Gene Mining And Mechanism Of Fertility Based On Transcriptome Of Ms1 Mutant In Gossypium Hirsutum L.

Gossypium hirsutium is an important economic crop which can provide natural textile fiber materials,protein and oil.Cotton has obvious heterosis,the hybrid offspring are stronger than the parents in growth vigor,biomass and fiber yield,the utilization of heterosis can significantly improve cotton yield and fiber quality.The utilization of heterosis is mainly carried out through the matching system of three lines(male sterile line,restorer line and maintainer line)for breeding.However,the research progress of heterosis utilization and fertility related genes in cotton is relatively slow,and the functional genes of fertility and the molecular mechanism of sterility are still unclear.In this study,the anthers at three different development stages: prophase,metaphase,telophase were collected used for RNA-sequencing in wild type C312 and male sterility mutant ms1(G.hirsutum L.),the molecular mechanism of male sterility in cotton was analyzed by using transcriptome,bioinformatics,genetics and molecular biology.Real-time quantitative PCR was conducted to validate the differentially expressed genes identified from the transcriptome and the candidate genes related to sterility to mine the sterility genes.For the candidate Gh OLE9 gene,bioinformatics was used to analyze the properties,structure and function of protein encoded by Gh OLE9.According to this,we used the CLCr V-based vector system to construct the virus induced silence vector p CLCr VA-Gh OLE9,and the viral silence vector was transformed into WT C312 to silence of endogenous Gh OLE9 gene by agrobacterium infection.We observed the phenotype(pollen activity detection,anther development,self-pollinate and so on)and analyzed gene expression in transgenic interfered lines.At the same time,PK7GWIWG2(I)-Gh OLE9 interference vector was constructed to infect hypocotyl and embryogenic callus of YZ1 and S1 lines of G.hirsutum mediated by agrobacterium and established a transgenic regeneration system for cotton.The main research results are as follows:(1)Compared wild type C312 with male sterility mutant ms1,there was almost no difference in flower organ morphology during anther development of prophase.However,at the later stage of anther development,the stigma of the mutant was obviously longer than wild type,filaments were shortened,and the stigma was exposed.When it was flowering,the mutant anther indehiscenced and pollen was rare and could not be released.(2)Anther transcriptomes of wild type C312 and male sterile mutant ms1 at different development stages were analyzed,and 3355,7002,7415 differentially expressed genes(DEGs)were identified in the early,middle,and late stage of anther development,respectively.In gene ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,DEGs were mainly related to carbohydrate metabolism,metabolism of amino acid metabolism,flavonoids metabolism and signal transduction and so on,indicating that many genes and complex metabolic pathways were involved during the anther development stage.(3)Verification of differentially expressed genes by q RT-PCR showed that the tested DEGs was the same with transcriptome,indicating that transcriptome was reliable.At the same time,there were genes related to pectin lyase and pectinesterase,which were identified the expression level in male sterility mutant was significantly lower than that in WT.We speculated that the down-regulation of these genes in the mutant affected the degradation of pectin,which possibly affected pollen development.(4)Gh OLE9 gene was analyzed by bioinformatics,found that the Gh OLE9 gene is 504 bp,encoding 134 amino acids,there was an X8 domain of Gh OLE9 from position 49 to position 133.The domain can be recognised and combined with the carbohydrate domain of beta-1,3-glucan,which is the conserved domain of glycosyl hydrolase family GH-17.The protein belonged to glycosylated hydrolases family GH-17,which is beta-1,3-glucanase and has the function of hydrolyzing beta-1,3-glucan.Present studies had shown that beta-1,3-glucanase regulates pollen development through the callose metabolic pathway during anther development,or involved in pollen tube wall re-formation during germination,so the function of Gh OLE9 gene was studied in the paper.(5)The expression pattern of Gh OLE9 gene was analyzed,found that this gene was specifically expressed in the reproductive organs of XC20 and C312(G.hirsutum),such as stamen and stigma.What’s more,the expression level of gene in sterile anther was significantly lower than that in fertile anther,suggested that it played an important role in the development of cotton anther.(6)Virus induced gene silencing(VIGS)was used to silence the Gh OLE9 gene,and a total of 10 positive plants were obtained.We found that the six of Gh OLE9 interfered lines had few stamens,stigma was longer,filaments were shortened,pollen was rare or no activity,malformed and inactive in the flowering,during the tetrad period,microspore development was abnormal and callose thickened.Real-time quantitative PCR was used to detect the expression level of obvious and stable phenotype in the 4 interfered lines.The results showed that,compared with WT,the gene expression level in interfered lines were significantly and extremely significantly reduced.The results showed that Gh OLE9 gene plays an important role in cotton anther development.Gh OLE9 interfered lines showed no significant difference from WT plants in vegetative growth,indicating that silenced Gh OLE9 gene could not affect other agronomic traits of cotton plants except fertility.(7)The PK7GWIWG2(I)-Gh OLE9 interference vector was constructed to infect the hypocotyls and embryogenic callus of cotton YZ1 and S1 mediated by agrobacterium,transgenic embryogenic callus in YZ1 and six transgenic regenerated plants in S1 were obtained.The analysis of molecular biological identification and agronomic character for regenerated cotton plants are being carried out.The above results indicated that DEGs between upland cotton WT C312 and mutant ms1 at different stages were mainly concentrated in carbohydrate metabolism,amino acid metabolism,flavonoid metabolism and other pathways,indicating that these metabolic pathways were involved in the development of cotton anthers and affected cotton fertility.At the same time,the downregulation of Gh OLE9 gene expression in upland cotton would lead to the thickening of callose and abnormal development of microspores in the tetrad period of cotton.It may affect plant fertility through callose metabolism.