The Research On The Ubiquitination Of Histones And Specific Expression Of Rad6in Physiological Male Sterility Wheat

Wheat as the main food crop, studying its utilization of heterosis and male sterility is the matter that has been tried to overcome in the world. The wheat male sterility can be devided into physiological male sterility and genetic male sterility. Physiological male sterility is induced by chemical hybridization agents to achieve high percentage of male sterility and saturated pollination rate at appropriate times. So far, it has already played an important role in cultivating hybrid wheat. The mechanism of wheat genetic male sterility has been reported in the past few years, however, the research has not been able to make substantive progress due to the complicacy of fertility genes. This research avoided the complexity of the genetic male sterility genes and based on the features of the physiological male sterility formation. The physiological type is not result of its fertility function gene mutation, but closely related to the variability in the process of transcription and translation because it is not inherit to its offspring. Moreover, based on the epigenetic research progress in recent years,"histone code" that is including modification of histone ubiquitination, methylation, acetylation, etc., plays a crucial role in the regulation of gene transcription. Many research results have proved that histone ubiquitination played a key role in spermiogenesis, the cell cycle during mitosis, inhibition of stem cells prematurely mature as well as premature differentiation of gene expression. In terms of plants, studies over years have confirmed that male sterility and microspore abortion were directly related to small spores abnormal division, and recent studies have also pointed out that there was close relationship between histone H2B ubiquitination and cell cycle during mitosis. Another study found that in rat, RAD6which was histone H2B ubiquitin-conjugating enzyme had two homologous proteins-HR6A and HR6B. Knocked out HR6B and rat exhibited male sterility, the cause of infertility was the defective in the process of histone H2B ubiquitinaton. It can be reasoned that there is a close relationship between RAD6, histone H2B ubiquitinaton and fertility.The study based on the research developments above, Used physiologically male sterility induced with chemical hybridizing agent SQ-1and corresponding normal lines of complete fertility as experimental materials in wheat, applied western blot technique to detection the histones ubiquitination level in different developmental stages of wheat flowers, meanwhile, combined the expression level of RAD6at different times to further analyse the relationship between the histone H2B ubiquitination and physiological male sterility in wheat. The main results obtained are as follow:(1) Applied acid extration method to isolate histones from the wheat nucleoprotein. The histone concentration and purity is higher, besides, the method is reliable and effective, therefor, it can be used to other plants histone extraction.(2) The ubiquitination levels of the histones were monitored with Western Blot. The results showed that there were ubiquitination of histone H2A, H2B and H3in the different developmental stages of the wheat floret from the mononuclear period to the trinucleate stage and there were big differences between the male sterile and fertile lines. Especially during mononuclear period, the ubiquitination of histone H2A was seemed to be existing in fertility, but disappeared in the sterility ones; the ubiquitination level of histone H2B in physiological male sterile wheat was seemed to be significantly less than that in fertile ones. Thus it can be seen that induced by Chemical hybridizing agents SQ-1, the ubiquitination level of histone H2A, H2B was significantly suppressed.(3) By cloning RAD6gene and using bioinformatic technique found that the RAD6belongs to ubiquitin ligase family and is involved in the process of protein ubiquitination. It Contains184amino acids and its molecular weight is23kDa. It has highly conserved sequence or the same activity center among different species, its26～137amino acids are its conserved sequence and the sequence of73～89amino acids (YHPNVDEMSGSVCLDVI) is the active site of ubiquitin-conjugating enzyme. The Cysteine residues located in the N-terminal85th has a catalytic role in the formation of ubiquitin-E2thioester bond.(4) Applied qRT-PCR to analyse the expression of RAD6gene and the results showed that the variational expression level of RAD6gene was consistent with the shifty ubiquitination level of histone H2B in various periods and the RAD6gene expression in sterility wheats was apparently suppressed especially during mononuclear period.These data indicated that SQ-1distinctly restrained the expression of the RAD6gene as well as the ubiquitination levels of histones H2A, H2B especially in mononuclear stage, which may be influence the translation of some genes and evently result in physiological male sterility in wheat. Thus, there is a close relationship between the ubiquitination of the histones and the physiological male sterility in wheat.