Cloning And Functional Analysis Of Soybean Male Sterility Gene

The study of male sterility gene of soybean?Glycine max?Linn.?Merr?plays an important theoretical role in soybean heterosis utilization and breeding,and has practical significance in greatly increasing soybean yield.The related research on soybean male sterility gene can not only deepen the understanding of soybean infertility mechanism,but also contribute to the utilization of heterosis.In order to study the function of the candidate gene MSA,this paper firstly narrowed the targeting range of the soybean male sterility candidate gene to reduce the number of candidate genes.Secondly,the meiosis of wild-type and mutant pollen was carried out.The observation may analyze the abnormality of meiosis and cause the change of fertility.The third full-length cDNA of soybean male sterility candidate gene is homologously cloned and analyzed by biological information according to the gene sequence.The structure and function of the protein were predicted by amino acid sequence.Then the expression characteristics of the target gene in soybean tissues?root,stem,leaf and flower?were analyzed by qRT-PCR.Finally,the CRISPR gene editing vector was constructed in this experiment.PBI121 functional complementation vector and successfully transferred the functional complementation vector into Arabidopsis male sterile mutant to obtain F1generation seeds and observed target phenotype.By constructing functional mutual vectors,genetically transforming Arabidopsis male sterile mutants,and observing the phenotype,the function of the gene MSA is to regulate soybean fertility.The above test content provides a theoretical basis for further research on the function of soybean male sterility gene.The main results of the study on gene mapping and candidate gene function of infertility mutant ms12 are as follows:1.Mapping of soybean male sterility genesWe used 600 individuals in the F₅ and F₆ populations of the hybrids of Zhonghuang13?purple?and ms12 mutants,and analyzed all the SSR markers in the chromosome 10location.The results showed that there were 6 SSR markers in the polymorphism between the parents,but the screening population failed to find the exchange of individual plants.According to the Phytozome genome annotation,the interval was found in the centromere region.2.Cytological observation of meiosis of wild type and mutantWe took 1200 and 3600 unopened flowers of wild-type middle-aged 661 and ms12sterile mutants,cytological observations of 9 meiotic stages,and comparison of meiotic stages of wild-type and mutants..The wild type showed normal at all times,but the dispersion of chromosome non-aggregation occurred in the second division of the meiosis was observed in the mutant.It is speculated that this is likely to cause the change of plant fertility.3.Candidate gene MSA editingFour editing vectors of the MSA gene were constructed,and the wild type 661 wild type was transformed,and no positive seedlings were obtained.4.Expression analysis of candidate gene MSATissue-specific expression analysis of MSA gene by qRT-PCR showed that the gene was expressed in all tissues of ZP661 and ms12 mutants?root,stem,leaf,flower?,but the expression level was extremely low.There was no significant difference in the expression of ZP661 and ms12 mutants in roots,stems and flowers,but the expression levels in leaves were significantly different?p<0.05?.The expression of MSA gene in ZP661 wild type leaves was the highest.And significantly higher than other tissues?roots,stems,flowers?,the expression level in the tissue from the largest to the smallest,followed by leaves,flowers,stems,roots,the highest expression in the leaves;the highest expression in the ms12 mutant It is a flower,and the expression level in the tissue is from flowers to flowers,leaves,roots and stems,and the flower has the highest expression.5.Functional complementation verification of candidate gene MSAThe Arabidopsis male sterile mutants CS3678 and CS8704 were transformed with the wild-type fertile gene of ZP661 to observe whether there was phenotypic recovery?functional complementation?.The functional complementation vector has completed genetic transformation of Arabidopsis thaliana,and transgenic screening by resistance Arabidopsis thaliana mutants were transgenic F₁ positive vaccines,and the target gene was judged to have a fertility function based on the positive seedling phenotype and the number of pods.