CA2192550A1 - Chimeric genes and method for increasing the threonine content of the seeds of plants - Google Patents

Abstract

A chimeric gene encoding a bifunctional feedback-insensitive aspartokinase-homoserine dehydrogenase (AK-HDH), is operably linked to a plant chloroplast transit sequence, and to plant seed-specific regulatory sequences. The chimeric gene is transformed into plants wherein increased levels of free threonine accumulate in the seeds.

Description

W096101gOS r~""., ~ TITLE
CHLMERIC GENES AND METHOD FOR INCREASING THE
THREONINE CONTENT OF THE SEEDS OF PLANTS
TECHNICAL FIELD
Tllis invention relates to a chimoric gene encoding a bir.~ feedback-insensitive ,..~ u,l~ homoserine d~llJJ~ D~ (AK-HDH), which is operably linked to a plant chloroplast transit sequence, and to plant seed-specific regulatory sequences. A method tor its use to produce increased levels of threonme in the seeds of LI~IDÇU~ plants is provided.
BACKGROUND OF THE INVENTION
Human food and animal feed, derived from many grains, are deficient in essential amino acids, such as Iysine, the sulfur amino acids methionine and cysteine, threonine and tryptophan. In corn, threonine is tne third most limitmgamino acid, after Iysme and the sulf Ir amino acids, for the dietary 1.,.. of 15 many animals.
While ~ - 1... ,.l L effort has been directed at increasing the Iysine and sulfur amino acid content of crops, little has been done to attempt to increase the threonine content. Mutant com and barley Imes that had elevated whole-kernel threonme ...~ - I ,a;~ ; were isolated from ceUs grown in culture by selecting for 20 growth in the presence of inhibitory of Iysime plus threonme ~Hibberd et al (1982) Proc. Natl. Acad. Sci. USA 79:559-563, Bright et al. (1982) Nature 299:278-279]. However, &~,lu~lv~ lly-acceptable cultivars have never been derived from these lines and, "
A preferable approach would be to incre&se the production and -25 of the threonine in .~v~v~ic~llly-acceptable cultivars, via genetic ---y ;~
technology. Threonine, along witll , Iysirte and isoleucine, are atnmo acids derived from asDpartate. The first step in the V;VD,~I~L.,IiC pathway is the l~hv~lJLv~ yl~l;vll Of aspartate by the enzyme ~ ~u Lvhi~ D~. (AK), and this enzyme has been found to be an important target for regulation of the pathway in many 30 organisms. me aspartate family pathv. ay is also believed to be regulated at vhe branch-pomt reactions. Thus, for threonine the reduction of aspartyl ~_~rm' ' ' ' ~d~ by hvlllv;~ d~,LyJIvv (HDH) may be an important point of control.
Galili and co-workers have reported on the il-~-UJU~,~iV-~ of an E. coli IvsC
35 gene that encodes a Iysine-sensitive AK enzyme into tobacco cells via I ~llDrVl~ lU [Galili et al. (1992 i Eur. Patent Appl. 91119328.2; Sllaul et al.
(1992)PfamPhysiol. 100:1157-1163]. E~pressionoftheE. colierrzymeresulted in small imcreases in the levels of free tlrreonine im the leaves and seeds of wo 96~019~
fran~ff~nnefl plartts, but effocts on the total threonine content were too small to be detected. Falco isolated a mutant of the E. Cf.~/i Ivsf gene, which encoded a Iysinc-insensitive AK. Falco Imked the gene to the bean phaseolin promoter and a plant cEtloroplast transit sequence, and t,.u.~r~ ' tobacco and canola with the chimeric gene. E~pression of multiple copies of this chimeric gene in the seeds of n ~ f( ,~ .i plants lead to a modest hlcrease in the total threonine content of the seeds [World Patent Publication U'O 93119190]. Although the last reference provides a means to achieve a small tbreonnne nncrease m seeds, there is a need for bener chimeric genes and methods to increase the threonine content of seeds I O further.
SUMMARY 0~ THE INVI~ION
Tbis inventiorls concerns a chimeric gene wherein a nucleic acid fragment encoding a bi-functional protein with ~ u- ~k; 1 1 ~ ' and hf fmf~Cf'rinf' d~llyll!U~
activities, both of which are .,~ islly insensitive to end-prodnct mhibition, isoperably Imked to a plant chloroplast transit sequence and to a seed-specrfic regulatory sequence. In a preferred . I-u~ , the nucleic acid fragment comprises tne E. col~ ~L gene. This invention aLso concems a piant comprising in its genome the chimeric gene described above and seeds obtained from that plant.
l'his invention further relates to a method for increasing tbe threonine content of the seeds of plants ~U~
(a) 1- h - ~ - fv ~ ~ ~ d 1 1~ plant cells with one of the chimeric genes described a'oove;
(b) growing fertile mature plants from the n ~ f~ d plant cells obtamed from step ~a~ under conditions suitable to obtam seeds;
and (c) selecting from the progeny seed of step ~b~ for tbose seeds containing increased levels of tEtreorrtne compared to , ,I,,,I,~r.,,,,,r.1 seeds.
30 Also disclosed are seeds obtained by this method and plants obtained from such seeds.
BRIEF l)ESCRIPIION OF THE
DRAWINCS AI~D SEOUENCE D~CRIPTIONS
The invention can be more fuliy undetstood from the following detailed 35 description and the .I ~,vu~ yi-lb~ drawings and tht seqnutce descriptions which form a part of this application.
E'igure I sEtows a map of plasrnid pBT718.
Figure 2 shows a map of plasmid pBT726.

WO 96101905 2 ~ ~ 2 5 ~ 0 P~
~ Figure 3 shows a map of plasmid pBT727.
Figure 4 shows a map of plasmid pBT728.
Figure 5 shows a map of plasmid pBT733 Figure 6 shows a map of plasmid pBT766.
5SEQ ID NOS: 1-2 were used in Example 1 as PCR pritners to isolate and modify the E. coli metL gene.
SEQ ID NOS:3-8 were used in E~ample 2 to create a com chioroplast transit sequence and lmk the sequence to the ~. col; meti_ gene.
SEQ ID NO:9 was used in Example 4 to create a soybean chioroplast 10transit sequence and link the sequence to the E. coli met gene.
DETAiLED DESCRIPTlON OF TilE INVENTION
The teachmgs below describe nucleic acid fragments, chimeric genes and procedures useful for increasing the ~ ' of threonine in the seeds of ~". . r "" d plants, as compared to levels of threonine in ~ r ~ plants.
15in the context of tùis disclosure, a number of terms shall be utilized. As used herein, the term "nucleic acid" refers to a large molecuie which can be smgle-stranded or double-stranded, composed of monomers (~ v~ ) containing a sugar, phosphate amd either a purine or pyrimidme. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. i n higher plants, ~I~.v~ ibu. .~l~ , acid 20 (DNA) is the genetic materiai whiie ribonucleic acid (RNA) is involved in thetransfer of the i - - ~ in DNA imto proteins . A "genome" is the entire body of genetic materiai contained in each ceii of an organism. The term "nucleotide seqmence" refers to a polymer of DNA or RNA which can be smgle- or double-stranded, optionaily containing synthetic, non-natural or aitered nucleotide bases 25 capable of ;~ n~ ~ . into DNA or RNA poiymers.
As used herein, "essentiaily sirniiar" refers to DNA seyuences that may involve base changes that do not cause a chamge in the encoded arnimo acid, or which involve boase chamges which may ater one or more amino acids, but do not affect the functionai properties of the proteim encoded by the DNA sequence. It is 30 therefore understood that the mvention ~ 1 more than the specific exemplary sequences. ~\f ..i;l;~ to the sequence, such as deletions, insertions,or ' - - in the sequence which produce siient changes that do not substantially affect the functional properties of the resulting protein molecuie are aiso .. . ' 3 For example, alteration m the gene sequence which refiect the 35 degeneracy of the genetic code, or which result in the production of a chemically equivaient amino acid at a given site, are ~ul-t..-.~' t; thus, a codon for the amino acid alanine, a Ly~Lv~ Jb;C amino acid, may be substituted by a codon encoding another less l..~.Lulul-vi,ic residue, such as glycime, or a more I-.~VIV~)h~
residue, such as vaiine, leucine, or isoleucine. Simiiarly, changes which resuit in 7lq25~
wo96/0190~ - ~I~J; 1~.II,J..,... .
substitution of one negatively charged residue for another, such as aspartic acid for Elutamic acid, or one positively charged residue for another, such as Iysine foratginine, can also be e~pected to produce a l~iuiugi~lly equivalent product.
Nucleotide changes which result in alteration of the N-termmal and C-tetminal 5 portions of the protein molecule would also not be e~pected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activr~ of the protein. E~ach of the proposed ~ is well withtn the routine skill in thean~ as is .1~ ~n of retention of biological activity of the encoded products.
10 Moreover, the skilled artis~m recognizes that "essentially sitnilar" se~:lences .1 by this invention are also defined by their ability to hybrid~7e, ;mder stringent conditions (O.IX SSC~ O.IS7f SDS. 65~C), with the ~sequences ~ d;ri~
herein.
"Gene" refers to a nucleic acid fra~ment that e~presses a specific protein, 1~ includmg regulatory sequences preceding (S' non-coding~ and follovving (3' non-coding) the coding region. "Nat;ve" gene refers to the gene as found in nature with its own regulatory sequences. "Chirneric" gene refers to a gene comprising y,rl1. ~. - regulatory and coding sequences. "F.. ~1.. ~ .. ~" gene refers to the native gene nonnally found in its natural location in the gen~me. A "foreign" gene 20 refers to a genc not nonnally found in the host organism but that is introduced by gene tramsfer.
"Coding sequence" refers to a DNA ~quence that codes for a specific protein and e~cludes the non-coding ~quences.
"Initiation codon" amd "h ti codon" refe.r to a unit of three adjacent 25 nucleotides in a codmg sequence that specifie.s initiation and chain Irmlin~inrl~
.;y, of protcin synthesis (mRNA translation~. "Open reading frame" refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
"RNA transcript" refers to the product resulting from RNA polymerase-30 catalyzed ~ of a DNA sequence. When the EiLNA transcript is a perfect ~ .. 1 .l.. a ~ y copy of the DNA sequence, it is refd to as the prunary transcript or it may he a RNA sequence derived from ~u~ ,dyLi~
processing of the primary transcript. "Messenger RNA ~mRNA) tcfers to RNA
that can be translated into protein by the cell. "cDNA" refers to a double-stranded 35 DNA, one strand of which is ~ J~ y to and derived frorm mRNA by reverse u ~ .. . "Sense" RNA refers to RNA transcript that includes the mRNA.

WO 96/OIgo~ 219 2 5 5 0 r~
~ As used herein, suitable "regulatory sequences" refer to nucleotide sequences located upstream (5'), within, and/or vu .. Ilot~ l (3'~ to a codmg sequence, which control the n ~ d 1 a ;~ and/or e~pression of the coding sequences, potentially in with the protein ~iuoyllL~h~lic apparatus of the cell. These regulatory 5 sequences include promoters, translation leader sequences, n ~ ;v termination sequences, and pvly ~dc.l ~ latiull sequences.
"Promoter" refers to a DNA sequence in a gene, usually upstream (S') to its codmg sequence, which controls the expression of the codmg sequence by providing the recogmtion for RNA pul~ and other factors required for 10 proper n i~ ;pl ;- -- - A promoter may also contain DNA sequences that are involved nn the binding of protein factors v.~hich control the ~;~f ~Li ~ DO of m ...~ ;l";,~.initiationinresponseto~ O;ulo~;i-,dlor~.'rl 'conditions. It may also contain enhancer dements.
An "enhancer" is a DNA sequence which can stimulate promoter activity. It 15 may be an innate element of the promoter or a h~,tl..ulv~J~O element inserted to enhance the level and/or tissue-specificity of a promoter. "Constitutive promoters"
refers to those that direct gene expression in all tissues and at all times. "Organ-specific" or "d~,~.lulu...~ -specific" promoters as referred to herein are those that direct gene expression almost e~cclusively in specific organs, such as leaves or20 seeds, or at specific v~,~. '(, stages m an organ, such as in early or late lyut~ll~ol~ tir~ly~
The term "operably linked" refers to nucleic acid sequences on a single nucleic acid molecule which are associated so that the function of one is affected by the other. For example, a promoter is operably linked with a structure gene 25 (i e, a gene encoding ~~ that is Iysine-insensitive as given herein) when it is capable of affecting the expression of that sttuctural gene (i e., that the structural gene is umder the ~ d 1~ control of the promûter).
The term "expression", as used herein, is intended to mean the production of the proteim product encoded by a gene. More I ' '~" "expression" refers to 30 the ( ~ ;1 a ;~ and stable ~ of the sense (mRNA~ or tha antisense RNA derived from the nucleic acid fragment(s) of the invention that, in conjuction with the protein apparatus of the cell, results m altered levels of protein product.
''Antisense inhibition" refers to the production of antisense RNA transcripts capable of preventmg the expression of the target protein "O~ ~LC~ oiUn~ refers 35 to the production of a gene product in transgenic organisnns that exceeds levels of production in normal or non-l----, r,--. -~d organisms. "Altered levels" refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non~ r. ~ ~ ", d organisms.

wo 9~ gO5 ~ 1 q 2 ~) 5 () The "3' non-coding se~ences" refers tO the DNA sequcnce pOltiOIl of a gene that contains a polyadenylation signaL and any other regulatory signai capable of affecvng mRNA processing or gene e~pression. The i~vl,~al~~ ivll sigllal is usually ~ h 1 hy affecting the addition of lvoly ad~ "y lic acid tracts to the 3' 5 end of the mRNA precursor.
The "translation leader sequence" refçrs to that DNA sequence pvrtion of a gene betwecn the promoter aod coding sequence that is transcribed into RNA and is present in the fully proces~d mRNA upstream (5') of the translation start codon.
The translation leader sequence may affect processing of the primary transcript to 10 mRNA, mRNA stability or translation efficiencyy.
"Mature" protein refers to a post-i ' ~ "y processed poly~ iJ~;
without its targeting slgnal. "Precursor" protein refers to the primary product of translation of mRNA. A "chioroplast targcting signal" is an amimo acid sequence which is translated in ~ with a protein and directs it to the chloroplast.
15 "Chloroplast transit ~qucnce" refers to a nuclcotide sequence that encodes a chloroplast targeting sigmal.
"End-product inhibition" or "feedback inhibition" re.fers to a biologicai regulatory mechanism wherein the catalytic activity of an enyme m a b;~,,.,.. ~li.
pathway is reversibly inhibited by bindmg to one or more of dle end-products of 20 the pathway when the ~ ;- -- - of the end-productls) reaches a sufficiently high level, tnus slowing the b;~D,~ h.,Li, process amd preventing over-_ of tbe end-product.
"T, i.. r~ .. , .. - ;.. " herein refers to the vransfer of a foreign gene into the genome of a host organism aofl its geneticaLly stable inheritance. E7~amples of methods of plant ll~bfv~ dvll include A~lub~.iGIi.. ' ~ n r~r~
amd particle-accelerated or "gene gun" tr:mcfnrr q~i-m technology.
"llost cell" meims the ce~l that is ll.lllbrv.,l,~l with the mtroduced genetic material.
Isolation of AK-HDH Geneiis This invention concerns chimeric genes encodmg bi-functional AK-IHDH
enzymes, wlIerein both catalytic activities are insensitive to end-product inhibition.
Over-e~pression of ~eedback-msensitivc AK incrcases flux through thc entire pathway of aspartate-derived amino acids even hl the presence of high ,, .u .,n. ,l, ~ of the pathway end~products Iysine, threor,ine and methionine.
Over-e~pression of a I r ~ ' feedback-husensitiYe AK-i:!DH enzyme dhects the increased flux throogh thc threonine branch of the aspaltate-derived arnino acid pathway, increasing the rate of threonine bi.Jb,yllll~b;b.

WO 96/0190~ 2 ~ 9 2 5 5 0 r .,~
A number of AK and AK-HDH genes have been isolsted ~md sequenced.
These include the thrA gene of E. coli ~Katiuka et al. (1980) Proc. Natl. Acad. Sci.
VSA 77:5730-5733], the met gene of E. coli (Zakui et al. (1983) J. Biol. Chem.
258:3028-3031J,thelvsCgeneofE.cc~/irCassanetal.(1986)J.Biol.C,hem.
5 261: 1052-10571, and the HOM3 gene of S. cer e~isiae IRafalski et al. (1988) J.
Biol. Chem. 263:2146-2151~. The thrA gene of E. coli encodes a l,;r...,. ~
protem, AKI-HDHI. The AK md HDH activities of this protem is inhibited by ~ threonine. The metL gene of E. coli also encodes a I r ' ~ I protein, AKI:I-HDHII, and both the AK and HDH activities of this protein are insensitive to 10 all pathway end-products. The E. coli IY~ gene encodes AKm, which is sensitive to Iysme inhibition. The HOM~3 gene of yeast encodes an AK which is sensitive tothreonme.
Among these microbial genes the E. coli rnetL gene encoding AKII-HDHII
is preferred. As indicated above, this gene has been isolated and sequenced. Thus, 15 it can be easily obtained from E. coli genornic DNA by a variety of techniques well known to those skilled in the at t, for e~ample via PCR using primers based on the published DNA sequence.
Plant mutants that e~press Iysine-insensitive AK-HDH are known. In barley, Iysine plus threonme-resistant mutants bearing mutations in two unlinked genes 20 that result in two different lysine-insensitive AK isoenzymes have been described rBright et al. (1982) Nature 299:278-279, Rognes et al. (1983) Planta 157:3Z-38,Arluda et al. (1984) Plarm Phsiol. 76:442~46]. }n com, a Iysine plus threonine-resistant cell line had AK activity that was less sensitive to Iysine inhibition than its parentlinerHibberdetal.(19S0)Plantal48:183-187]. A ~ tiyisolated 25 lysme plus threoninc .~ com mut~m is altered at a different genetic locus amdalso produces Iysine-insensitive AK IDiedrick et al. (1990) Theor . Appl. Genet.79:209-215, Dotson et al. (1990) Planta 182:546-552]. In tobacco there are two AK enzymes m leaves, one Iysine-sensitive amd one threonine-sensitive. A Iysine plus i' -resistant tobacco mutant that e~pressed completely Iysine-30 insen~sitive AK has been described I Frankard et al. (1991) Theor. Appl. Genet.82:273-282] .
These plant mutants could serve as sources of genes encoding feedback-insensitive AK-HDH and used, based on the teachings herein, to mcrease the a~ iull of threonine in the seeds of n ~ r -., . .~ ~i plants. A partial amino acid 35 sequence of AK from carrot has been reported rWilson et al. (1991 ~ Plant Physiol.
97:1323:1328]. Using this ;- r.. ~;.. a sot of degenerate DNA G, could be desigued, synthesized and used as a l~ iu~ probe to permit the isolation of the catrot AK gene. Recently the carrot AK gene has been isolated 2 ~
wo 96~ 0 and i~s nucleotide sequence has been derermined [Matthews et al. ~1991 ) U.S.S .N.
07/746.705J. This gene can be isolated based upon its sequence using PCR and used as a l,rl.lid;~ z probe to isolate the genes encoding Iysine-insensitive AK-HDH described above.
C.mc ruction of C.himeric Genes for Expression of Hr-H in the Seeds of plantc In order to rncrease b;~Jor~ of threonine in seeds, suitable regulatory sequences are provided to create chimeric genes for high level .seed-specrfic exprcssion of the AK-HDH coding region. The ~C~ t of the native regulatory sequences: . ' ' two t~mgs: I ) any pleiotropic effects that tlle ac~ of e~cess free th-reonine might have on the vegetative growth of plants is prevented because the chimeric gene(s) is not e~tpressed in vegetativetissue of the ~I~.Of~ ' plants 2) high level expression of the enzyme(s) is obtained in the seeds.
The expression of foreign genes in plants is ~. _11 e.,1.ltLi,l.~.J ~e Blaere et al.
(1987) Meth. En .~ol. 143:277-291]. Proper level of e~cpression of AK-HDH
mRNA may requrre the use of dtfferent chimeric genes utilizrng different promoters. Such chimeric genes can be trari5ferred into host plants either togethcr in a single e~pression vector or .,~ d~liy usrng more thsm one vector. A
preferred class of l-~t~)lor~ ,O hosOE for the cxpression of AK-HDH genes are eukazyotic hosts, particularly the oells of higher plants. Particula~y preferredamong the. higher plants and the seeds derived frorn them are soybean, rapeseed (Brassica napus. B. campestris~, sunflower (Helianthus arnus), cotton (Gossypium l!irsutum), corrl, tobacco ~Nicotiana Tubacum~, alfalfa ~Medicago sativa), wheat (T~itzcum sp), barley (Hordeunz vulgare), oats (Ai~ena sativa, L), sorghum (Sorghzm bicolor), rice (Ory~a satzval, and forage grszsses. Expression in plants will use regulatory sequences functional m such plants.
The origm of the promoter chosen to drive the expre.ssion of the codrng sequer.ice is not critical as long as it has sufflcient ~ activity to accomplish the invention by e~pre.ssing translatable mRNA for AK-HDH genes im the dcsired host tissue.
Preferred promoters slre tho5e that allow expression of the protein specifically m seeds. This may be especially useful, smce seeds are the prircarysource of vegetable amino acids and also since ~ed-spccific e~::pression wiLI avoid aly potential deleterious effect in non-seed organs. E~amples of seed-specific promoters mclude, but are not limited to, the promoters af seed storage proteirLs.
The seeo storage protems are strictdy regulated, being e~pressed almost exclusivcly in seeds in a highly organ-specific and stage-specific manner [HigginLs et al.~l984) 21 g2550 WO 9610190~ P~ v."r t ~ Ann. Rev. Plant Physiol. 35:191-221; Goldberg et al.(l989) Cell 56:149-160;
Thompson et al. (1989) BioEssa!s 10:108-113]. Moreover, different seed storage proteins may oe e~cpressed at different stages of seed d~
There are currently numerous e~amples for seed-specific e~pression of seed storage protein genes im transgenic di~vi yl_du~luuO plants. These include genesfrom dicuL~L,Iullv~.~ plants for bean ,B-phaseolin [Sengupta-Goplalan et al. (1985) Proc. Natl. Acad. Sci. USA 82:332V-3324; Hoffman et al. (1988) Plant Mol. Biol.
~ 11 :717-729], bean lectin [Voelker et al. (1987) EMBO J. 6: 3571-3577], soybean lectin [Okamuro et al. (1986) Proc. Natl. Acad. Sci. USA 83:8240-8244], soybean kunitz trypsin inhibitor rPerez-Grau et al. (1989! Plant Cell I :095-1109], soybean ~-conglycinin [Beachy et al. (1985) EMBO J. 4:3047-3053; Barker et al. (1988) Proc. Natl. Acad. Sci. USA 85:458462; Chen et al. (1988) EMBO J. 7:297-302;
Chenetal.(1989)De~.Genet.10:112-122;Naitoetal.(1988)PlantMol.Biol.
11:109-123],peavicilinrHigginsetal.(1988)Plan~Mo/.Biol. /1:683-695],pea convicilin rNewbigim et al. (1990) Planta 180:461], pea legumin [Shirsat et al.
(1989) Mol. Gen. Gene~ics 215:326]; rapeseed napin rRadke et al. (1988) Theor.
Apl71. Genet. 75:685-694] as well as genes from nio~vcu~Jl~,dul-uu~ plants such as for maize 15 krt zein [Hoffman et al. (1987) EMBO J. 6:3213-3221; S~ ' ' et al. (1988) EMBO J. 7:1249-1253; Williamson et al. (1988) Plant Physiol.
88:1002-1007], barley ~-hordein [Marris et al. (1988) Plant Mol. Biol.
10:359-366] and wheat glutenin [Colot et al. (1987) EA~BO J. 6:3559-35641-Moreover, promoters of seed-specific genes, operably linked to L~t~,.ulu~,v..v codrng sequences in chimeric gene constructs, also maintam their temporal and spatial e~pression pattern im transgenic plants. Such el~amples imclude liuking either the Phaseolin or Arabidopsis 2S albumin promoters to the Brazil nut 2S
albumin coding sequence and e~pressing such ~ h~ in tobacco, Arabidopsis, or Brassica nap~s [Altenbach et al., (1989) Plant Mol. Biol.
13:513-522; Altenbach et al., (1992) Plant ~fol. Biol.18:235-245; De Clercq et al., (1990) Plant Physiol. 94:970-979], bean lectm and bean ~-phaseolin promoters to e~press luciferase rRiggs et al. (1989) Plant Sci. 63:47-57~, and wheat gluteninpromoters to e~;press, ~ ' , ' ' acetyl transferase [Colot et al. (1987) EMBO J. 6:3559-3564].
Of particular use in the e~pression of the nucleic acid fragment of the invention will be the L~ vlvlsv~o promoters from several extensively-~ h ~, ., . ;,. A soybean seed storage protein genes such as those for the Kunitz trypsitl inhibitor [Jofuku et al. (1989) Planf Cell 1:107g-1093; Perez-Grau et al.
(1989) Plam Cell 1:I095-1109], glycinin [Nielson et al. (1989) Plant Cell 1:313-328], ~o~ - [Harada et al. (1989) Plant Cell 1:415425]. Promoters ~7 9~55 r~
of genes for a~- and ,B-subunits of ~soybean ~-conglycinin storage protein will be particularly useful in expressing AK-HDH mRNA~ in tbe cotyledons at mid- to late-staEes of soybean seed ~ 1U~ It rBeachy et a1. (1985) EMBO J.4:3W7-3US3;
Barkeret al. (1988~Proc. Natl. Acad. Sci. USA ~5:458462; Chen et al. (Ig88) E111BOJ.7:297-3U2;Chenetal.l1989!Del~.Genet.10:112-122;Naitoetal.
~1988)Plan~Mol. Biol. 11:109-123] in transgenic plants, since: a~therois very little position effect on dleir e~pression in transgenic seeds, and b) the two promoters show different temporal regulation: the promoter for the a'-subtrnit gene is e~:pressed a few days before that for the ~-subunit gene.
Also of particular use rn d~e e~:pression of the nudeic acid fragments of the ilvention wi] I be the promoters from several e,stensively . l, ~ - .;, ~ ~ I com seed storage protein gencs such as endospemm-specific promoters frorn the 10 kD zein rKrrihara et al. (lg88) Gene 71:359-370], the 27 kD zein lPrat et al.11987) Gelle 52 51-49;Gallardoetal.tl988)P/antSci.54:211-281].andthe 19kDzein IS rMar}!.s et ol. (1985'~J. Biol. Chem. 260:16451-1O459]. Therelative iUI~] activities of these promoters in com have been reported rKodrzyck etal.~l989)P/antCell/:105-114]providrngabasisforchoosimgapromoterfor usr in chimeric gene constructs for com. For e~pression in com cmbryos, a strongemhryo-specific promoter, e.g. the promoter from the GL13 L gene [Kriz (1989) BiochemicalGenetics27:239-251,Wallaceetal.(1991)PlarttPhysiol.
95:973-975] can be used.
It is envisioned that the illLIudu~liull of enhancers or enhancer-like elements rnto other promoter constructs will also provide increased levels of primsry u .l~ ll for AK HDII genes to accomplish the invention. These would include vnral enhancers such ss that found in the 35S promoter rOdell et al. (1988) Plant Mol. B~ol. 10:263-2721, enhsncers from the opine genes [~romm et al.
(1989) P/a~t Cell /:977-g841, or e.nhancers from any other source that result rnmcreased ~ ... when placed mtû a promoter operably linked to the nucleic acid fragrnent of the invention.
Of particular hllportance is the DNA sequence element isolated frorn the gene for tke al-subunit of ,~-conglycinin that caul confer 40-fold seed-specifice,l~- to a constitutive promoter IChen et al. (1988~ EMBO J.7:297-302, Chen et al . ~ I 9~9~ De~ Genet. 10: 112- 122] . One skilled in the att can readily isol3te tkis elemcnt and in~sert it witltiul tbe promoter region of any gene in order to obtain seed-specific enh3nced expression with the promoler in transgenic plants.Insertiml of such an element rn any seed-specific gene that is e~pressed at different times than the ~3-conglycinin gene w i71 result in e~cpression in transgenic plants for a longer period during seed d. ~

7 ! 92~50 Wo 9610190~ P~IIUJ~
~ Any 3' non -coding region capable of provi&g a pc,l ~ .J~.~ y L.~iVI~ signal and other regulatory sequences that ma~ be required for the proper expression of theAK-HDH coding regions can be used to accomplish the mvention. This would include the 3' encl from any storage protein such as the 3' end of the bean phaseolin 5 gene, the 3' end of the soybean ~-conglycinin gene, the 3' end from viral genes such as the 3' end of the 35S or the 19S cauliflower mosaic virus transcripts. lhe 3' end from the opine synthesis genes, the 3' ends of ribulose 1,5-' '~
bu~LIo~ or chlorophyll alb bmding proteim, or 3' end sequences from any source such that the sequence employed provides the nvcessary regulatory 10 information within its nucleic acid sequence to result in the proper expression of the ~ulwllulvlJLuvi-lg region cv~ , to which it is operably linked. There are numerous examples in the art that teach the usefulness of different 3' non-coding regions rfor example, see Ingelbrecht et al. (1989) Plant Cell 1:671 -680].
DNA sequences codmg for ~ " ' lorS~li7 lti- n sequences may be added 15 to the AK-HDH codulg sequence if required for the proper e~pression of the proteins to accomplish the invention. Plant amino acid l/iu~ ic enzymes are known to be localized m the ~LIulo~ , and therefol:e are synthesized with a chloroplast targetmg signal. Bacterial proteins such as E. coli AKII-HDHII have no such signal. A chloroplast transit sequence could, therefore, be fused to the20 coding sequence. Preferred chloroplast transit sequences are those of the small subunit of ribulose l,5-l . '- ~' carbo;sylase, e..g. from soybean ~erry-Lowe et al. (1982) J. Mol. Appl. Gene,t. 1 :483-498] for use Ul IJi~ulyL~lulluuD plants and from com [Lebrun et al. (1987) IYucleic ,qcids Res. 15:4360] for use in IIIUIIU~UI~h~dUIIU~.~ plants.
h- u udu~liull ûf Chimeric Genes into Plants Various methods of introducing a DNA sequence into eukaryotic cells (i.e., of tr~nrf~ ti~-n) of higher plants are available to those skilled in the art (see E,PO
0 295 959 A2 tmd 0 138 341 Al ~. Such methods include those based on u ,. . . ~ f~ . vectors utilizing the Ti and Ri plasmids of A~s, ul,.ll, t.., i,,,,, spp .
It is I ' ly preferred to use the binary type of these vectors. Ti clerived vectors transform a wide variety of higher plants, including Illullu~,ulyhJulluuO and di~ol~l dulluuD plants, such as soybean, cotton and rape [Pacciotti et al. (1985) Bio/Tccl1nolog~ 3:241; Bvme et al. (1987) Plant Cell, Tissuc artd f~t,~ar1 Culture 8:3; " ' ' 1: ' et al. (1987~ Plant Mo/. Biol. 8:209-216: Lorz et al. (1985) Mol.
Gcn. Genct. 199:178; Potrykus ~1985) Mol. Gcn. Cenet. 199:183].
Other ll~lolvllll~liiull methods are available to those skilled in the alt, such as direct llptake of foreign DNA constructs [see EPO publication 0 295 959 A2], tec,',niques of ~,L,~llu~)ul~liull [see Fromm et al. (1986) Nature (London) ~9:791]

7 ~ ~2~iO
WO 96101905 r~ a.
or high . Iv- ;Iy balliseic ' ' ' with metal particlos coated w ith thc nucleic acid construns [see Kline et al. (1987) Nalure (London) 327:70, and see U.S. Pat.
No.4,945,050]. Once ~r.qncform~ the cells can be regeneratecl by those skiUed inthe art.
Ot particular relevance are the recelltly described methods to transform foreign genes into commerciaUy important crops, such as rapeseed [see De Block et al. (1989) Plam Pi)vsiol. gl:694-701], ~sunflower [Everett et al. ~1987) BiolTechnology 5: 1201], soybean tMcCabe et al. (1988) Bio/Technolc1gy ~:923;
Hrncheeetal.(1988~BiolTech~ology6:915;Cheeetal.(1989)PiantPhysiol.
91: 1212- 1218; Christou et al. (1989) Proc. Narl. Acad. Sci US~ 86:7500-75W;
EPO Publication 0 301 749 A2]. and corn [Gordon-Kamm et al. (1990) Plant Ce~l 2:603-618; Fromm et al. (1990) Biotechnology 8:833-839].
E!~spression sf Chimeric Genes in Trqn~fr nn~rl Pl~ ~
To snalyze for e~pression of the chimeric AK-HDH gene in seeds and for the ~, of e~prcssion on the amino acid content in the seeds, a seed meal can be prepared by &''y of a number of suitable methods knr,~wn to those skillod in the art. The seed rneal can be partially or completely defatted, via he~cane e~traction for example, if desired. Protein extracts cam be prepared from the meal and analyzed for AK or HDH enzyme activities. Altematively the presence of any of the proteins c&n be tested for ' ' ~ , by mcthods well-hlown to tttosc skitled in the &rt. To mc&surc free &~nino acid . ~ . of the seeds, frec amimo acids c&n be e~tractcd from the me& and &nalyzcd by methods known to those skilled in the art [Bieleski el ai. (1966'~ Anal. Bioc~hem. 1~:278-293]. Ammo acid ~~J~ can then be detennined using any ~ '~ available &mino ac;d ar~lyzer. To measure total amino acid ~ l"J~ . of the seeds, meal containing both protein-bound amd frce &mino acids c&n be acid hydrolyzed to rele&se the protein-bound &mino acids &nd the ~"1'~~ c&n then be determined usmg &ly r~mm~-rri~1iy available ammo acid &nalyzer. Seeds acpressing thc AK-HDH
-iO protein and with higher tttrconine content th tn the wiid type secds can thus be id~ntrfied &nd propagated.
EXA~LES
The present invention is further defineci in the followirlg E~amples, in which &I parts &nd p~.. , are by weight and degrees &e Celsius, unless otherv~ise 35 stated. It should be understood that these i~&mples, while indicatmg preferred .... 1.~,.I;.. 1:- of the invention, are given by way of illustration ollly. Prom Ihe above &cussion and these E~amples, one skilled in the art can &scertain thc essenti& < h ~ m i~ of this invention, and witttout departing from the spirit and .7i ~255~
WO 9Gl01905 1~
~ scope thereof, can make various changes and ~lifirq~i~ms of the invention to adapt it to various usages and conditions.
F~AMPLE I
Isolation of the E. coli metL Gene amd Over-E~pression if AKII-l lDHII in E. coli The metL gene of E. coli encodes a birull~liullalA protein, AKII-H[DH~; the AK and HDH activities of this enzyme are msensitive to all pathway end-products.The metL gene of E. coli has been isolated and sequenced previously [Zakin et al.
(1983) J. Biol. Chcm. 258:3028-3031]. For the present invention a DNA fragment containmg the metL gene was isolated and modified from E. coli genomic DNA
obtamed from strain LE392 using PCR. The following PCR primers were designed and DylllL.,D~I;

CP23 = SEQ ID NO:l:
5'-GAAACCATGGCCAGTGTGATTGCGCAGGCA-3' CP24 = SEQ ID NO:2:
5'-GAAAGGTACC TTACAACAAC TGTGCCAGC-3' These primers add an Nco I site which includes a translation initiation codon at the amino terminus of the AKII-HDHII protein. In order to add the restriction site and additional codon, GCC coding for alanme, was also added to the amino terminus of the protein. The primers also add a Kpn I site " ~y following the translation stop codon.
PCR was performed using a Perkin-Elmer Cotus kit according to the instructions of the vendor on a tL,.lllv-y~ , r, ~ l by the sarne company.
The primers were at a . ~ A. of l O ,uM and the i ' y~Llg conditions were:

30 94~ 1 min,50~ 2 min, 72~ 8 min for 10 cycles followed by 94~ I min, 72~ 8 mm for 30 cycles.

Reactions with four different ~ -u ~ of template DNA all yielded the expected 2.4 kb DNA fragment, along with several other srnaUer fragments. The 35 four PCR reaction mi~es were pooled, digested with Nco I and Kpn I ald the 2.4 kb fragments were purified and isolated from an agarose gel.
To achieve high level e~pression of the ~_ gene in E. coli an e~pression vector base upon pET-3a [Rosenberg et al. (1987) Gene 56:125-135] which employs the l ~ ,-, T7 RNA IJoly ~ /T7 promoter system was 71~5~
wc) 9Uolgo5 r~
concrn-rte~ First the EcoR I and Hind III sites in pET-3a were destroyed at their origrnal positions by cutting, filling the ends using the Klenow fragment of DNApol~merase a~ld religating. An ~ adaptor containing EcoR I and Hind 111 sites was in~serted at the BamH I site of pEiT-3a. This created pPT-3aMS w ith additional Imique cloning sites for insertion of genes into the e~pression vector. Then, the Nde I site ut the position of translation Initiation was converted to an Nco ~ sitc using, ' ~ i ' -directed l~ The DNA ~queoce of pET-3aM in tbis region, 5'-_TATGG, was convezted to 5'-CCCATCIG, creating plasrmid pBT430. This plasmid was furtller modified by additon of a Kpn I
10 site dv .. ,~ ~ of tdle Nco I site using olic~- ~ucl~,vti~ adaptors.
The 2.4 kb Nco 1 ald Kpn 1 metL fragments described above were inserted into the modified pBT43U e~pression vector cut with Nco I and Kpn 1. DNA was isolatcd from ~ clones carrying the 2.4 kb fragment in the e~spression vector and u ,.. .i~rl .. . d into the e~spression host strain BL21~DE3 ).
Cultures were grown rn TB medium containing ampicillin ( 100 mg/L~ at 37~Covemigbt. Thecellswerecollectedbye ~l ;r ~ andlr~ ~in 1125th the original culture volwme in 50 mM NaCI: 50 mM Tris-Cl, pH 7.5; I rnM
EDTA, and frozen at 20~C, thawed at 37CC and sonicated, m an ice-water bath, to Iyse whe cells. The ]ysate wa~s centrifuged at 4~C for 5 mim at 12,000 rpm. Tlte20 supemata~tt was rcmoved and wle pellet was ~ ..L .1 in thc above bwffer.
The supernatant fractions were assayed for HDH enzyme activitics to identify clones e~pressing fimctional proteiws. HDH activity was assayed as sho vn below:
HDH ASSAY
St~rL ' l Q~LL2QmLE~~ C9L-0.2 M KPO4, pH 7.0 500 ~LL100 ,uL 100 rnM

3.7 MKCI 270 ~IL54 ,ItL l.UM
0.5 M EDTA 20 ~LL4 ~LL10 mM
1.0 M MgCI~ 10 ,uL 2 ~LL 10 rnM
2 mM NADPH 100 ~IL 20 ~uL 0.20 mM
Make Mi~ture of above reagents with amownts multiplied by number of assays.
IJse 0.9 mL of mi~; for Iml assay; 180 ~LI of mu; for 0.2 mL assay in micn:~titer dish.

WO 96/01905 ~ 1 9 2 5 S ~1 r~
Add 1.0M ASA in 1.0N HCI 1,uL 0.211L 1.0mM
to ]12 the assay mi~; remaining 112 lacks ASA IO serve as blank enzyme e~tract 10-100 IlL 2-20 ~LL
H20 to 1.0 mL to 0.20 mL

Add enzyme e~tract last to start reactivn. Incubate at ~30~C; monitor NADPH o:~idation at 340 nh~l. I unit o~idizes 1 ~Lmol NADPH/mm at 30~C in the I ml reaction.
S Four of eight e~tracts showed HDH activity well above the c~
E. coli host. These four were then assayed for AK activity. AK activity was assayed as shown below:

AK ASSAY
Assay mi~ (for 12 X 1.0rnL or 48 X 0.25mL assays):
2.5 mL H20 2.0 mL 4~1 KOH
2.0 mL 4M NH2OH-HCi 1.0 mL IM Tris-HCI pH 8.0 0.5 mL 0.2M ATP (121 mg1mL in 0.2M NaOH) 50 ,uL lM MgSO4 pH of assay mi~ should be 7-8 E~ach 1.5 mL eppendorf assay tube contains:
MACRO assay micro assay assay mr~ 0.64 mL 0.16 mL
0.2M L-Aspartate 0.04 mL 0.01 mL
e~tract 5-120 IlL I-30 ~ L
M20 to total vol. 0.8 mL 0.2 mL
Assay tubes are incubated at 30~C for 30-60 min Add to develop color;
FeC13 reagent 0.4 mL 0. I mL
FeCI~ reagent i.s: 10% wlv FeC13 50 ~
3.3~,c TCA 15.5 g 0.7% HCI 35 mls HCI
H2O to 500 mls Spin for 2 min in eppendorf centrifuge tobe.
20 Read OD at 540 nm.

wos6/o1gn5 21 925 50 ~.I/U~
Two e~ctracts also had high levels of AK enzyme activity. These two actracts werc then tested for inhibition of A~ or HDEI activity by the pathway end-products, lys, thr and met Neither ;he ~K nor the HDH activity of the e~tract from clone 5 Wl S inhibited by 30 mM ~ ,.. u A l ;- " 1.~ of any of the end-products.
Tttc supematant and pellet fractions of several of the e~tracts were also auu iy~d by SDS l~vly .~ yl~l~ide gel c~ ,Ll u~uhu~c2,~s~ In the e~tract from clone 5, the major protein i isible by Coomassie blue staining in both the pellet and supematant fractions had a molecular weight of about 85 kd, the el~pected size for AKIl-HDHlI. Tite metL gene inplasmid pBT718 (Figure 1~ from clone 5 was used fol aU subsequent wolk. AKII-llDHII protein derived from clone 5 was sent to Hazelton Research Facility (310 Swampridge Road, Denver, PA 17517~ to have rabbit antibodies raised agamst the protein.

Cons~r.~ n of Chimeric Genes for E~pression I S E~ co1i metL m the Emhrvo and Endosperm of Transfommed Com The follolh ing chimeric genes were made for il~f~ ' into com:

globulm 1 ln~"~ /globulin 13' region 20 glutelin ~ NOS 3' region The globulin I promoter ;uld 3' sequences were isolated from a Clontech COIIl genomic DNA library usimg ~ . - L v~ probes based on the published sequence of the globulin I gene [Kriz et al. (1989) I'lalz~ Physiol. ,~1:636]. The 25 cloned segment includes the promoter fragrr ent e~tending 1078 nucleotides upstream from the ATG translation start codon, the entire globuiin coding sequence includmg mtrons r.nd the 3' sequence e~tending 803 bases from the ' stop. To a low ~ JIa~ L of the globulin I coding sequence with other coding sequences an Nco I site was introduced at the ATG start codon, and 30 Kpn I and Xba I sites were introduced following the u ~ ~~:. ",l stop codon qia PCR to create vector pCC50. There is a second Nco I site witllin the ,~Lobulin Ipromoter fragment. 1'he gloonlm I gene cassette is danked by Hind 111 sites.
The glutelin 2 promoter was cloned from conn genomic DNA Usilg PCR
witit prirr.ers base.d on the published sequence [Retna et al. ( 1990i ~'uc1eir Aci~fs Res. 1~:6426-642t-]. The promoter fragmem includes 1020 nucleotides up~stream from the ATG translation st~rt codom An Nco I site was irltroduced via PCR at theA1'C'startsitetoallo~Yfordirectl ~ lfusons. ABamHIsitew.s introduced on the 5' cnd of the promoter. The 1.02 kb BamH I to Nco I promoter fragment was cloned into the BamH I to Nco I sites of a previously constf uctec' ~ ~ q;~5 5(~
wos6101sns r~o....
plant e~cpression vector replacing the 35S promoter to create v~ctor pML90. Thisvector contains the glutelm 2 promoter iioked to the GUS codmg region and the NOS 3'.
Plant amimo acid bivDy~ , enzymes are icnown to be locairzed in the 5 ~ I k" u~ and therefore are Dylltil~ .-d with a chloroplast targeting signai.
- Bacterial proteins have no such signai. A chioroplast transit sequence (cts) was therefore fused to the E. colf ~L coding sequence in the chimeric genes described below. For cotn the cts used was based on the the cts of the small subunit of ribuiose I ,5-; , ' . ' ~albu~yla~ from com ~Lebrun et ai. (1987) 10 I~ucleic Aculs Rcs. 1~:4360J and is designated mcts.
~l ;v~ Ol ;~ l - SEQ ID NO: 3 and SEQ ID NO:4, which encode the carbo~cy terminal part of the com chloroplast targeting signai, were anneaied, resuiting in Xoa I and Nco I cornpatible ends, purified via luvlya~ly- ~ ' gel ek~llu,ullulvDiD, and inserted into Xba I plus Nco I digested pBT718 (Figure 1).15 Tne insertion of the correct sequence was verified by DNA sequencing yieldingpBT725. To complete the com chioroplast targeting signai, the amino terminai part of the com chioroplast targetimg signai from a previous construct, pBT580, was inserted.
The plasmid pBT580 was constructed as follows: ~ 'iv ' ' SEQ ID
20 NO:3 and SEQ ID NO :4 ~above) were annealed, resulting in Xba I and Nco I
compatibleends,purifiedviaAuulya~ - ~ gel~ IIV~hU~ andinsertedmto an Xba I plus Nco I digested plasmid containing the E. coii IvsC gene, thus fusing the carboxy terminal part of the com chloroplast targeting signal to the AKIII
protein, and destroying the Nco I site. The insertion of the correct sequence was 25 verified by DNA sequencmg yieldmg pBT556. ~ ~ I ~ u~ SEQ ID NO:5 and SEQ ID NO:6, which encode the middle part of the chloroplast targetmg signal, were annealed, resulting in Bgl II and Xba I compatible ends, purified via IJulya.Jy- ~ ' gel CI~ UIJIIU~ and in~rted into Bgl II and Xba I digestod pBT556. The insertion of the correct sequence was verified by DNA sequencimg 30 yielding pBT557. ~' v ' ' SEQ ID NO:7 and SEQ ID NO:8~ which encode the amino termimal part of the chloroplast targeting signal, were annealed.
resulting in Nco I amd Afl lI compatible ends, purified via luuly~,l yl~lfil~ gel el~nU~I.U.~ and inserted into Nco I and Afl :lI digosted pBT557. The msertion of the correct sequence was verified by DNA sequencing yiel&g pBT558. Thus 35 the complete mcts was tused to the IvsC gene.
The mcts/lvsC-M4 coding sequence was isolated from plasmid pBT558 and inserted mto Nco I plus Sma I digested pML90 ~aoove) creating plasmid pBT580.
pBT580 was digested with BamH 1 auld Xba I yielding a 1.14 kb fragment ~o 96/0190~ 2 ~ ~ 2 5 5 ~
containing the glutelin 2 promoter plus tl-o ammo terminal part of the com chloroplast targeting signal. This fragmerlt was insened into pBT725 digested with Bgl 11 and Xba I, creating pBT726 (Figure 2) wherem the complete mct.s was fused to the m~tL coding sequence.
To construct tlle chuneric gene:
globulir~ tc/m~/globrllin 1 3' region the 2.o kb Nco I to Kpn I fragment containing the mcts/~L codmg sequence was isolated from plasmid pBT726 and inserted UltO Nco I (partial digest~ plus Kpn Idigested pCC50 creating plasmid pBT727 (Figure 3).
To cvnstruct the chimeric gene:
glutelin 2 ,VlUI~lVt~ /Ul'~ Nos 3' region the 2.6 kb Nco I to l~pn I fragment contaming tbe mcts/me.tL codin~s sequence was isolated fr~ plasm id pBT726 imd inserted mto pML90 (described above) di~ested with the same enzymes creating plasmid pBT728 ~Figure 4~, EXA~LE 3 T.... ~r....... , .~ -n~ of com with ~him~rir~ Genes for F.~r~ ~einn of E. coli, in rhr r ~ andEndos,n~rm Com was l-~f -- ' d with the chimeric genes:
~looulin 1 ~u~u.. .v~ 'r.~ globulin 1 3' region or glutelin 2 ~vmvt~ ./NC)S 3' region The bacterial bar gene from Su r / r~u. . .JIr~ r~ J~ , v~i~ u~ that confers resistance to the herbicide glufosinate l'lllompson et al. ~ 1987) The EUBO
Jvun~al 6:2519-25~!3] was used as tlle selectable marker for com tt ' ~~
The ~}~ gene had its translation codon changed from GTG to ATG for proper translationtllitiationinplants[DeBlocketal.(1987)TheEMBOJournal 6: 2513-2518] . The bar gene was driven by the 35S promoter from Cauliilower Mosaic Virus amd uses the termmation and uul~GI~I~lGliu~l signal from the octopine syntbase gene from A~. ,,l~acrc,i~
Ll~blyur~ callus cultures were mitiated frvm immature embryos (about 1,0 to 1,5 mm~ diss~cted from kemels of a com line bred for giving a "type II
callus" tissue cultu.re response. The embryos were dissected lU to 12 d after pollrnation and were placed with the a~is-side down and m contact witll agaro~-solidified N6 medium [Chu et al. ~I Y74) Sci Sin f 8: 659-668~ ~ -rr 1~ ~ v~ith 1.0 mg/L 2,4-D (~'6-1.0). The embryos were kept in the dark at 27~C, Friable tll8vlyub~ , callus consisting of ~ lirr~.. ' ' masses of cell.s with somatic ~u~ lyvs and somatic embryos bome on suspensor structures ~ om WO 96101905 ~ 1 9 2 5 5 ~
~ the scuteUum of the immature embryos. Clonal c.. blyur_lli. caUi isolated from inoividual embryos were identif~ed and sub-cultured on N6-1.0 medium every 2 to 3 weeks.
The particle bUlllb ' ' method was used to transfer genes to tho callus S culture cells. A Biolistic7M PDS- I O00jHe (BioRAD Laboratories, Hercules, CA)- was used for these . ~ 1~ .; . u ~
Circular plasmid DNA or DNA which had been lineanzed by restriction - digestion was 1 " c~ ' ' 1 onto the surface of gold particles. DNA
from two or tbree different plasmids, one containing the selectable marker for com 0 ~an~fnml~tjnn, andonecontainingthechimericgeneformcreasedthreonine ar~ inn im seeds were CO-AUlc~ uild~ . To accomplish this 2.5 ~g of each DNA (in water at a c~ of about I mg/mL) was added to 25 ~LL of gold particles (average diameter of 1.0 pm) suspended in water (60 mg of gold per mL)Calcium chloride (25 '~IL of a 2.5 M solution~ and spermidine (10 ,uL of a 0.1 Msolution) were then added to the gold-DNA suspension as the tube was vorte~ing for 3 mm. The gold particles were centrifuged in a microfuge for I sec and the supematant removed. The gold particles were then A ' ~ in I mL of absolute ethanol, were centrifuged again and the supematant removed. Finally, the gold particles were I , ~ ' in 25 IlL of absolute ethanol and sonicated twice for one sec. Five pL of the DNA-coated gold particles were then loaded on each macro carrier disk and the ethanol was allo-ved to evaporate away leaving the DNA-covered gold particles dried onto the disk.
L.~ Y~ C caUus was arranged in a circular area of about 4 cm in diameter in tbe center of a 100 X 20 mm petri dish contatning N6-1.0 medium A ~
with 0.25M sorbitol and 0.25M marulitol. The tissue was placed on this medium for 4-6 h prior to bUllll/ald~ as a IJlCtlCdtl~l~.llt and remained on tbe mediumduring the ' I ' procedure. At the end of the 4-6 h ~ n~ n ~.1 period, the petri dish containing the tissue was placed in the chamber of the PDS-1000/He.
The air in the chamber was then evacuated to a vacuum of 28-29 mch of Hg. The O-,~li~ was accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1080-1100 psi. The tissue was placed ~ , 8 cm from the stoppmg screen. Five to seven plates of tissue were bombarded with the DNA-coated gold panicles. Following bUl~lllaU- t, dle caUus tissue was transferred to N6-1.0 medium without ~ .I,I,i~ ~- . " ~I sorbitol or mannitol.
Within 3-5 days after b ' ~ the tissue was transferred to selective medium, N6-1.0 medium that contained 2 mglL bialaphos. AU tissue was transferred to fresh No- 1.0 medium ;, ~ d with bialaphos every 2 weeks.

Wo 96101905 2 1 9 2 5 5 Q PCTI[IS95/08501 Aftcr 6-12 weeks clones of actively growmg cailus were identi~ied. CaUu~s was the.n t~.msferred to an hiS-based medium that promotes plamt 1~ r.
EiXA~LE 4 Const~ruction of a Chimeric Gene for E~pr~ n ~. coli metL in the Seed of T ~.( ' Soybeam The following chimeric gene was made for ~nsforrnq~inn into soyboan:
phaseolm 5' region/cts/~L/phaseolin 3' region A seed-specific expression cassette composed of the promoter and ~U~ iU~I terminator from the geDe encoding tho 13 sub~mit of the secd storage 10 protein phaseolin from the bean Pf~seolus vulgaris [Doyle et al. ( 1986) J. Biol.
Chem.261:9228-g238]wasusedfore~pressiûniml.,...~fi,... dsoybean. The phaseolin cassette includes about 500 nucleotides upstrcani (5') from the translation initiation codon and about 1650 nucleoddes ~IUWII~ (3'~ from the translation stop codon of phaseolin. Bet~een d~e S' and 3' regions are the unique restriction ~ ' ' sites Nco I (which includes the ATG translation initiation codon~, Sma I, Kpn I and Xoa I. The entire cassette is flimked by Hind m sites.
To constmct the chimc}ic gene:
phaseolin S' l~-r O-4'~,lr.~'h~LL/phaseolm 3' region the 2.4 kb Nco I to Kpn I fragment containing the ~ _ coding sequence was isolated from plasmid pBT7 18 ~aoove) and inserted into a pUC18 vector carrying the seed e~prcssion cassette also digestcd with Nco I plus Kpn I creating plasrnid pBT733 (Figure 5).
Piant amino acid i ;u~y.A~h_lic enzymes are known to be locallzed in tbe lulu~l~ib and therefore are synthesized with a cbloroplast targeting sigr~i. The~5 bacterial protein AlCII-HDHn has no such signal. A chloroplast transit seqnence (cts) was therefore fuscd to the E. eoii metL coding sequence in the chirneric gene.
The cts used, SBQ ID NO:9:, was equivalent to tbe the cts of the sruali subunit of ribulose 1,5-1 , ' A ~ ' C~UIlUAyi~ frorn soybean [Berry-Lowe et al. (1982) J.
Mol. Appl. Gene~. 1:483 i98]. The cts was fianked with Nco I and inserted into the mç.~L seed expression cassette of pBT733 creating plasrnid pBT766 (Figure 6).
EXAMPLE ~
Ti,..,~r " . ~ ... of SoYbean with a pl ~ L C,himeric Gene To induce somatic embryos. cot~,ledons, 3-5 mm in len&th dissectcd from 35 .surface steri'iized~ immature seeds of the soybe~n cultivar A2~g72, vere cultured iUl the li~ht or dark at 26~C on an agar medium (SB I or SB2~ for 6-10 weeks.
Somatic embryos, which produced secondary emblyos were e~cised ~md placed into a liquid medium (SB55i. After repeated selection for clustcrs of somatic WO9610190!5 ,~) ~ 92 5 5 0 F~l/U.~..,' I
embryos which multiplied as early, globular staged embryos, the . were maintained as described below.
Soybean ~ ' yu~,_u. suspension cultures we}e maintained m 35 mL liquid media (SB55} on a rotary shaker, 150 rpm, at 26~C with florescent lights on a 5 16:8 hour day/night schedule. Cultures were ' ' ~,d every two weeks by inoculating ~ 35 mg of tissue into 35 mL of liquid medium.
Soybean ~Illblyur~_uu suspension cultures were n,...~r~.... d by the method of particle gun l ~ ~---l ~ -- 1~ ~ .1 [Klme et al. (1987) Nature (London) 327:70, U.S.
Patent No. 4,945,050]. A Du Pont BiolisticrM PDS1000/HE instrument (helium 10 retrofit) was used for these I.,~r~
The selectable marker gene for soybean L-. ' ~ wa~s a chimeric gene composed of the 35S promoter from Cauliflower Mosaic VirLls [Odell et al.(l985) Nature313:810-812],thel~ul~ly~ull~Lu~ genefromplasmid pJR225(fromE.coli)[Gritzetal.(1983)Gene25:179-liY8]andthe3'rogionof the nopaline synthase gene from the T-DNA of the Ti plasmid of A~, uZ~u~
~UrnelL/Ci.~.~. The seed ei~pression cassette, phaseolin 5' region/cts/metL/phaseolin 3' region, (Er.ample 4) was isolated as an alu,u~ 4.5 kb Hind m fragment from pBT766 (Figure 6). This fragment w as inserted into a unique Hind 111 site of the vector carrymg the marker gene creating plasmid pBT767.
To 50 ~LL of a 60 mg/mL 1 ~Lm gold particle suspension was added (m order); S ~LL DNA (1 ~lg/~L), 20 111 spermidine (0.1 M), amd 50 ~LL CaC12 (2.5 M).
The particle preparation was agitated for three minutes, spun in a microfuge for10 seconds and the , removed. The DNA-coated particles were then washed once m 400 ,uL 70% ethamol and ~G~ .ld~d in 40 ~LL of anhydrous ethanol. The DNA/particle suspension was sonicated three times for one second each. Five llL of the DNA-coated gold particles were then loaded on each macro carrier disk.
A,u~ , 300~00 mg of a two-week.-old suspension culture was placed in an empty 60x 15 mm petri dish and the residual liquid removed from thetissue v,~ith a pipette. For each u ~ ru~ A~]. ' t, ~
5-10 plates of tissue are. normaUy ln ' ' ' Membrane rupture pressure was set at 1100 psi and the chamber was evacuated to a vacuum of 28 imches mercury.
The tissue was placed ~ul.., ~ 'y 3.5 inches away from the retaining screen and bombarded three times. Following l,. ,,,~ h~ .1 tlle tissue was divided in half 35 and placed back imto liquid and cultured as described above.
Five to seven days post ' ' .' the liquid media was exchanged with fresh SB55, and eleven to twelve days post ' ' ' with fresh SB55 containing 50 mg~mL L, ~IUUIJ ~UI. The selective media was refreshed woekly.

WO 9610190S ~ 5 ~ r~
Seven to eight weeks post 1,- ' ' t, green. u ~ r .. ~ 1 tissue was observed gro-h~ing from ~ u, - ~fiv. Il. ~1, necrotic c"~blyye,~ clusters. Isolated gro~n tissue was removed and inoculated mto individual flasks to generate new, clonally propagated. '.c 1 ~L~Ab~Y~Ig~ C suspension cultures. Each new line was S trcated as an ;~ ti--n event. These , could then be subcultured and maintained as clusters of irnmature embryo.s or reeen~t~d itito whole plants by mamration and ,~ ; n ~ - of individual somatic ombryos.

Media:
l O SB55 Stock Solutions ~grams per liter):

MS Sulrate IOQX Stock MS H~ fl~ IOOX StocL
MgSO4 7H2O 37.0 CaCI2 2H2O 44.0 MnSO4 H20 1.69 KI 0.083 ZnSO4 7H20 0.86 CoC12 6H2o U.00125 CuSO4 5H2O 0.0025 MS P.B.Mo lOOX Stock MS FeEDTA IOOX Stock ICH~PO4 17.0 Na2EDTA 3.724 H3BO3 0.62 PeSO4 7H20 2.784 Na2MoO4 2H20 0.025 B5 Vitimin Stock SB55 (per literl 10 g m-inositol 10 mL each MS stocks 100 mg nicotinic acid 1 mL B5 Vitamin stock 100 mg pyrido~ine }ICI 0.~ g NH4NO3 1 g thiamhle 3.033 g KNO3 1 mL 2,4-D (10 mglmL stock) 60 g sucroso 0.667 g asparagiue pH5.7 21 925~0 WO96/0190~ r~l~u., SB103 (perliter~ SBI (perliter) MS Salts MS Salts ~% maltose B5 Vitamins 750 mg MgC12 U. 175 M glucose 0 2'3'o Gelrite 20 mg 2~4-D
pH 5.7 0.8% agar pH58 same a~s SB 1 e~cept 40 mg/L 2,4-D

~ ~ q255~ .
W 096/0190~ r~
ST~lr~rlErrcE LT.qTING
il) GENERAL INFORMATION:
~i) APPBICANT: E. I. DU PONT DE N~MOURq AND COMPANY
~ii) TITLE OF INVENTION: CHIMERIC GENES ~D
METHOD FOR INCREASING THE
THREONINE COUTEI~T OF
T~E SEEDS OF PLANTS
BER OF SEQUENCES: 9 ~iv) CORR~u~ .J~liC~ ADDRESS:
~A) ADDRESSEE: E. I. DU PONT DE NEMOURS AND COMPANY
~B) STREET: 1007 MAR~ET STREET
~C) CITY: WILMINGTON
(D~ STATE: DELAWARE
(E) COUNTRY: U.S.A.
~F) ZIP: 19893 (v) COMPlrTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC corrlpatiblo (C) OPERATING SYSTEM: PC-DOS~MS-DOS
(D) SOFTWARE: Microso~T Word 6.0 (vi) CUBE~ APPLICATION DATA:
(Al APPLICATION Nr3MBER:
(B) FILING DATF.:
(C) CLASSIFICATION:
(viil) ATTORNEY/AGENT INFORMATION:
(Al NAME: BAREARA C. SIEGELL
(B~ REGISTRATION NrMBER: 30 684 (C~ REFERENCE/DOCXET Nr3MBER: EB-1063-A
(i~) TTT~ uN INFORMATION:
(A) TELEPHONE: 302-992-4931 ~B) TELEFAX: 302-773-0164 (C) TELEX: 835420 ~1 92~5~' WO96101905 r~1/L1 ~ (21 INFORMATION FOR SEQ ID NO:l:
1i~ SEQUENCE C~ARACT5PISTIC.S:
(A) LENGT~: 30 base pairs ~B~ TYPE: nucleic acid ~C) sT~ANnr~NF~ : sil~gle ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomlc) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

~2) INFORMATION FOR SEQ ID NO:2:
(i~ SEQUENCE CRARA~~
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STP~ .q: single (D1 TOPOLOGY: linear ~i1 MOLECULE TYPE: DNA (genomic) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
rDDDrrT~r-r TTACAACAAC TGTGCCAGC 29 ~2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE rH~R~rT~RrsTIcs:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic aaid (C) sTRD~rn~-~q: single ~D) TOPOLOGY: llnear (ii) MOLECULE TYPE: DNA ~genomic) ~xi1 SEQUENCE DESCRIPTION: SEQ ID NO:3:
CTAGAAGCCT CGGCAACGTC Drr~D~r~rr. GAAGAATCCG GTG 43 (21 INFORMATION FOR SEQ ID NO:4:
~i) SEQUENCE r~DpDrT~RTqTIcs:
~A) LENGTH: 43 base pairs ~B) TYPE: nucleic acld ~C) STRANDEDNESS: single (D1 TOPOLOGY: Linear (ii) MOL,ECULE TYPE: DNA (genomic) (xl) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CATGCACCGG AT~.c~ CGTTGCTGAC GTTGCCGAGG CTT 43 ~ T 92550 ~ :
W OY61~1905 1 (2~ INFORMaTION FOR SEQ ID NO:S:
~i~ SEQUENCE CRARACTERISTICS:
(A) LENGTH: SS oase pairs ~B) TYPE: nucleic acid ~C~ STP~DEDNESS: s}ngle ~D~ TOPOLOGY: linear ~ii) MOLECULE TYPE: DliA Igenomic) ~xi~ SEQUENCE DESCRIPTION: SEQ ID NO:S:
G~TCCCATGG CGCCCCTTAA GTCCACCGCC AGCC.TCCCCG l~bC~bC~G CTCCT 55 (2~ INFORMATION FOP. SEQ ID NO:6:
(i~ SEQUENCE mp~R5~RrcTIcs:
(A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) sTp~l~EnN~cc single (D) TOPOLOGY: linear (ii) UOLECULE TYPE: DNA Igenomic) (~i, SEQ~E~CE DESCRIPTION: SEQ ID NO.6:
~r~ c n~.m~C~.~m GGGGAGGCTG GCGGTGGACT TT~CC~C~ CATGG 55 ~2~ INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE ~H~R~ERT.qTICS:
(A) LENGTH: S9 base pairs (B~ TYPE: nuclelc acid (Cl S~P~ ~nN~CC: single ID~ TOPOLOGY: linear (ii) MO~ECULE TYPE: DliA Igencmic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CATGGCGCCC ACCGTGATGA ~ b~U GGr~AHr~H b1~bUlU~bl TCCAGGGGC S9 (2) I1iFOPMATION FOR SEQ ID NO:8:
~i) SEQUENCE rR~RA~T~.RT.CTICS:
A~ LENGTH: 59 base pairs ~B~ TYPE: nucleic acid IC) Srp~Nn~n~qq~ ringle ~D~ TOPOLOGY: linear (iii MOLECULE TYPE: DNA ~genomi~~
~ SEQUENCE DESCRIPTION: SEQ ID NO.8:

2~

2 ~ q25~

~ (2~ INFORMATION FOR SEQ ID NO:9:
~i1 SEQUENCE ~R~ RTSTICS
~A) LENGTH: 174 base pairs ~B) TYPE: nucleic acid ~C) STPhNDEDNESS: doubLe ~D) TOPOLOGY: linear OLECUEE TYPE: DNA ~genomic) ~xi~ SEQUENCE DESCRI~TION: SEQ ID NO:9:
CCATGGCTTC CTCAATGATC TCCTCCCCAG CTGTTACCAC CGTCAACCGT ~ ~ 60 GCATGGTTGC TCCATTCACC GGCCTCAAAA GCATGGCTGG CTTCCCCACG A~.~AA~ 120 ACA~TGACAT TACCTCCATT GCTAGCAACG GTGGAAGAGT ACAATGTGCC ATGG 174

Claims (10)

What is claimed is:

1. A chimeric gene wherein a nucleic acid fragment encoding a bi-functional protein with aspartokinase and homoserine dehydrogenase activities, both of which are substantially insensitive to end-product inhibition, is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatorysequence.

2. The chimeric gene of Claim 1 wherein the nucleic acid fragment comprises the E.coli metL gene.

3. A plant comprising in its genome the chimeric gene of Claim 1.

4. Seeds comprising in their genome the chimeric gene of Claim 1.

5. A method for increasing the threonine content of the seeds of plants above the level found in seeds of untransformed plants comprising:
(a) transforming plant cells with the chimeric gene of Claim 1;
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds;
and (c) selecting from the progeny seed of step (b) for those seeds containing increased levels of threonine compared to untransformed seeds.

6. A plant produced by the method of Claim 5.

7. A plant comprising in its genome the chimeric gene of Claim 2.

8. Seeds comprising in their genome the chimeric gene of Claim 2.

9. A method for increasing the threonine content of the seeds of plants comprising:
(a) transforming plant cells with the chimeric gene of Claim 2;
(b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds;
and (c) selecting from the progeny seed of step (b) for those seeds containing increased levels of threonine compared to untransformed seeds.

10. A plant produced by the method of Claim 9.