Plant Molecular Biology 19: 1049-1055, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

1049

Update section Short communication

Molecular analysis of a cruciferin storage protein gene family of Brassica napus John P. Breen and Martha L. Crouch Biology Department, Indiana University, 513 E 2nd, Bloomington, I N 47405, USA Received 3 October 1991; accepted in revised form 10 April 1992

Key words: Brassica napus, rapeseed, gene expression, nucleotide sequence, storage proteins

Abstract We have isolated a five-member gene subfamily which encodes cruciferin, a legumin-like 12S storage protein of Brassica napus L., and have analyzed the structure and expression of the family members in developing embryos. Sequence analysis has shown that the coding regions of all five genes are highly similar, with the two most divergent members of the family retaining 89 ~o sequence identity. The analysis of this cruciferin gene family's expression indicates that the developmental pattern of expression of each gene is similar, and the steady-state mRNA levels of each gene are approximately equivalent to each other at all developmental stages.

Introduction Cruciferin is the major storage protein of Brassica napus, and its expression is developmentally regulated. Cruciferin mRNA is first detected in developing embryos of Brassica napus at 23 days after anthesis, accumulating to peak levels at approximately 38 days after anthesis and then declining to barely detectable levels in dry seeds [5,31. Although the overall pattern of cruciferin mRNA accumulation has been well defined in

Brassica napus, little is known about the expression of the individual members of the gene family. Peptide mapping of the cruciferin precursor peptides suggests there are three distinct precursor peptide subfamilies (P1, P2 and P3). Two cruciferin cDNA clones (pCRU 1 and pC 1) have been isolated, and based on nucleotide sequence analysis are thought to encode members of the B. napus subfamilies P1 and P2, respectively [ 10, 12]. This paper describes the isolation and characterization of the P2 subfamily of cruciferin storage protein genes.

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X59294 (BnC1) and X59295 (BnC2).

1050 Materials and methods

RNA dot blots

Plants

Dot blots were performed essentially according to Thomas [ 14], using 2/~g of denatured RNA per well. Corn embryo RNA (isolated from developing embryos of Illini Supersweet) was added to each dilution of the in vitro cruciferin transcripts to give a final total RNA concentration of 0.4 #g/ #1. Corn embryo RNA was also loaded separately as a control for background levels of hybridization. Relative concentrations of rRNA in each B. napus RNA sample were determined by probing with Raphanus sativum 18S ad 25S rRNAs sequences [2].

Brassica napus L. cv. Tower plants were grown as described by DeLisle etal. [3]. On the day of anthesis, the flowers were self-pollinated using bee sticks [ 15] and tagged with the date. Developing B. napus embryos were collected and staged according to their age and morphology.

Isolation of clones and sequence analysis

Genomic Southern blots were done using the procedure described by Brown and Crouch [ 1]. Genomic clones were isolated by screening three separate genomic libraries (Charon 4A, Charon 35 and 2gtl0) using the cDNA clone pC1 as a probe. Subclones used for sequencing templates were generated by three techniques: subcloning of restriction fragments, exo III-generated sequential deletions (Exo/Mung deletion kit, Stratagene, LaJolla, CA), and transposon-mediated deletions (SequenestlI deletion system: Gold Biotechnology, St. Louis, MO). DNA sequencing was carried out by using the Sequenase sequencing kit (United States Biochemical, Cleveland, OH) according to the manufacturer's instructions for 35S sequencing.

RNA isolation, synthesis and quantification

Total NA was isolated from B. napus tissues by phenol extraction as described by Finkelstein et al. [5]. RNAs used as standards in the dot blot analysis were synthesized in vitro from inserts subcloned into the plasmid vector Bluescript (Stratagene, LaJolla, CA). All RNAs were quantitated spectrophotometrically by assuming that a 40 #g/ml solution yields 1 A260unit. In addition, the in vitro transcript concentrations were confirmed using an ethidium bromide fluorescence assay [6].

Results

Southern blots of Brassica napus genomic DNA probed with the cruciferin cDNA clone, pC1 [ 12], indicated a small gene family of about five members. Using pC1 as a probe, members of this gene family were isolated from genomic libraries and mapped using several restriction enzymes (Fig. 1). All five genes have a Sal I restriction site located at the 5' end, a Hind III restriction site located in the center, and a Kpn I restriction site located in the 3' half of the gene. In addition to the similar location of restriction sites, sequence analysis revealed a high degree of sequence similarity between these genes. Both BnC 1 and BnC2 have been completely sequenced, and share 89.7~ identity within the coding regions (Fig. 2). Partial sequencing ofBnC3, BnC4, and BnC5 revealed that each of these three genes shares extensive sequence similarity with BnC1. The high degree of sequence similarity between the five cruciferin genes extends into the 3' untranslated region and the 5' flanking region of all five genes (Fig. 2). No sequence differences were detected between the 3'-untranslated regions of BnC3, BnC4 and BnC5, and these sequences differ from the 3'-untranslated region of BnC1 only at three sites. The 3'-untranslated region of BnC2 has diverged from the other four cruciferin genes but still retains approximately 83 ~ sequence similarity.

1051

BnC1 '

It

BnC2

BnC3

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X I

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SO0~9 Fig. 1. Restriction map of the cruciferin genomic clones. The arrow indicates the location of the cruciferin coding sequences and the regions where the synthesized oligonucleotides hybridize is shown below BnC5. Both C2-specific and C345 oligonucleotides hybridize at region 1, the Cl-specific oligonucleotide hybridizes at region 3 and the ALL oligonucleotide hybridizes at region 2. Bg, Bgl II; E, Eco RI; H, Hind III; K, Kpn I; S, Sal I; Xh, Xho I; Xb, Xba I.

Expression of individual cruciferin genes as quantitated by isolating total RNA from embryos at specific stages of development, and hybridizing the RNA with each of four distinct oligonucleotide probes (C1 = 5'-CTGTTGCTCGCGTCCTT-3'; C345 = 5'-CTGTTTCTCTCGTCCTT3'; A L L = 5'-CTGTCACGTAGAGAACC-3'; C2 = 5 ' - C C T A T G T A G C T T C C T T T T A C A G T AGAGGC-3'. Under the appropriate wash conditions (2 x SSC, 0.5~o SDS at 27 °C for the ALL oligonucleotide, 35 °C for the C345 oligonucleotide, 43 °C for the Cl-specific oligonucleotide, and 60 °C for the C2-specific oligonucleotide) these oligonucleotides would only hybridize to their specific target mRNA. In vitro synthesized cruciferin transcripts were dotted along with the total embryo RNA in each experiment to confirm the specificity of the probe (Fig. 3). Transcripts were synthesized from a collection of subclones that cover the entire transcribed regions of each of the five cruciferin genes described in this

paper: clone 3'C1 contains the Hind III/Taq I restriction fragment of BnC1 (the fist 700 bp of BnC1); clone 5'C1 contains the Hind III/PstI restriction fragment of pC 1 (the remaining 800 bp of pC1); clone 5'C2 contains the Eco RI/SalI restriction fragment of BnC2 (all but the last 300bp of BnC2); clone 3'C2 contains the Eco RI/Hind III restriction fragment of BnC2 (the last 300 bp of BnC2); clone C3 contains the Eco RI/Sal I restriction fragment of BnC3 (the entire coding region of BnC3); clone C4 contains the Sal I/Sal I restriction fragment of BnC4 (the entire coding region of BnC4); clone C5 contains the Barn HI/Sal I restriction fragment of BnC5 (the entire coding region of BnC5). (Note that due to the size of the 3'C2subclone the ALL oligo will hybridize to this fragment of BnC2, but hybridizes to the 5'C1 subclone of BnC1.) To measure the amount of cruciferin mRNA in total embryo RNA, a standard curve was constructed for each oligonucleotide probe by hybrid-

1052 ~1

r~ar;~cGemc~i~:z.z~rc~c~rc~r?rc~r~ccGem~rAr~ccAr~cr~~TrA~cc~,

BnC2

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

100

BnC2

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

Bnc1

AT-- ........ ~ACCAACATTAG*AAAACTATCA~AA~GAGCAOTrCG~FJAAACGTAATT~CrA~A~TGA~G~TATTGT~T

BnC2

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

Bncl

~GTT~ACTGATCAAA~AAAGAATAATACAA~*ATATATA~GA~TCAG*ArATATA*ACAAGAAA~A~ATA~F2-IG2~C~ATT~

BnC2

400

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

SnC3

BnCl

300

500

ArA~GCC*A~rCAXA~r~GArGTGTA&-AGACA~GC~**~AA*A~GCCATGCAAAr~3AGA~GT~~TA~AC *******************************************************************-****************************

~nc3

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

BnCl

AA~*.~IGCCA,U~'C~.A~AAGAAC~C*rA~AC~rAGC,~C~FA~r

SnC 2

************************************************************************ ..... ****tttt***tt ......... ***********************-**************************************************************************

Bnc 3

BnCl

.....

AG~.ACTCTATATACC~

gO0

700

BnC2

c~Arc~CA~Tc~A~ITA~CAA~A~ArCAAC~CTArAC~r~AGT~-A~TA~ATA~c~cATcr~G~T~c~ ...................................... ******************************************************

Bnc5

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

BnCl BnC2 BnC5

CCACA~CA~CTA~AAAGAGAAA/L1~TCGGCTCTcATcTCTTcTCT~TTTTTc~TTAGCAcTT~TT~*C&~T~C~T ************************--************************************************************************ **************************

Bnc1 E41C2

TTCCA&ACGAGTGTCAGC TAGACCAGCTCAATGCACTGGAGCCGTCACACGTACTTAAGGC TGAGC-C TGG T C ~ * ~ T ~ C ~ ****************************************************************************************************

BnCI BnC2

TCAGC TACGTTGC TC TGGTGTC TCCT TTG TACGTTACATCATCGAG T C T ~ 3 G G T C TC TAC T T C ~ C C ~ T ~ T A G ~ ~ ~TCC ~G~ **t***te****e****tt***t****C~ -t*-te-t**A*~****-**C****t**At *********~***-**&*AT,****et *CG****T***eeT

i000

BnC1 BnC2

GCTAAAGGTACGTG ................................................ AA~CTGATTTTGATACTATATGAGTATCG ****~******~**TT2TC~TGTAC~FyTCT~ACCAAATAGTTTy~G2TT2TGGTA~CrT*TAr*A**GA*G***G*G*T*AC**~*~C***A

1100

BnCl BnC2

AATTCG~ATCTTTAAGG~n~AG~CTTrTC,~ . . . . . . . . . . . . . . . . . . . . . . . . . . . A A A A G ~ G ~ I G T A G ~ A A G * A * A T C A C * A r A C A C G r G ...... **C**AA**----************c*C*G**A~AGTGfTA*AGAcT~g*AAGA~TAC*****c***T**~A*G**T*TG*GT****r**A**TT~CA*

1200

BnC1 BnC2

.............. - C r A A G G T T T T G A T ~ A A A * A C A T r A T A . . . . . . . - A T A T T I T ~ T T G . . . . . . . . . . . . . . . . . . . . ~ I T A A ~ T T A * A A C C T A A A * ~EGyrTAGATTGGGC**C*~A*************ACA**C**A*ATA*A*****A**G***ATTCTTTC?TAGGAG*AGAAC******ACAA~*A~*C**

1300

BnCl

A~A~C~TCGATG?FCACAGAAC~CGCAC

BnC1 B/IC2

AGAGTGGTCCCTGGATGCGC C@AGAC&TTCC/I~AC TCATCAGTG TTTCA&CCAAGCGG~G GTAGCCCC ~ G ~ G ~ ~ ***************************************************************************************************

B00

~C

900

TTCA *

GAGAAGGTCTTATGGGG

..................... TAAA~ZTTTYTTT2T~GTTTG~ACAT~A*AG

1400

1500

BnCl BnC2

GTCAGGGCCAAGGCCA---CCAAGGTCA~GGCCAAGGACAAC ........ - - - - A G G G C ~ G T C A - - - G C A A G G A C A A C A G A G T C A A G G C C A G G G * * • ** * *G* • ***T* ~GGGT* ,G**** * • ***A~G**C**~*AGGGTCAAGGCA** * **** ***G* ** * *ATCC**G~ *C**** ~*G***-* **T* *A**

1600

BRCI

C TTCC.G TGATATGCACCAGAAAGTGGAGC&CATJUtGGAC

1700

BnC1

CCACTTGTCATCGTTTCCGTCCTCGATTTAGCCAGCCACCAGRATCAGCTCGA~CC~&TA*~*~~~

BnC2

**T********t***G*******Ge*******************•*******************-************cc**************TA

TGGGGACACCATCG C T A C A C A T C C C G G T ~ T A G C C ~ T ~ T * T A C ~ G ~

1800 ***--

BLOC1 A G G G C ` A A A T C ~ C C A A A A * A G C ~ A T * ~ * A A A ~ A ~ A ~ y . A ~ * A G C ~ A A ~ C * A A A A T C ~ A C ~ A A ~ A ~ A C ~ * ~ T * ~ T BnC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1~00

BnCl

TTATCCTTrGAAAArTTTAAYF~TTTTA~TTTTCAAAATTTGAAA~C~"A~JCCCAAAACCTCAT~C*T~*~

2000

BnC2

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

BnCI BnC2

A~C~A~ACC~A~ACCC*A~ACCCC~1AACcC*AAACCCTA~ACCCTAA~TCCFAAA~CCCAGCCTT~AAC*T~C~G~C*~*~

Bnc1 BnC3

AACATTAAG~C*ATTTTTG~GA~TT2TGACC~GGGTGC~AGTT~`AGAACA*A~AC~GA~TAGTGCrA~~TAT~

BnCI

C~TATAA~FACAGAATA2~AAAAA*A~g~zx'fz~z~fTTTATCTG?AG

BnC2

......

Bnc1

ACGCGAGCA~CAGCCACA.%AAGAACA*CTTAATGGCTTCACACC~%GAGGT*y~`GC~AAAGCTTTCA`%~TT~C~GC~C~G

2400

BnCI BnC2

AA~AGCAAGACAA~CGTGGAAAcATTATCC~AGTCCAAGGCCCAT*AGTGT~ATTAGG~CGC~TTTGAGGAGTCAGAG~CG~A~A ***************************************************************************************************

2500

BnCI BnC2

ACGG TTTAGAAGAGACCATATGCAGCGCGAC-GTGCACCGATAACC TCGATGAC CCATC T ~ ~ TAT~CCA~ *~ TT~A~G~C ****************************************************************************************************

2600

BnCl BnC2

*TGAACAGCTATGATCTCCCCATCCTTCGCTTCCTTCGTC*TC~GCCCTCCGTGGATCTATCCGTCAAGTAAGTAC ...... A T A T G C A T T T T A A T A G ******************************************************************************--****************

2700

BnCl BnC2

GAATTAACATTTGAA~GTTTGGT~AGTATTA~TA~GC~T~CGG~T~GGCA~GTGCCGTA~*A~rTc~A*AA~---~T~ A***C**~GC***C*~A*G*~TAA*AGGGAT*G*CA~*G*AA****G*T2~G**T~A~*T~*GG**A*~CGG2TTAA*~AA*****C*A**A*AA**AT

BnCl

BnC2

GG~TC~C ......... TA*AAACA~JAC~A~TCUIAACAT~TA~TGTAG ********--***********************************

BnC1 BnC2

~TAcG~A~AGAcG~GGAAGCCCATGTG~AGGTGGTTAAcGACA&~GGTGA~AGAGTGTTc~ACGGACAAGTCTC*AAGGAC~TAcTTTC~TA~CA ***************************************************************************************************

5000

BnCl

~TTTC*CGTGGTGAAACGCGCAN:~AGTTCCOGTGGATCOAGT~CA~ACGCA~ACGCACAGATCA~A~G~ ****************************************************************************************************

3100

BnC2 BnC1 ]]nC2

CCTCGGTCT*GAGAGGTTTACCATTAGAGGTCAT~TCCAATGGGT&C~ * ~ * ~ G ~ T T ~ ****************************************************************************************************

]~nC1 ]~nC2 ]~nC3

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

~nCl

AGGAAGT~AAA*AG~A~AAAAA----GAGTAArAATAArG~cGCA~GTGAC~GGT~T~G*AGAGG~I~---~AGAA~G~-AC~TT~

2100

.................................................................................................... 2200

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

***4****2"**********--******************

CCATTTTAC TTRGCCGGARACAACCCACA&GGCC RAGTATGGATAGAAGG

TTTC-.ACGCACAGCAGTGGCCCAGCTAGCTACGGAGGGC~AAGGCI'GATGC~CTTA . . . . . . . . . . . .

* * - - * * * * * * * *. . . . . . . . . . . . .

~

C

~

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

BnCI

~*AAC~CTTTTCrATC *AAA~.AAAr

230~

3200

3500

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

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

*CAGGC2~GC AACAAAAACA

~ C ~

-CC~AGTC-,AACCTC*ACTGTA,e.A

BnC2

*********A**C*CG*C*A

2800 *

AACGCGATGGTGCTTCCACAGTGGAACGCAAACG~CGCGG~TCT ***********************************************

~nc3

BnC2

230(

*A************A*****A********************C**C*C**

3400

1053

Fig. 3. Quantitation of cruciferin mRNA. The right half of the figure is a schematic representation of the RNA samples spotted onto the nitrocellulose filters. Each nitrocellulose blot contains three rows of RNA samples. The first row of each blot contains RNA samples extracted from B. napus embryos at the various stages in development (days post anthesis, dpa), CR being a negative control (corn RNA). Each dot of the second row represents 25 ng of an in vitro synthesized cruciferin transcript from one of the cruciferin subclones (see Materials and methods, and Fig. 1). The third row contains the dilution series used to establish a standard curve, the numbers representing the ng quantity of transcript present. Each of these four filters was hybridized with a different labelled oligonucleotide: panel W with C2-specific, panel X with Cl-specific, panel Y with C345, panel Z with ALL.

Fig. 2. Nucleotide sequence of BnC1, BnC2 and BnC3. The two sequences were aligned by adding gaps to achieve the maximum number of matches [4]. Asterisks represent identical residues between the sequences and the BnC1 (master) sequence. Initiation codons, stop codons and polyadenylation signals are underlined. The 5' and 3' flanking and intron nucleotide sequences are italicized.

1054 ization to a dilution series of in vitro synthesized cruciferin transcripts (third row, Fig. 3). Background counts were subtracted from all cpm data, and normalized relative to rRNA content, with the assumption that rRNA concentrations were equivalent in all embryo samples. The adjusted cpm values were compared to the standard curve to determine the quantity of cruciferin mRNA present per 2/~g of total embryo RNA. The expression data reveal that cruciferin genes BnC1 and BnC2 are both expressed, although the steady-state levels of BnC1 mRNA are consistently higher than steady-state levels of BnC2 mRNA. The quantity of m R N A measured with the C345 oligonucleotide, which detects transcripts made from genes BnC3, BnC4, and BnC5, is approximately three times the level of BnC1 at all stages examined.

Discussion

Each member of this cruciferin gene family contains the expected structural elements necessary for efficient transcription and translation in a eukaryotic cell, including a consensus promoter element, TATAAATA, 57 bp upstream of the initiation codon, and contain a good plant consensus translational start site (AACAAUGGC [6]). The five cruciferin genes also share a high level of nucleotide sequence identity. Even the two most divergent family members, BnC 1 and BnC2, share 89.7~o nucleotide sequence identity within the coding region. However, only the genomic clone BnC1 matched the 3' untranslated region of the cDNA clone pC1 identically, and therefore we conclude that BnC1 encodes the message represented by the cDNA clone pC1. The high level of sequence identity between the five cruciferin genes is not just limited to the coding regions. Although the sequence of the BnC2 introns have diverged significantly from those of the other four genes, there is a strong similarity between the introns of genes BnC1, BnC3, BnC4 and BnC5. The fact that cruciferin gene introns as well as the amino acid coding region are highly conserved between the family members implies

that the sequence identity shared between the cruciferin genes is probably not due to functional constraints. Instead, the high level of sequence identity may indicate that the gene duplication events responsible for the creation of this gene family occurred relatively recently in time. Sequence analysis also revealed that the sequence previously reported for pC1 by Simon etal. contains two errors. At nucleotide 1485 there is an adenosine instead of guanosine and between nucleotides 901 and 902 the triplet 'ACA' should be inserted [12]. Thus the sequence for clone pCRU2/3 as reported by SjOdahl et al. is actually identical to pC1 and not an additional member of the P2 subfamily [13]. In terms of timing and quantity of expression we believe that all five members of the P2 subfamily are expressed and that each of the five genes is expressed identically. This result was not unexpected since the structural analysis indicates that these five cruciferin genes, including the promoter regions, are extremely similar. In future work it will be interesting to determine if the cruciferin genes which encode the protein precursors P1 and P3 show similar patterns of expression.

References 1. Brown SM, Crouch ML: Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase. Plant Cell 2:263-274 (1990). 2. Delseny M, Cooke R, Penon P: Sequence heterogeneity in radish nuclear ribosomal RNA genes. Plant Sci Lett 30:107-119 (1983). 3. DeLisle AJ, Crouch ML: Seed storage protein transcription and mRNA levels in Brassica napus during development and in response to exogenous abscisic acid. Plant Physiol 91:617-623 (1989). 4. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12:387-395 (1984). 5. Finkelstein RR, Tenbarge KM, Shumway JE, Crouch ML: Role of ABA in maturation of rapeseed embryos. Plant Pysiol 78:630-636 (1985). 6. Lutcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differs in plants and animals. EMBO 6:43-48 (1987). 7. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning:

1055

8.

9.

10.

11.

A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). Needleman SB, Wunsch CD: A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48:443-453 (1970). Pang PP, Pruitt RE, Meyerowitz EM: Molecular cloning, genomic organization, expression and evolution of 12S seed storage protein genes of Arabidopsis thaliana. Plant Mol Biol 11:805-820 (1988). ROdin J, Ericson ML, Josefsson L, Rask L: Characterization of a cDNA clone encoding a Brassica napus 12S protein (cruciferin) subunit. J Biol Chem 265:2720-2723 (1990). Scofield SR, Crouch ML: Nucleotide sequence of a member of the napin storage protein family from Brassica napus. J Biol Chem 262:12202-12208 (1987).

12. Simon A, Tenbarge KM, Scofield SR, Finkelstein RR, Crouch ML: Nucleotide sequence of a cDNA clone of Brassica napus 12S storage protein shows homology with legumin from Pisum sativurn. Plant Mol Biol 5:191-201 (1985). 13. Sj0dahl S, ROdin J, Rask L: Characterization of the 12S globulin complex of Brassica napus. Eur J Biochem 196: 617-621 (1991). 14. Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205 (1980). 15. Williams PH: Bee-sticks, an aid in pollinating Cruciferae. HortScience 15:802-803 (1980).