Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ 2181418
Irlventor (s): ~rian A. 1arkin~
L~rry R. ~
Craig ~. Colemzm
ll~ANSGENES W~I~ FLOURY~ ~EN13 SIGNAL ~
P~C~~ OF S~ TNV~n~
The present invention ~-slateb to a L~ th~t
contains a polynucleotide fiegm~nt encoding at least a
portion of a sign~l se~uenc~ fro~ a floury2 g-ne. For
5 eYample, a transgene within the inv~ntlon al&o cah.
contain a ~ecand segmQnt ~:oding rOr ~n al,~L~ iCIlly
high-value protein, such th,~t the l~_ , - - e ~ ~,ies a
fusion protein cosprised of' the si~nal s~u~:...,e moiety
and a~ino acid sequenc~ of ths high-value protein. ~Ae
O present inventlon also rel,~tes to cereal plant3 that
contain a transgene, such as millet or 60r~hum plants
c~ ihin~ ~ naize or other heterologou~ flou~y2 gene.
~ etween 50~ and 60% of the ~rotein Ln ~aize kernels
consi6ts of a D~iYt-~re Or pr~la~ain storago proteins known
15 as "zeins," which are D~ nt;Ally devoid Or lysine. This
makes the seed nutritionally inferior for v.,~,~J~ ic
Anis~ls The lysiné d~ficiency of saize spurred
extensive efforts to id~ntify rutants with higher levels
of thi6 e~sential amino acid. The mai2e floL~ry2 ~flZ~
20 mutant ~as first described by Emerson et al., and was
reported to re6ult ~rom zl 5D~;~ ihant mutation that
causes ~ ~ioft, starchy emiosper;3. CO~N_~ U~ S
AGXICULTORAL EXPRR~MENTAI STA~ON ~PORT 180 (l935~.
~The content~i of this docu~lent and all other6 ~ntioned
25 herein ~re incorpor~ted by ~eferencQ. ~ fl2 allele
occurs on the short arm o~ 4, ~nd for ~any
years it served as ~ u~eful genetic sarker for the ~hort
arm o~ LI ~~ ~ 4.
In lg64, 1'12 ~nd anothl~r ~ut~tion in ~ize, Op~qUGZ
30 ~o2~, took on special in~erest when it wa6 reporte~ that
both of theso ~utations leall to a ~ubstantial increase in
lysine content of naize geed8. Nelgo- et al., Sci~c~
2'i81418
--2--
150; i46~3-7~ 11965), and ?~ert~ e~ ~1., soience l- S:
27g-80 (1964~ . Kernels in normal maize ~Qn~yy 3 average
around 0.20 to 0.25~ ly~ine, whlle kernels rrO~ o2 and
fl2 mutations hav~ lysine contents of 0.3 to 0.35%. I~ut
S the soft starchy endu~ a~sociated ~ith thQ fl2 and o2
~L~ Ly~ causes ths lc~n~ to be susceptible to
r- --nir~ll das~age, whicn cr~ates a higher s~F~eptihi1ity
to in~cct and ~ungal da~age. co~s2ciusn~1y, neither
mutant gain~d Yide~pread co~ercial applic~tion.
For many years, o2 ancl rl2 were considered to be
de~ects of genes reclulating zein synt~e~i~. This
conc~usion was based on the significant and fairly
specific efiect these r~utations have on storage protein
synthesis. Both o2 2nd fl2 reduce zein synthesis by
15 about 50S of the wild type ~evel, with thQ o2 ~utation
specifically a2'~ecting the 22-kDd 4-zeins, and th~ ~12
mutation ec~lly affecting synthesis of all classe21 of
zelns .
Other distinctiv~ hi~sh~m~r~l di~erences have be2n
20 reported for o~ and fl2 mutant~. Proteln bodies in both
o2 and fl2 are smallar than normal, but fl2-encodad
protein bodi~s are asy~ettical and m~hAr~r~ co~parad to
the spherical protein boc~i~s o~ nor~al and o2 ~I~A~perm.
Lending ~ Larkins, Pli~nt C811 1: 123-133 (1989). ~he o2
25 mutation is recessi~e, ~hile th~ ~12 _utation is
semiao~inant, with thc~ seYerit~ of the p~_..o~y~"e
correlated to dosage of t~-e ~tant allele.
qh~ hy~othesis that o;! is a zein regulatory qen2 was
conf irmed eventually, fc~llo~ing its tagging with a
transposable el21nent. Ihis led to the ~ r cloning
of 02 and the A ' ~tion that it encc~des a leucine
z~ppQr-type tr~n~cription factor that binds th~ yL. ~e.
o~ certain ~--2Qin genes and controls e~cpression o~ tbe
2Z-~cDa fa~ily of c~-zein genes. Schmidt et ~1., Science
23~: 96~-63 (1987). N~erous atte~pt~ to tag rl2 by a
~imilar strat~cly were ~ . ~ce,~rul, and the ba~i~ of the
~12 defect r2mained unknc~-n.
~ 2181418
--3--
The failure to discern the nature of t3a ~12 defsct
c(~nt~ despite ~airly ext~!nsive study of this ~hutant.
Several studie~ not~d the appearanc~ of an unusual ~-zein
protein in rl2 with a nolecu3.ar w~ight of 24 kDa, higher
s than normal zein. Lee et A7., Biocham. Genet. ~: 641-50
(1976); Soave et ~ ydic~l 23: 145-5~ (l97a); GalantQ
~t al., ~ol. Gen. G n~t. ~92: 316-21 ~1983). ThQ level
o~ this protein w2s found to be dependent on the dosage
o~ the f l Z allele . A high ~ lon of b-70, the
10 maize ho~D.ologus of the BiP ch.~peronin, al~o Wd5 reported
to be associated with protein botie~ in fl2. Zhanq ~
Boston, Protoplasma 171: 142--52 ~1~93~. 8iP is a ~e~ber
of the hsp-70 protein faslily that bindG malfolded
polypeptide chdins. The levQl of b-70 is a~ected by the
15 dosage of flZ double m~tants, as is the degree to whic
the protein bodieS become ~i~5h~r~". It al2~0 was
reported th~t in o2/~12 douo:le 7~utants, thc unu~ual 24-
kDa ~-z~in W~5 not synthesize~ and the Dorphology of the
protein bodios was ~imilar to that ln o2. ~hus, the o2
20 gene was reported to be epist.atic to rl2. Lopes et ~1.,
Mol. ~;en. Genst. 2~5: 537-47 (lY94~.
Lopes et al. report~d three ~-~eins protein~ in
~ddition to the abnormal Z4-kDa proteln in thQ storage
proteln fraction of fl2, Witll molecular ~.reights ranqing
2~ ~rom ~3~out 25 to 27 kDa~ ~hey also detected 2
restrictioh ~ragment length polymorphism (RPIP~ linked to
th~ ~12 locus with a 22-kDa ~-zein 3?robe. They
hypothe~ized th~t the characteristiCs ;o~ fl2 might be a
response to the Al lAt;on of the defective 24-kDa
30 protein, but were unable to 3?rove that the acc~ ulation
of this protein wa6 regpon~ or the rlz ph_.~oty~ ~.
~IMAR~ OF ~
It is sn object o~ the p~esent invention to provide
35 ~ fusion protein Or ~ 21 amino acid Glgnal seguenc- from
fl2 ~ith a desired protein.
~ 2 1 8 1 4 1 8
It i8 a ~urther obiect of th~ inventlon to pro~ride
plants that contain an r~V~ g DNA sec,~lenc~ comprising
this fusion protein, in wh/~:h expre~ision o~ the desired
protein i~ increased ln seecls Or the plant.
It also i~ an object Or the present inventian to
provide a ~Dethcd Of increasing the content ot Ps~n~
amino acids in an ani.al fe~d vithout ~ppl~ ~nll.
In accompllshing these and other objectives, there
has been prcvided~ in ~ccortlance vith on~ aspect of the
present inventlon, a cereal plant thzt contains a
C.Ipnr compri5ed of ~ first polynucleoeid~ ~eg~ent
co~prisinq a nucLeotide seq-lence that encodes the amino
acid secuence MATKTTArr1~rTATrvsATNv j~md tii) a second
polynucleotide segment coding for a protein. In on~
preferred '~ , polyn~cleotide seg_ent (il) ha~
high content o~ an amino at:id selected from th~ group
consisting of _ethionine, ly~ine, LLy~L, ' 'n and
threonine, snch th~t the amount o~ said amino aoid in
seeds Or said cereal plant i5 increased a~ compared to
zo seeds fro_ other~ise idenl:ical plant~ that are not
trans~ormed. In another preLerred ~mh~ the fir~t
and seccnd pclynuclectide ~egments are cperably linked to
a promoter, such as the fl2 prcmoter, SO that s~id cer~l
plant ~AiJ~ ._.; both segmQnt:s under the contrcl of said
prcmo~er. Pursuant to other . ' ~1 L~, the cereal
plant can be a maize plant, wht~re segment (ii) is not
native to naize, or can be rice, :rheat, barley, millet cr
sorghum, for examplQ.
In acocrdance with cther aspects c~ the pres~nt
inventicn, there alsc is provided seed produced by a
plant ~s descrlced abcve and a feed product cc_prising
~eal obtained fro~ s~ch seed.
Pur3uant tc ancther aspe~:t of the present in~ention,
a transgene is provided that co~prise~ (i) a first
pol~nuclectidQ segment ccmpri.sing the nuoleotide seguenc~
Coding ror the amino acid ~eSluence MA~rTr~Tr~rT~rTVSATNV
and ~ii) a second polynuclectidQ seg~ent ccding for a
protein. In a prefQrred ~mho~lir , th~ L.e.,,s~n~
~ 2181418
~dditLonally cocpriDes ~ ~e~ence of fl2 ~!lected fro~
one or bcth of nucleotides 761-3824 and e61~-8335 o~
Figure 1, described in g~-eater d,tail belo,r. In
addition, a transgene i5 prolrided that co211pri~-s thn fl2
5 pro~ote~ op-r~bl~ lin~A~ed to a polynucleot$t~ 6egrAent.
other obiects~ features and advantages of the preScnt
invention will ~ecome apparent frcm 'ch~ follo~ing
detailed de~cription. It shc~uld be understood, however,
that the detailed descriptioll and the ~pecific ex~pl~,
10 while ind~catLng preferred ~--'i 1,5 0~ the inYention,
re given by way of illustration only, 3ince various
changes and modifications wil:hin the spirit and scope of
the invention will beco~le aE)parent to t:hosa skilled in
the art frosl this detailed dencription.
~3RI~F ~8CRIP~I0~l OF ~ DaAN~lG
E~igure 1 is the nucleotide fiequence of a clone of
fl2. Positions 1-760 and 8,336-~0,539 are vector
sequences, and positions 761-~3,33 are the coDIplete
nucleotide seoyence o~ ~12.
Figure 2 sho~ the ~u~le~n~ sequence and deduced
a3nino acid seiuence of th,e coding region of fl2,
i nr~ the gignal secuence. Nu~bers on thc left
correspond to position of the f irst ~II;ino acid o~ eaoh
llne beginning witb -21 to re~lect the signal peptide, so
th~t the -1 position is ol:cupied by the C-terminal
residue of the signal peptide and the +1 position is
occupied oy the first a~ino acid of the predicted ~ature
polypeptide .
It has been discovered that a gene encoding a ZZ-kDa
~-ze$n protein, an,d not a regulatory gene, i~ r~vpnr~qihl~
for the fl2 ~utant ph~2not~p~ diAr~lr~n~d above. In
~ -6- 2181418
particular, it has be~n fo~md th/~t the Z4-kDa protein
identified in fl2 mutants cn2priass th- amino acid
~:e~uence o~ a 22-k3~ a-zein plus ar; unclQaved, 2~-amino
acid signal peptide. I
The ccmpl~tQ nucleotide sequencs of the rlz i9 sb.cwn
in Figurc 1, while Figure 2 depictR the nucleotide
seguence and deduced amino acid 6equence o~ th~ codinq
region of the 24-kDa prote:Ln. Th~ deduc~d amino acid
sequence of the sign~l pept~de begins a'c poRltion -21 in
Figure 2, so that the -1 })osition iL occupied by the
C-terminal residue of the sign~ pQptide and the +1
position is occupied by the first anino aoid o~ the
predicted ~ature polypeptide ~he lieq;uence of the signlal
peptide is MhrKTTJ~T-A~TAr.rV~AT23V. A co~parison of thi~
deduced U-terminal amino acid sequence of th~ 24-kD~
a-zein protein with other a-~eins haR revealed an alanine
to valine substitution at the C-termin~l position of the
Signal peptide, a histidine insertion within the sevsnth
a-helical repeat, and ~n ~lanine to threonine
substitution Yith the same a-helical repeat of the
protein. Iihen an alanin~ codon is substituted for ths
valine codon Or the mutan~ zein gene, the in vivo
translated protein product is procesoed corrsctly in the
presence o~ maize microsome~.
The ~ional peptide targets th~ a-zein protein to thq
lu~en of the rough ~n~lorlR~mic reticulum (R~. The
signal peptide is retainled on the 24-kDa ~-zein
p~L~ r; that is, the Z4-kDa a-zein is not pl cessed in
l'lZ endosperm. Tho Z4-kDa ~-zein is 'b~lieved to remain
anchored to the RE~ me~brane, disrupting the normal
h~ n~ of protein bodies. In normal protein body
developr~ent, zein protein ~r~ retained within the ER
where they coalesce into spherical bodies in which
a-zeins ~re lor:~l;2ed to the interior Or a shell Or
cross-linked ~- zmd ~-zein. At~r I o~ an a-~ein to
the RER membrane i~hibits its nlovement into the interlor
of th~ protein body. Xore particularly, inter~ction o~'
h- h~--ttA~hed ~ 1n vitll the ~hu11 of ,~ y-~
,~ 2~814~8
--7--
disrupts the spatial or~anl~ation of developing protein
oodie~ ~y forlDing multiple foci ~or ~-z~in aggregation
n~ar the sur~ace Or thQ ~ ~eDbrane, whic~ th~ irr~gular
budding that occurs in fl.2 protein bodiGs. The retention
o~ the signal peptide on the Z2-kD4 ~-z~in also provide~
an explanation for the ~ y~ ion o~ 8iP in fl2,
since thi~ would afrect the notmal folding Or t~h-
prot~in.
The conversion of valine to 21anine in the sign~
peptide of this 3-zein l~rovides an explan~tion for it~
retention on the protein, and rOr 1;2ny o~ the phenotypic
e~ects of the fl2 mutation. According to von Neijne'~
"-3- rule" for signal peptides, the -1 positi3n is
critical for recognitic~n ~y signal peptidase ~nd i~
gen~rally occupied by ~n uncharged amino ~cid with a
s~all side chain. 5~e von Heijne, ~ur. J. ~ioc~e~. 133:
17 -27 ( 198 3 ) .
A point mutation in ~:he signal peptide is consistcnt
with the fact that onl~r one fl2 allole has been
identiried. A point mutation algo helps to explain the
difficulty in conventional ~Ipproaches to tag fl2 by
transposon mu~es~cic.
The ~o~plete ~12 sequence contains 7575 b~se pairs,
nucleotides 761-8,335 of Figure l. The flZ coding region
(open reading fra~e), inrl~ in~ t~e stop codon, comprlse3
nucleotides 3,825-~,613. This sequence has be~n
trans~ormed into maize. Transgenic seed that contained
t4e gene expressed the fl2, 24-kDa zein, and seed
segregAting r~hich did not have the rl2 protein did not
have the gene
Since the coding region matches the sequence o~ fl2,
24-kDa protein, it is understood th2t the sequence shown
in Figure 1 include~ the pro~oter for fl2, Nucleotide~:
oetween nucleotides 761 and al~out 3,8z4 in the sequence
Or ErLgure 1 e4code the rlz pro~oter. 6everal motifs
comrlon t~ 22-kr)a 2ein promoters are found in this region
of the fl2 sequence of Figure 1. For example, locatQd
up~tream of the start o~ initiatiot~ is a ~equence
218t418
-a-
5 ' -~;~CA~CCAC-3 ' . ~he rirSt nucleotide is at -300 with
respect to the start of inltiatl~n. ~is uur.., '- to
part of the sequence rsco~Jnized by the 02 gQnR pro~Uct,
also locate~ 300 bp upstrea3L of th~ start of initiation,
ar ~1 i Crl oq~d in Figura S of Sch~idt et al ., Plant CQ~1
~: 68 9 ~ 199 2 ~ .
Similarly, Morton et al, rerer to a prolamin-speciric
5 '-TGTAAAG-3 ' motif co3u~on to a~l zein genes of ~aiz~,
comn~only rQferred to as the U_300 boY" by virtue o~ its
location 300 bp fro~ the start o~ tr~nslation.
"Regula~ion of Seed Stor~ge Protein Gene ~xpr~ssion," in
sEEl~ D~vEr3~ENT hND ~ N ~Kigel and Ga~ili, eds. J, Ncw
~ork: ~farcel Dek3cer, Inc. ~1995). A ~.L'~7~J ~in~
sequence ig found at nucleotide 3500 in the fl2 segu~nce
o~ Figure 1. Mort~n Qt al. also disclo~a a
S~-CATGCATG-3 ' elenent co~Don to r.any ~La ~,. ci~i¢
gqmes. Thi5 sequencQ i91 sirilar to thQ sequence
5'-CATGCG~G-3' of fl2, vhich beqins at nucleotida 3517 in
~igure l
The retention o~ the Z4-kl~a prot-in on the RER and
its ~ lation in the endosper~ leads to a concollitant
decrease in the levels o~ other zein proteins and, hQn~a,
to a decreased lcvel of total 6torag~ proteins. The
reduction in Sotal storage protein lead~ to the soft,
st rchy ph~",~r~e of fl2 and the reductio;n of the zein
~ractiOr, as a percf~ntage of total 3torag~ protein lead~
to an apparent increase in lysine content, sinc~ the
otller storage proteins in ~aize, suo~ as globulins and
albu~Ding, have higher lys,ine contents.
~he discovery or t~le nature of the fl2 defect
provides the basis rOr seed progcny , i ri-l ly
ongineered to Qxpress various phL~ y~.. o~ a~, ; r
interest. That is, a fusion ~orotein of the 21 a~ino acid
~lqnal GequenC~ from fl2 with a desired polypeptide can
35 ~e u~ed to provide increased ~xpression of the desired
polypeptide in seedG of a host plant. ~be r~gion fro~
3, 888-4, 613 ls replaced ~ith the ooding region ~open
read~ng rralle) of tha desired polypeptid~.
2181418
g
Alternatively, rl2 ~equonces upstrea~ ~nd d~ ,~ o~
the coding region a~ ~la can be used to drive expre~sion
of a desired polypQptide. In tbis case, the region ~rom
3,825-4,613 in ~12 18 r2placed by the coding rQgion o~
5 the desir~d polypeptide.
A wide variety of polypeptide6 csn be fu6ed to th-
rl2 signal seguenc~ and/or to t~e r~gions upstrealD or
do~rnstrea~ of th~ flZ coding region, in or~er to achleve
higher levels o~ expre~aion andJor accumula~ion o~ the
10 polypeptide ~han might otherwise be attained. The
palypeptides may or ~Ry not be native to the plant in
which they are being ~i.ee~e.l. IlluYtrative of suc4
polypeptides are short polypeptides o~ less th~n about 40
~ino acids, polvpeptides which are ~ither unuGually
15 hydrophilic or ~Iy~Jpl~obic or amphip~thic, polypeptides
which have unioue solubility characteristlcs,
polypeptides which have a unique thre~-dir~ension~l
structure, polypeptides ~hich have ~Otir~ which lnclude
binding domains, polypepl:ld~ which have either vsry few
20 or many disulfide bonds, polypeptides which have a high
content of charged amino ~cidY, polyp~ptide6 ~hich have
a particularly high cont~nt of specific amino acids, ~
~stnionine, ly~ine, tL-yptophan or thrsonine, and
polypeptides which have been altered rrO:G their native
25 structure such that th~y ~ight otherwis~ not be stable.
DNA coding for the polypeptide may be modified to reflect
preferred codon usag~ in the partlcular crop that is the
target of the transfor~ation.
A fusion protQin of tlle slgn~l p~aptid~3 with a protein
30 other than 8 zein protein should not afrect the total
amount of zein storaqe proteins that are accumulated by
the plant. Inas~uch a~ it i~ the decrease in storag~
protein~ in fl2 that is reported to give rise to the
starchy ~ rr~m characteriYtic Or the ~Z mutant, zl
35 phenotype like that o~ the fl2 ~utant is not expected
when plants are tran~or~Ded with i fusion prot~-in o~ thQ
fl2 signal peptide and a protein other than a zcin
protQin .
2181418
--~.o--
Alternatlv~ly, the entlr- ~12 gene say b-7 tran~rorr~ed
into a plant that produc~s s~eds that are - .nic:~7 ly
stronger than mai2e. Fo~- exa31pl4, sorg~um arld r~ t
produc~,7 smaller and rounder seeds which are less a~ected
5 by shear force~ and, bence, to oy any .7~ernel ~ ~k~nir~
as~ociate~ ~ith flZ expr~ssion. ~xpression o~ rl2 in
these crops can ~rovide ~e~.ds ~Irlth improved digestibility
~ithout unacceptable leve~.s of kernel ~ -keni~q. It is
expected that rice, wheat and barley liJcewisQ could
lC ~- - te rl2 expres~ion in accordance with the pres~nt
invehtion,
In accordanCe with the present invention, a DNA
raolecule comprising a transformation/expresSion vector i~
enqineered to inolude th4 21 amino acid signal sequence
from ~12 and/or regions up~,tteam or down3trea~ Or thc ~12
coding region, and eithqr the rer~ainder of the fl2 gene
or a DNA segment ~ncoding a high-value protein a8
described abova. A copy of the sequence of fl2 or o~ the
21 ar~ino aoid signal segue:~c~ fro~ rl2 coupl~d to a
20 desired high-value protein is plaoed into nn exp~ession
vector by st~ndard ~ethods. The r~~t~ ~r1 of an
appropriate expression vect:or will depend upon the blethOd
of in~roducing the eXprefisiOn vector into host cl311s.
A typical expres~ion vectcr contains: prokaryotlc DNA
ZS elehlents coding for a b~cterial rep7ication origin and an
~ntibiotic resistanc~ gene to provide for the gro~th and
tiorl Of the expres~ion vector in the baoterial ho!~t;
a cloning site ~or insertion of the ~ J-?nv.l~ DNA
sequence; eukaryotic DNA el,~ments that control initiation
30 of transcription o~ the t,~g- cvu, DNA seqUenGe, such as
a promoter and an optional enhancer; and DNA elements
that control th~ processir~g Or tran~cripts, such as a
transcription termination-polyadenylation ~equence. ~h-
vector also could contaln .~dditional 5_, ~' that arQ
35 necessary to allow ror the eventual lntegration o~ th~
vect or into a chror~osoms. ~or a gen~ral de~oriptlon o~
p7ant expression vector~ ~e Gruoer t7t al., "Vectors ror
2181418
11-
Plant ~ransformation, " in ~T~ODS IN P~N~ MOLE~ULAR
sIO~GY AND BIOTECE~OLOGY 89-119 (CRC Prefis, 1993).
~xprQsslon of the g~lls sequence i~ under the ccntrol
of n promoter . EYampl-t~, o~ suitabl- p. tc~ J are the
S promotor ~or the sr~ll 6ubunit Or ribulose-1,5-bi~-
phosphate carboxylas~ pro20ters fro~D t~or-inducing
pl~smids o~ Agrobacteriun ~e~aci~ns, ~uch ~8 the
nopaline synt~ase and ocrtopin~ synthase pro~ot~rs, and
viral pro~otQrs such as th~ oauliflower ~osaic virus
(CaXV~ l9S and 35S pL~ or the flg~ror'c r,losaic viru~
35S promoter. The promoter can b~ constitutive or
;n~lr~ihl~, I
Fc:p~ri:~lly preferred is a "seed tissue-preferredn or
~'seed tissue-specific" prouoters, that is, pror~ot~rs that
drive hiqh eYpression o~ the hQterclogous DNA sagslent in
E eed tlssue where control of genes that ar~- involved in
seed metabolism is desir~cd, and little ar no expression
in other pa~s o~ the plant. I~anufacture of the protein
encoded by the heterolog~us D:~J~ segment $n other parts of
the plant n~ A~.c~:1y expends the plant's energy.
Ex~mples of known seed tis6ue-prererred or seed tissue-
specific pro~oters include thQ seed-directed ~/L~
from the zein qenes o~ maize ~-Y~ rh,. Pederfien et al.,
Cell 2g: lolS ~1982). The flZ promoter is particularly
preferred.
In addition to a suitable promoter, one or more
Qnhancers ar~ useful Ln the invention to increa~e
transcription of the intr~duced DNA segment. ~he
enhancer or enhancer-like elemen~ can~ be i~serted into
the promoter to provide 'higher levels of transcription.
Exalsples of such c-nhancers include, inter alia, viral
enhancerfi like those wit,h,in the 35S promoter, as sho~rn by
od--ll et al., Pl~n~ Mol. ~lol. 10: Z63-72 (1988), and an
enhz~ncer ~rorD an opine gene as desoribed by Tror~ et ~7.,
Pla~t Cell 1: 977 ~1989~.
selectable ~arker ~erles, in phy~ic~l proximity to th-
int~u, ~ NA seglsent, are used to allow transforlse~l
c~ 9 to be Lcco~clcd by either positiv~ gen~tlc
~ -~2- 2181418
sel~ction or screQnin~. The ~electable marker genes alsO
allow for ~aintainihg sel~ction pressur- on ~ , ic
plant population, to 6~n6ure that the i-d,L~' ' D~A
~eg:;ent, and its controlling promotars and enhancQ~s, are
retain~d by the transgel~ic plant.
Many o~ the comronl~ used positive ~olectAbl~ nlarker
g~nQs itor plant transformation have 'oeen isolated ~ro
bacteri~- and code itor enz~es that ~etabolically d~toxii~y
a selectiv~ chemical ay~nt which 2ay be an antLbiotlc or
a herblcide. Other p~sitive ~ ;nn ~arker genes
encodQ an a ltered 'carget which i8 insensitive to the
ihhibitor~
A preferre~ sele~tion marker gene for plant
tranGforration is the BAR or PAT gene, which i8 used with
ths selecting aqent h~ ~1 arhrs Spencer ~t al ., ~E~R.
APpL. GENEr., Berlin: Sp~.ingcr Int~rnational, vol. 79, pp
62S-631, 1990. Another usefUl s~lecticn marker gQne is
the neomycin phosphotranafer2se II (~IptII~ gen~, isolated
fro~ ~n5, which conferl~ resl~tance to Xanan~ycin ;rhen
placed under th(3 control Or plant regulatory signals.
Fraley et ~l., Pr~c. NAt'l Acad. sci. 17SA ao: 4803
119~33~. 'rhe ~Iy~r~ n ph~Jayl~ U.~r~LCISe qene, ~hich
con~ers resistance to the antibiotic hygro~ycin, ~s a
further example of a us~ful s~l~ct~hl~ ~;arker. Vanden
El2en Qt al., Plan Mol. Riol 5: 299 (1985~. Additional
po~itive select2ble ~ar3cers qenes Or bacterial origln
that confer resistahce to antibiotics include ~ nt~rnir;n
acstyl transfera8e, str~pto:llycin phosphotransrer~Q,
a~inoglycoside-3 ~-adenyl tran6feras~ and thc ~lecmycin
resistance determinant. Hayford et ~ lant Physiol.
~C: 1216 (1988~; Jone~ et al., Mol. Gen. Genet. 21~: 86
(1987~; Svab et al., Piant ~ol. 13iol. 1~: 197 (l990);
Hill- et 21., loc. cit, 7: 171 (1986~.
other positLve selectable ~arker gones i~or pl~nt
transformation are not oi' ~acterial oriqin. I'hes~ genes
include 130U8e dlhyc~roi'olate reduatase, plant
5-enc~lpyruvylshikimate-~ pl,o~ c.te ~y~thase and pl~nt
acetolactate synthase. Elchh~ltz et ~l., SocatiC C~ll
, ~ 2181418
--13--
~ol. Genet. ~3: 67 (lge~); Shah et al., Scier~ce 233: 478
~19~6); Charest et al., Plant C311 R~p. 8: 643 (1990).
Another clas~ o~ useful ~a.rker gen~s ~or plant
tn~nsrOr~ation witll the DNA ~equ~ncc re~lrea screehing
of presu~Dptively tranbrori~ed pl~nt cells rather than
dir~ct genetic 8 ~~~t'iOIl 01~ ~L~I:irU ~ cells for
resistance to a toxic ,ui,,t~n-.~ such as ~n antibLotic.
~hese genes i~re particularly userul tc quantitat~ or
visua1izQ the spatial ~attern o~ sxpres610n Or th~ DNA
sequence in speclric tissues and are ~requently rererred
to as reporter genes because they can ~e rused to ~ gene
or qene regulatory sequence ~or the investigation of qene
expression. Com~only used genes ~or screening
pres~ ptiv~ly transfor~ed cells includ~ glucuronidase
(G~7S), ,B-gdlactosid~s~, luciferase, ~nd ehl~L ,' ;CO1
acetyltransrerase Jef~erson, ~lant Hol. ~iol. R~p. 5:
3B7 (igs7); Teeri et ~ S30J. 8: 343 ~1989~; Konc~ e~
al., Proc. Nat~ Acad. ,sci. U~;A 8~: 131 (1987~; De Block
et ~1., E~30 J. 3: 1681 ~1984~. Another approach to the
identi~ication of relatively rare transrormatlon events
has been use c~ a gene that encodes a dc~inant
con6titutive regulator of the z~a mays anthocyanin
pigi~cntation pathway. 1udwig et al., Sclence 2~7: 449
tl990) .
~n order to create an e~cpres5ion vector cnn~aln;ng
the gene or DNA segment Or interest, an expre~sion
cassette first i6 i~ade ~>y inserting a cloned fl2 gene, or
a D~IA segl;ent comprising the fl2 signal sequence ru~ed to
a de6ired high-valu~ protein d5 described above, into ~
plas~id under the control Or a regulatory sesIuence. l~e
resulting expression cassette can be ligated back to
itself to produce an e~pression cassettc with a tandem
repeat Or the cloned g~n~. A rurther ligation can be
psr~orr~ed to generate a ~ L.LU''t that contalns four
tdnde~ copies of the gene.
one or more COpl~L of the ~xpression cassetto
rr~n~ n; nq the introduced DNA segment CO~L - AL V~ ; n7 to
thc rl2 g-nQ or to thq ~NA seg~ent compri~ing th~ fl~
~ 2~8~418
--14--
!
Gignal s-quence fused tc a deslred protein i~ transferred
to an e~pression vector. In a preferred ' ~; ', thQ
vector also contains a gene ~ncodirlg a sel~ction ~arlcer
which is functlonally llnked to pro3l0tor~ that control
S tr~nscription initiatio~l.
To creats a transg~!~nic plant, an ex.oression vector
cnntAinln~ the fl2 g-ne or the DNA seg~ent co3lp~ising th-
rl2 signal ~iequenc~ fu~,ed to a desired protein can be
in~ into protopla8ts; into intact ti~r~ue~, such a~
i~nature embryos and me~-LstelD~; into calluri c~lture~ or
into isolated cells. Preferably, expression vectors aro
inserted into intact tissues, such as explants derived
from hypocotyl or cotyledonary nodes of a yer3cinat~d
seed. (In this regard, an explant is a pieco of tissu-
that is taken from a dono~ plant and i5 capable of
producing callu6 in cultu~r~. ~ypocotyl tis~e. i8 thAt
portiOn of th~ ste~ of a plant enbryo or seedling below
tho cotyledonL and above the root. A cotyledon is an
~mbryonic loaf, and a cotyledoni~ry nodo is that part of
~0 the seedllnq between the embryonic axis and the
cotyledons which ~otanically de~inos the divisian of the
h~pocotyl and the epicotyl, or elrbryonic shoot. )
General ~ethods of culturing plant tissuos are provid~d,
for exa3~ple, by ~5iki ~t ~ Plv.~duY~s ~or Introducing
Foreign DNA into Plants," in ~ETHODS ~N Pl,ANT Mor~-rrr.
~IOLOGY AND ~Io~ECE~OLOGY 67-aa ~CRC Pre3s 1993) .
Prefera~ly, the ~12 gene or DNA aegment co~prisir~g
tbe flZ siqnal sequence ~u.sed to a desired high-value
protoin is transforned into ~ v~ ic naize callus by
p~rticle ~ - ~ t. Tran~genic ~aize pl~nts are
produced by bonbarc~ent of c ' y~enically responsive
ir~mature e~ryos T;ith tullgsten p~rticles associ2ted ~ith
DNA plasmids. Th~ plaslnids consist of a ~e~ t~hle and
an unselectable ~rker gene.
The present invention is described fUrther by
refer~nc~ to the ~ollot~irl~, illustrative exaD~ples.
-- 21~418
-15-
F 1 ~ 1. Chnracteri~r~tion of 24-~a p~ote.Ln of fl2
Wild-type maize, W64A+, W15 crossed with W6~A~12 to
gl~Q an F2 population, according to Lopes ~t ~1., 19~4,
thQ contents of whil:h are inco.~ tad hereln 3~y
S referenc~. The proqeny were sorted into floury, semi-
floury And norm~l kernl21s, and DNA was isolated fro~ 30
individuals in each pbenotypic Cl~fiS. To ldentify DNA
polyrorphisms li~ked ~ith the fl2 mutation, a bulked
seqreqant analysls w~ uaed. ~i~h~~ e et ~-1., P~oc.
Nat'l Acat. USA 8a: 9~28-32 ~19gl~. A~t~r ext~nsiv-
~creening with different DNA prcbe~restricticn enzym~
combinations, an RF3.P was detectcd iD SstI-di~ested DNA
vith a 22-kDa ~-zein c~NA. A 7.7 3cb DNA frsgment was
pres~nt in t31e ~ ~U.Il~ fl2 bulked sample and the
15 heteLvzy~,a~ semi-floury bulX, but not in the norm21
buLk .
The ~. 7 kb Sstl fragment wa~ i~olatea from genomic
DN~ of ~64~12, And t:he resulting clone, pCC515, was
mappet by restrictlon enzyme digestion, as described in
Z~ Cole~an et al., Proc. Nat'l Acsd. sci. USA 92: 6828-31
~19~5~, the contents ol. ~hich are inc-JL~-oL~ed herein by
rererence. Clone pCC515 was found to contain a single
22-kDa ~-zein coding sequence, vhich was obtained ag ~
1. 6 kb EcoR1 fragment. Upon nucleotide se~Iuence
25 analysis, the deduced ami~o acid sequence vas found to
coL~ .o,ld to a 22-kDa ~I-zein. The protein contains Z62
amino acids, including a Zl a~ino ~oid fiignal peptide.
Comparison of the ~educed a~ino acid sequence of the
slgnal peptide with th~ signal peptides o~ other ~-z-ins
3û reveals an alanine to valine substitution at the
C-terminal (-13 residue of the signal peptide, insertion
of a hifitidine following the seventh re3idue in the
seventh ~r-helical repeat and an alanine to thoureonin-
sub~titutlon Ln the sa~e ~-helical repeat. N-terninAl
35 sequence analysis of th~ purified 24-kDa protein ~ron ~12
~.dos3J~ Ghowed an Ldentical match ~or the f ir~t ~.
amino acid residue~ oet~een pCC~515 and the 24-kDa
pol~peptide~. ~he fiignz~l peptide that targets the protein
218~418
--16--
into the lu~en of the rough ~ndoplas~ic r~ticulum ~ras
found to be attzch~d to thc protein.
r le 2. ~rransfor~tion of ~i~:e vltb the rl2 ~7e~e
To prove that pCC515 contains th_ fl2 gen-, the gen~
~as transformed into ~v~ ic maize callu~ by p~rticl~
b,ombardment. ~ran~genic llaize plants wer~ produced by
bombardment Or ~ Lr~ ir~lly re6ponsive i~ature
embryos with tungsten partlcles a~Lociatsd wit~ DNA
plasmid¢. The plasmids consi6t of a ~electaole and an
unselPct~hle ~arker qene.
Prop~ra~cion Or ti~ue
Immature eDroryos of maize variety High ~ype II were
the target for particlo ~ ~ ~ ,t-mediated
transformation. ~his yenotype is the F~ of two purebred
genetic lines, parents P. and B, derived rrol3 the cross of
two know maize inbreds, A188 and B73. Both parents were
~elected ror high competenae of so~atic . y~gd~ 3iU,
according to Armstrong et al., ~ais~e Genetlc~ Coop. News
65: 92 ~1991). The High q'~'pQ II genotype does not
pos~ess the native mutant r12 gene.
Ears from ~, plasts were selfed or sibbed, and
er~oryos were aseptically ~l~cc~c~d ~rom developing
caryOpseS when the scul ellum ~irst became opaque. ~his
stage occurred about 9-13 days post-pollination, and ~ost
~ener~lly about lO days post-pollinatlon, clPr~n~lin7 on
growth conditlons . ~he e~bryo~ were about 0 . 75 to 1. 5
milli~eters long. Ears were surface sterilized with
20-50% Clorox for 30 Dlinutes, ~ollo~red by three rinses
with 6terilo distilled water.
~ature embryo-~ were cultured with the sc~t~l ~
oriented upward, on ~ vy~l,ic induction mediu~n
comprised Or N~ ba~-al salts, EriXsson VitaD~ins, 0.5 mg/l
thlamin~ }}CI., 30 gm/l ~ucrose, 2.88 gm/l L-proline,
1 mg/l 2,4-dichlorophenoxyacetic ACid, Z q~/l Gelrite,
ana 8.S mgll Ag~103. Chu et al., Scl. Sin~ 18: 659
(1975); ~rlksson, Physiol. Plant 1~: 976 ~1965~. The
medium was sterilized by autoclavinq at 121-C for 15
~ 2 1 ~
--17--
mlnuSes and ~iFr~n~q~ cl into lC0 X 25 mm PQtri dishes .
Ag~03 is rilter-sterilized and added to the mediun aftQr
autoclavlng. ~ha tisLiues wOEe cultured in cosplete
darkneJs At 28~C. Ar'cer about 3 to 7 ~lay3, most usually
5 n~out 4 days, the scuto~lu~ of ehe embry-o had fiw-lled to
about double it~ origin,~l size and th- protu~er~nces at
t~e colPnrhi~ surface of the ~ at~llu~ indlc~ted the
inception of _~.y;~nic ti~sua. 1~ to 100~ o~ the
embryo~ di5played this respons-, but nost co3;~only, the
10 e2lb y ,_ ~ responsc rL~ y~ was about 80t.
Wnen the ~_JLyc~ ic response was observlad, the
ellbryos ~ere trans~erred to a nedium co~prised o~
induction ;~lediU~ modi~ied to contain 120 g~l suoro~e.
The enoryos wsrs orient~d with thc coleorhizal pole, th~
15 allLbryo~n1t~11y responsive ti~sue, upwards from the
culture medium. $en e~ry-os per Petri dir~ were located
in the center of a Petri Aisn in an area about 2 cn in
diametsr. The emb~yos wlare naintaineq on thls ~edium rOr
3-1~ hour, prererably 4 hours, in complete darXness at
20 28-C just prior to bombard2ent with pArticles zssoclated
with plasmid DN~s ~ontaining the selectAble and
unselectable marker gane~.
To effect particle! bombardr~ent of e~bryos, the
particle-DNA ~gglomerates were accelerate~ using a DuPont
25 prlS_1000 particle acceleration devicd. The partlcle-~NA
agqlomeration was briefly sonicated and 10 ~1 were
depositad on ~acrocarriers and the ethanol was allowed to
Qvaporat~. The macrocarrier was accalerat~d onto a
stainless-steel Ltopping screen by the rupture of a
30 p~lymer diaphrag~ (rupt~rQ disk). Rupture was e~fectad
by pressurize~ helium ~ha veloclty o~ parti~le-~NA
~ccelaration was c~t~r~inD~I ba~ed on the rupture dLslc
~reaking pressure Rupture dis~c lJL._ ~Du~Ls of 200 to
1800 psi ~ere used, with ~50 to 110~ psi being preferred,
35 and ~bout 900 psi bcing IDost highly preferred. Nultiple
diDks were used to ~ffeot a r~nge of rupture ~ r~lL~_L~
~ rhe shelf cnnt ~1 n j nq th~ plate with e~bryos ~as
placed 5.1 c~ belo~ the botto~ o~ the ~L~ L.ier
2181418
-lB -
platrorm ~shelS' t3~. 'ro effect particl~ bo~b2rd~ t Of
cultured i~ature ~bryos, a rupture difik 1nd a
~cacrocarrlsr with driecl particle-DNA agglo~erates wer-
installcd in tne device. The E~e pressure delivsred to
5 the device wa~ adju~lted to 200 psi above tl~ rupturQ disX
breaking pre~sure. A Pe:tri di~h with the t~rget embryo~
was placed into the va~:uu~n chamber and loc~ted in the
pro~cted path o~ ~ccel~rated ~nrticles. A vacuuD was
created in tl~e chan~er, preferably about 2a in Hg. I~fter
10 operatioh Qf the devlce, the vacuu~o wa~ relea3ed ~nd the
Pet~i dish was removed.
~ ' .l.-d embryos re~lained on the osmotically-
ad~usted mediu~ durinq bo~bardlDent, and 1 to 4 day~
subsequently. The e31bryos were transferred to selectlon
15 Dediu~ conpriscd of t~6 ~asal s~lts, Erik~on vita~ins,
0.5 ~g/l thia~ine HCL, 30 g~3/l sucros~ /l 2,4-
dichl~,L~ .4~acetic acid, 2 gu/l Gelrit~, 0.85 mg/l Aq
NO3 and 3 mg/1 bialaphos tElerbiace, ll~ rhr~
was ;~dded f ilter-steriliz~d. ~he enbryos were
20 subc~ltured to fresh se~ection medium at 10 ~o li d~y
int~rvals. A~ter aoaut 7 weeks,: ~., tc tiss~e,
putatively trans~orr~ed for both selectable and unselected
~arXer genes, proliferaced fro:~ about 7~ of the bo~ba~ded
embryos. Putative transgenic tissue ~ras rescued, and
25 that tissue derived ~ro~ individual ellLbryos was
CQne 1 ' ~t d to be an eve~t and Wl18 propagated
lnA~-r~nd..r~tly Oh so~ t~ ~edi~. rwo cycles o~ clonal
propagation were acnieved by visual s~lection ror the
slDa~llest contiquous ~ragrents of organized: ~-,g~"ic
3r~ ti55Ue.
A sa~ple Or tl~sue ~rQm each even~ ~a~ y~Occ~ d to
recover DNA. ~he DNA was restricted with a re~triction
endonuclease and probed witb pri~er 5~LaCllC._3 desisned to
anpli~y ~lrA ~ n~ s overlapping thc rl2 and non-flZ
35 portion of the plasnid. r yot, I) ~ tissue with
anpllfiable ~equence wag advanced to pl~nt rc4~...aL~tion.
~ or Lt~,. rl~iOn of transgenic pl~nts, y~...ic
tissue was ~ubculturcd to ~ ~edi~ conprising ~l-e salt~
~ -19- 2181418
~nd vit~inq (lSura6hi~- L Skooq, PhyJiol. Plant ~5: 473
1196ZJ), 100 mgtl myo-ino~itol, 60 gm~l sUcrO5~, 3 g~tl
Gelrite, 0.5 3~gJl z~a~ in, 1 n~g/l lndole-3-acetic acid,
Z6.4 ng~1 ~cLs-trans-~c~ jcqic acid, nd 3 21lgJ1 bial~phos
s ln 100 X 2~ 3UII P~tr~ dishe~, and w~ lnc~ated ln
darkness at 28-C unti l the de~elopD~nt of v~ll-for~ed,
m:-tur-d so~atic e~ryos coult be se-n. ~his requir4d
about 14 day~. ~iell-formed so~atic enb~yos Yere opaqu~
and cream-colored, and ~cre coDlprised o~ ~n identi~iable
scutellum ~nd coleoptile. Ths embryos wer~ individually
su'ocultured to a ~er~ination DediUDI co~prising HS sAlts
and vitamins, 100 mgll myo-inositol, 40 g~/l sucrose and
1.5 g~/l G~lrite in 1~0 X 25 ~ Petri dishes and
incuba~ed under a 16 hl~ur liqht;8 hour dark photoperlod
and 40 ~einstein~ sec-~ ~roill cool-white i'lUv,.3~
tubes . Af ter about 'J days, thc 50natio e~bryos had
germinated and produced a well-dcrined shoot and root.
T'Ae individual plants were subcultur~d to gP~;n~tior~
modlum in 125 X 25 ~ gl~ss tube~ to allo~r furt~-r plaAt
dev~l:, n~ The plants were Daintained under a 16 hour
light:8 hour dark E,h~lu~_~lod and 40 ,U~;nstP~n~--'7sec-
fro~ cool-wh$te fluore~;cent tubes. After about 7 d~ys,
the plants were well-esta~lished and were tran~planted to
h~rticultural soil, hardened o~f, and potted lnto
Z5 coDmercial greenhouse soil mixture and grOwn to ~exual
maturity iA a greenhouse. An elite inbred lina wa5 used
as a male to pollinate regenerated transgenic plants.
Prep~r~tio~ of p~r1:iclell
Firtcen ;llg of tungsten particlos ~Gener~l Electrlc~,
0.5 to 1.8 Il, pr~ferably 1 to 1.~ IL, and 3~o_t pre~erably
1 ~, were added to 2 ~l of COnCG~lt.La~l nitric acid
This s~lsponci~n was ~ohioated ~t 0~C ~o~ 20 ~inutes
~Bran~on Sonl~ier Mod~l ~,50, 40% output, constant duty
cycle). Tungs~en particles wGro pelloted by
centrifuSI~tion at lOoOO rp!l (Hio~uqe) ~or one ~inute, ~nd
the supcrhatant wa3 re311oved. T~o milliliter~ Or stG~rilc
dit;tilled wat~r werc added to the pellet, and brief
~onication wa-~ used to r~ ., ~ the particle~. The
2l8l4l8
--20--
suspen?,ion wa~ pslleted, one ~illiliter of Ab~olute
ethanol va6 added to the pellet, ~nd bri~f F~ 'at ~ was
used to resusp-nd the p,~rticles. Rlj~slng, pell~ting, and
r~Cucp~n~ ~ n~J 0~ the particles was p~rformed two sar-
S ti~es with sterile di.stilled water, ard rin~lly th-
~particles w~re r~C~C~ in tYo nillilit~rs of sterile
dittilled water. The particles w~re suhdivided into
250~ a~iquots ~md stored frozen.,
ic~ of particl~-pl~ia ~IA a-90ci~tior.
~hc ~tock ol tungst,~n particles ~Jas sonicat2d briefly
in a water bath sonicator ~firanson Sonifier .~lod~l ~.50,
2-~% output, constant duty cycle) and 50 ~ as.
transferred to a microfuge t~loe. ~qui~nolar anounts o~
selectable and unselectable plas~id DNA ~ere atded to the
particles for a final DNA anount o~ 0.1 to 10 llg in 10 yl
total volune, a~ d brierly sonicated. Preferably, 1 ~Lg
total DYA was used. Specifically, 3 . 5 ,ul o~ DP~810
(U~2~:ubiint~ :pinIr~, 5.6 kltp) plus 6.5 ~1 Or DP6645
(flZ?::fl2!:fl21, 10.2 kbp), both at 0.~ ,ug/~l in Tl:
buffcr, were added to the particle suspension. Firty
~icroliters of sterile a.queous 2 . 5 M CaCll were added,
and ths ~ixture ~as l riefly sonicated and vortexed.
Twenty microliters o~ sterile aqueous ~.1 M sr~
wsre added and the ~ ture was ,~rief ly sonicated and
Z5 vortexed. ~he nixture ~i/as inc.Loated at roolL te~nper~ture
f:or 20 minutes with interlrittent brief 30nication. The
particle su~pe--,sion wa8 centrifuged, ~nd the gl~r~2i2tAnt
wa3 renoved. Two hundred fifty nicroliters of absolute
ethanol wer~ added to the pellet, rollowed by brief
82r~2n;~?tir~n. ~le 8~ pen~1r~ was pelleted, the supcrnatant
~as re~oved, and ~t ILl of absolute ethanol were added.
The suspension was sonic~ted briefly before loading the
particla-DNA agglo:~eration onto Dlacrocarriers.
F ~ tn 3 . ~ractic~n a~td ~ t;~n of p~otcLn
3 5 rrO~ tr~r~ en ~ c seed
ED~ryos wcre hand-d1~ s~to~ fron dry, ~atUrQ kernel~
ss~pled fron Siully d~eloped ears snd ~ I w~re
2~814~8
-2 1--
pulveriz~d to a ~ine ~eal ~ith a b~ll nill. Alph~-zeina
were extracted overnight in 70% ~v/~ ethanol with
con6tant sh~kiDg at 37'C. Arter centrlfugation ~or 15
minute$ at 12, 000 rlp3~, the aupernatant waY ro~ t~,
5 vacuu~ dried, and stored at 4~C until use. ~ot~l ~ein~
and non-zein proteinl; were isolaSed ~cco~ding to Wallace
et al, Pla~.7t Pl~ysio.~. 92: 191-96 (1950~.
SDS-polyacrylamide gels ~10 and 12.~9~, w/v) and
qradient gels ~7.5~ %, ;r/v) wera prepa~ed according to
I,ae~D~li, Naturo 22,1: 680-8~ 70~, but the TR~S
~,OI~ e"~._tlons used in the re~olving g~l and running
ouffer ~ere doubled. P~otcin samples were diluted ln
I,ae~li sa~ple bu~rer and boiled for 3 31inutes be~ore
loadlng. Gradient and 12 . 5% gels were run at room
t, at,uL~:: at a constant current until th~ dye front
~iqrat~d throught the stacking gel, ar~d then at 25G ~A
through the resolvillg gel. Gels were stained with
C_ sCie overni~ht, and destained in 40% (v/V) ~ethanol
antS lû% fv/v) oc~tic acid ~or at lea~t 8 hour~.
7 ~70ttlnq analy6es were Lsed specifically to deteat
Q-~eins in protein extracts. ~rotein e~acts were
separated by SDS-P~ as described above, transferred to
nitrocellulose f llters, and treated with a rabbit anti-
~-zein polyclonal antibody. Lending et al., Protopla~:3a
~3: 51-62 ~19~8) . Go~t anti-ra~bit alkaline ph~oph~ta8e
con~ugate waa u3ed rOr indirect detection of ~-~ain, as
de~cribed by l~necht et al., A~al. ~iocl~e~. ~36: 180-84
(1984). The 24 kDa ~--zein was detected in the seed fr~m
the tran~igenic plants.
