Studies On Cytoplasmic Male Sterility And Restoration In Cotton (Gossypium Hirsutum L.)

Plant cytoplasmic male sterility (CMS) is a maternally inherited trait that is unable to produce viable pollens, which plays an important role in utilization of hybrid vigor. As cotton cytoplasmic male sterility mechanism is unclear and the restore gene has not been cloned, the study of cotton CMS is far behind of maize and rice,etc.therefore,it has theoretical and practical significance for investigating the molecular mechanism of cotton CMS in order to utilize the cotton hybrid vigor better. A upland cotton CMS with the cytoplasm of G harknessii is controlled by cytoplasmic genes and repressed by nuclear restorer genes. Sterile-line anthers begin to degenerate in the sporogenous cell division stages and reach abortion peak in the pollen mother cell meiosis stage, so that it can not form tetrad, which is named as non-pollen male-sterility,and is appropriate for the hybrid seed production and study of the CMS mechanism.In this paper, the anthers of CMS, maintainer, restorer and Fi were employed to investigate the cotton CMS-associated and fertility restoring genes and proteins by using differential display of anthers cDNA-AFLP and mitochondrial proteome, the results were shown as follows: 1. Using sucrose Percoll and Nycdenze gradient ultracentrifugation technique to isolate anthers' mitochondria from CMS, maintainer, restorer and F1. then identificate them by measuring mitochondrial marker enzyme and transmission electron microscopy,At last we establish a way to isolate highly qualified mitochondria from frozen cotton anthers.2. In the present research, the cDNA-AFLP technology was employed to analyze the differential gene expression profiles among the anthers of 6 days before anthesis of CMS line, maintainer line, restorer line, and F1, the results showed:that there is tremendous difference of the gene expression profiles of the anthers between the CMS line and its "isonuclear alloplasmic" maintainer line, suggesting that organelle gene may play important roles in modulation of the nuclear gene expression; the distorted nuclear genome expression triggered by CMS gene must contribute more directly to cotton cytoplasmic male sterility. Gene expression profile of the restored fertile plants of F1, i.e. the CMS line after introducing restorer gene, exhibited similar to that of the fertile lines including maintainer and restorer, and almost same as that of the restorer, implying that nuclear genes may counter the effect of CMS gene in regulating nuclear gene expression, thereby restore the fertility. The regulatory roles of CMS gene in modulating nuclear gene expression and the fact that the restoring gene may compromise this modulation effect must contribute a lot to explain the molecular mechanism of sterility and its restoration. Furthermore, we identified 36 amplified cDNA fragments which predominantly expressed in restorer line and F1, while showed no or very slight expression in CMS and maintainer lines, which observation shed lights on cloning of the restorer or restorer-related genes in cotton.3. By comparing mitochondrial proteome of CMS line with fertile lines and CMS line with restorer line, we obtain 46 CMS-associated proteins and 1 fertility restoring protein. Then, we identify 34 CMS-associated and 1 fertility restoring protein by MALDI-TOF/TOF.as cotton mitochondrial genome is hidden so, we only obtain 17 CMS-associated and 1 fertility restoring protein functional verification in the end.We divided the 34 CMS-associated proteins into five categories according to function, they were:energy metabolism related proteins, protein processing related protein, hormone synthesis related proteins, and toxin related proteins and other proteins. They confirmed that cotton CMS was related to energy metabolism, enzymatic activity, hormone and active oxygen.ATPase is an important enzyme in energy metabolism of living organisms. It is ATPase beta subunit related CMS fertility restoration. Therefore,We speculate that the ATP synthase beta subunit can restore the CMS mitochondrial energy metabolism By regulating the activity of the ATP synthase.Then restored the CMS fertility.