Plant MolecularBiology5: 191-201, 1985 © 1985 Martinus NijhoffPublishers, Dordrecht - Printed in the Netherlands

Nucleotide sequence of a c D N A clone of Brassica n a p u s 12S storage protein shows homology with legumin from P i s u m s a t i v u m Anne E. Simon, Karen M. Tenbarge, Steve R. Scofield, Ruth R. Finkelstein & Martha L. Crouch Department of Biology, Indiana University, Bloomington, IN 47405, U.S.A.

Keywords: Brassica napus, cDNA clone, legumin, nucleotide sequence, proteolytic processing, seed storage proteins

Summary The most abundant protein in seeds of Brassica napus (L.) is cruciferin, a legumin-like 12S storage protein. By in vitro translation of embryo RNA, and pulse-chase labelling of cultured embryos with 14C-leucine, we have shown that the 30 kd o~ polypeptides and 20 kd ~ polypeptides of cruciferin are synthesized as a family of 50 kd precursors which are cleaved post-translationally. One member of the cruciferin family was cloned from embryo cDNA and sequenced. The nucleotide sequence of the cruciferin cDNA clone, pC1, contains one long open reading frame, which originates in a hydrophobic signal peptide region. Therefore, the complete sequence of the cruciferin mRNA was obtained by primer extension of the cDNA. The predicted precursor polypeptide is 488 amino acids long, including the 22 amino acids of the putative signal sequence. The amino acid composition of cruciferin protein is very similar to the predicted composition of the precursor. Comparison with an amino acid sequence of legumin from peas, deduced from the nucleotide sequence of a genomic clone, shows that the a polypeptide precedes the ~ polypeptide on the precursor. Cruciferin and legumin share 40070 homology in the regions which can be aligned. However, cruciferin contains a 38 amino acid region high in glutamine and glycine in the middle of the o~ subunit, which is absent in legumin. Legumin has a highly charged region, 57 amino acids long, at the carboxyl-end of the o~ subunit, which is not found in cruciferln. Both of these regions appear to have originated by reiteration of sequences.

Introduction Brassica napus (rapeseed) synthesizes two major seed storage proteins in the cells of both axis and cotyledons during embryonic development. One of the two storage proteins, napin, comprises approximately 20°70 of the protein in the seed, and has previously been characterized at both the protein (9, 1) and cDNA level (2). The Brassica 12S seed storage protein, cruciferin, is an oligomeric protein with a molecular weight of about 300000. Like other 11-12S globulins (3), cruciferin is composed of six 50 000 d subunits, each subunit containing an interpeptide disulfide-bonded 2 6 - 30 kd (a) and 20-21 kd (/~) polypeptide (21). Cruciferin constitutes 60°7o of the seed protein at maturity (1). There is considerable N-terminal protein sequence homology reported among the/3 polypep-

tides of the 12S seed proteins from soybean (16), pea (4), pumpkin (18), broadbeans and oats (22). It has been suggested that homology which extends over such diverse plants, both monocots and dicots, must be due to a constrained protein structure necessary to fulfill the basic physiological role of storage proteins as a source of nitrogen for the developing seedling. In this paper, we report the cloning and sequencing of a cDNA clone for cruciferin. We present evidence that both o~ and ~ polypeptides of cruciferin are synthesized on a single precursor polypeptide. By comparing the deduced amino acid sequence of cruciferin with the amino acid sequence of pea legumin predicted from a genomic clone (10), we have found regions of extensive homology as well as regions composed of repeated sequences which are highly diverged.

192 Materials and methods

Plants Plants of Brassica napus cv. Tower were grown under greenhouse conditions (2). Embryos were removed from seeds, staged, and cultured as described (1). They were then frozen in liquid nitrogen and stored at - 70 °C. Radioactive labelling of embryo proteins Immature embryos (26 d post anthesis) were cultured on agar medium with 10-5 M ABA for 2 d (1). They were then transferred to liquid medium containing 14C-leucine (Amersham, 12.2 GBg/ mmol) for 15 min. The leucine had been dried under vacuum and resuspended in liquid medium to 33 #Ci/ml. Approximately 100 embryos were labelled in 1.5 ml medium with gentle agitation. After labelling, the embryos were washed by vacuum filtration with unlabelled medium (2 ml/embryo), and put back on agar medium (12 embryos/ 60×13 mm Petri plate) for the times indicated in Fig. 2. Each sample of 12 embryos was frozen in liquid nitrogen, and stored at - 7 0 ° C until use. Embryo extracts were prepared as described for immunoelectrophoresis (2), except the buffer was 40 mM sodium phosphate (pH 7.2), 0.25 M sodium chloride, and 0.1 mM phenylmethylsulfonylfluoride. Extracts containing 104 acid-precipitable cpm were subjected to SDS-polyacrylamide gel electrophoresis on 12-17°70 linear gradients and fluorographed. Molecular weight markers were included (14C-labelled high molecular weight range: Bethesda Research Labs). Antiserum against cruciferin was used to immunoprecipitate polypeptides from the embryo extracts, as described (1).

Two-dimensional polyacrylamide gel electrophores& In vitro translations using rabbit reticulocvte lysate, and immunoprecipitations with Staphylococcus aureus cells were performed as described (2). Immunoprecipitated cruciferin and its precursor were displayed on two-dimensional gels. The first dimension was nonequilibrium pH gradient electrophoresis (NEPHGE), as described by Jones (7), run for 1 950 volt hours to maximize the range of the pH gradient (17). After electrophoresis, tube

gels were equilibrated with SDS sample buffer and electrophoresed on 12-17% SDS-polyacrylamide slab gels. The second dimension gels were stained, subjected to fluorography and exposed to Kodak XAR-5 film at - 7 0 °C. Construction of cDNA clones from embryo RNA Total RNA from embryos cultured on ABA medium was prepared by phenolchloroform extraction (2). After reverse transcription and synthesis of the second strand of the cDNA (2) the cDNAs longer than 900 bp were eluted from an agarose gel to exclude cDNAs of napin (850 bp). These size-selected cDNAs were tailed with dCTP, annealed with G-tailed pBR322 plasmid, transformed into E. coli HB101, and colonies were selected for growth on tetracycline (2). Filter colony screens, plasmid DNA isolation, and RNA blots were performed as described (2). DNA sequencing DNA sequencing was carried out by the chemical degradation procedure of Maxam & Gilbert (12). Primer extension of pC1 cDNA Primer extension of pC1 cDNA was performed as described by Ghosh et al. (5). The primer was prepared by digesting 20 #g of pC1 insert with Nco I followed by treatment with bacterial alkaline phosphatase and radio-labelling of the 5 ' end with 32p ATP using T4 polynucleotide kinase. After secondary digestion with Dde I, the DNA was separated on the 8°70 strand-separating gel, the fragments visualized by autoradiography, and the single-stranded 21 b primer electroeluted from the gel. The radiolabelled primer was hybridized to 3 mg of total RNA isolated from 35-40 d postanthesis embryos (2), in a total volume of 250/A containing 50070 formamide, 0.4 M NaCl, 10 mM Pipes pH 6.4, and 0.1070 SDS for 16 h at 40 °C. The hybridized primer DNA was extended with AMV reverse transcriptase (Life Sciences) as described for the synthesis of cDNA (2). The primer extended DNA was separated from the template and unextended primer on a 15070 sequencing gel (12). The extended sequence was visualized by autoradiography, electroeluted from the gel and the DNA sequence determined.

193 Results

Cruciferin is synthesized as a precursor polypeptide Analysis of in vitro translation products shows that cruciferin is synthesized as a group of precursors of about 50 kd. Embryos (25 days postanthesis) which had been cultured for 3 d on ABAcontaining medium to increase storage protein levels were used as a source of poly A ÷ RNA for in vitro translation in a rabbit reticulocyte system with 3H-leucine. The protein products were immunoprecipitated with antiserum directed against purified cruciferin holoprotein isolated from mature seeds (1). The immunoprecipitates are displayed on two-dimensional O'Farrell gels in Fig. 1. Native cruciferin protein (Fig. 1A) is composed of five major sizes of polypeptides; 30, 26, 22, 21, and 20 kd; each containing one or more isoelectric species. The total number of major spots detected on these gels is 12. The 30 and 26 kd polypeptides migrate between 6 and 7 in the pH gradient, and show less variability in charge than the 2 0 - 2 2 kd polypeptides, which span the acidic and basic regions. This pattern is similar to that observed on 2-dimensional gels of cruciferin from B. napus cv. Jet Neuf (8). The polypeptides characteristic of the mature protein are not present in immunoprecipitates of in vitro translation products (Fig. 1B). Instead, there are three size classes o f polypeptides; 51, 48, and 43 kd; with two major spots at 51 and 48 kd and some minor spots in each class. These data suggest that the a and/3 polypeptides of the mature seed are synthesized as precursors. The cruciferin precursors are completely processed in vivo over a 4 h period, as shown by labelling cultured embryos with 14C-leucine for 15 min, and then chasing with unlabelled medium for the times indicated in Fig. 2. The total embryo extracts are shown in Fig. 2A. The predominant band in the 0 h and 0.5 h lanes is the napin precursor (18.5 kd), which is processed by 9 h to the mature napin subunits (9 and 4 kd). A pulse of 14C-leucine at the end of the 10 h experiment has a polypeptide pattern which is indistinguishable from the pattern at the beginning (Fig. 2B). This confirms that the changes in polypeptides seen during the chase are due to processing events, and not the result of changing synthesis patterns. The cruciferin precursor is difficult to distinguish from other polypep-

tides in the total extract, so immunoprecipitates of the extracts in Fig. 2A are shown in Fig. 2C. After 15 min, the first immunoprecipitated proteins have apparent molecular weights of 48, 43, 30, 22, and 21 kd. During the chase, label disappears from the 48 and 43 kd bands and increasingly appears in the polypeptides of mature cruciferin. Cruciferin cDNA cloning and sequence analysis From the in vitro translations and the in vivo labelling experiments we predicted that cruciferin was encoded by family of related mRNAs, each long enough to encode a polypeptide of approximately 50 kd. We therefore size-selected the cDNAs used in making an embryo library to enrich for full-length cruciferin cDNA clones and to exclude clones of the other major storage protein, napin, known to have a 850 b mRNA (2). A differential colony hybridization screen was performed to identify colonies containing inserted sequences homologous to cruciferin mRNA. Duplicate filters were probed either with 32p-cDNA from embryos cultured either with or without ABA in the medium. Colonies which showed a stronger hybridization signal in the set of filters probed with cDNA from ABA-cultured embryos were isolated from a triplicate plate and grown for analysis of the inserted DNA. The longest plasmid had a DNA insert of 1 620 bp, and hybridized to a 1 750 b RNA on a Northern blot of embryo RNA (Fig. 3). The hybridizing mRNA was also more abundant in embryos grown on ABA, as predicted. We chose this putative cruciferin cDNA clone, pC1, for DNA sequencing. The restriction endonuclease map of pC1 along with the sequencing strategy is shown in Fig. 4, and the DNA sequence is presented in Fig. 5. The sequence of the cDNA begins at nucleotide 39 (see below), and contains one long open reading frame of 1 439 bp. An initiating methionine is not evident in the translated cDNA sequence, so the complete mRNA sequence was determined by the technique of primer extension. A primer consisting of a single-stranded 21 bp Nco I-Dde I fragment located 12 bp downstream of the 5' end of the cDNA clone was hybridized to total embryo RNA and the primer DNA extended using reverse transcriptase. The sequence of the primer-extended DNA is shown in Fig. 5, nucleotides 1-38. The presump-

194

Fig. 1. Polyacrylamide gel electrophoresis (PAGE) of immunoprecipitated cruciferin from B. napus embryo extracts (A) and cruciferin precursor from in vitro translation products (B). Embryos were labelled by culturing on 14C-leucine-containing medium !n the presence

of 10 5 M ABA. In vitro translation of embryo RNA using rabbit reticulocyte lysate was done in the presence of 3H-leucine. In both cases, extracts were immunoprecipitated with cruciferin antiserum. The precipitates were run on NEPHGE in the first dimensio~ and SDS-PAGE in the second dimension, using a 12-17% linear polyacrylamide gradient. The gels were dried and fluorographed. Onedimensional SDS-PAGE of the immunoprecipitates and the apparent molecular weights determined from standard proteins (not shown) are at the left in each panel.

195

Fig. 2. Fluorographs of B. napus embryo extracts and cruciferin immunoprecipitates after labelling with ~4C-leucine. Embryos (26 d postanthesis) were cultured for 2 d on 10-5 M ABA. After incubation for 15 min in medium containing ~4C-leucine, the embryos were transferred to medium without label for the times indicated. Panel A: Proteins were extracted and electrophoresed on SDSpolyacrylamide gels with ~4C-labelled molecular weight markers (mw). The predominant band in the fluorograph of total extracts is the napin precursor (18.5 kd), which is processed by 9 h to mature napin subunits (9 and 4 kd). Panel B: Total extracts of embryos pulsed with ~4C-leucine after 2 d in culture or 2 d 10 h in culture are indistinguishable from each other. Panel C" Immunoprecipitation with cruciferin antibody of the extracts in panel A shows the disappearance of the 48.4 and 43.0 kd bands and the accumulation of bands that comigrate with subunits of mature cruciferin ( • ). The immunoprecipitate in the lane from the 0 h chase contains 1/3 the cpm of the other lanes due to loss of sample during preparation.

196 ing involved with polyadenylation in the 3 ' untranslated sequence, one near the stop codon and another 24 b upstream from the site of poly A addition. Analysis of the amino acid sequence deduced from the cruciferin c D N A shows several interesting features. Starting at the N-terminus of the predicted precursor polypeptide, there is a hydrophobic region with the characteristics of a typical signal peptide. Using the rules for determining the cleavage site between signal peptides and the remaining polypeptide (22), we have assigned the cleavage site between alanine-23 and glutamine-24. This predicts a precursor with a molecular weight of 53.7 kd, cotranslationally processed to 51.3 kd. The amino acid composition of the predicted protein, excluding the signal peptide, corresponds closely with the known composition of purified cruciferin from B. n a p u s (Table 1). Although cruciferin has been reported to be a glycoprotein by some researchers (Goding et al., 1970), analysis of the predicted amino acid sequence shows no glycosylation sites (AsnoX-Ser/Thr, 19), consistent with the data of Schwenke et al. (20). Two areas of the predicted protein have repeated sequences. Beginning with amino acid residue 126 and extending until amino acid residue 160 is a region in which 33 out of 35 amino acids are either glutamine or glycine (Fig. 6A). The region may have originated by reiteration of a 21 bp sequence followed by internal duplications of a 9 bp region, as diagrammed in Fig. 6B. The second clustering of amino acids is a series of asparagine-alanine doublets (Fig. 6C). Three doublets are encoded between nucleotides 1 0 9 8 - 1 129, and two doublets are encoded between nucleotides 1 2 7 8 - 1 3 0 9 . Although there is no homology at the amino acid level except for the

Fig. 3. Autoradi0gram of hybridization of cruciferin cDNA clone (pC1) with B. napus embryo RNA. Total cellular RNA (5 ttg) from R napus embryos cultured for 3 d with 10-~ M ABA in the medium (ABA) or on basal medium (bas), was resolved on a formaldehyde 1% agarose gel. The gel was blotted onto nitrocellulose using the Northern procedure (3), and hybridized with nick-translated pC1 cDNA insert. Size of the cruciferin mRNA was calculated from mobilities of the rRNA and fragments of pBR322 digested with Eco RI and Hinf I (mw).

tive initiating methionine, which is preceeded by two stop codons, is 17 b from the 5 ' end of the message and is followed by an open reading frame of 488 amino acids. As in other plants m R N A s (14), there are two consensus sequences for process-

P

N ,I I I,,, DS

TV TF ",1 i" I I I D D D

V F I(

F I

I

TFT 31(

A

Ik

DA



=~.

H I

I

S

FF I I

I

C

T I

I Ik

I

S DS

C

T I

)11

ASS



TT I I

P I

I

S

), ~

IOObp I

!

Fig. 4. Restriction map of the cDNA insert in pCl. The horizontal arrows indicate the extent of fragments sequenced and the direction

of the sequence. Sequencing was done using the method of Maxam & Gilbert (12). H=Hind III, N=Nco I, P=Pst I, C=Hinc II, V=Ava II, D=Dde I, F=Hinf I, A=Hpa II, S=Sau 3A, T=Taq I.

197 f met aLe a r g Leu e s r s e t Leu Leu a e r ACAC TAG TAA GAG AAA ATG GET C~G CTC TCA TCT CTT C ~ T C T pC7 2 0 6 o r t h r oLo~GLn gLn phe p r o esn gLu iLo phe Leu h i s gLy ATC TTT CTC CAT GGC TCT ACA GCT CAA GAG TTT CCA AAC GAG

phe s e r l e u oLo Leu l e u TTT TCC TTA GCA CTT TTG 61 cys gLn Leu asp gLn Leu TGT CAG CTA GAC CAG CTC 121

~O asn oLo Leu gLu p r o s e t h i s vaL Lou Lye aLe gLu a l e gLy a r g i L e bLu vaL t r p asp AAT GCA CTG GAG CCG TCA CAC GTA CTT AAG GCT GAG GCT GGT CGC ATC GAG GTG TGG GAC 161 60 h i s h~s aLe p r o gLn Leu a r g c y s s e t gLy vaL s o t phe v a t a r g t y r i L e i L s g t u s e t CAC CAC GCT CCT CAG CTA CGT TGC TCT GGT GTC TCC TTT GTA CGT TAC ATC ATC GAG TCT 241 80 LyS gLy Leu t y r l o u p r o s e t phe phe s e r t h r e t a Lys Leu s e r phe veL aLa l y s g t y GGT CTC TAG TTG CCC TCT TTC TTT AGC ACC GCG AAG CTC TCC TTC GTG GCT AAA GGA 301 I00 gLu g t y Leu met gLy a r g vaL v a t Leu c y s aLa gLu t h r phe gLn asp s e t s e t vaL phe GAA GGT CTT ATG GGG AGA GTG GTC CTG TGC GCC GAG ACA TTC CAG GAC TCA TEA GTG TTT 361 120 gLn p r o s a t gLy gLy s e r p r o phe gLy gLu gLy gLn g l y g l n g l y g l n gLn g t y g l n g l y CAA CCA AGC GGT GGT AGC CCC TTC GGA GAA GGT CAG GGC CAA GGA CAA CAA GGT CAG GGC 42l gLn gLy h i s gLn gLy gLn gLy gLn gLy gLn gLn gLy g t n gLn gLy gLn gLn gLy g t n gLn CAA GGC CAC CAA GGT CAA GGC CAA GGA CAA CAG GGC CAA CAA GGT CAG CAA GGA CAA CAG 461 160 s e r gLn g t y gLn g t y phe a r g asp met h i s 9Ln Lys vaL gLu h i 5 i L e a r g t.hr g t y asp AGT CAA GGC CAG GGC TTC CGT GAT ATG CAC GAG AAA GTG GAG CAC ATA AGG ACT GGG GAC 541 180 ~ h r tLe a t a t h r h i s p r o gLy v a t aLe gLn t r p phe t y r asn asp g l y ash gLn p r o Leu ACC ATC GCT ACA CAT CCC GGT GTA GCC CAA TGG TTC TAC AAC GAC GGA AAC CAA CCA CTT 50] 200 vaL t t e vaL s e t vaL Leu asp teu aLa s e r h i s g t n asn gLn Leu asp a r g ash p r o a r g GTC ATC GTT TCC GTC CTC GAT TTA GCC AGC CAC CAG AAT CAG CTC GAC CGC AAC CCA AGG 661 220 p r o phe t y r Leu aLa g t y ash asn p r o gLn g t y gLn vaL ~rp iLe gLu g t y a r g gLu gLn CCA TTT TAC TTA GCC GGA AAC AAC CCA CAA GGC CAA GTA TGG ATA GAA GGA CGC GAG CAA 721 2~0 g i n p r o gLn l y s ash i L e Lau asn g l y phe t h r p r o g t u vaL Leu a t a t y s aLe phe Lys CAG CCA CAA AAG AAC ATC CTT AAT GGC TTC ACA CCA GAG GTT CTT GCT AAA GCT TTC AAG

78~ 260 i L e asp vaL a r g t h r aLe gLn gLn Leu gLn ash gLn gLn asp ash a r g gLy asn i l e i L e ATC GAT GTT AGG ACA GCG CAA CAA CTT rAG AAC GAG CAA GAC AAC CGT GGA AAC ATT ATC 641 280 a r g vaL gLn g l y p r o phe s e t v a t i L e a r g p r o p r o Leu a r g s e t gLn ar'g pro gLn gLu C,~A GTC C~A GGC CCA TTC AGT GTC ATT AGG CCG CCT TTG AGG AGT CAG AGA CCG CAG GAG 90l gLu vaL asn~g ly 300 Leu gLu g t u 1,hr i L e c y s see a l e a r g cys t h r asp a6n Lau asp asp GAA GTT AAC GGT TTA GAA GAG ACC ATA TGC AGC GGG AGG TGC ACC GAT AAC CTC GAT GAC 961 320 p r o s e r asn aLa asp vaL CCA TCT AAT GCT GAC GTA 3~0 asp Leu p r o 1te teu a r g GAT CTC CCC ATC CTT CGC

~;yr Lys p r o gLn Leu gLy l:yr i L e s e t t h r Leu asn s e t t y r TAG AAG CCA CAG CTC GGT TAC ATC AGC ACT CTG AAC AGC TAT 1021 phe teu a r g Leu s e r a l e t s u a r g gLy s e r i t s a r g gLn ash TTC CTT CGT CTC TCA GCC CTC CGT GGA TCT ATC CGT CAA AAC 1061

360 a l e met v a l l e u p r o g t n t r p ash a l e asn oLo asn a l e v a l l a u t y r v a l t h r asp g t y GCG ATG GTG CTT CCA CAG TGG AAC GCA AAC GCA AAC GCG GTT CTC TAC GTG ACA GAC GGG 114~ 38O g t u a l e h i s vaL gLn v a t vat asn asp asn gLy asp a r g vaL pho asp g t y gLn vaL s e t GAA GCC CAT GTG CAG GTG G]l" AAC GAC AAC GGT GAC AGA GTG TTC GAC GGA CAA GTC TCT 1201 ,~OO g l n gLy 9 l n Leu l o u s e r CAA GGA GAG CTA CTT TCC ~20 gLn phe a r g t r p i L e g t u CAG TTC CGG TGG ATC GAG

i l e p r o gLn gLy pho s e t v a l v e l l y s a r g a l a t h r s e r g l u ATA CCA CAA GGT TTC TCC GTG GTG AAA CGC GCA ACA AGC GAA 1261 pho l y s t h r a s n a l e esn e t a g t n i L e asn ~ h r Lau a l e gLy TTC ~ ACA AAC GCA AAC GCA CAG ATC AAC ACA CTT GCT GGA 1.~2I

a r g t h r e a r v a l teu a r g g l y l e u p r o Lau g l u v e l i t e e a r ssn g l y t y r gLn i L e s e r CGA ACC TCG GTC TTG AGA GGT TTA CCA TTA GAG GTC ATA TCC AAT GGG TAC CAA ATC TCA 1361 teu g t u g t u aLa e r g a r g v a l Lys phe asn t h r i L e gLu Chr t h r teu I;hr h i s s e t s e t CTC GAA GAA GCA AGA AGG GTT AAG TTC AAC ACG ATC GAG ACC ACT TTG ACG CAC /~C AGT 1441 gLy p r o aLe s e r t y r gLy gLy p r o a r g Lys a l e asp a t a and GGC CCA GCT AGC TAC GGA GGG CCA AGG AAG GCT GAT GCT TAA GGGCTTACCC AGTGAACCTC

1503

TACTGTAAAA GGAAGTTA/~ TAGTAATAAA AAGAGTAATA ATAATGTACG EA/~TG'rGAC TGGTTTTGTA

1573

GAG6TTTTAGA ATGTTACTCCT TTTCTGAATA AAATAACTCT TTFCTATCT(AJ31T(A}16

Fig. 5. Nucleotide sequence of inserted D N A of cruciferin c D N A clone, pC1, including the primer-extended 5' sequence. Nucleotides are numbered at the right margin, and deduced amino acids are numbered above each line, starting with the presumptive initiating methionine. The c D N A insert sequence begins at nucleotide 39. The arrow between amino acids 23 and 24 denotes the predicted signal sequence cleavage site. The gly- and gln-rich repeats beginning at amino acid 126 are underlined, as are the regions containing asn-ala pairs at amino acids 361 and 421. These regions are shown in more detail in Fig. 6. An arrow at amino acid 298 indicates the proteolytic cleavage site between the c~ and/3 polypeptides. The underlined sequences in the 3' untranslated region are two consensus processing signals.

198 Table 1. C o m p a r i s o n of the a m i n o acid compositions of cruciferin with the predicted precursor.

A m i n o acid residue

mol A A per 50000 g protein Cruciferin a

Ammoniac Glu Gin Asp Asn Trp Lys His Arg Cys Thr Ser Pro Gly Ala Val Met Ile Ley Phe Total

Precursor b

72 >72

> 45 3 13 8 24 5 20 22 25 43 30 30 7 23 39 20 439

Mol%

-

-

78

Nucleotide sequence of a cDNA clone of Brassica napus 12S storage protein shows homology with legumin from Pisum sativum.

The most abundant protein in seeds of Brassica napus (L.) is cruciferin, a legumin-like 12S storage protein. By in vitro translation of embryo RNA, an...
2MB Sizes 0 Downloads 0 Views