Cloning And Functional Analysis Of Flower Development Related Genes In Soybean (Glycine Max L.)

Soybean(Glycine max (L.) Merri.) is an important oil crop. To understand themechanism for soybean flower development, a soybean genome chip (GeneChip(?) SoyGenome Array, Affymetrix) was used to compare the transcriptoms of roots, leaves andflowers and 347 flower-predominant genes were identified. According to putative functions,the flower-predominant genes are classified into 9 groups involved in cell structure,metabolism, transcription, defense, signal transduction etc. All genes encoding putativetranscription factors were selected for function annotation and cluster analysis. It was foundthat most of flower-predominant transcription factors genes(41%) encode MADS-boxproteins. Other genes encode MYB, bHLH, zinc finger or NAC transcription factors,suggesting most types of transcription factors play roles in soybean flower development.Based on the cluster analysis, transcription factor genes form two groups. The genes ingroupⅠare highly expressed in flowers and also expressed in leaves and roots, such as twoNAC protein genes; groupⅡtranscription factor genes are expressed specifically in flowersand may specifically play roles in flower development, such as three MYB genes. Thecluster result also suggested some genes with similar expression profiles might co-playroles. To confirm gene chip hybridization result, 7 flower- predominant transcription factorgenes were selected for real-time RT-PCR analysis. The similar results were obtainedbetween gene chip and real-time RT-PCR. During real-time RT-PCR, we also analyzedexpression of such genes in pods and some genes were found with higher expression levelin flowers and pods, such as Gma003239(encoding for a MADS-box protein), Gma012717(MYB protein).Two putative MADS-box gene fragments(Gma010711, Gma003239) were selectedfor further analysis. By rapid amplification of cDNA ends (RACE) approach, we obtainedthe full length genes of two frgments and designated as GmMADS28 and GrnMADS29respectively. GmMADS28 gene amplified in this paper is 1026bp in length and contains acomplete ORF of 732bp which encodes a protein with 243 amino acids. The full sequenceof GmMADS28 has been desposited in GenBank under accession number AJ878424.Similarily, full length of GmMADS29 was obtained by RACE approach. GmMADS29 gene is 1021 bp in length, containing a complete ORF of 747bp which encodes a polypeptide of248 amino acids. GmMADS29 gene has been registered in GenBank under accessionnumber DQ159905. Database searching suggested GmMADS28 and GmMADS29 sharedhigh sequence similarity with plant SEPALLATA (SEP) proteins. GmMADS28 share 59%,60%, 74% and 47% sequence identity with Arabidopsis SEP1, SEP2, SEP3 and SEP4.GmMADS29 shows 74%, 71%, 59% and 52% identity to SEP1, SEP2, SEP3 and SEP4,respectively. Thus we concluded GmMADS28 gene might be the homolog of SEP3 whileGmMADS29 was SEP1 homolog in soybean.With transient expression system in onion epidermal cells, we found GmMADS28-GFPand GmMADS29-GFP fusion proteins were localized into nucleus, respectively. To analyzestructure of GmMADS28 gene, we cloned separately the genomic DNA of GmMADS28with four parts. The assembled complete genomic sequence of GmMADS28 gene contains 8exons and 7 introns and all of the exon-intron boundaries follow the GU...AG rule.Moreover, comparisions among structures of GmMADS28, Arabidopsis SEP3, lotus LjSEP3and maize ZAG2 showed GmMADS28, SEP3, and LjSEP3 were structurally conservedexcept the intron sizes, suggesting introns of MADS-box genes might bear the most ofmutaions during evolution.Semi-quantative RT-PCR was used to determine expression patterns of GmMADS28and GmMADS29. The expression analysis showed both GmMADS28 and GmMADS29 wereexpressed mainly in reproductive organs including flowers, seeds, pods, but not invegetative organs such as roots, leaves. To further analyze two genes expression in flowers,we prepared total RNA from sepals, petals, stamens and pistils. The results revealed thatGmMADS28 and GmMADS29 mRNAs were more abundant in petals and pistils. Insoybean NJS-10Hfs mutant whose petal numbers are increased and some stames convertinto petals, we found GmMADS28 and GmMADS29 were highly expressed in petals andstamens as compared with expressed in normal plants revealed by real-time RT-PCR assay.We suggested the cause of mutant formation might be the inhibitor of GmMADS28 andGmMADS29 was mutated. This hypothesis will be validated by latter ectopic expression ofGmMADS28 gene in tobacco. Moreover, the expression analysis showed GmMADS28 andGmMADS29 genes were expressed with higher levels in early and later stages of seeddevelopment.To further address function of GmMADS28, full-length GmMADS28 sequence wascloned into plant binary vector pBI121, creating pBIGmMADS28 used for tobacco transformation. By Agrobacterium mediated transformation, 35S:GmMADS28 transgenictobacco plants were generated. Extopic expression of GmMADS28 in tobacco showed novelphenotypes by flowering early, decreasing plant height, increasing numbers of stamens,petals and sepals, as well as conversation from sepals and stamens into petals. Somephenotypes of 35S:GmMADS28 transgenics are similar with soybean mutant NJS-10Hfs,suggesting high level of GmMADS28 expression might direct increasing petal number andconversation from stamens into petals. Besides, some 35S:GmMADS28 transgneicsproduced carpel-like sepals, cleavaged petals and belt stamens. Duing to the bending ofstamens, the stames of transgenics could not touch pistils, resulting failed pollination.Moveover, anthers of transgenics could not open successfully and the pollen vigour wasalso largely reduced. All the factors eventually resulted sterile of most transgenicsectopically expressing GmMADS28.An Arabidopsis SUPERMAN-like gene (GmZFP1) was cloned in soybean. Expressionanalysis showed GmZFP1 was expressed mainly in reproductive organs but not expressedin vegetative organs except stem tips, and expressed in four whorls flower organs withhigher level in petals and stamens. Real-time RT-PCR assay showed GmZFP1 was moreaccumulated in petals of NJS-10Hfs mutant than in those of wild-type plants. Further, wefound that the expression of GmZFP1 was higher in late seed development than in earlyseed development. To our knowledge, GmZFP1 is the first characterized gene encoding forsingle zinc finger protein in soybean, and may play a role as a transcriptional repressor inreproductive organs development, especially functioning in petals, stamens and latedeveloping seeds.