The Cloning And Function Analysis Of Related Genes Of A Wheat K-type Cytoplasmic Male Sterile Line

Wheat (Tritium aestivum L.) is one of the major crops which plays a very important role in human beings’life and society development. The utilization of heterosis is the main approach to improve the yield and quality of wheat. Many efforts have been made in studying the mechanism of wheat male sterility, but the mechanism of wheat male sterility is still unclear. In this study, we carried out the envestigations from physiology and molecular biology levels to study the mechanism of wheat K-type cytoplasmic male sterility (CMS), the main results are as following:Firstly, we detected O2-production rate and H2O2content in the flag leaves and anthers at different developmental stages of wheat K-type cytoplasmic male sterile line (T. aestivum cv Yumai3), its maintainer line (T. aestivum cv Yumai3), F1line and restorer line (T. aestivum cv Yumai2). No obvious difference of O2-production rate and H2O2content was observed in the flag leaves of the four lines. But in the anthers at different developmental stages, the O2-production rate and H2O2content of the four lines showed different levels. O2-production rate in the uninucleate stage and dinucleate stage of CMS line was higher than that of the maintainer line. H2O2content of the CMS line was also higher than the maintainer line at all the three stages. Indicating dynamic imbalance of active oxygen metabolism in anthers of the CMS line may damage the cell membrane of plants, thus resulting in the phenomenon of male sterility.Secondly, we cloned an important nuclear gene (a MADS-box gene, closely associated with flower development) which expression level was higher in the CMS line than in the maintainer line in our previous microarray analysis. Its chromosomal localization, expression patterns analysis and functional studies were also carried out.Two MADS-box genes were identified from wheat K-type CMS line using homologous cloning, their predicted amino acid sequences had typical conserved domains MADS-MEF2-like and K-box of MADS-box proteins. The two genes were respectively localized on wheat chromosomes3A, and3B by using nulli-tetrasomes of Chinese Spring, thus they were, named TaMA and TaMB, Expression patterns of TaMA and TaMB in Chinese Spring leaves, spikes, roots and stems were analysed using quantitive RT-PCR. The results showed both TaMA and TaMB had different expression levels in different tissues of Chinese Spring. TaMA had relatively high expression levels in leaves and spikes, and low levels in roots; while TaMB had relatively high expression levels in spikes, and low levels in roots, stems and leaves, indicating TaMA and TaMB may play different roles in wheat different tissues during the development processes, TaMA may play a major role in leaves and TaMB may play a major role in spikes. Expression analysis in anthers of the four lines at different developmental stages showed that the two genes showed upregulated trends from dinucleate stage to trinucleate stage in CMS line, while downregulated in maintain line. The transcripts of the two genes were obviously higher in male sterile line than in maintainer line at trinucleate stage. These different expression patterns of the two genes may be associated with the CMS line sterility.Overexpressing TaMA and TaMB in Arabidopsis resulted in obvious phenotypes that the plants showed earlier reproductive development, slower growth or premature mortality; the bud, stamen, and stigma were abnormal. The higher TaMA and TaMB expressed, the more deformity was found, so TaMA and TaMB may closely related to male sterility of the CMS line. Silencing TaMA and TaMB in wheat fertile line was also carried out with VIGS method to clarify the functions of TaMA and TaMB. The silenced TaMA and TaMB plants showed green and yellow striped leaves, emaciated spikes, and decreased selfing rates. These results further demonstrate that TaMA and TaMB are closely related to the fertility of wheat CMS line, and silencing TaMA makes fertility change is more obvious, thorough.Thirdly, we respectively cloned three COI1genes from diploid wheat relative species, Triticum urartu Thum.ex Gandil, Aegilops speltoides, Aegilops tauschii Coss and wheat K-type CMS line. The full length of COI1were2.8kb containing three exons and two introns. The three genes were respectively localized on wheat chromosomes4A,4B, and4D by using nulli-tetrasomes of Chinese Spring, thus they were named COI1A, COI1B, and COI1D.The expression patterns of COI1genes in Chinese Spring leaves, spikes, roots and stems were analysed using quantitive RT-PCR. The results showed that the three genes were constitutively expressed in all the tested tissues, but the expression level of COI1A was significantly higher than COI1B, and COI1D in the spike, indicating COIIA may play a major role in wheat spikes.Expression analysis of COI1genes in anthers of the four lines at different developmental stages showed that the transcripts of the three genes in CMS line at uninucleate stage were higher than that of the maintainer line. COI1B and COI1D had same trends in CMS line and maintainer line, but COI1A showed up-regulated from dinucleate stage to trinucleate stage in CMS line, and down-regulated in maintainer line.These results indicate that COI1A may also play a role in the male sterility of wheat K-type CMS line.