Cloning And Differential Expression Analysis Of Candidate Gene From Cytoplasmic Male Sterility Of Allium Fistulosum

Explorating and cloning the candidate genes of cytoplasmic male sterility from Allium fistulosum is a key for clarifing the molecular mechanism of CMS.The mtRNA were isolated from cytoplasmic male sterility(CMS)line and maintainer of Allium fistulosum.Then the full-length of atp6,atpA,nad9,nad5,orfl92,orf111,orf100,mat-R genes were obtained by combining homology clones and CR-RT-PCR methods,comparing their sequences,finding the differences between of them.Subsquently,the expression level of them in the CMS line and maintainer were analysed by real-time quantitative PCR method.In addition to combine the results of a comprehensive analysis of the above, the final confirmation cytoplasmic male sterility candidate genes for further study.The main results of obtainningthe academic dissertation were as follows:1.The optimization of CR-RT-PCR techniqueIn order to obtain the mitochondrial genes of full length sequence,this study optimized reaction conditions in CR-RT-PCR (the ring of RNA reverse transcription polymerase chain reaction) technology. The optimized technology was used to obtain the Allium fistulisum mitochondria cob gene full length. In our experimental conditions, optimized the reaction requirements were:RNA 65℃ for 5 min, then added reaction reagent in 15℃ for 2 h, and 70℃ for 10 min to inactivate the enzyme. The reaction system without adding a catalyst PEG6000. The results showed that the length of the cob gene was 1522bp which encoded 397 amino acids, it contains the complete N-terminal and C-terminal. Phylogenetic reconstruction with other published cob gene from diverse plant species indicates that Allium fistulisum cob gene clustered with the monocots clade, and most similar to the Allium cepa. The research showed that we can obtain the mitochondria cob gene complete information through CR-RT-PCR technology, this information was suitable for subsequent gene functional studies.2.Filtrating candidate genes of cytoplasmic male sterility (CMS) from Allium fistulosum..According to the early stage of the laboratory experiment dimensional electrophoresis of protein difference results of male sterile line and its maintainer line, the male sterile line and its maintainer line RNA-seq results,and RFLP analysis the result of the sterile line and its maintainer line, designing the specific primers of the genes by primer5.0 software. The candidate genes of CMS from Allium fistulosum can be screened by the technology of CR-RT-PCR.The full-length of atp6 of the sterile line and its maintainer line are respectively 2836bp and 2828bp; The full-length of atpA of the sterile line and its maintainer line are all 2337bp; The full-length of nad9 of the sterile line and its maintainer line are respectively 1013bp and 1014bp; The full-length of nad5of the sterile line and its maintainer line are respectively 1453bp and 1486bp; The full-length of orfl92of the sterile line and its maintainer line are all 712bp; The full-length of orf111 of the sterile line and its maintainer line are all 956bp,The full-length of orf100 of the sterile line and its maintainer line are respectively 1498bp and 1501bp;The full-length of mat-R genes were of the sterile line and its maintainer line are all 2249bp.3.The candidate genes of the expression analysis from Allium fistulosum.The real-time quantitative PCR (qRT-PCR) technology as a kind of precise gene expression analysis technique and is widely used in a variety of plants. To analyze the gene cloning,the study of using real-time quantitative PCR technology from the Allium fistulosum cytoplasmic male sterile line and maintainer line.Results show that,at the medium-term, the atpA,nad9,nad5,orfl92,orflll,orf100, mat-Rof sterile lines gene expression are raised, sterile lines were maintainer lines6.35,1.35,2.05,1.06,1.29,1.09,1.19 times;The atp6 of sterile lines gene expression are declined,sterile lines were maintainer lines 0.99 times.