The Cloning And Expression Analysis Of Mitochondrion Atp9 Gene From Cotton (Gossypium Hirsutum L.)

Cytoplasmic male sterility(CMS) of cotton is a maternally inherited trait.lt has unique advantages on utilizing heterosis in the case of the hybrid cotton production. Through sterile line, maintainer and restorer lines of "three lines" complementary and the "three lines" system to produce seed,enabling large-scale production of hybrid cotton seed.In theory,it is also a good model system on the reaserch of pollen development and nucleo-cytoplasmic interaction.A new cytoplasmic type P30A (originated from derivative lines of 104-7A) has been developed in three-line hybrid combination and applied in production-scale. According to many research reports,cytoplasmic male-sterility related factors have a direct relationship with mitochondrial genome, but there are few report published about the CMS-realated gene of cotton.In this reaserch, we used P30A and its maintainer P30B, restorer line Y18R as the main experimental materials,and the methods of RFLP(Restriction fragment length polymorphism), Northern blot and Genome walking were performed to screen the mitochondrial gene associated with CMS in cotton.Meanwhile ,further analysis of the different expression pattern of atp9 gene between CMS line and its maintainer have been done. The results provide a pilot platform for further investigating the mechanism of CMS. Here are the main results and conclusions of the research:1. In this study,atp9 gene were cloned from the mitochondrial genome of cotton CMS line and its maintainer line by the method of homologous cloning. Partial sequence of atp9 gene was taken as probe in RFLP analysis between CMS line P30A and its maintainer P30B, restoring line Y18R. EcoRâ… restriction polymorphism was detected in sterile cytoplasm and fertile cytoplasm.Atp9 gene identified by the RFLP was a single copy in the mitochondrial genome.2. Northern blot was performed to further confirm the different expression pattern among the young bud of three lines for atp9 gene. The result demonstrated that two transcripts of atp9 gene were detected in three lines of cotton. One band showed clear hybridizing signal and another was weak. There were no obvious differences in the level of transcription. The clear band was cloned by cRT-PCR and identified the full length by sequencing.3. We have first cloned and sequenced the Hindâ…¢restriction enzyme fragment of atp9 gene between sterile cytoplasm and fertile cytoplasm via the method of inverse PCR (IPCR).On this basis, with the application of Tail-PCR method, we also obtained flanking sequences of atp9 gene in sterile cytoplasm and fertile cytoplasm.4. By comparing DNA with cDNA sequences of atp9 gene, we found that within the conservative area of atp9 gene transcripts there were 7 editing sites in the N,S and R cytoplasms respectively.The 7th editing site was found to change an arginine codon into a termination codon,shortening the protein of ATP9 to the "standard" size.Furthermore,detailed RNA editing for atp9 gene in young buds of "three lines" has been analyzed.The results showed that the atp9 gene editing frequency of maintainer line was significantly lower than sterile line and restorer line in the organ of young bud. This is the first report on the editing of mitochondrial atp9 gene transcript of cotton.We found that the editing frequency of atp9 have close relationship with the form of cytoplasm of cotton.Nuclear background also affect the editing frequency, Our study show that mitochondrial atp9 gene may be related to cytoplasmic male sterility, but needs further analysis.