Study On Molecular Mechanism Of Male Sterility For Wheat V-type Cytoplasmic Male Sterility Line

Cytoplasmic male sterility(CMS), a maternally inherited phenotype of pollen sterility in higher plants, is one of the main ways to use heterosis, and also a classical model of nuclear-cytoplasmic interaction. It is generally accepted that sterility gene is located in mitochondria genome, but the mechanism of the male gamete abortion which is a complex process involving in many nuclear genes is still unknown. This paper explored the molecular mechanism of cytoplasmic male sterility in wheat V-type CMS line from nuclear and cytoplasmic genes.. Firstly, candidate genes for male sterility sequenced and analyzed from mitochondrial genome of wheat V-type CMS line. Secondly, MSAP method was used to detect nuclear DNA methylation differences between wheat V-type CMS line and the maintainer line. The main results are as follows.(1) Three large fragments(>10kb) of mitochondrial DNA(mtDNA) from wheat V-type CMS line were cloned, and sequenced, sequence analysis results showed that the three fragments of mtDNA from wheat V-type CMS line shared 98% identity with K-type CMS line, indicating mtDNAs are conserved among Aegilops species. In addition, we also cloned and sequenced 33 mitochondrial genes and 22 orfs(which were believed as candidate CMS genes of wheat K-type CMS lines) from wheat V-type CMS line, the results showed that except rps13 and orf256, other mitochondrial genes and orfs were highly conserved between the two CMS lines. These results suggested that they may have similar CMS gene or mechanism of male sterility in wheat V- and K- type CMS lines.(2) Methylation analysis showed that the two CMS(KA\VA) lines which had same nuclear genetic background had higher methylation or permethylation levels than its maintainer line(B). In terms of single methylation site, the CMS lines(KA/VA) had two types of methylation and demethylation modification compared to the maintainer lines,demonstrating that DNA methylation might inhibit the expression of genes which affected the process of meiosis, and interacted with sterility genes, thus resulted in male sterility in wheat V-type CMS line.(3) Using homology based cloning, 11 TSK1 gene sequences and four coding sequences were obtained from wheat V-type CMS line. Sequence analysis results showed that there were several SKP1- like genes in common wheat; To further explore the function of TSK1 in wheat CMS line(VA) and the maintainer line(B), the expression of TSK1 was investigated at different development stages of anthers in wheat. The results indicated that TSK1 was significantly suppressed at the early development stages of anther in wheat V-type CMS line,,suggesting TSK1 might be involved in the male sterility process or affected by the process. In addition, prokaryotic expression of TSK1, and mass spectrum identification of TSK1 were also carried out.In summary, the above results indicate that wheat V-type CMS line has similar mitochondrial genes as K-type CMS line, DNA methylation and TSK1 may be involved in male sterility in wheat V-type CMS line.