No.:132.
Figure 67 shows nucleic acid SEQ. ID. No .:133 and amino
acid SEQ. ID. No.:134.
Figure 68 shows nucleic acid SEQ. TD. No .:135 and amino
acid SEQ. ID. No.:136.
Figure 69 shows nucleic acid SEQ. TD. No .:137 and amino
acid SEQ. ID. No.:138.
Figure 70 shows nucleic acid SEQ. ID. No .:139 and amino
acid SEQ. ID. No.:140.
Figure 71 shows nucleic acid SEQ. ID. No .:141 and amino
20acid SEQ. ID. No.:142.
Figure 72 shows nucleic acid SEQ. TD. No .:143 and amino
acid SEQ. ID. No.:144.
Figure 73 shows nucleic acid SEQ. ID. No .:145 and amino
acid SEQ. ID. No.:146.
Figure 74 shows nucleic acid SEQ. TD. No .:147 and amino
acid SEQ. ID. No.:148.
Figure 75 shows a proced ure r cytochrome
used cloning
fo of
P450 cDNA fragments by PCR. SEQ. s. 49-156
ID. 1 are
No shown.
Figure 76 illustrates amino identity of groin
acid
members.
DETAILED DESCRIPTION
DEFINITIONS
Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood
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by one of ordinary skill in the art to which this invention
belongs. Singleton et al. (1994) Dictionary of Microbiology and
Molecular Biology, second edition, John Wiley and Sons (New
York) provides one of skill with a general dictionary of many
of the terms used in this invention. All patents and
publications referred to herein are incorporated by reference
herein. For purposes of the present invention, the following
terms are defined below.
"Enzymatic activity" is meant to include demethylation,
hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-
and 0- dealkylations, desulfation, deamination, and reduction
of azo, nitro, and N-oxide groups. The term "nucleic acid"
refers to a deoxyribonucleotide or ribonucleotide polymer in
either single- or double-stranded form, or sense or anti-sense,
and unless otherwise limited, encompasses known analogues of
natural nucleotides that hybridize to nucleic acids in a manner
similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence includes the
complementary sequence thereof. The terms "operably linked",
"in operable combination", and "in operable order" refer to
functional linkage between a nucleic acid expression control
sequence (such as a promoter, signal sequence, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence affects
transcription and/or translation of the nucleic acid
corresponding to the second sequence.
The term "recombinant" when used with reference to a cell
indicates that the cell replicates a heterologous nucleic acid,
expresses said nucleic acid or expresses a peptide,
heterologous peptide, or protein encoded by a heterologous
nucleic acid. Recombinant cells can express genes or gene
fragments in either the sense or antisense form that are not
found within the native (non- recombinant) form. of the cell.
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Recombinant cells can also express genes that are found in the
native form of the cell, but wherein the genes are modified and
re- introduced into the cell by artificial means.
A "structural gene" is that portion of a gene comprising a
DNA segment encoding a protein, polypeptide or a portion
thereof, and excluding the 5' sequence which drives the
initiation of transcription. The structural gene may
alternatively encode a nontranslatable product. The structural
gene may be one which is normally found in the cell or one
which is not normally found in the cell or cellular location
wherein it is introduced, in which case it is termed a
"heterologous gene". A heterologous gene may be derived in
whole or in part from any source known to the art, including a
bacterial genome or episome, eukaryotic, nuclear or plasmid
DNA, cDNA, viral DNA or chemically synthesized DNA. A
structural gene may contain one or more modifications that
could effect biological activity or its characteristics, the
biological activity or the chemical structure of the expression
product, the rate of expression or the manner of expression
control. Such modifications include, but are not limited to,'
mutations, insertions, deletions and substitutions of one or
more nucleotides. The structural gene may constitute an
uninterrupted coding sequence or it may include one or more
intron~s, bounded by the appropriate splice junctions. The
structural gene may be translatable or non-translatable,
including in an anti-sense orientation. The structural gene
may be a composite of segments derived from a plurality of
sources and from a plurality of gene sequences (naturally
occurring or synthetic, where synthetic refers to DNA that is
chemically synthesized).
"Derived from" is used to mean taken, obtained, received,
traced, replicated or descended from a source (chemical and/or
biological). A derivative may be produced by chemical or
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biological manipulation (including, but not limited to,
substitution, addition, insertion, deletion, extraction,
isolation, mutation and replication) of the original source.
~~Chemically synthesized", as related to a sequence of DNA,
means that portions of the component nucleotides were assembled
in vitro. Manual chemical synthesis of DNA may be accomplished
using well established procedures (Caruthers, Methodology of
DNA and RNA Sequencing, (1983), Weissman (ed.), Praeger
Publishers, New York, Chapter 1)~ automated chemical synthesis
can be performed using one of a number of commercially
available machines.
Optimal alignment of sequences for comparison may be
conducted by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology
alignment algorithm of Needleman and Wunsch, J. Mol. Biol.
48:443 (1970), by the search for similarity method of Pearson
and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection.
~ The NCBI Basic Local Alignment Search Tool (BLAST)
(Altschul et al., 1990) is available from several sources,
including the National Center for Biological Information (NCBI,
Bethesda, Md.) and on the Internet, for use in connection with
the sequence analysis programs blastp, blastn, blastx, tblastn
and tblastx. It can be accessed at
htp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to
determine sequence identity using this program is available at
http://www.ncbi.nlm.nih.gov/BLAST/blast help.html.
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The terms "substantial amino acid identity" or
"substantial amino acid sequence identity'° as applied to amino
acid sequences and as used herein denote a characteristic of a
polypeptide, wherein the peptide comprises a sequence that has
at least 70 percent sequence identity, preferably 80 percent
amino acid sequence identity, more preferably 90 percent amino
acid sequence identity, and most preferably at least 99 to 100
percent sequence identity as compared to a reference group over
region corresponding to the first amino acid following the
cytochrome P450 motif GXRXCX(G/A) to the stop codon of the
translated peptide.
The terms "substantial nucleic acid identity" or
"substantial nucleic acid sequence identity" as applied to
nucleic acid sequences and as used herein denote a
characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence that has at least 75
percent sequence identity, preferably 81 percent amino acid
sequence identity, more preferably at least 91 to 99 percent
sequence identity, and most preferably at least 99 to 100
percent sequence identity as compared to a reference group over
region corresponding to the first nucleic acid following the
cytochrome P450 motif GXRXCX(G/A) to the stop codon of the
translated peptide.
Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each
other under stringent conditions. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Generally, stringent conditions are selected to
be about 5°C to about 20°C, usually about 10°C to about
15°C,
lower than the thermal melting point (Tm) for the specific
sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength and pH) at which 500
of the target sequence hybridizes to a matched probe.
1S
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Typically, stringent conditions will be those in which the salt
concentration is about 0.02 molar at pH 7 and the temperature
is at least about 60°C. Four instance~in a standard Southern
hybridization procedure, stringent conditions will include an
initial wash in 6xSSC at 42 °G followed by one or more
additional washes in 0.2xSSC at a temperature of at least about
55°C, typically about 60°C and often about 65°C.
Nucleotide sequences are also substantially identical for
purposes of ,this invention when the polypeptides and/or
proteins which they encode are substantially identical. Thus,
where one nucleic acid sequence encodes essentially the same
polypeptide as a second nucleic acid sequence, the two nucleic
acid sequences are substantially identical, even if they would
not hybridize under stringent conditions due to degeneracy
permitted by the genetic code (see, Darnell et al. (1990)
Molecular Cell Biology, Second Edition Scientific American
Books W. H. Freeman and Company New York for an explanation of
codon degeneracy and the genetic code). Protein purity or
homogeneity can be indicated by a number of means well known in
the art, such as polyacrylamide gel electrophoresis of a
protein sample, followed by visualization upon staining. For
certain purposes high resolution may be needed and HPLC or a
similar means for purification may be utilized.
As used herein, the term "Vector" is used in reference to
nucleic acid molecules that transfer DNA segments) into a
cell. A vector may act to replicate DNA and may reproduce
independently in a host cell. The term "vehicle" is sometimes
used interchangeably with "vector." The term °'expression
vector" as used herein refers to a recombinant DNA molecule
containing a desired coding sequence and appropriate nucleic
acid sequences necessary for the expression of the operably
linked coding sequence in a particular host organism. Nucleic
acid sequences necessary fox expression in prokaryotes usually
16
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WO 2003/078577 PCT/US2003/007430
include a promoter, an operator (optional), and a ribosome
binding site, often along with other sequences. Eucaryotic
cells are known to utilize promoters, enhancers, and
termination and polyadenylation signals.
For the purpose of regenerating complete genetically
engineered plants with roots, a nucleic acid may be inserted
into plant cells, for example, by any technique such as in vivo
inoculation or by any of the known in vitro tissue culture
techniques to produce transformed plant cells that can be
regenerated into complete plants. Thus, for example, the
insertion into plant cells may be by in vitro inoculation by
pathogenic or non-pathogenic A. tumefaciens. Other such tissue
culture techniques may also be employed.
IS
"Plant tissue" includes differentiated and
undifferentiated tissues of plants~ including, but not limited
to, roots, shoots, leaves, pollen, seeds, tumor tissue and
various forms of cells in culture, such as single cells,
ZO protoplasts, embryos and callus tissue. The plant tissue may be
ire planta or in organ, tissue or cell culture.
"Plant cell" as used herein includes plant cells in planta
and plant cells and protoplasts in culture. "cDNA'° or
25 "Complementary DNA" generally refers to~a single stranded DNA
molecule with a nucleotide sequence that is complementary to an
RNA molecule. cDNA is formed by the action of the enzyme
reverse transcriptase on an RNA template.
30 STRATEGIES FOR OBTAINING NUCLEIC ACID SEQUENCES
In accordance with the present invention, RNA was
extracted from Nicotiana tissue of converter and non-converter
Nicotiana lines. The extracted RNA was then used to create
,, I7
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cDNA. Nucleic acid sequences of the present invention were
then generated using two strategies.
In the first strategy, the poly A enriched RNA was
extracted from plant tissue and cDNA was made by reverse
transcription PCR. The single strand cDNA was then used to
create P450 specific PCR populations using degenerate primers
plus a oligo d(T) reverse primer. The primer design was based
on the highly conserved motifs of P450. Examples of specific
IO degenerate primers are set forth in Figure 1. Sequence
fragments from plasmids containing appropriate size inserts
were further analyzed. ~ These size inserts typically ranged
from about 300 to about 800 nucleotides depending on which
primers were used.
In a second strategy, a cDNA library was initially
constructed. The cDNA in the plasmids was used to create p450
specific PCR populations using degenerate primers plus T7
primer on plasmid as reverse primer. As in the first strategy,
sequence fragments from plasmids containing appropriate size
inserts were further analyzed.
Nicotiana plant lines known to produce high levels of
nornicotine (converter) and plant lines having undetectable
levels of nornicotine may be used as starting materials.
Leaves can then be removed from plants and treated with
ethylene to activate P450 enzymatic activities defined herein.
Total 'RNA is extracted using techniques known in the art. cDNA
fragments can then be generated using PCR (RT-PCR) with the
oligo d(T) primer as described in Figure 1. The cDNA library
can then be constructed more fully described in examples
herein.
', _18
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The conserved region of P450 type enzymes can be used as a
template for degenerate primers (Figure 75). Using degenerate
primers, P450 specific bands can be amplified by PCR. Bands
indicative for P450 like enzymes can be identified by DNA
sequencing. PCR fragments can be characterized using BLAST
search, alignment or other tools to identify appropriate
candidates.
Sequence information from identified fragments can be used.
to develop PCR primers. These primers are used to conduct
quantitative RT-PCR from the RNA's of converter and non-
converter ethylene treated plant tissue. Only appropriate
sized DNA bands (300-800 bp) from converter Lines or bands with
higher density denoting higher expression in converter Lines
were used for further characterization. Large scale Southern
reverse analysis were conducted to examine the differential
expression for all clones obtained. In this aspect of the
invention, these large scale reverse Southern assays can be
conducted using labeled total cDNA's from different tissues as
a probe to hybridize with cloned DNA fragments in order to
screen all cloned inserts.
Nonradioactive Northern blotting assay was also used to
characterize clones P450 fragments.
Nucleic acid sequences identified as described above can
be examined by using virus induced gene silencing technology
(VIGS, Baulcombe, Current Opinions in Plant Biology, 1999,
2:109-113).
In another aspect of the invention, interfering RNA
technology (RNAi) is used to further characterize cytochrome
P450 enzymatic activities in Nicotiana plants of the present
invention. The following references which describe this
technology are incorporated by reference herein, Smith et al.,
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Nature, 2000, 407:319-3200 Fire et al., Nature, 1998, 391:306-
311; Waterhouse et al., PNAS, 1998, 95:13959-139640 Stalberg et
al., Plant Molecular Biology, 1993, 23:671- 683e Baulcombe,
Current Opinions in Plant Biology, 1999, 2:109-113; and
S Brigneti et al., EMBO Journal, 1998, 17(22):6739-6746. Plants
may be transformed using RNAi techniques, antisense techniques,
or a variety of other methods described.
Several techniques exist for introducing foreign genetic
material into plant cells, and for obtaining plants that stably
maintain and express the introduced gene. Such techniques
include acceleration of genetic material coated onto
microparticles directly into cells (US Patents 4~945,050 to
Cornell and 5,141,131 to DowElanco). Plants may be transformed
using Agrobacterium technology, see US Patent 5,177,010 to
University of Toledo, 5,104,310 to Texas A&M, European Patent
Application 013162482, European Patent Applications 120516,
15941881, European Patent Applications 120516, 15941881 and
176, 112 to Schilperoot, US Patents 5, 149, 645, 5, 469, 976,
5, 464, 763 and 4, 940, 838 and 4, 693, 976 to Schilperoot, European
Patent Applications 116718, 290799~ 320500 all to MaxPlanck,
European Patent Applications 604662 and 627752 to Japan
Nicotiana, European Patent Applications 0267159, and 0292435
and US Patent 5,231,019 all to Ciba Geigy, US Patents 5,463,174
and 4,762,785 both to Calgene, and US Patents 5,004,863 and
5,159,135 both to Agracetus. Other transformation technology
includes whiskers technology, see U.S. Patents 5,302,523 and
5,464,765 both to Zeneca. Electroporation technology has also
been used to transform plants, see WO 87/06614 to Boyce
Thompson Institute, 5,472,869 and 5,384,253 both to Dekalb,
W09209&96 and W09321335 both to PGS. All of these
transformation patents and publications are incorporated by
reference. In addition to numerous technologies for
transforming plants, the type of tissue which is contacted with
the foreign genes may vary as well. Such tissue would include
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but would not be limited to embryogenic tissue, callus tissue
type I and II, hypocotyl, meristem, and the like. Almost all
plant tissues may be transformed during dedifferentiation using
appropriate techniques within the skill of an artisan.
Foreign genetic material introduced into a plant may
include a selectable marker. The preference for a particular
marker is at the discretion of the artisan, but any of the
following selectable markers may be used along with any other
gene not listed herein which could function as a selectable
marker. Such selectable markers include but are not limited to
aminoglycoside phosphotransferase gene of transposon Tn5 (Aph
II) which encodes resistance to the antibiotics kanamycin,
neomycin and 6418, as well as those genes which code for
resistance or tolerance to gl.yphosatet hygromycin;
methotrexate~ , phosphinothricin (bar)1 imidazolinones,
sulfonylureas and triazolopyrimidine herbicides, such as
chlorosulfuron; bromoxynil, dalapon and the like.
In addition to a selectable marker, it may be desirous to
use a reporter gene. In some instances a reporter gene may be
used without a selectable marker. Reporter genes are genes
which are typically not present or expressed in the recipient
organism or tissue. The reporter gene typically encodes for a
protein which provide for some phenotypic change or enzymatic
property. Examples of such genes are provided in K. Weising et
al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated
herein by reference. Preferred reporter genes include without
limitation glucuronidase (GUS) gene and GFP genes.
Once introduced into the plant tissue, the expression of
the structural gene may be assayed by any means known to the
art, and expression may be measured as mRNA transcribed,
protein synthesized, or the amount of gene silencing that
occurs (see U.S. Patent No. 5,583,021 which is hereby
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WO 2003/078577 PCT/US2003/007430
incorporated by reference). Techniques are known for the in
vitro culture of plant tissue, and in a number of cases, for
regeneration into whole plants (EP Appln No. 88810309.0).
Procedures for transferring the introduced expression complex
S to commercially useful cultivars are known to those skilled in
the art.
Once plant cells expressing the desired level of P450
enzyme are obtained, plant tissues and whole plants can be
regenerated therefrom using methods and techniques well-known
in the art. The regenerated plants are then reproduced by
conventional means and the introduced genes can be transferred
to other strains and cultivars by conventional plant breeding
techniques.
The following examples illustrate methods for carrying out
the invention and should be understood to be illustrative of,
but not limiting upon, the scope of the invention which is
defined in the appended claims.
- EXAMPLES
EXAMPLE I: DESTELOPMENT OF PLANT TISSUE .AND ETHYLENE TREATMENT
Plant Growth
Plants were seeded in pots and grown in a greenhouse for 4
weeks. The 4 week old seedlings were transplanted into
individual pots and grown in the greenhouse for 2 months. The
plants were watered 2 times a day with water containing 150ppm
NPK fertilizer during growth. The expanded green leaves were
detached from plants to do the ethylene treatment described
below.
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Cell Zine 78379
Tobacco line 78379, which is a burley line released by the
University of Kentucky was used as a source of plant material.
One hundred plants were cultured as standard in the art of
growing tobacco and transplanted and tagged with a distinctive
number (1-100). Fertilization and field management were
conducted as recommended.
Three quarters of the 100 plants converted between 20 and
1000 of the nicotine to nornicotine. One quarter of the 100
plants converted less than 5% of the nicotine to nornicotine.
Plant number 87 had the least conversion (20) while plant
number 21 had 100% conversion. Plants converting less than 30
were classified as non-converters. Self-pollinated seed of
plant number 87 and plant number 21, as well as crossed (21 x
87 and 87 x 21) seeds were made to study genetic and phenotype
differences. Plants from selfed 21 were converters, and 990 of
selfs from 87 were non-converters. The other 10 of the plants
from 87 showed low conversion (5-150). Plants from reciprocal
crosses were all converters.
Cell Zine 4407
Nicotiana line 4407, which is a burley line was used as a
source of plant material. Uniform and representative plants
(100) were selected and tagged. Of the 100 plants 97 were non-
converters and three were converters. Plant number 56 had the
least amount of conversion (1.20) and plant number 58 had the
highest level of conversion (96%). Self-pollenated seeds and
crossed seeds were made with these two plants.
Plants from selfed-58 were segregating in about a 3:1
converter to non-converter ratio. The 58-33 and 58-25 were
identified as homozygous converter and nonconverter plant
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lines, respectively. The stable conversion of 58-33 was
confirmed by analysis of its progenies of next generation.
Ethylene Treatment Procedures
Green leaves were detached from 2-3 month greenhouse grown
plants and sprayed with 0.3% ethylene solution (Prep brand
Ethephon (Rhone-Poulenc)). Each sprayed leaf was hung in a
curing rack equipped with humidifier and covered with plastic.
During the treatment, the sample leaves were periodically
sprayed with the ethylene solution. Approximately 24-48 hour
post ethylene treatment, leaves were collected for RNA
extraction. Another sub-sample was taken for metabolic
consituents analysis to determine the concentration of Leaf
metabolites and more specific constituents of interest such as
a variety of alkaloids.
As an example, alkaloids analysis could be performed as
follows. Samples (0.1 g) were shaken at 150 rpm with 0.5 ml 2N
NaOH, and a 5 ml extraction solution which contained quinoline
as an internal standard and methyl t-butyl ether. Samples were
analyzed on a HP 6890 GC equipped with a FID detector. A
temperature of 250°C was used for the detector and injector.
An HP column (30m-0.32nm-1°m) consisting of fused silica
crosslinked with 5o phenol and 95o methyl silicon was used at a
temperature gradient of 110-185 °C at 10°C per minute. The
column was operated at a flow rate at 100°C at l.7cm3min~'1 with
a split ratio of 40:1 with a 2~1 injection volume using helium
as the carrier gas.
EXAMPLE 2: RNA ISOLATION
For RNA extractions, middle leaves from 2 month old
greenhouse grown plants were treated with ethylene as
described. The 0 and 24-48 hours samples were used for RNA
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WO 2003/078577 PCT/US2003/007430
extraction. In some cases, leaf samples under the senescence
process were taken from the plants 10 days post flower-head
removal. These samples were also used for extraction. Total RNA
was isolated using Rneasy Plant Mini Kit (Qiagen, Inc.,
S Valencia, California) following manufacturer°s protocol.
The tissue sample was grinded under liquid nitrogen to a
fine powder using 'a DEPC treated mortar and pestle.
Approximately 100 mg of ground tissue was transferred to a
sterile 1.5 ml eppendorf tube. This sample tube was placed in
liquid nitrogen until all samples were collected. Then, 450.1
of Buffer RLT as provided in the kit (with the addition of (3-
Mercaptoethanol) was added to each individual tube. The sample
was vortexed vigorously and incubated at 56° C for 3 minutes .
The lysate was then, applied to the QIAshredder spin column
sitting in a 2-ml collection tube, and centrifuged for 2
minutes at maximum speed. The flow through was collected and
Ø5 volume of ethanol was added to the cleared lysate. The
sample is mixed well and transferred to an Rneasy mini spin
column sitting in a 2 ml collection tube. The sample was
Centrifuged for 1 minute at 10, OOOrpm. Next, 700,1 of buffer
RW1 was pipeted onto the Rneasy column and centrifuged for 1
minute at 10,OOOrpm. Buffer RPE was pipetted onto the Rneasy
column in a new collection tube and centrifuged for 1 minute at
10,000 rpm. Buffer RPE was again, added to the Rneasy spin
column and centrifuged for 2 minutes at maximum speed to dry
the membrane. To eliminate any ethanol carry over, the
memebrane was placed in a separate collection tube and
centrifuged for an additional 1 minute at maximum speed. The
Rneasy column was transfered into a new 1.5 ml collection tube,
and 40 ~.1 of Rnase-free water was pipetted directly onto the
Rneasy membrane. This final elute tube was centrifuged for 1
minute at 10,OOOrpm. Quality and quantity of total RNA was
analyzed by denatured formaldehyde gel and spectrophotometer.
!. 25
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Poly(A)RNA was isolated using Oligotex poly A RNA
purification kit (Qiagen Inc.) following manufacture's
protocol. .About 200 ~,g total RNA in 250 ~,1 maximum volume was
used. A volume of 250~Z1 of Buffer OBB and 25 ul of Oligotex
suspension was added to the 250 ~,l of total RNA. The contents
were mixed thoroughly by pipetting and incubated for 3 minutes
at 70°C on a heating block. The sample was then, placed at
room temperature for approximately 20 minutes. The
oligotex:mRNA complex was pelleted by centrifugation for 2
minutes at maximum speed. All but 50 pl of the supernatant was
removed from the microcentrifuge tube. The Sample was treated
further by OBB buffer. The oligotex:mRNA pellet was resuspended
in 400 ul of Buffer OW2 by vortexing. This mix was transferred
onto a small spin column placed in a new tube and centrifuged
for 1 minute at maximum speed. The spin column was transferred
to a new tube and an additional 400 ~Zl of Buffer OW2 was added
to the column. The tube was then centrifuged for 1 minute at
maximum speed.. The spin column was transferred to a final
l.5ml microcentrifuge tube. The sample was eluted with 60 ul
of hot (70 C) Buffer OEB. Poly A product was analyzed by
denatured formaldehyde gels and spectrophotometric analysis.
EXAMPLE 3: REVERSE TRANSCRIPTION-PCR
First strand cDNA was produced using Superscript reverse
transcriptase following manufacturer's protocol(Invitrogen, Carlsbad,
California). The poly A enriched RNA/oligo dT primer mix consisted of
less than 5 ~.g of total RNA, 2 ~Zl of IOmM dNTP mix, 1 p.l of Oligo
d (T) 1~_1$ (0.5ug/p.l) , and up to 10 pl of DEPC-treated water. Each
sample was incubated at 65° C for 5 minutes, then placed on ice for at
least 1 minute. A reaction mixture was prepared by adding each of the
following components in order: 2 ~.1 10X RT buffer, 4 ul of 25 mM
MgCl2, 2p.1 of 0.1 M DTT, and~1 ul of RNase OUT Recombinant RNase
;, 26
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WO 2003/078577 PCT/US2003/007430
Inhibitor. An addition of 9 ul of reaction mixture was pipetted to
each RNA/primer mixture and gently mixed. It was incubated at 42° C
for 2 minutes and 1 ~1 of Super Script II RT was added to each tube.
The tube was incubated for 50 minutes at 42° C. The reaction was
terminated at 70° C for 15 minutes and chilled on ice. The sample was
collected by centrifugation and 1 ul of RNase H was added to each tube
and incubated for 20 minutes at 37° C. The second PCR was carried out
with 200 pmoles of forward primer (degenerate primers as in Figure 75,
SEQ.ID Nos. 149-156) and 100 pmoles reverse primer (mix of l8nt oligo
d(T) followed by 1 random base) .
Reaction conditions were 94°C for 2 minutes and then performed 40
cycles of PCR at 94°C for 1 minute, 45° to 60°C fox 2
minutes, 72°C
for 3 minutes with a 72°C extension for an extra 10 min.
IS
Ten microliters of the amplified sample were analyzed by
electrophoresis using a 1% agarose gel. The correct size fragments
were purified from agarose gel.
E~~AMPLE 4: GENERATION OF PCR FRAGMENT POPULATIONS
PCR fragments from Example 3 were ligated into a pGEM-T Easy
Vector (Promega, Madison, Wisconsin) following manufacturer's
instructions. The ligated product was transformed into JM109
competent cells and plated on LB media plates for blue/white
selection. Colonies were selected and grown in a 96 well plate with
1.2 ml of LB media overnight at 37°C. Frozen stock was generated for-
all selected colonies. Plasmid DNA from plates were purified using
Beckman's Biomeck 2000 miniprep robotics with Wizard SV Miniprep kit
(Promega). Plasmid DNA was eluted with 100~.lwater and stored in a 96
well plate. Plasmids were digested by EcoR1 and were analyzed using 1%
agarose gel to confirm the DNA quantity and size of inserts. The
plasmids containing a 400-600 by insert were sequenced using an CEQ
2000 sequencer (Beckman., Fullerton, California). The sequences were
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WO 2003/078577 PCT/US2003/007430
aligned with GenBank database by BLAST search. The P-450 related
fragments were identified and further analyzed.
EXAMPLE 5; CONSTRUCTION OF CDNA LIBRARY
A cDNA library was constructed by preparing total RNA from
ethylene treated leaves as follows. First, total RNA was extracted
from ethylene treated leaves of tobacco line 58-33 using a modified
acid phenol and chloroform extraction protocol. Protocol was modified
to use one gram of tissue that was ground and subsequently vortexed in
5 ml of extraction buffer (100 mM Tris-HC1, pH 8.5; 200 mM NaCl;
lOmM EDTA; 0.5o SDS) to which 5 ml phenol (pH5.5) and 5 ml chloroform
was added. The extracted sample was centrifuged and the supernatant
was saved. This extraction step was repeated 2-3 more times until the
supernatant appeared clear. Approximately 5 ml of chloroform was
added to remove trace amounts of phenol. RNA was precipitated from
the combined supernatant fractions by adding a 3-fold volume of ETOH
and 1/10 volume of 3M NaOAc (pH5.2) and storing at -20° C for 1 hour.
After transferring to Corex glass container it was centrifuged at
9,000 RPM for 45 minutes at 4° C. The pellet was washed with 700
ethanol and spun for 5 minutes at 9,000 RPM at 4° C. After drying the
pellet, the pelleted RNA was dissolved in 0.5 ml RNase free water.
The pelleted RNA was dissolved in 0.5 ml RNase free water. The
quality and quantity of total RNA was analyzed by denatured
formaldehyde gel and spectrophotometer, respectively.
The resultant total RNA was isolated for poly A+ RNA using an
Oligo(dT) cellulose protocol (Invitrogen) and MiCrocentrifuge spin
columns (Invitrogen) by the following protocol. Approximately twenty
mg of total RNA was subjected to twice purification to obtain high
quality poly A+ RNA. Poly A+ RNA product was analyzed by performing
denatured formaldehyde gel and subsequent RT-PCR of known full-length
genes to ensure high quality of mRNA. In addition, Northern analysis
was performed on the poly A+RNA from ethylene treated non-converter
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WO 2003/078577 PCT/US2003/007430
leaves, zero hour ethylene treated converter leaves and ethylene
treated converter leaves using the full-length p450 as probe. The
method was based on the protocol provided by the manufacturer's
instructions (KPL RNADetector Northern Blotting Kit, Gaithersburg,
Maryland) using 1.8 ~,g of polyA+RNA for each sample. RNA containing
gels were transferred overnight using 20X SSC as a transfer buffer.
Next, poly A+ RNA was used as template to produce a cDNA library
employing cDNA synthesis kit, ZAP-cDNA synthesis kit, and ZAP-cDNA
Gigapack III gold cloning kit (Stratagene, La Jolla, California). The
method involved following the manufacture's protocol as specified.
Approximately 8 ~.g of poly A+ RNA was used to construct cDNA library.
Analysis of the primary library revealed about 2.5 x 106 - 1x 10' pfu.
A quality background test of the library was completed by
complementation assays using TPTG and'X-gal, where recombinant plaques
was expressed at more than 100-fold above the background reaction.
A more quantitative analysis of the library by random PCR showed
that average size of insert cDNA was approximately 1.2 kb. The method
used a two-step PCR method as followed. For the first step, reverse
primers were designed based on the preliminary sequence information
obtained from P450 fragments. The designed reverse primers and T3
(forward) primers were used amplify corresponding genes from the cDNA
library. PCR reactions were subjected to agarose electrophoresis and
the corresponding bands of high molecular weight were excised,
purified, cloned and sequenced. Tn the second step, new primers
designed from 5'UTR or the start coding region of P450 as the forward
primers together with the reverse primers (designed from 3'UTR of
P450) were used in the subsequent PCR to obtain full-length P450
clones.
The P450 fragments were generated by PCR amplification from the
constructed cDNA library as described in example 3 with the exception
of the reverse primer. The T7 primer located on the plasmid downstream
of cDNA inserts (see Figure 75), was used as a reverse primer. PCR
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fragments were isolated, cloned and sequenced as described in Example
4.
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Numerous modifications and variations in practice of the
invention are expected to occur to those skilled in the art upon
consideration of the foregoing detailed description of the invention.
Consequently, such modifications and variations are intended to be
included within the scope of the following claims.
EXAMPLE 6e CHARACTERIZATION OF CLONED FRAGMENTS - REVERSE SOUTHERN
BLOTTING ANALYSIS
Nonradioactive large scale reverse southern blotting assay was
performed on all P450 clones identified in above examples to detect
the differential expression. It was observed that the level of
expression among different P450 clusters was very different. Further
real time detection was conducted on those with high expression.
Nonradioactive southern blotting procedures were conducted as
follows.
1) Total RNA was extracted from ethylene treated converter (58-
33) and nonconverter (58-25) leaves using the Qiagen Rnaeasy kit as
described in Example 2.
2) Probe was produced by biotin-tail labeling a single strand
cDNA derived from poly A enriched RNA generated in above step. This
labeled single strand cDNA was generated by RT-PCR of the converter
and nonconverter total RNA (Invitrogen) as described in example 3 with
the exception of using biotinalyted oligo dT as a primer (Promega);
These were used as a probe to hybridize with cloned DNA.
3) Plasmid DNA was digested with restriction enzyme EcoR1 and
run on agarose gels. Gels were simultaneously dried and transferred
to two nylon membranes (Biodyne B). One membrane was hybridized with
converter probe and the other with nonconverter probe. Membranes were
UV-crosslinked (auto crosslink setting, 254 nm, Stratagene,
Stratalinker ) before hybridization.
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Alternatively, the inserts were PCR amplified from each plasmid
using the sequences located on both arms of p-GEM plasmid, T3 and SP6,
as primers. The PCR products were analyzed by running on a 96 well
Ready-to-run agarose gels. The confirmed inserts were dotted on two
nylon membranes. One membrane was hybridized with converter probe and
the other with nonconverter probe.
4). The membranes were hybridized and washed following
manufacture's instruction with the modification of washing stringency
(Enzo Diagnostics, Inc, Farmingdale, NY). The membranes were
prehybridized with hybridization buffer (2x SSC buffered formamide,
containing detergent and hybridization enhancers) at 42°C for 30 min
and hybridized with 10,1 denatured probe overnight at 42°C. The
membranes then were washed in 1X hybridization wash buffer 1 time at
room temperature for 10 min and 4 times at 68°C for 15 min. The
membranes were ready for the detection.
5) The washed membranes were detected by alkaline phosphatase
labeling followed by NBT/BCIP colometric detection as described in
manufacture's detection procedure (Enzo Diagnostics, Inc.). The
membranes were blocked for one hour at room temperature with 1x
blocking solution, washed 3 times with 1X detection reagents for 10
min, washed 2 times with 1x predevelopment reaction buffer for 5 min
and then developed the blots in developing solution for 30-45 min
until the dots appear. All reagents were provided by manufacture (Enzo
Diagnostics, Inc).
In some cases, one step RT-PCR (Gibco Kit, Carlsbad, California)
was performed on,the total RNA's from non-converter (58-25) and
converter (58-33) lines using primers specific to the P-450 fragments.
Comparative RT-PCR was conducted as follows:
1) Total RNA from ethylene treated converter (58-33) and
nonconverter (58-25) plant leaves was extracted as described
in example 2.
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2) Poly(A) RNA from total RNA was extracted using Qiagen kit as
described in example 2.
3) One step RT-PCR was conducted using primers specific to
cloned P450 following the manufactures procedure(Invitogen). The poly
A enriched RNA was added to the reaction mix, along with, 25~,~ of 2X
Reaction Mix, 1~,1 of 10~,1VI Sense Primer, 1~,1 of 10 ~.tSVI Anti-sense
Primer,
1 ~,l of RT/ Platinum taq Mix, and up to 50 ~,1 of water. Reaction
conditions were 50°C for 20 minutes and then 94 C for 2 min, performed
40 cycles of PCR at 94°C for 30 sec, 55° to for 30 sec,
70°C for 1
minute with a 72°C extension for an extra 10 min. Ten microliters of
the amplified sample were analyzed by electrophoresis using a 10
agarose gel.
EXAMPLE 7: CHARACTERIZATION OF CLONED FRAGMENTS - NORTHERN BLOT
ANALYSIS
Alternative to Southern Blot analysis, some membranes were
hybridized and detected as described in the example of northern
blotting assays. Northern Hybridization was used to detect mRNA
differentially expressed in Nicotiana as follows.
First step, probe preparation: the random priming method was
used to prepare probes from cloned p450 DNA fragments (Random Primer
DNA Biotinylation Kit, KPL). The following components were mixed:
0.5~,g DNA template (boiled in a water bath for 5-10 minutes and
chilled on ice before use); 1X Random Primer Solution; 1X dNTP mix;
10 units of Klenow and water was added to bring the reaction to 50,1.
The mixture was incubated in 37 °C for 1-4 hours. The reaction was
stopped with 2~,I of 200 mM EDTA. The probe was denatured by incubating
at 95°C for 5 minutes before use.
Second step, sample preparation: The RNA samples were prepared
from ethylene treated and non-treated fresh leaves, and senescence
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leaves. In some cases poly A enriched RNA was used. Approximately
15~,g total RNA or 1.8~Cg mRNA (Methods of RNA and mRNA extraction are
described in Example 5) was brought to equal volume with DEPC H20(5-10
~.1). The same volume loading buffer (1 x MOPS; 18.5 ~ Formaldehyde; 50
o Formamide; 4 o Fico11400; Bromophenolblue) and 0.5 ~,1 EtBr (0.5~.g
/~,1) were added. The samples were heated at 90 °C for 5 minutes, and
chilled on ice.
Third step, separation of RNA by electrophoresis: Samples were
subjected to electrophoresis on a formaldehyde gel (1 o Agarose, 1 x
MOPS, 0.6 M Formaldehyde) with 1XMOP buffer (0.4 M
Morpholinopropanesulfonic acid; 0.1 M Na-acetate-3 x H20; 10 mM EDTA;
adjust to pH 7.2 with NaOH). RNAs were transferred to Hybond-N+
membrane (Nylon, Amersham Pharmacia Biotech) by capillary method in 10
X SSC buffer (1.5 M NaCl; 0.15 M Na-citrate ) for 24 hours. Membranes
with RNA samples were UV-crosslinked (auto crosslink setting, 254 nm,
Stratagene, Stratalinker ) before hybridization.
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Fourth step, hybridization: The membrane was prehybridized for 1-
4 hours at 42 °C with 5-10 ml prehybridization buffer (5 x SSC; 50 0
Formamide; 5 x Denhardt's-solution; 1 % SDSs 100~.g/ml heat-denatured
sheared non- homologous DNA). Old prehybridization buffer was
discarded, and new prehybridization buffer and probe were added. The
hybridization was carried out over night at 42 °C. The membrane was
washed for 15 minutes with 2 x SSC at room temperature, followed by a
wash with 2 x SSC, 0.1 % SDS at 65 °C for 2 times, and a final wash
with 0.1 x SSC, or more wash with 0.1 x SDS at 65 °C (optional).
Fifth step, detection: AP-Streptavidin and CDP-Star were used to
detect the hybridization signal( KPL's DNA Detector Northern blotting
Kit). The membrane was blocked with 1X Detector Block Solution for 30
minutes at room temperature. The blocking buffer was discarded and the
membrane was incubated in new 1X detector Block Solution with 1:10,000
AP-SA at room temperature for 1 hour. The membrane was washed in 1X
Phosphatase Wash Solution for 3 times, followed by a wash with 1X
Phosphatase Assay Buffer for two times. The signal was detected with
CDP-Star Chemiluminescent Substrate. The wet membrane was exposed to
X-Ray film under saranT"~ wrap. The results were analyzed and recorded.
A major focus of the invention was the discovery of novel genes
that may be induced as a result of ethylene treatment or play a key
role in tobacco leaf quality and constituents. As shown in the table
below, Northern blots were useful in determining which genes were
induced by ethylene treatment relative to non-induced plants.
Interestingly, not all fragments were affected similarly in the
converter and nonconverter< The cytochrome P450 fragments of interest
were partially sequenced to determine their structural relatedness.
This information was used to subsequently isolate and sequence full
length gene clones. Functional analysis utilizing down-regulation
methods was performed in whole plants with the fragments genes.
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Induced mRNA
Fragments Expression
Ethylene Treatment
Converter w Nonconverter
D186-AH4 +
D56-AC7 + +
D56=AG12 +
D56-AC12 + +
D70A-AB5 + +
D73-AC9 + +
D70A-AA12 + +
D73A-AG3 +
D73A-AE10 +
D35-AG11 +
D58-AD4 + ~ +
D34-52 +' +
D56-AG6 + +
EXAMPLE 8: NUCLEIC ACID IDENTITY AND STRUCTURE RELATEDNESS OF
ISOLATED NUCLETC ACID FRAGMENTS
Over 100 cloned P450 fragments were sequenced in conjunction with
Northern blot analysis to determine their structural relatedness. The
approach used utilized forward primers based either of two common P450
motifs located near the carboxyl-terminus of the P450 genes. The
forward primers corresponded to cytochrome P450 motifs FXPERF or
GRRXCP(A/G) as denoted in Figure 1. The reverse primers used standard
primers from either the plasmid, SP6 or T7 located on both arms of
pGEM plasmid, or a poly A tail. The protocol used is described below.
Spectrophotometry was used to estimate the concentration of
starting double stranded DNA following the manufacturer's protocol
(Beckman Coulter). The template was diluted with water to the
appropriate concentration, denatured by heating at 95° C for 2
minutes, and subsequently placed on ice. The sequencing reaction was
prepared on ice using 0.5 to l0ul of denatured DNA template, 2 lZl of
1.6 pmole of the forward primer, 8 ul of DTCS Quick Start Master Mix
and the total volume brought to 20 ul with water. The thermocycling
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program consisted of 30 cycles of the follow cycle: 96° C for 20
seconds, 50° C for 20 seconds, and 60° C for 4 minutes followed
by
holding at 4° C.
The sequence was stopped by adding 5 ul of stop buffer (equal
volume of 3M NaOAc and 100mM EDTA and 1 ul of 20 mg/ml glycogen). The
sample was precipitated with 60 ~1 of cold 95o ethanol and centrifuged
at 6000g for 6 minutes. Ethanol was discarded. The pellet was 2
washes with 200 ul of cold 70o ethanol. After the pellet was dry, 40
~1 of SZS solution was added and the pellet was resuspended. A layer
of mineral oil was over laid. The sample was then, placed on the CEQ
8000 Automated Sequencer for further analysis.
In order to verify nucleic acid sequences, nucleic acid sequence
was re-sequenced in both directions using forward primers to the
FXPERF or GRRXCP(A/G) region of the P450 gene or reverse primers to
either the plasmid or poly A tail. All sequencing was performed at
least twice in both directions.
The nucleic acid sequences of cytochrome P450 fragments were
compared to each other from the coding region corresponding to the
first nucleic acid after the region encoding the GRRXCP(A/G) motif
through to the stop codon. This region was selected as an indicator
of genetic diversity among P450 proteins. A large number of
genetically distinct P450 genes, in excess of 70 genes, was observed
similar to that of other plant species. Upon comparison of nucleic
acid sequences, it was found that the genes could be placed into
distinct sequences groups based on their sequence identity. It was
found that the best unique grouping of P450 members was determined to
be those sequences with 75o nucleic acid identity or greater (shown in
Table I). Reducing the percentage identity resulted in significantly
larger groups. A preferred grouping was observed for those sequences
with 81% nucleic acid identity or greater, a more preferred grouping
91% nucleic acid identity or greater, and a most preferred grouping
for those sequences 99o nucleic acid identity of greater. Most of the
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groups contained at least two members and frequently three or more
members. Others were not repeatedly discovered suggesting that
approach taken was able to isolated both low and high expressing mRNA
in the tissue used.
Based on 75o nucleic acid identity or greater, two cytochrome
P450 groups were found to contain nucleic acid sequence identity to
previously tobacco cytochrome genes that genetically distinct from
that within the group. Group 23, showed nucleic acid identity, within
the parameters used for Table I, to prior GenBank sequences of
GI:1171579 (CAA64635) and GI:14423327 (or AAK62346) by Czernic et al
and Ralston et al, respectively. GI:1171579 had nucleic acid identity
to Group 23 members ranging 96.90 to 99.50 identity to members of
Group 23 while GI:14423327 ranged 95.40 to 96.90 identity to this
group. The members of Group 31 had nucleic acid identity ranging from
76.7% to 97.8% identity to the GenBank reported sequence of
GI:14423319 (AAK62342) by Ralston et al. None of the other P450
identity groups of Table 1 contained parameter identity, as used in
Table 1, to Nicotiana P450s genes reported by Ralston et al, Czernic
et al., Wang et al or ZaRosa and Smigocki.
As shown in Figure 76, consensus sequence with appropriate
nucleic acid degenerate probes could be derived for group to
preferentially identify and isolate additional members of each group
from Nicotiana plants.
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Table I: Nicotiana P450 Nucleic Acid Sequence Identity
Groups
GROUP FRAGMENTS
1 D58-BG7 (SEQ ID No.:1), D58-AB1 (SEQ ID No.:3); D58-BE4
(SEQ ID No.:7)
2 D56-AH7 (SEQ ID No.:9); l3a-5 (SEQ ID No.:l1)
D
3 D56-AG10 (SEQ ID No.:l3); D35-33 (SEQ ID No.:l5); D34-
62 ) (SEQ ID No.:l7)
4 D56-AA7 (SEQ ID No.:l9); D55-AE1 (SEQ ID No.:22); 185-
BD3 (SEQ ID No.:143)
5 D35-BB7 (SEQ ID No.:23); D177-BA7 (SEQ ID No.:25) D56A-
AB6 (SEQ ID No.:27); D144-AE2 (SEQ ID No.:29)
6 D56-AG11 (SEQ ID No.:31); D179-AA1 (SEQ ID No.:33)
7 D56-AC7 (SEQ ID No.:35); D144-AD1 (SEQ ID No.:37)
8 D144-AB5 (SEQ ID No.:39)
9 D181-AB5 (SEQ ID No.:41); D73-AC9 (SEQ ID No.:43)
10 D56-AC12 (SEQ ID No.:45)
11 D58-AB9 (SEQ ID No.:47); D56-AG9 (SEQ ID No.:49); D56-
AG~ (SEQ ID No.:51); D35-BG11 (SEQ TD No.:53); D35-42 (SEQ
ID No.:55);
D35-BA3
(SEQ ID
No.:57);
D34-57
(SEQ ID
No.: 59); D34-52 (SEQ ID No.:61);
D34-25 (SEQ ID No.:63)
12 D56-AD10 (SEQ ID No.:65)
13 56-AA11 (SEQ ID No.:67)
14 D177-BD5 (SEQ ID No.:69); D177-BD7 (SEQ ID No.:83)
15 D56A-AG10 (SEQ ID No.:71) ; D58-BC5 (SEQ ID No.:73);
D58-AD12
(SEQ ID
No.:75)
16 D56-AC11 (SEQ ID No.:77); D35-39 (SEQ ID No.:79); D58-
BH4 (SEQ ID No.:81); D56-AD6
(SEQ ID No.:87)
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17 D73A-AD6 (SEQ ID No.:89); D70A-BA11 (SEQ ID No.:91);
D7 OA-BB5 ( SEQ ID No . : 93 )
18 D7 OA-.ABS { SEQ ID No > : 95 ) ; D7 OA-AA8 ( SEQ ID No . : 9'7 )
19 D70A-AB8 (SEQ ID No.:99); D70A-BH2 {SEQ ID No.:101);
D70A-AA4 (SEQ TD N0.:103)
20 D70A-BAl (SEQ ID No.:105); D70A-BA9 (SEQ ID No.:107);
D17 6-BG2 ( SEQ TD No . : 141 )
21 D70A-BD4 (SEQ ID No.:109)
22 D181-AC5 (SEQ ID No.:111); D144-AH1 (SEQ ID No.:113);
D34-65 (SEQ ID No.:115)
23 D35-BG2 (SEQ ID No.:117)
24 D73A-AH7 (SEQ ID No.:119)
25 D58-AA1 (SEQ ID No.:121); D185-BC1 (SEQ ID No.:133);
D185-BG2 (SEQ ID No.:135)
26 D73-AE10 (SEQ ID No.:123)
27 D56-AC12 (SEQ ID No.:125)
28 D177-BF7 (SEQ ID No.:127); D185-BE1 (SEQ ID No.:137);
185-BD2 {SEQ ID No.:139)
29 D73A-AG3 (SEQ ID No.:129)
30 D70A-AA12 (SEQ ID No.:131); D176-BF2 (SEQ ID No.:85)
31 D176-BC3 (SEQ ID No.:145)
32 D176-BB3 (SEQ ID No.: 147)
33 D186-AH4 (SEQ ID No.:5)
EXAMPLE 9: RELATED AMINO ACID SEQUENCE IDENTITY OF ISOLATED
NUCLEIC ACID FRAGMENTS
The amino acid sequences of nucleic acid sequences obtained for
cytochrome P450 fragments from Example 8 were deduced. The deduced
region corresponded to the amino acid immediately after the
'' 40
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GXRXCP(A/G) sequence motif to the end of the carboxyl-terminus, or
stop codon. Upon comparison of sequence identity of the fragments, a
unique grouping was observed for those sequences with 70o amino acid
identity or greater. A preferred grouping was observed for those
sequences with 80o amino acid identity or greater, more preferred with
90o amino acid identity or greater, and a most preferred grouping for
those sequences 99o amino acid identity of greater. The groups and
corresponding amino acid sequences of group members are shown in
Figure 2. Several of the unique nucleic acid sequences were found to
have complete amino acid identity to other fragments and therefore
only one member with the identical amino acid was reported.
The amino acid identity for Group 19 of Table II corresponded to
three distinct groups based on their nucleic acid sequences. The
amino acid sequences of each group member and their identity is shown
in Figure. 77. The amino acid differences are appropriated marked.
At least one member of each amino acid identity group was
selected for gene cloning and functional studies using plants. In
addition, group members that are differentially affected by ethylene
treatment or other biological differences as assessed by Northern and
Southern analysis were selected for gene cloning and functional
studies. To assist in gene cloning, expression studies and whole
plant evaluations, peptide specific antibodies will be prepared on
sequence identity and differential sequence.
Table II: Nicotiana P450 Amino Acid Sequence Identity Groups
GROUP FRAGMENTS
1 D58-BG7 ( SEQ ID No . : 2 ) , D58-.AB1 ( SEQ TD No . : 4 )
2 D58-BE4 (SEQ ID No.:8)
3 D56-AH7 (SEQ ID No.:lO); Dl3a-5 (SEQ ID No.:l2)
__ - 41
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4 D56-AG10 (SEQ ID No.:l4); D34-&;
(SEQ ID No.:l 8)
D56-AA7 (SEQ ID N0.:20); D56-AEl (SEQ ID N0.:22); 185-
BD3 (SEQ ID
No.:144)
5 6 D35-BB7 (SEQ ID No. :24) ; D177-BA7 (SEQ ID NO. :26)
;
D56A-AB6 ID No.:28); D144-AE2 (SEQ ID No.:30)
(SEQ
7 D56-AG11 (SEQ ID No.:32); D179-AA1 (SEQ ID No.:34)
8 D56-AC7 (SEQ ID No.:36); D144-AD1 (SEQ ID No.:38)
9 D144-AB5 (SEQ ID No.:40)
10 DI81-AB5 {SEQ ID No.:42); D73-AC9 {SEQ ID No.:44)
11 D56-AC12 (SEQ ID No.:46)
12 D58-AB9 {SEQ ID No.:48); D56-AG9 {SEQ ID No.:50); D56-
AG6 (SEQ ID
No.:52);
D35-BG11
(SEQ
ID No.:54);
D35-42
(SEQ
TD No.:56);
D35-BA3
(SEQ ID
No.:58);
D34-57
(SEQ ID
No.: 60); D34-52
{SEQ
ID No.:62)
13 D56AD10 (SEQ ID No.:66)
14 56-AA11 (SEQ ID No.:68)
15 D177-BD5 (SEQ ID No.:70); D177-BD7 (SEQ ID No.:84)
16 D56A-AG10
(SEQ
ID No.:72);
D58-BC5
(SEQ
ID No.:74);
D58-AD12 ID No.:76)
(SEQ
17 D56-AC11 (SEQ ID No.:78); D56-AD6 (SEQ ID No.:88)
18 D73A-AD6 (SEQ ID No.90:); D70A-BB5 (SEQ ID No.:94)
l9 D70A-AB5 (SEQ ID No.:96); D70A-AB8 (SEQ ID No.:100);
D70A-BH2 ID No.:102); D70A-AA4 (SEQ ID No.:104); D70A-
{SEQ
BA1 (SEQ ID
No.:106);
D70A-BA9
(SEQ
ID No.:108);
D176-BG2
(SEQ ID No.:142)
20 D70A-BD4 (SEQ ID No.:110)
21 D181-AC5 {SEQ ID No.:112); D144-AH1 (SEQ ID No.:114);
D34-65 (SEQ
ID No.:116)
22 D35-BG2 {SEQ ID No.:118)
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23 D73A-AH7 (SEQ ID No.:120)
24 D58-AA.1 (SEQ ID No.:122); D185-BC1 (SEQ ID No.:134);
D185-BG2 (SEQ ID No.:136)
25 D73-.AE10 (SEQ ID No.:124)
26 D56-AC12 (SEQ ID No.:126)
27 D177-BF7 (SEQ ID No.:128); 185-BD2 (SEQ ID No.:140)
28 D73A-AG3 (SEQ ID No.:130)
29 D70A-AA.12 (SEQ ID No.:132); D176-BF2 (SEQ
ID No.:86)
30 D176-BC3 (SEQ ID No.:146)
31 D176-BB3 (SEQ ID No.:148)
32 D186-AH4 (SEQ ID No.:6)
EXAMPLE 10: CLONING OF FULL LENGTH cDNA P450 CLONES
A cDNA library was constructed by preparing total RNA from
ethylene treated leaves as follows. First, total RNA was
extracted from ethylene treated leaves using a~ modified acid
phenol and chloroform extraction protocol. Protocol was
modified to use one gram of tissue that was ground and
subsequently vortexed in 5 ml of extraction buffer (100 mM
Tris-HC1, pH 8.5; 200 mM NaCl; lOmM EDTA; 0.5o SDS) t0 Which
5 ml phenol (pH5.5) and 5 ml chloroform was added. The
extracted sample was centrifuged and the supernatant was saved.
This extraction step was repeated 2-3 more times until the
supernatant appeared clear. Approximately 5 ml of chloroform
was added to remove trace amounts of phenol. RNA was
precipitated from the combined supernatant fractions by adding
a 3-fold volume of ETOH and 1/10 volume of 3M NaOAc (pH5.2) and
storing at -20° C for 1 hour. After transfering to Corex glass
container it was centrifuged at 9,000 RPM for 45 minutes at 4°
._ 43
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C. The pellet was~washed with 70o ethanol and spun for 5
minutes at 9,000 RPM at 4° C. After drying the pellet, the
pelleted RNA was dissolved in 0.5 ml RNase free water. The
pelleted RNA was dissolved in 0.5 ml RNase free water. The
quality and quantity of total RNA was analyzed by denatured
formaldehyde gel and spectrophotometer, respectively.
The resultant total RNA was isolated for poly A+ RNA using
an Oligo(dT) cellulose protocol (Tnvitrogen) and
Microcentrifuge spin columns (Invitrogene) by the following
protocol. Approximately twenty mg of total RNA was subjected
to twice purification to obtain high quality poly A+ RNA. Poly
A+ RNA product was analyzed by performing denatured
formaldehyde gel and subsequent RT-PCR of known full-length
genes to ensure high quality of mRNA. In addition, Northern
analysis was performed on the poly A+RNA from ethylene treated
non-converter leaves, zero hour ethylene treated converter
leaves and ethylene treated converter leaves using the full-
length p450 as probe. The method was based on the protocol
provided by the manufacturerPs instructions (KPh RNADetector
Northern Blotting Kit) using 1.8 ug of polyA+RNA for each
sample. RNA containing gels were transferred overnight using
20X SSC as a transfer buffer.
Next, poly A+ RNA was used as template to produce a cDNA
library employing cDNA synthesis kit, ZAP-cDNA synthesis kit,
and ZAP-cDNA Gigapack III gold cloning kit (Stratagene). The
method involved following the manufacture s protocol as
specified. Approximately 8 ug of poly A+ RNA was used to
construct cDNA library. Analysis of the primary library
revealed about 2.5 x 106 - 1x 107 pfu. A quality background
test of the library was completed by a- complementation using
IPTG and X-gal, where recombinant plaques was expressed at more
than 100-fold above the background reaction.
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A more quantitative analysis of the library by random PCR
showed that average size of insert cDNA was approximately 1.2
kb. The method used a two-step PCR method as followed. For
the first step, reverse primers were designed based on the
S preliminary sequence information obtained from p450 fragments.
The designed reverse primers and T3 (forward) primers were used
amplify corresponding genes from the cDNA library. PCR
reactions were subjected to agarose electrophoresis and the
corresponding bands of high molecular weight were excised,
purified, cloned and sequenced. In the second step, new
primers designed from 5'UTR or the start coding region of p450s
as the forward primers together with the reverse primers
(designed from 3'UTR of p450) were used in the subsequent PCR
to obtain full-length p450 clones.
Full-length p450 genes were isolated by PCR method from
constructed cDNA library. Two steps of PCR were used to clone
the full-length genes. In the first step PCR, unspecific
reverse primer (T3) and specific forward primer (generated from
the downstream sequence of P450s) were used to clone the 5'end
of the P450s from cDNA library. PCR fragments were isolated,
cloned and sequenced for designing the forward primers in next
step PCR. Two specific primers were used to clone the full
length p450 clones in the second step PCR. The clones were
subsequently sequenced.
Numerous modifications and variations in practice of the
invention are expected to occur to those skilled in the art
upon consideration of the foregoing detailed description of the
invention. Consequently, such modifications and variations are
intended to be included within the scope of the following
claims.
