Cloning And Expression Of Genes Related To Cytoplasmic Male Sterility In Sugar Bee

In order to test molecular mechanism of the Sugar beet CMS and improve its efficiency in sugar beet CMS heterosis. in this study, using cytoplasmic male sterility line and its maintainer line as material, based on the genomic DNA sequences in public databases (GenBank) and other CMS related gene sequences in crops, apply the homologous sequences cloning method, we amplified the CMS-related sugar beet gene sequences including atp6, atpA, coxⅠ, coxⅡ, orfl87, orf324 and orf129. Then through the serios of bioinformatics and comparative genomics analysis method, analyzed the difference of these DNA sequences in the cytoplasmic male sterile line and maintainer line, and speculated the structural features of amino acids of these genes or fragments. At the same time, we compared and analyzed the difference expression of related genes and fragments of the cytoplasmic male sterile line and maintainer line in the phase of bud development by the semi-quantitative PCR and real-time quantitative PCR technology. The main results are as follows:1.At the level of mtDNA:None of the gene atp6 and gene orfl29 has been amplified bands in cytoplasm male sterile line and maintainer line; Gene orf324 has been amplified successfully in cytoplasm male sterile line while not been in its maintainer line, which indicates that the gene may be one of the leading candidate genes of cytoplasmic male sterility (CMS) in sugar beet; Gene atp A, Gene coxl, Gene coxⅡ and Gene orf187 all have obvious difference in the amplification bands of these two lines.2.The comparison of gene fragment in different band to gene atpA、coxⅠ、coxⅡ、 orf187and orf324 in cytoplasm male sterile line and maintainer line shows that there have been some transformations like the base of insertions, deletions and mutations in the sterile lines nucleotide compared to maintainer line. The coding sequence of amino acids has been changed accordingly.3.The expression analysis of different gene in three development bud stages shows that the expression of gene atpA to small bud and moderate bud in cytoplasm male sterile line was significantly lower than in maintainer line, but there was no significant difference in large bud, while gene coxⅡ still presents a low expression in all the bud stages compared to the maintainer line. The expression of gene orf187 in small bud was significantly higher than in maintainer line and gradually increased, while significantly lower than in moderate and large bud and gradually declined. More interesting thing is that the expression of gene orf324 has been increased gradually and then declined with the growing of bud in cytoplasm male sterile line.