Primary Functional Analysis Of Two Mads-Box Genes From Soybean

MADS-box genes are known to be involved in many important processes during plant development.They are key components regulating flowering transition and play an important role during the development of lateral root,fruit and seed-coat.Genome-wide identification of MADS-box genes has revealed I07 genes in Arabidopsis,but little is known in the soybean.GmMADS28 and GmAGL15 are two important members of MADS-box genes family.GmMADS28 has high expression in flowers.Analysis of sequence alignment and phylogenetic tree indicated that it belongs to SEP subfamily. GmAGL15 expresses only in embryos.This paper made primary research on the two MADS-box genes.In order to investigate the function of GmAGL15,subcellular localization by transient expression of GmAGL15-GFP fusion protein on onion epiderm showed that GmAGL15 was localized in nucleolus and membrane.Pairwise comparison of GmAGL 15 protein with AGL15 revealed a similarity of approximately 69%.An alignment of the genomic and cDNA sequences revealed that GmAGL15 contained eight exons and seven introns,which was similar to AGL15.N-terminal and C-terminal of the soybean sequence displayed overall homology to the AGL15 proteins.Therefore,it is likely that GmAGL15 is the soybean ortholog of AGL15.In order to study the rule of GmAGL15 gene expression and regulation in soybean,we cloned a 1kb upstream of the translation start site from soybean genome in silico.Promoter sequence analyzed by PLACE showed that it had TATA-box,CA.AT-box and some cis-acting element which regulated specific expression in different organs and responses to stresses,light,and self-feedback.Therefore,GmAGL15 may be regulated by sucrose,auxin and ethylene.Compared the promoter of soybean GmAGL15 with homologue of other plants by FootPrinter analysis,we found that these promoters had conservatism and diversity and the distributing of transcription factor-binding sites had similarity and difference.It implied the accuracy and diversity of GmAGL15 gene expression and regulation.GmAGL15 was transferred into tobacco by Agrobacterium-mediated method.Real Time RT-PCR assay revealed GmAGL15 expressed in all these lines.But there is no noticeable phenotypic change to the wild-type plants.In order to investigate the function of GmMADS28,GmMADS28 was transferred to soybean through Agrobacterium-mediated co-transformation system to enhance its expression.62 resistant plantlets were obtained through antibiotic screening,and a few of them were found to be early flowering.RNAi technology has been widely used in resent years.In this paper,Gateway technology was used to replace the conventional method to construct GmMADS28 RNAi vectors.Then the vector was transferred to Agrobacterium C58C1 for its further transformation in plants.