Cloning And Preliminary Functional Analysis Of The Photoperiodic-related Gene Gmms From Soybean

Soybean,[Glycine max (L.) Merr.], whose seeds are rich in protein, belongs to family Fabaceae and is the main food of humans and domestic animals around the world. Soybean is one of the typical short-day plants. It is difficult to introduce and extend the improved varieties for the photoperiod sensitivity. In my study, the photoperiod sensitive soybean "Zhongdou24" under different photoperiods treatment were used as materials. The differential gene fragments were screened from soybean leaves of different photoperiods using cDNA-AFLP. Then the gene fragments which has a significant of the differential expression were cloned, sequenced and analyzed. Primers were designed according to the latest results of the soybean genome sequencing, RT-PCR were used to obtain the full-length gene. Then the soybean methionine synthase gene was transformed into tobacco and soybean, respectively,which we analysis the preliminary function of the soybean methionine synthase gene combining bioinformatics. This study was conducted to further clarify the molecular mechanism of soybean photoperiod response..The main results are as follows:1. The differential gene fragments were screened from soybean leaves growing under short day-length (SD), neutral photoperiod (NP) and long day-length using cDNA-AFLP.36significantly photoperiod-regulated TDFs ranging in length from150to500bp were cloned and sequenced, including21up-regulated TDFs by LD and15up-regulated TDFs by SD, of which,26gene fragments were homolog to function-known genes, seven with function unknown proteins, and three with no match by BLAST searching the NCBI database.14differentially expressed cDNA fragments in which the total36significantly photoperiod-regulated TDFs were proved the expression patterns via semi-quantitative RT-PCR analysis.2. Search14differentially expressed cDNA fragments of homologous genes in the tobacco, which be choosen the homologous sequences to build the VIGS vector. This gene fragment GmPR9make a significant phenotypic in tobacco by VIGS, which causing tobacco homologous gene silencing compared with the control, tobacco premature aging, stunted growth. Sequence analysis showed that the homologous gene belongs to the methionine synthase gene, the gene named GmMS (Methionine Synthetase).3. Choose the soybean methionine synthase gene fragments, blast with the soybean genome database. Then the primers of full-length GmMS cDNA sequence were designed, PCR the Methionine Synthetase. Sequence analysis showed that the total length of the gene is2292bp, encoding763amino acids. Semi-quantitative RT-PCR analysis of the methionine synthase gene expression in the different tissues of soybean, results showed that the highest expression levels in stem, significantly higher than the expression in roots and leaves.4. The bioinformatics analysis of the methionine synthase gene indicates that GmMS is a highly hydrophilic protein, which has neither trans-membrane protein, signal peptide nor disulfide. GmMS is located in the cytoplasm, mitochondria and chloroplasts, which need for protein synthesis. GmMS is highly conserved in the evolution which contains two domains, tetrahydrofolate binding domain and catalytic domain, which is consistent with its biological function of the basal metabolism.5. Overexpression vectors was constructed based on GmMS full-length cDNA sequences. Then it has been delivered into tobacco via Agrobacterium-mediated transformation using leaf disk as explants. Finally20the kanamycin resistant plants were obtained,13of those were identified by the PCR.6. Transgenic tobacco statistical analysis of the physiological and biochemical indexes. We found over-expression of transgenic tobaccos showed glutamic and cysteine content increased, serine, histidine, glycine, arginine, tyrosine and phenylalanine content decreased, methionine etc. have not consistency trend, an upward trend of Chlorophyll, prolamin and methionine syntheses activity, and a downward trend of content of anthocyanin. 7. Overexpression vector been delivered into soybean via Agrobacterium-mediated transformation using embryonic tips as explants. Eight plants were identified by PCR.