Identification Of Mutants Induced By EMS, Cloning And Functional Analysis Of Flowering Regulating Genes GmSVP1 And GmOFP1 In Soybean [Glycine Max(L.) Merri.]

Soybean [Glycine max （L.） Merr] is an important food and oil crop, which can provide sufficient protein, oil and other nutrition for human, and plays a critical role in food industury and agriculture. But in China, the planting area of soybean has reduced year by year because of its lower yield, and China has become the biggest country for soybean output rather than the biggest soybean imput one. The soybean genome contains about 975M base pair, and it has very complicated structure and lower transformation efficiency, these limiting factor hinder the fast development in functional genomics of soybean. Therefore, we can obtain lots of mutants with meanful trait variation induced by physical or chemical mutagen, this will provide significant meterials for well understanding the functional genomics of soybean. In this study, the soybean "Nannong 86-4"was treated by 0.4% EMS to construct a soybean mutant library, and we made a further research on the chlorophyll-deficient soybean mutant and early-flowering mutant, the results as following:1. In this study, until M6 （2009） after investigation of visible phenotype for 5 years, we obtained 34 mutants with significant traits variation, including the development of stem, leaf and other important traits. At most 4% SSR polymorphism between 11 mutants and control were detected, these mutants we obtained in this study will provide important materials for functional genomic research of soybean.2. We isolated a early flowering soybean mutant（elf1） in M3 progenies, named early flowering 1, which elrly flowering trait was controlled by a nuclear recessive gene. The differences of genomic sequences between elf1 and control were test by comparative genomic hybridization （CGH）. There were 135 probes has significant signal differences between them, but no base variation was detected.3. We cloned GmSVPl and GmSVP2 from soybean, bioinformatics analysis indicated that both genes have the same gene structure in their genomic regions, which has 8 introns and 9 exons, encoding the protein with conservative domain of MADS-box. Both genes have the nearest genetic relationship with PsSVP in pea; and the gene GmSVPl express mainly in cell membrane by subcellular localization; tissue specifity analysis showed that both genes were mainly detected in stamen and pistil of soybean flowers, but lowly in root, stem, leaf and sepal. Gene GmSVP1 display a regular expression model induced by the abiotic stress of cold and heat, but not in NaCl and cut, and GmSVP2 was insensitvie to all of these four abiotic stresses. Gene GmSVP1 was subcloned into a expression vector and transform them into tobacco to analysis their functions. Phenotype dentification of to and T1 positive transgenic tobacco plants indicated that most of them flwoering earlier than that of wild-type tobacco, and mutations in their floral organs, including stamen and petal were also detected among them, this mutations in flowering time and floral organs may be triged by the over expression of sobean gene GmSVP1 in the transformed tobacco.4. We cloned GmOFPl and GmOFP2 in soybean, and bioinformaitics analysis indicated that both genes have no intron in its genomic region, and phylogenetic analysis showed that they have the nearest genetic relationship with AtOFP17 in Arabidopsis thaliana, and we also detected the transcript expression of both gene in mainly detected in stamen and pistil of soybean flowers, but lowly in root, stem, leaf and sepal. Abiotic stress shows that GmOFPl gene performed a regular expression model induced by heat stress and NaCl stress, but GmOFP1 was insensitive to the stress of cold, heat, cut and NaCl, this result indicated that there maybe a "cross-talk" between the pathway of response to heat, NaCl stress and the pathway of floral organs regulation in soybean. Tranformation of GmOFPl into tobacco was preformed for identify the funtion of GmOFPl, six To positive transformed tobaccoes were obtained and the expression of GmOFPl was also detected by qRT-PCR. Phenotype identification indicated that the mutations in petals were detected in all of these six positive transformation tobacco plants. All the results indicated that gene GmOFP1 perform its main biological function in the development of petal in soybean.5. We obtained the chlorophyll-deficient soybean mutant, named ’cd1’, in the progenies of M3 induced by EMS. After invesgating the main agricultural traits between cdl and its control, we found that the plant height,100-seed weight and seeds weight of a plant were significantly reduced comparied with those of control. And the total chlorophyll content in cdl at all development stage was 30-66% lower than that of its control. TEM analysis of ultrastructure of chloroplast and thylakiod between them indicated that the cdl had abnormal chloroplasts, which contain less lamella in grana. We also discovered that the cdl had better drought tolerance than that of control. The yellow-green trait was controlled by a nuclear recessive gene, and the gene was mapped on the linkage group O, flanking with the marker Satt633 and Sat₂91, and the correspondent genetic distance is 2.48 cM and 4.48 cM, respectively.