Plant Molecular Biology 11:683-695 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

683

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants Thomas J. V. Higgins l, Edward J. Newbigin 1, 2, Donald Spencer 1,*, Danny J. Llewellyn I and Stuart Craig 1

1CSIRO, Division of Plant Industry, GPO Box 1600, Canberra 2601, Australia (*author for correspondence); 2Dept. Botany, Australian National University, GPO Box 4, Canberra 2601, Australia Received 27 May 1988; accepted in revised form 25 August 1988

Key words: vicilin gene, transgenic tobacco, Agrobacterium tumefaciens Abstract

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5' and 3' regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number o f integrated copies of the vicilin gene. Pea vicilin was confined to the seeds o f transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M r - 50000 together with a range o f smaller polypeptides.

Introduction

The seed storage protein fraction of most dicotyledonous plants is dominated by its globulin components. The globulins fall into two categories, the llS legumin type and the 7S vicilin type. The most studied members o f these two groups are the legumins from Pisum sativum, Viciafaba and Glycine max and the vicilins from the same species together with the vicilin-like protein (phaseolin) from Phaseolus vulgaris. Within each group, there is a significant level o f sequence similarity between members from different plant species [7, 29]. The seed storage globulins have many characteristics in common. They are all synthesized on the endoplasmic reticulum (ER) and are transported in-

tracellularly via the lumen o f the ER and the Golgi apparatus to be deposited in the vacuole or vacuolederived protein bodies. An amino-terminal leader sequence is removed cotranslationally during the translocation of the growing polypeptide chains into the lumen of the ER, and all legumins and some vicilins undergo varying degrees o f post-translational endoproteolytic cleavage after their deposition in the protein bodies (e.g. [11]). The legumins are cleaved to yield two polypeptides which remain linked by disulphide bonds [22]. The vicilins vary with regard to post-translational proteolytic processing. Phaseolin (from P. vulgaris) and /~conglycinin (from G. max) are not substantially modified in this way [6, 4], whereas vicilins from P. sativum, V. faba and Phaseolus aureus undergo ex-

684 tensive modification to yield a series o f smaller polypeptides which form a part of the oligomeric proteins (see [22] for review). The biological function, if any, of these proteolytic cleavages o f the original translation product is not understood. This paper reports the isolation and sequencing of a vicilin gene from P. sativum. As part of a programme to study the regulation of this gene and the fate of storage protein genes in a new host species, the vicilin gene was then stably introduced into the tobacco genome and a number of parameters of its expression were studied.

Methods

Construction and screening of a pea genomic library and sequencing of a vicilin gene DNA was isolated from light-grown pea seedlings (10 days old) as described [37]. The DNA was partially digested with Sau3A and fractionated by agarose gel electrophoresis and a size fraction corresponding to 14 to 20 kb was eluted from the low melting point gel [27]. The DNA was ligated to BamHI-digested M059 arms packaged and propagated in E. coli strain C600 [25]. A library o f 10 6 plaque-forming units was obtained and was screened with a pea vicilin cDNA clone pPS15-84 [36] as described [5]. Three positive plaques were purified and characterized by restriction mapping. One (M0.2) was subcloned into pUC8 (pEN2) and further characterized by sequencing using the dideoxy chain termination method [30]. For this purpose the 5.5 kb EcoRI fragment was sonicated and the subfragments subcloned into M13 mp8 [14].

Transfer of the pea vicilin gene into tobacco The plasmid, pEN2, was linearized at the unique Bam HI site in the polylinker and the entire clone was ligated into the Bgl II site o f the Agrobacterium tumefaciens/E, coli shuttle vector pGA471 [1]. This plasmid (pAGAB1) was transferred from E. coli (HB101) by conjugation to Agrobacterium tumefaciens (LBA 4404) using triparental mating methods

[15]. Transconjugants containing the binary vector were selected on LB medium containing rifampicin (50/~g/ml) and tetracycline (5/~g/ml). The structure of the gene in Agrobacterium was verified by Southern blotting [34] and tobacco (Wisconsin 38) leaf pieces were transformed as described [23] with modifications. The leaf pieces were incubated with the Agrobacterium for 15 min and were transferred, without blotting and without feeder layers, to agar culture dishes containing basal MS medium [28]. After two days the leaf pieces were transferred to agar culture dishes containing medium (MS9) for shoot induction (basal medium containing 0.5 mg/l indole acetic acid, 1 mg/1 benzyl amino purine) and the antibiotics cefotaxime (300 mg/l) and kanamycin sulphate (100 mg/1). Shoots of about 1 cm in length formed within 21 to 28 days and were transferred to basal MS medium containing the same antibiotics for rooting. Plants that formed roots (10 to 14 days) were later transferred to soil in the glasshouse (26 °C). Flowers were tagged at anthesis just as the petals opened and began to turn a pink colour.

Isolation and characterization of nucleic acids from tobacco DNA was isolated from young tobacco leaves as described by Sutton (1974). After digestion with restriction enzymes the DNA was fractionated on 0.80/0 agarose gels and transferred to nitrocellulose membrane (Schleicher and Schuell, Keene, NH). Hybridization was carried out using the 5.5 kb vicilingene fragment from the clone pEN2. The fragment was labelled by nick translation using a kit from Bresa (Adelaide, South Australia). Hybridizations were performed at 68 °C without formamide in the buffer described by Singh and Jones [32]. Heparin was replaced by polyanetholesulphonic acid, sodium salt (Sigma, St. Louis, MO). Blots were washed f o r 2 x 10 m i n w i t h 2 x S S C a t 2 5 ° C , then for 3 x 20 min at 68 °C in 2 x SSC containing 0.1°70 SDS and 0.1% sodium pyrophosphate, and finally for 3 x 20 min in 0.1 × SSC containing 0.1070 SDS and 0.1% sodium pyrophosphate at 60°C. The blots were fluorographed at - 70 °C using a Hi-Plus (Du Pont) intensifier screen.

685 Total RNA was isolated from frozen tobacco seeds by grinding in a mortar with a pestle using conditions described earlier [21] except that 1 M Tris, p H 9.0 was used instead o f 0.1 M Tris. The integrity o f the RNA was monitored by agarose gel electrophoresis in the presence o f formaldehyde [27]. S1 nuclease mapping was performed as previously described [38] using a single-stranded DNA probe [24]. The probe extended about 1000 bases upstream of position +210 (see Fig. 1). The protected fragments were separated on urea/acrylamide gels using, as size markers, 32p-labelled Msp I fragments of pBR322.

were prepared as described earlier [13]. In brief, tissue was fixed at 0°C in 0.1% glutaraldehyde-2% paraformaldehyde for 1.5 h, post-fixed with 1% OSO4, dehydrated through a series of ethanol solutions and embedded in LR White resin at 55 °C. Sections were treated with NalO4 and HCI [12], prior to saturating non-specific binding sites o f protein absorption with bovine serum albumin (10 mg/ml in 0.15 M NaCI, 10 mM sodium phosphate, pH 7.4 and 0.1% Tween-20). Anti-vicilin (20 #g/ml) and protein A-gold (15 nm) were used as described [13]. All steps were carried out for 10 min at 22 °C. Labelled sections were treated with uranyl and lead salts before viewing in a JEOL 100S electron microscope.

Western blotting Total protein was isolated by grinding fresh tissue with a mortar and pestle in 0.1 M N-Tris (hydroxymethyl)methyl-2-amino-ethanesulphonic acid (TES), p H 7.8, 0.2 M NaC1, 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, 2070 /3mercaptoethanol. The extracts were centrifuged at 10000 x g for 15 min. The concentration of protein in the supernatant was measured with the Bradford reagent [8]. Protein was fractionated by SDS-PAGE [35] and electroblotted onto nitrocellulose membrane as described [36]. Vicilin was detected with primary antibodies generated in rabbits by using an immunodetection kit (Bio-Rad, Richmond, CA) which incorporates a second antibody linked to alkaline phosphatase for colorometric detection.

Densitometry Fluorographs and negatives were scanned using a Joyce-Loebl double beam micro densitometer (MK111 CS). The relative contribution o f individual bands was monitored by cutting and weighing the peaks.

Immunocytochemistry Developing seeds from non-transformed (Wisconsin 38, W38) and transformed (B3, see Fig. 3) plants

Results

The structure of a pea vicilin gene A pea genomic library, constructued in X1059, was screened with a vicilin cDNA clone, pPS 15-84 (kindly provided by Peter Chandler). Three positive clones were mapped by restriction endonuclease digestion and a 5.5 kb EcoRI fragment from one clone (X10.2) was selected for detailed study. Part of the EcoRI subclone (4.2 kb of the 3' end) was sequenced on both strands and the sequence was compared (Fig. 1) to both the pea vicilin cDNA clone (pPS 15-84) and the gene sequence for an analogous 7S seed storage protein, phaseolin from Phaseolus vulgaris [16]. Over 99% of the nucleotides in the coding region of the isolated pea vicilin gene were identical to the vicilin cDNA clone used to isolate it (Fig. 1, lines b and d). The vicilin and phaseolin nucleotide sequences were aligned for maximum similarity by the placement of gaps in both sequences. As a result, the numbers used in Fig. 1 refer to position rather than to consecutive nucleotides within either sequence. The pea vicilin gene contained a direct repeat sequence in the 5' flanking region. It consisted of 54 bases (positions - 466 to - 519) which were repeated with only four mismatches between positions - 378 and -431 (note the two overlined regions in Fig. 1). No similar sequences were identified in the phaseo-

686

b. A A A A A C A T A A A T A C T A G A A T T A G T A T T T A T A T T T A T G A A C A T A T A T A T C A A A A T T A A T A T T A A T A A A A G T T G A C A A T T C C T A A A A T A A A T T A T A T T T T A T A T T T A T T A A A T A T T A T T A T T

-1435

b.

AATTAAAATATTTGACTACTATTTTAATTTTAATAAACATTTAAGCTACAATATCATTAAAAGATATTATCTTCAATTTTTATATATAAAAAAAAATGTTAGAGAGGAA-%CATTTCCCTC

-1315

b.

ACAAAATTCATTACAATGACATCTTGCACATTTTTGATGGAATTTGTGAGGAAAATGTTAAAGATAGATAAAATTCATCTCAAAC.CTCTTCACAAAATTTACTTTTATCATATATTTAGG

-1195

b.

GAGTTTGTGAGGGATTATTTGTGACTGAATATTTTCATAACAAATTTCATCGCTAATGTTCATCCATTTAATTTTTTTTATTTGTAGTAAAAATACTATTCTCTCTCTTCCATTTATTAT

-1075

b. A T TT TT T A A A A C T T T GTCdtAATGATCAACTA T G A C A A T T A T T T T G G G A C G G A G T G A G T A T C A T G G T T T b.

TT T T A G G A A C A G T T T T A C A A A C T C A T T T G T T A T T G G A C A C A G A T A A C A T

TGATCGTGTCTAATTTCTTAACTGTTCT

c.

.T T T . . A C A .

b.

GATAGAACGATC

c.

TG, TATG.A

T TGGTGTGATAAAACATAATAATTACTAATAT

b.

AC T A A T A A T G C C C T G A G A A T A C G G A T A T A

C.

TA..TC..ATA.

.AA.. CTATAAT.

TAAATTAATATTAGTTTTAAATGTT

C C C .TT. G. C. C. A C G G . . G T A A C T G A A G . .

TAGT TATCATGTGTAGATAAAT

TT TAT TAAAAAATAATTGTATGTTTTATCCTTATTACTCCATCTTCTC

C. G C T . . . A C . T G C G A G A C .

T.AAA.A...C.TT

b.

TAAAAG TAGAT TATAATATATATTATATTTT

c.

A . . T T . . T T T A . . G. T A G . T . . A A G . . A A . A . A A G . A A .

TTT

T . A A . . ATAG, TACTA, A T T C A A G A .

CATCTTCTA..G.AAT.

b.

T A C A T TT T T A T A T A T T A A G T A T A T T A T A G T A T A T A T C A A G T A G A C G G A T A A A T A A T A G A

C.

.T T . C C . . . T C T . C ~ G . . T . . . A G . C C G T A A C . .

.... A T T . T A A G T . T . A T T G T T G A A T .

TGTGACTAT.GA,,.

T T C T T TT T T A A T A T A T T A A A T A A A G T A T A G T A T A T G T A A A G T A A A C C - G A T A A A T A A T A G A T A A A T A A T GT. GTAGAGTGT..

G . . . G. T A C C C T A A A C C .

G. A T T . C A G T G G . T T G C C . .

-835

-715

T.CA

A. T . . . C T A C T A .

-595

, .TT

TAAATGACATATATCTACAAT

-475

T A .. C T A T .AC. T. T . . G. T G G . C . . . . . T T C A T . T . . T . C . TATTC-C.

TAGATAATTAAATGACGTA

T A T G T A G A A T T A C A T T TT T C A C A T G A C A A G T A C A A A C C T A T G T C . . G . G C A A G A A . A. G A C A . . . A . . . . . GAA. A A

-355

GGC. CTCT. TGGTC. TTTG. TTCATGCATGG.

b.

C A C TT C T A A G T G C A A G T T T A T G G A G T T A T T T G C A T G T C T T A G A G C T T G T G T T G T A G G A T A C A A C A C T T G T T A A A A T T C T C T A G T C A A T T C A T T A A T

c.

AGACAAA.

C.

CATCCATCCAGAGTACTACTACTCT.

GACAA. AC. CAA. CACACA.

-955

T G . . A A T . T C . C C A A A . . A. C A . T. G T . G . . . T T C T G A A G C . A G T C A

TT T G G A C A T G A C A A A T A T A A C C G T T T T A A T A A TT T A T C A TCT T T G T G A T G G A G A A T T A T G T A A C T A T A TTT TT T T T A A T T G A G G A T T G T A A

T C A . T . T ,.. T T C . . A . A A A T T . A T . A G A T , T A . .

CAGAG,

TGGACT TAAAAAACATTTAAATTT

T TAAAAATT TTAATTCACAT TTTAACAAT TATAAAATTGTCATTAGTAT

CCAAC. CA. AT.A.. CACTGC-CTGAT...

G. T C G C C C - C G T C C A T G T A T . . .

C T A . T. T A . T. C C C C A . C C C A . C. C A T . T .... T . C T A . . *

T. A A T G C C .

...... TG .... G...AAGGG

TCATATACACATGGCCGAAGACAA T G C A . AC-C. A C A C G T . .

....... CC.,T

-235

T T . . C. T G C

.... G*** ...... CT ....

d ................................................................................................

C.

. . G . . . . . . C . T. C . . C C . . A . T. G C C A . . T C A . T C . G G G . G G A G G A A G A G A G .

C . A G A .... C C C . . C T . C T T C A A C .

C... C.. CTCCTGGAACACTCTATTCAAAAACCAAT

.... T

d .........................................................................................................................

C .........

T G . C .. C .... G G . . C . . . . . . . A A . . C . . . C G A C .

d ...............

c.

. .T..G.TAC

A ................

TC ....... TG..G ...........

G .... G.TC. G .........

CG.A..CC.CC

....... T ..... GG. T..,

T ........................................................................................

. . . . A . . . . . C . G T . . . . . . . . G , A . . . G C . A C . G G T A . C .CT, G . . . C T T C T . G C A G . A A T

.AT. G . A . . G A G T T T T T T .

*****************************

d ........................... TCAAACACTCAC

TT TT TTAT TGGT TGAAACATGTGTGGGTCCC

TTTGCAACAA

606

TAAAT TCAGTTATAAAATCTAATGTCATG

726

b.

AACAC TC TC TT TT CAACACTC

c.

************************************************************************************************************************

T CGCTT TGGAAA TAGGTCCCACATAAAGTGGTAAGAG

TCACACATA

d. b.

ATGAAAGAGTGTGTGTTT

c.

************************************************************************************************************************

C~GAGAGTGT TGAAAAGAGAGTATTATTAGCATT

TCTC T T A T T A C T T T A C A T A T T T G T T T T G T A A C ~ T A A A T

d.

::

TT

cc

oLL

o

GT

LL-.

cc

ToL

A

LA

...............

C ........ * . . . G . G C . . C . . . . . . G T C T . . G . . . . . . . T . . . . . . C. C. G A G A G . A .... T T .... A C . A ... A T A ~ C C . . . . . T T C T C T G A T C A C C A G , d ...............................................................................................................

c.

TTC

. . . . . . . . . . . .

CCT...CC...A

d . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.....

T..C...A.

,A.CC.A

........

G .....

T..C.AC.

~ .***..GA

....

T...C.

*************

846 . .A. C . . . . . A . . A . . C . . .

~*************~***

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

b.

TATGGTATGCAT TCTAATGTACA~C~TTTAGC-C~CCTCTACCATAACATCACAACA~T

C. d.

** * * * * * * * * * * * * * * * * * * * * * * *

**"****

* * * ** * * * * * * * * * * * * * * G .

CTTTTGT..

TGCGCCTGTACATAT TACCGAA.

TT TTGCTGTR.~.TAT T T T C C T A A T A T T T T C T T T A T T T T T T T T

,A A T T T . T . G . . A T T . T A . C. T G C A A T T .

CTCTCCAAA.

G. G A . G A . A A A

1086

687

a. b.

S

F

L

L

S

G

N

Q

N

Q

Q

N

Y

L

S

G

F

S

K

N

I

L

E

A

S

F

N

GTCCTTTTCAA~GT~TTTCTTATTGTCTGGAAATCAAAACCAACAAAACTA~TTATCTGGGTT~GT~GAA~TTCTA~GGcTTCCTTCAATGT~GCA******************

c.~TT.G.C.TGT..GAA..T..CC.A...A.C.~G..~

.......

d.

TC ......

GCAA.A

...... C...C.T

...........

1206

C ..............

A.AGAAAA~GCATCT~CT

.A ..........................................................

a. b.

T

.... TG.AT..GT.GAAT~.A.~TT..AT

Y

E

E

I

E 1326

.... TTGCATG.TTTT

...... ~.A.TC..G

d.

a.

D

***T~CA~CAATTTTTTTTT~TTTATGTATGTATTAGTTT~TATTGTATATGTT~TACT~CTTTGTCAATGCA~TACTGTAAAAAAATATA~CT~TTATGAA~TAGAA

c.A~..TTT~GTTGC~...AG.TAG..CT.TGTCT~.TG.~C

..... ~.C

• ...................

K

V

L

L

E

E

H

E

K

E

T

Q

H

R

R

S

L

K

D

K

R

Q

Q

S

Q

b.

AAGGTTCT

TT TAGAAGAGCATGAGAAAGAGACACAACACAGAAGAAGCCTTAAGGATAAGAGGCAGCAAAGTCAA~TGT~TAGT~TTAT~~

c.

.G ......

G..T

......

G.G.GAC.GC.AGAGGG.GTG.TTGTG.A.A.

E

E

N

V

I

V

K

L

S

R

G

Q

I

E

E 1446

T T G A ~

****************************************************************

.....

d .........................................................................................................................

a.

L

S

K

N

A

K

S

T

S

K

K

S

V

S

S

E

S

E

P

F

N

L

R

S

R

G

P

I

Y

S

N

E

F

b.

TTGAGTAAAAATGCAAAGTCTACCTCCAAAAAAAGTGTTTCCTCTGAATCTGAACCATTCAACTTGAGAAGTCGCGGTcCTATCTATTcCAACGAGTTTC`GAAAATTcTTTGAAATCACC

c.

C .... C...C

.......

A .... GT..A.GG...TCCC

..... AAAC..GA.A.CA.

****--***************************

G

K

F

F

E

I

T 1566

..... A ........

CC.GAC...G.GG...

d .........................................................................................................................

a.

P

E

K

N

P

Q

L

Q

D

L

D

I

F

V

N

S

V

E

I

K

E

b.

CCAGAGAAAAATCCACAGCTTCAAGACTTGGATATATTTGTCAATTCTGTAGAGATTAAGGAGGTATGATAAAATTATTTTATAATATAGGAAATT~TTA~T~GATTTC

c.

**************************

.... A..G.G..AA...G

.... A .......

GG.A

...... AATAC...

1686

GA.AAACCAT...G.CA..C.CAG.A.TTGAGTTCT..T.TTCACT

d ............................................................. b.

ACTTGATCAAATTACAATTGTTCTAAATGATTTGATTTTTGTCCTTTGAAGTTATAATGTCAAAC

c.

GTCGTC.

TGGT.ACA..A.C..AGT.T...GACT..AA..AAATAA..GTT.

TTTTGTTACTAACTTGACATCTCATACACAACAAGTTTTACATACTCAATAACAT

1806

********************************************************************

d. a.

G

S

L

L

L

P

H

Y

N

b.

GTTTTATTTATAG~TATAT~T~TGTATTTATTT~TTATTCTTTCAAATTAAATATTAC-C-GATCTTTATTGTT~CTACAATTCAAC-GGC~TAGT~TAGT~GTT~C

c.

******************************************************

d .

a.

.

E

G

K

.

G

D

F

E

.

L

V

G

Q

.... * .......

G..C.T..TG

.

R

N

E

N

Q

S

R

A

I

V

I

V

E

Q

R

K

E

D

D

E

E

E

E

Q

G

E

V

N 1926

...........

T .... T.A ......

T..T...C..GTG

C ................................

Q

T

E

E

I

N

..... T

T .................

K

Q

V

Q

N

b.G~GGAAAAG~TTTTGAACTTGTGGGTCAAAGAAATGAAAACCAAC~C-CA~GAAAAGAA~T~C~GGAA~GGAACAAGGAGAA~GGA~TAAATAAA~GT~T c .......

G...C.C..G

..........

d ............................ a.

Y

K

A

K

L

S

S

~

G

2046

T..C.C..A.GGAA.T..~..ACCTT.G.ATATG.GA~T.~.CT..~T~CTA...AC******************************

D

V

........................................................................................... F

V

I

P

A

G

H

P

V

A

V

K

A

S

S

N

L

D

L

L

G

F

G

I

N

A

E

N

N

Q

b.TACAAAGCTAAATTATCTTCAGGA~TGTTTTTGT~TTC~GCAGC-C~Tc~GTTGCCGTAAAAGCTTCCTCAAATCTT~TTT~TTGGGTTTGGTATT~T~T~GAAC~TCAG c.

************************

..... A ..... A..C

.......

AT

...........

A.C..G...A

.... C..CG.GA

2166

.... ~C...T..C

..... C ...... A.T ..... CA.T

d .........................................................................................................................

a. b.

R

N

F

L

A

AGGAACTTTCT~TATATTATATTAT~CC~GTCTCTGT~CTATTTATTCATTTT~GTGTGTATTTTAAAAGTC~CTTCTATTTAAATCAAGC`GGAAAATATT~GATAT~

c .......

C.C .............

ATAT

2286

.... T.TAT.T~C.AT~.T~TGAATA.AGGG~.GTTC`A..G.A...TTT.T.TAT~.T~.~T~GTG.TT*******************

d ..............

a.

G

D

E

D

N

V

I

S

Q

I

Q

R

P

V

K

E

L

A

b.

TTATTATTTTGGTGATTAAAAATTTGAAGGCGATGAGGATAATGTGATTAGTCAGATA•AGCGACCAGTGAAAGAGCTTGCATTC•CTGGATCAGCTCAAGAGGT

c.

*******"***

...... GT .... A ......

TA.GAC...C

F

P

N

Q

K

Q

S

H

F

A

D

A

Q

P

S

A

Q

E

V

D

R

I

L

E

TGATAGGATACTAGA

2406

..... C..A..CAGC..CGGTA..G.TC..G.C.GTA~%.ACG.GTTG..GCTTA.GTTCTCT.GGTC.G.TGACGA..T

d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a.

G

Q

Q

R

E

R

G

S

R

E

T

R

b.

GAATCAGAAACAATCCCACT

c.

T.TGA..CTGATCAA.A.ACAGAGT.GAT.GT.CTT.GTGG.TGCAC.CCATCACCAA.AG...CAGCA.A.GG.AAG.AAGGGT.CA.

D

R

L

S

A ...............................

S

V

TTGCAGATGCTC•ACCTCAACAAAC`GGAGAGAGGAAGTCGTGAAACAAGAGATCGTCTATCTTCAGTTTGAAATGTTTCTTAATGAGTGGACAAAATACT

2526

TTGTGTAC.GAAT.A.TA..A..T...ATGC

d .........................................................................................................................

b.

ATGTA

C ......

TGTATGCTATCAAGAGATATATCTCACGGGGAGCAATGAATAAAACA G..G.

******

..... ******

.... T..A

..... TG ..... TTGT.

d ....................................................

* * * * * * *ATGTTATCTTATAACTATAATTATATATCCACTTTTCTACTATGAATAAATAAATC;~%GAG TCCGACC

.... A.CAG

..... TA.CTGAGC.CC...

* ...... A ................

TCAC*

.... T ...........

2646 C... GG.T.

TT

(A) n

b.

TATTTTGGTGTTCCTCTTAGATATTATATTATTTTAAGAAAGTCAATAACTAAAAGTACAAAACACTCATTAAATTTAAACTAATGTAAAGACTTAATATCTCATATCCATGATTCATGA

c.

ATGA

b.

TATACAATCATCAATCAATTAATAGAATTC

2766

Fig. 1. The gene, c D N A and protein sequences of a pea vicilin. A phaseolin gene sequence [16] is included for comparison, a = protein sequence deduced from the vicilin gene; b = vicilin gene sequence; c = phaseolin gene sequence; d = vicilin c D N A sequence. * = gaps introducted to maximize nucleotide matching; • = identity of bases. In line d, a continuous line indicates the position o f introns. Direct repeats in the vicilin sequence are overlined while the regions conserved between the vicilin and phaseolin genes around the "TATA" box and in the upstream region ( - 6 2 to - 1 5 3 ) are boxed. The two downward arrows indicate the positions of post-translational processing in vicilin. Note that, because gaps have been introduced to maximize matching, the numbers refer to position only and not to actual nucleotide numbers in the sequence.

688 lingene at the 60% matching level. The pea vicilin and phaseolin gene sequences showed an overall similarity of 39°7o in the sequences covering about 900 bp in the 5' regions plus the coding regions and about 150 bp 3' to the stop codons. However, there were short regions of much closer identity, particularly in certain parts of the 5' flanking sequences and in some of the exons. For example, there was notable sequence conservation in the 5' flanking sequences of the two genes between positions - 2 0 0 and +1 (the cap site), particularly around the "TATA" box (position - 3 3 ) and cap site and again further upstream (the boxed positions between -153 and - 6 2 ) , covering a sequence of about 80 bases (Fig. 1). Although the 5' untranslated sequences were divergent there was sequence conservation in the 3' untranslated region and this was especially obvious upstream (starting at position 2620) of putative poly(A) addition signals which occur at positions 2570 and 2630 (Fig. 1). Both vicilin and phaseolin genes contained five introns located at identical positions with respect to the amino acid sequence. The introns were all small but neither their precise size nor their sequence was conserved between the genes except around the splice junctions where they conformed to the consensus sequence described for other plant genes [9]. Nucleotide sequence similarity in the six exons was highest in the third exon (70o7o)and lowest in the fourth (40070) and sixth (36%) exons of the gene. Sequence comparison at the amino acid level in general reflected the nucleotide comparison. Overall, there was 50o70 identity between the amino acid sequences of vicilin and phaseolin (data not shown), the third exon showed highest conservation (63o7o) while the fourth and sixth exons were lowest (32 and 340/0, respectively). In pea seeds, some of the vicilin polypeptides undergo post-translational cleavage at one or both o f two cleavage sites [19, 36] while, in bean, phaseolin is not cleaved [33]. The cleavage sites in vicilin are characterized by charged amino acids and cleavage occurs on the amino terminal side o f an aspartic acid residue (note the arrows at positions 1381 and 1996). When the vicilin and phaseolin amino acid sequences were aligned for maximum similarity the first processing site of vicilin was found to be deleted

in phaseolin and the sequence around the second processing site was not conserved in phaseolin and there were six and twelve amino acid deletions in phaseolin on either side of the processing site (data not shown).

The expression of a pea vicilin gene in tobacco A DNA construction containing the entire 5.5 kb EcoRI fragment of pEN2 together with a selectable neomycin phosphotransferase II gene was introduced into tobacco leaf pieces via an Agrobacterium tumefaciens binary vector transformation system. Kanamycin-resistant plants were regenerated from infected leaf pieces and the mature seed from these transgenic plants was analysed for the expression of pea vicilin by the Western immunoblot procedure (Fig. 2). Anti-serum against pea vicilin reacted with one major seed protein component (M r - 4 7 0 0 0 ) in seeds from untransformed tobacco (Fig. 2, W38). In addition to this vicilin-related

Fig. 2. Vicilin-related polypeptides in the seeds of nontransformed (W38) and transformed (B3) tobacco. Samples containing either 150/zg of protein from tobacco seed extracts, 3/zg o f protein from pea seed extracts or 0.2 or 2.0/~g of vicilin purified from pea seed extracts were fractionated by SDS-PAGE, electroblotted onto nitrocellulose and probed with antiserum prepared against pea seed vicilin. Numbers on the vertical axes give the approximate M r x l0 -3 of a number of major vicilin-related polypeptides.

689 sitometer tracings of a more extensive series of vicilin concentrations than shown in Fig. 2 with the level found in a known amount of B3 seed protein. These indicated that pea vicilin comprised about 0.5°70 o f the total seed protein.

tobacco protein, anti-vicilin serum reacted with a number o f other protein components in the seeds o f the transformed tobacco (Fig. 2, B3). The m a j o r new c o m p o n e n t had an Mr o f - 5 0 0 0 0 and comigrated with the m a j o r c o m p o n e n t of vicilin from pea seeds (Fig. 2, vicilin, pea). A number o f smaller cross-reacting polypeptides were also observed in the seeds of transgenic plants (Fig. 2, B3). The mobility o f some o f these polypeptides on SDS-PAGE is the same as that o f the post-translational processing products o f authentic pea vicilin ( M r - 50000, 34000, 27000, 25000 and 12000) (Fig. 2, vicilin, pea) while others are unique to the transgenic tobacco seeds (M r - 3 8 0 0 0 , 19000 and 10000). The level o f pea vicilin expression in the mature seed of a range o f nine independently transformed tobacco plants was examined by Western i m m u n o blot analysis (Fig. 3). All plants had been selected for kanamycin resistance, and this resistance was confirmed by at least one further passage through tissue culture. Eight out of nine transformants contained detectable levels o f pea vicilin, with similar levels in plants B4, B6, B7 and B8 and progressively higher levels in B10, B2, B1 and B3. One transformed plant (B9) failed to accumulate detectable levels of pea vicilin. A n estimate o f the a m o u n t o f vicilin accumulated in seeds o f the most highly expressing transgenic tobacco (plant B3) was obtained by comparing den-

D N A was isolated from the leaves of a selection o f transformed tobacco plants, digested with EcoRI endonuclease and analysed by the Southern blot procedure using the vicilin gene (5.5 kb EcoRI fragment o f pEN2) as probe (Fig. 4). The level of pea vicilin D N A in the transformants correlated closely with the level o f accumulation o f pea vicilin in the mature seeds o f the transformed tobacco plants. Transformant B9, which yielded no detectable pea vicilin in the Western blot assay (Fig. 3), did not contain the pea vicilin gene but remained kanamycinresistant. Transformant B3, which gave the highest level o f vicilin expression (Fig. 3), also contained the highest number of introduced genes. Densitometric comparison o f the signal obtained for the vicilin gene in the nine transformed tobacco plants with that given by a set of gene reconstructions (not shown) indicated that, transformants B6, B7 and B10 each have one to two copies o f the introduced gene, B1 has 6 - 8 copies and B3 at least 30 copies.

Fig. 3. The distribution of vicilin-relatedproteins in the seeds of

Fig. 4. The presenceof the pea vicilingene in six putative tobacco

nine putative transformants (BI to B10)and the non-transformed progenitor (W38). Seed extractscontaining approximately350/~g of protein were fractionated by SDS-PAGE, electroblotted onto nitrocellulose and probed with antiserum against pea vicilin. Numbers on the vertical axes indicate the approximate Mr x 10-3 of the polypeptides resulting from the expression of the introduced pea vicilin gene.

transformants (Bl, B3, B6, B7, B9, BI0). Total DNA (15 /~g)from leaves of each tobacco plant and a sample of total pea DNA (15/~g) was digested with EcoRI, fractionated by electrophoresis on agarose,blotted onto nitrocelluloseand probed with the vicilin gene (5.5 kb EcoRI fragment from pEN2). Figures on the vertical axis show the size in kilobases of known DNA markers (hDNA digested with HindIII).

Vicilin gene copy n u m b e r

690 In all cases studied there was a close correlation between the number of vicilin gene copies detected and the level of vicilin protein in the transgenic tobacco. This suggests that in these cases the actual position o f insertion of the gene in the tobacco genome did not have a major influence on the level of gene expression.

Intracellular localization o f pea vicilin in tobacco seeds Developing seeds o f transformed (B3) and nontransformed (W38) tobacco were fixed, embedded and sectioned before challenging with anti-vicilin serum followed by gold-labelled protein A. Examination of these sections in the electron microscope revealed that vicilin-related proteins were accumulated in the protein bodies of the developing seed of transformed tobacco (Fig. 5A). In seeds of non-

transformed tobacco there was a very low level of immunolabelling (Fig. 5B), presumably due to the vicilin-like tobacco proteins detected by Western blotting (Fig. 2, W38). The greatly increased level o f labelling in both the cotyledon and embryonic axis o f the seeds of the transformed plant reflects the accumulation of pea vicilin in these seeds. Tobacco protein bodies contain a distinct crystalloid structure within a background matrix. Both pea vicilin and the tobacco vicilin-related protein were largely confined to the matrix of the protein bodies (Fig. 5A, B). Tobacco seeds differ from pea seeds in that they contain a significant volume of endosperm as well as cotyledon tissue in the mature seed. Although protein bodies in both storage tissues appear structurally similar (Fig. 5A, C), immunolabelling indicated that pea vicilin was largely confined to the cotyledons and embryonic axes of the transgenic tobacco seeds. Out of ten embryos (cotyledons plus

Fig. 5. Electron micrographs of protein bodies in cells of cotyledons(A) and endosperm (C) tissues from the tobacco transformant B3 and in cotyledoncells of non-transformed tobacco (B). Sections were immunogold-labelled for vicilin. A and C were recorded from a single section in whichboth cotyledonand endosperm tissue had been fixed in situ. Vicilin is sequestered within the proteinaceous matrix of the protein bodies but was not detectedin the crystalloid, labelled (C). All micrographsare from developingseed at 19daysafter flowering. Micrographswereprinted to enhancethe contrast of the colloidal gold. As a result the lipid bodies, whichfill the adjacent cytoplasm, are not evident.

691 embryonic axes) examined, vicilin was detected in eight cases. In contrast, it was detected, at a much lower level, in two out of eleven endosperm sections.

Organ specificity of vicilin gene expression Western immunoblot analyses for vicilin in extracts of roots, stems, seeds and leaves of transformed tobacco indicated a high level of specificity in the expression of the vicilin gene (Fig. 6). In both transgenic tobacco and in peas, vicilin gene expression was confined to the seeds, although at much higher levels of loading of total protein, faint cross-reacting polypeptide bands were detected in stems and leaves of peas (Fig. 6, lanes 2 and 3) and in leaves of transformed tobacco (Fig. 6, lane 7).

Temporal pattern of vicilin expression in developing transgenic tobacco seeds

vicilin-related protein of non-transformed tobacco were determined by Western immunoblot assay (Fig. 7). Vicilin-related polypeptides (Mr -55000) were detected in non-transformed seeds at the earliest harvest (11 days after flowering), although the particular polypeptide which is characteristic of mature seed (Mr - 4 7 000) was not detected until later in development (17 days after flowering). This transient appearance of polypeptides of higher molecular weight earlier in development suggests a possible precursor relationship with the smaller, later product. In the seeds of transgenic tobacco, pea vicilin (Mr - 50000) was detected at the earliest harvest (11 days after flowering) and increased t o a maximum proportion of total seed protein by 17 days after flowering, by which time all the cleavage products were evident. The early synthesis of the pea vicilin in tobacco suggests strongly that transcription of the vicilin gene is regulated in the same temporal fashion in transgenic tobacco as it is in peas [10]. This conclusion was supported by the changing

The patterns of pea vicilin accumulation in developing transgenic tobacco and of the endogenous,

Fig. 6. The distribution o f viciiin-related polypeptides in the organs o f pea a n d transformed tobacco plants. Extracts o f roots (lanes 1 and 5), stems (lanes 2 and 6), leaves (lanes 3 a n d 7) and seeds (lanes 4 a n d 8) were fractionated by SDS-PAGE, electroblotted onto nitrocellulose a n d probed with antiserum to pea vicilin. Lanes 2, 3, 5 a n d 6 were loaded with 150 #g protein, lanes 1 and 7 with 75 #g protein, lane 8 with 38/zg protein and lane 4 with 5 #g protein. N u m b e r s on the vertical axis give the approximate M r × 10 -3 of vicilin-related polypeptides.

Fig. 7. The pattern of accumulation of vicilin-relatecl polypeptides during seed development in transformed (B3) and nontransformed (W38) tobacco. Seeds were harvested at intervals after flowering, extracts containing 150 #g protein were fractionated by SDS-PAGE, electroblotted onto nitrocellulose and probed with antiserum against pea seed vicilin. Numbers on the vertical axis show the approximate M r x 10 -3 of the major vicilinrelated polypeptides.

692 levels o f pea vicilin mRNA throughout development o f transgenic tobacco seeds as measured by S1 protection assay (Fig. 8). Pea vicilin mRNA was detected at 11 days after flowering and the levels increased to a maximum at 15 (or 17) days and thereafter declined sharply to 19 and 21 days after flowering. Vicilin mRNA was not detected in non-transformed tobacco seeds (W38) at 17 days after flowering. The sizes of the two DNA fragments (183 and 180 bases) which were protected from the action of S1 nuclease by the mRNA were the same in both transgenic tobacco and in pea (Fig. 8) indicating that the start site(s) of transcription of the pea vicilin gene is/are the same in both these situations. From densitometric measurements, the maximum level o f pea vicilin mRNA in the transgenic tobacco plant, B3, at 15 days after flowering was estimated to be approximately 1.5070 of the level found in pea RNA

Fig. 8. The level o f vicilin m R N A in seeds of a transformed tobacco (B3) during seed development as determined by SI protection assay. Lane 1 contains radioactive size markers generated by labelling pBR322 digested with MspI endonuclease. Lanes 4 to 10 contain 50 t~g of total R N A isolated from developing tobacco seeds at 11, 13, 15, 17, 19, 21 and 17 days after flowering, respectively. The R N A was first hybridized to a 32p-labelled probe comprising 183 bases of the coding region and cap site together with a further one kb 5' to this region. The hybridized mixture was then digested with S1 nuclease and the products fractionated by electrophoresis on acrylamide gels and the protected fragments detected by autoradiography. Lanes 4 to 9 contain total R N A from seeds of a tobacco plant (B3) transformed with a pea vicilin gene, lane 10 contains R N A from a non-transformed plant (W38). Lanes 11 and 12 contain 1.0 and 0.1/zg total RNA, respectively, from developing pea seeds at 17 days after flowering. Lane 2 contains undigested 32p-labelled probe and lane 3 contains labelled probe and 50/xg t R N A digested with SI ribonuclease.

from developing seeds at 15 days after flowering.

The fate of pea vicilin in transgenic tobacco seeds during germination Pea vicilin in transgenic tobacco seeds was degraded early in seedling development (data not shown). Breakdown products of pea vicilin were apparent in transgenic tobacco seed extracts one day after imbibition and the original pea vicilin components were almost entirely degraded after six days. This breakdown of pea vicilin followed the same time course as the breakdown of the vicilin-related tobacco protein (M r - 4 7 000) during germination.

Discussion

We report here the isolation, DNA sequence and expression of a pea vicilin gene in a foreign host. Based upon restriction map comparisons, this gene probably belongs to the vicilin gene family at locus Vc-4 described by Ellis et aL [17]. This family of closely related vicilins (over 95O7o base matching) contains at least four members (P. M. Chandler, personal communication). Although it is not known whether this particular gene is expressed in pea seeds, it clearly has the potential to do so, because it can be expressed in the transgenic host, tobacco. The cDNA used to isolate this pea vicilin gene is over 99°7o identical, at the nucleotide level, to the corresponding sequence of the isolated genomic clone. Of the nine nucleotide changes in the coding region, six of these result in amino acid changes, some of which are not conservative in nature. No changes were found in the (short) 5' untranslated region and the cDNA contained a single 7 bp insertion in the 3' untranslated region. Both the isolated gene and the cDNA probe contained a putative polyadenylation signal (AATAAA) 31 bp upstream of the site of addition of the poly(A) tract, but there was a second putative signal further downstream in the gene (position 2634) which, if used, would result in two populations of m R N A with slightly different lengths of 3' untranslated regions. Lycett et al. [26] have reported the isolation and sequence of two partial cDNA clones

693 (pDUB2 and pDUB7) encoding vicilin polypeptides. These clones showed 86°70 base similarity in their overlapping regions. The sequence of the vicilin cDNA reported here (pPS15-84) shows 84070 and 85070 base similarity to pDUB7 and pDUB2, respectively, where their coding regions overlapped. Thus, all three c D N A clones show substantial sequence divergence from one another, consistent with the findings o f Ellis et al. [17] who showed at least three different loci for the vicilin genes in pea. All genes for 7S seed storage proteins from a range of genera have similar basic structural features in that they all contain five introns [16]. There are also strong nucleotide sequence similarities particularly within the coding regions o f the 7S genes [7]. Intron size and sequence are not conserved except for sequence conservation around the splice junctions. The 5' flanking sequences of the 7S genes in pea, Phaseolus vulgaris and soybean are divergent except for three regions o f conservation. Two are short sequences around the cap site and "TATA" box [16] and the third is a sequence o f about 80 bp located about 100 bases 5' to the "TATN' box in all three genera but occurring between positions - 6 2 and 153 in the pea vicilin gene [16, 19], this study). The role, if any, o f this "7S or vicilin box" in regulating gene expression has yet to be determined. When the pea vicilin gene was introduced into tobacco, it was expressed in its new host with a high degree o f fidelity with respect to all the parameters studied. These included the temporal pattern o f vicilin m R N A (Fig. 8) and vicilin protein (Fig. 7) accumulation during seed development, the start site of transcription (Fig. 8), the organ and tissue specificity of its expression (Figs. 5 and 6), and its susceptibility to post-translational processing (Fig. 2). For several of these parameters, a similar high level o f fidelity o f gene expression was reported earlier when genes for two other vicilin-like proteins (phaseolin from P. vulgaris and/~-conglycinin from G. max) were introducted into tobacco or petunia [2, 3, 31]. The preferential accumulation o f the foreign protein in the cotyledons rather than the endosperm tissue o f the transgenic tobacco seeds is particularly striking with both phaseolin [20, 31] and pea vicilin. Both these tissues o f the tobacco seed contain protein bodies, but it appears that the pea vicilin and -

phaseolin genes contain regulatory sequences which specify embryo (cotyledon plus axis) expression. Only low and irregular accumulation of these foreign proteins was detected in the endosperm, which is maternal tissue. Using in situ hybridization, Barker et al. [2] have recently reported the strict localization o f mRNA for soybean #-conglycinin in the embryo of transgenic tobacco. The accumulation of newly synthesized vicilin in the protein bodies in transgenic tobacco seeds (Fig. 5) strongly suggests that the targeting signals inherent in the protein structure of vicilin elicit similar responses from the tobacco cell as from the pea cell. This in turn implies that, as is the case in pea [22], vicilin in transgenic tobacco is synthesized on the rough endoplasmic reticulum and transported via the Golgi apparatus to the protein bodies. However, we were unable to detect vicilin in either the endoplasmic reticulum or the Golgi bodies o f transgenic tobacco, presumably due to the low amounts present. Some differences were observed in the detail o f the post-translational cleavage of vicilin in transgenic tobacco in that not all the resultant polypeptides were the same size as in the pea seed (Fig. 2) and additional cleavage products were detected. This implies either that the conformation o f pea vicilin in tobacco differs from that in peas in a way that exposes other sites to proteolytic attack, or that proteases with different specificities are present in the tobacco seed protein bodies. A more extreme difference was seen in the case o f phaseolin in transgenic tobacco [31]. Phaseolin undergoes no major proteolytic processing in its natural host, but in transgenic tobacco about 70070 of the primary translation product was cleaved to form a group of smaller polypeptides. However, the overall pattern of behaviour of pea vicilin and bean phaseolin in transgenic tobacco, such as accumulation in protein bodies and early breakdown during germination, all suggest that these "foreign" legume storage proteins fulfil a similar functional role in their new, non-legume host. In peas, the vicilin polypeptides of M r - 50000 are the major products o f a multigenic family (P. M. Chandler, personal communication). In earlier work on the post-translational processing of vicilin in peas [26, 36], it was suggested that within the vicilin gene

694 f a m i l y s o m e m e m b e r s h a d diverged sufficiently to generate different c o m b i n a t i o n s o f the two m a j o r processing sites, while o t h e r m e m b e r s h a d n o processing sites. T h e s e latter w o u l d give rise to the m a j o r Mr - 5 0 0 0 0 p o l y p e p t i d e s o f m a t u r e vicilin. It was envisaged t h a t if processing sites were present t h e m o l e c u l e w o u l d be t o t a l l y p r o c e s s e d at t h a t site. T h e b e h a v i o u r o f a single m e m b e r o f t h e p e a vicilin f a m i l y in t r a n s g e n i c t o b a c c o c o n t r a d i c t s this n o t i o n . T h e a p p e a r a n c e o f b o t h a m a j o r M r - 50000 p o l y p e p t i d e a n d a series o f s m a l l e r p o l y p e p t i d e s as the p r o d u c t o f a u n i q u e vicilin gene shows t h a t p a r t i a l p r o c e s s i n g o f a single p r i m a r y p r o d u c t does occur. I n b o t h p e a a n d t r a n s g e n i c t o b a c c o , t h e ratio between the p r i m a r y t r a n s l a t i o n p r o d u c t a n d the processing p r o d u c t s is very consistent. This reprod u c i b l e level o f p a r t i a l processing c o u l d be due to the fact t h a t n o t all cleavable sites are accessible to p r o teolytic a t t a c k w h e n vicilin is in its o l i g o m e r i c (trimeric) c o n f i g u r a t i o n . Several cases have n o w been r e c o r d e d in w h i c h genes for 7S seed p r o t e i n s f r o m legumes have b e e n t r a n s f e r r e d into t o b a c c o a n d p e t u n i a ([2, 3, 31] a n d the p r e s e n t study). I n all cases these genes have b e e n t r a n s c r i b e d a c c u r a t e l y f r o m their correct start sites, i n t r o n s have b e e n spliced o u t o f t h e gene t r a n s c r i p t s a n d a u t h e n t i c p r o t e i n p r o d u c t s have a c c u m u l a t e d . F u r t h e r m o r e , expression o f these genes has closely p a r a l l e l e d t h a t in their o r i g i n a l h o s t with r e g a r d to o r g a n specificity a n d t e m p o r a l regulation. This c o m m o n a l i t y o f expression o f these genes in their new h o s t s suggests t h a t the f l a n k i n g , r e g u l a t o r y regions o f these genes c o u l d be used in c h i m a e r i c gene c o n s t r u c t i o n s to ensure the effective o r g a n specific synthesis o f " n o v e l " seed storage p r o t e i n s in g r a i n l e g u m e crops.

Acknowledgements We t h a n k D r G y n h e u n g A n for s u p p l y i n g t h e shuttle vector ( p G A 471), D r Peter C h a n d l e r for m a k i n g available the u n p u b l i s h e d sequence d a t a o n p e a vicilin c D N A ( p P S 15-84), Drs M a r j o r i e a n d A n t o n i u s M a t z k e for t h e i r h e l p a n d c o r r e s p o n d e n c e a n d D r C h r i s t i n e W a n d e l t for c o m m e n t s d u r i n g p r e p a r a t i o n o f t h e m a n u s c r i p t . T h e technical assistance o f C e l i a M i l l e r a n d m a n u s c r i p t p r e p a r a t i o n b y Denese

M c C a n n are a c k n o w l e d g e d with t h a n k s . E.J.N. is t h e recipient o f a C o m m o n w e a l t h P o s t - g r a d u a t e Scholarship.

References 1. An G, Watson BD, Stachel S, Gordon MP, Nester EW: New cloning vehicles for transformation of higher plants. EMBO J 4" 277-284 (1985). 2. Barker SJ, Harada J J, Goldberg RB: Cellular localization of soybean storage protein mRNA in transformed tobacco seeds. Proc Natl Acad Sci USA 85:458-462 (1988). 3. Beachy RN, Chen Z-L, Horsch RB, Rogers SG, Hoffmann N J, Fraley RT- Accumulation and assembly of soybean Bconglycinin in seeds of transformed petunia plants. EMBO J 4:3047-3053 (1985). 4. Beachy RN, Jarvis NP, Barton KA: Biosynthesis of subunits of the soybean 7S storage protein. J Mol Appl Genet 1: 19- 27 (1981). 5. Benton WD, Davis RW: Screening )~gtrecombinant clones by hybridization to single plaques in situ. Science 196: 180-182 (1979). 6. Bollini R, Vitale A, Chrispeels M J: In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps. J Cell Biol 96: 999-1007 (1983). 7. Borroto K, Dure L III: The globulin seed storage proteins of flowering plants are derived from two ancestral genes. Plant Mol Biol 8:113-131 (1987). 8. Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 (1976). 9. Brown, JWS: A catalogue of splice junction and putative branch point sequences from plant tissue. Nucleic Acids Res 14:9549-9559 (1986). 10. Chandler PM, Spencer D, Randall PJ, Higgins TJV: Influence of sulfur nutrition on developmental patterns of some major seed proteins and their mRNAs. Plant Physiol 75:651-657 (1984). 11. Chrispeels MJ, Higgins TJV, Spencer D: Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons. J Cell Biol 93:306-313 (1982). 12. Craig S, Goodchild DJ: Periodate-acid treatment of sections permits on-grid immunogold localization of pea seed vicilin in ER and Golgi. Protoplasma 122:35-44 (1984). 13. Craig S, Miller C: LR White resin and improved on-grid immunogold detection of vicilin, a pea seed storage protein. Cell Biol Internat Reports 8:879-886 (1984). 14. Deininger PL: Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem 129:216-223 (1983). 15. Ditta G, Stanfield S, Corbin D, Helinski DR: Broad host range DNA cloning system for gram-negative bacteria: construction of a genebank of Rhizobium meliloti. Proc Natl

695 Acad Sci USA 77:7347-7351 (1980). 16. Doyle J, Schuler M, Godette WD, Zenger V, Beachy R, Slightom J: The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris: structural homologies of genes and proteins. J Biol Chem 261:9228-9238 (1986). 17. Ellis THN, Domoney C, Castledon J, Cleary W, Davies DR: Vicilin genes ofPisum. Mol Gen Genet 205: 164-169 (1986). 18. Gatehouse JA, Lycett GW, Croy RRD, Boulter D: The posttranslational proteolysis of the subunits of vicilin from pea (Pisum sativum L.). Biochem J 207:629-632 (1982). 19. Gatehouse JA, Evans IM, Croy RRD, Boulter D: Differential expression of genes during legume seed development. Phil Trans R Soc Lond B 314:367-384 (1986). 20. Greenwood JS, Chrispeels M J: Correct targeting of bean storage protein phaseolin in the seeds of transformed tobacco. Plant Physiol 7 9 : 6 5 - 7 1 (1985). 21. Haffner MH, Chin MB, Lane BG: Wheat embryo ribonucleates. xii. Formal characterization of terminal and penultimate nucleoside residues at the 5'-ends of "capped" RNA from imbibing wheat embryos. Can J Biochem 56: 729-733 (1978). 22. Higgins TJV: Synthesis and regulation of the major proteins in seeds. Ann Rev Plant Physiol 35:191-221 (1984). 23. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227:1229-1231 (1985). 24. Hudson GS, Farrell PJ, Barrell BG: Two related but differentially expressed potential membrane proteins encoded by the EcoR1 Dhet region of Epstein-Barr virus B95-8. J Virol 53: 528-535 (1985). 25. Karn J, Brenner S, Barnett L, Cesarini G: Novel bacteriophage h cloning vector. Proc Natl Acad Sci USA 77: 5172-5176 (1980). 26. Lycett GW, Delauney A J, Gatehouse JA, Gilroy J, Croy RRD, Boulter D: The vicilin gene family of pea (Pisum sativum L.): a complete cDNA coding sequence for preprovicilin. Nucleic Acids Res 11:2367-2380 (1983). 27. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

28. Murashige T, Skoog F: A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473-497 (1962). 29. Plietz P, Drescher B, Damaschun G: Relationship between the amino acid sequence and the domain structure of the subunits of the llS seed globulins. Int J Biol Macromol 9: 161-165 (1987). 30. Sanger F, Coulson AR, Barrell BG, Smith AJH, Roe BA: Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161-178 (1980). 31. Sengupta-Gopalan C, Reichert NA, Barker RF, Hall TC: Developmentally regulated expression of the bean ~-phaseolin gene in tobacco seed. Proc Natl Acad Sci USA 82: 3320- 3324 (1985). 32. Singh L, Jones KW." The use of heparin as a simple costeffective means of controlling background in nucleic acid hybridization procedures. Nucleic Acids Res 12:5627-5638 (1984). 33. Slightom JL, Sun SM, Hall TC: Complete nucleotide sequence of a French bean storage protein gene: phaseolin. Proc Natl Acad Sci USA 80:1897-1901 (1983). 34. Southern E: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503-517 (1975). 35. Spencer D, Higgins TJV, Button SC, Davey RA: Pulse labeling studies on protein synthesis in developing pea seeds and evidence of a precursor form of legumin small subunit. Plant Physiol 66:510-515 (1980). 36. Spencer D, Chandler PM, Higgins TJV, Inglis AS, Rubira M: Sequence interrelationships of the subunits of vicilin from pea seeds. Plant Mol Biol 2 : 2 5 9 - 2 6 7 (1983). 37. Sutton WD: Some features of the DNA of Rhizobium bacteroids and bacteria. Biochim Biophys Acta 366: 1-10 (1974). 38. Weaver RF, Weissman C: Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' terminus of 15S/~-globin mRNA precursor and mature 10S ~-globin mRNA have identical map coordinates. Nucleic Acids Res 7:1175-1193 (1979).

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants.

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5' an...
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