Cloning And Expression Pattern Analysis Of GhOTUD5 Gene In Cotton

Cotton is an important economic crop with obvious heterosis,however,the research and utilization of cytoplasmic male sterile(CMS)of cotton in China develop relatively late,which makes the advantage utilization and basic research of cotton heterosis have lagged behind.The mechanism of CMS in plants is extremely complex and has not been fully resolved.Covalent modification of histone in higher eukaryotes is an important part of gene expression regulation,such as acetylation,methylation and ubiquitination Among them,ubiquitination as a strictly regulated dynamic reversible process is closely related to the reproductive development of plants.In this study,use materials for flower buds form different development stages of CMS cotton Jin A,Yamian A and their maintainers.Cloning and analysis expression of histone deubiquitinase enzymes GhOTUD5 gene,to investigate the relationship between histone deubiquitination enzyme GhOTUD5 and cytoplasmic male sterility of cotton,it lays the foundation for exploring the mechanism of cytoplasmic male sterility in cotton.The results are as follows1.Using homologous cloning,the full-length cDNA and genomic DNA of the histone deubiquitinating enzyme GhOTUD5 was cloned and bioinformatics analysis was performed.The full-length cDNA of GhOTUD5 of the cotton Jin A,Yam A male sterile line and its maintainer line was 2007 bp,there are two open reading frames,whose main effect open reading frame(mORF)is 1635bp,encoding 544 amino acids,the upstream open reading frame(CPuORFs)of the conserved peptides is 141 bp long and encodes 46 amino acids,the total length of genomic DNA is 6166bp,6165bp and 6158bp,which are composed of 8 exons and 7 introns.According to bioinformatics cluster analysis,the mORF protein of GhOTUD5 gene was direct line homologous with human HsOTUD5 protein,arabidopsis OTLD1 protein and rice Os04g0414100 gene encoding protein,the upstream conserved peptide sequence(CPuORFs)belongs to the uORFs homologous group polypeptide HG31.The analysis of protein multiple sequence alignment found that the conservatism of OTUD5 protein was relatively conservative,and conserved only in conserved domains.The sequence analysis of the upstream promoter of the GhOTUD5 gene showed that the expression of GhOTUD5 gene was regulated by the photoresponse and the biological hormones such as methyl jasmonate,gibberellin and so on.2.After repeated cloning experiments,three types of GhOTUD5 gene mutants were identified,which were SNP single-base mutants,fragment deletion mutants and fragment insertion mutants.The three mutants were coded 236aa,214aa and 348aa respectively,these mutations all lead to the absence or incomplete of the conservative domain of GhOTUD5 protein3.The GhOTUD5 gene expression was measured in the bud of cotton Jin A,subcotton A sterile line and its maintainer microspore development at different stages.The results showed that:The gene of GhOTUD5 in the sterile line of Jin A was significantly down regulated in the process of microspore abortion,while at the same time,the expression level of GhOTUD5 gene in Jin A sterile and maintainer leaves was almost the same.The GhOTUD5 gene of cotton A male sterile line was significantly higher than that of the maintainer line before microspore abortion,but the expression level of the flower bud after abortion and abortion and leaves in the same period of abortion were almost the same.It is suggested that GhOTUD5 gene is only differentially expressed in flower buds,and is related to the fertility of microspore,and the expression patterns of GhOTUD5 were different in different types of sterility.4.Construct GhOTUD5 gene subcellular localization vector,and It was observed that GhOTUD5 protein was mainly localized in the nucleus and cell membrane,presumably acting as a deubiquitinating protease and not only targeting histones,but also possibly having the same effect on membrane lipoproteins.5.Construct GhOTUD5 gene expression vectors,and the Arabidopsis thaliana was successfully transfected by Agrobacterium-mediated inflorescence infection.It lays the foundation for the subsequent epigenetic experiment of GhOTUD5 gene function-acquired mutant.