Analysis Of Differential Expressed Proteins And Genes Related To Thermo-Sensitive Male Sterility In BNS Wheat

BNS is a new type of thermo-sensitive male sterile lines in wheat, which has favorable sterility and conversion property, and be considered as a value material for hybrid wheat production. In order to explore the sterility mechanism of the line, this paper taking anthers of BNS male sterile lines and its conversion line in3periods, tetrad, mononuclear, and binuclear stages, as material, observed the morphology, cytological structure and abortion process of the microspores, and sepereted and identified differentially expressed proteins between both two lines using2DE and MALDI-TOF-MS, and analysed mRNA differential transcription levels of some important genes that was identified in2DE and MALDI-TOF-MS by the method of fluorescence real-time quantitative PCR. The main results are as followings:1. The microspores of BNS sterile lines were normal from meiosis to binuclear stage. They could complete the first nuclear devision and become binuclear pollens. Since then, the pollens developed abnormity, and some phenomena, such as cytoplasm substance light, nucleolus disintegrated, and empty pollen without nuclear, and so on appeared frequently in the process of the abortion. Abortion pollens are mainly spherical and stained with I2-K1in male sterile line. About1%pollen could develop fertile in male sterile line, and over50%develop fertile in conversion line.2. Differential expressed proteins were separated by2DE from anthers of BNS male sterile line and its conversion line at the stage of mononuclear.193protein spots in sterility and241spots in fertility were displayed on the gels. Among them,23differential expressed spots over2fold wrer recognized. Of the23spots,13spots expressed only in the fertile anthers,3up-regulated in conversion lines,7down-regulated in the sterile anthers. The16spots expressed only or up-regulated in conversion lines were identified by MALDI-TOF-MS. According to the function analysis,5of them were consided as crucial proteins correlated with BNS sterility.They were HSP23.5(heat shock protein), cMDH (cytoplasmic malate dehydrogenase), ALDH2(Aldehyde dehydrogenase2), ATP1(ATP synthase subunit alpha) and ATPβ(ATP synthase subunit beta).3. In order to validate these proteins’s differential expression, the mRNA expression quantity of atp1、 atpβ、hsp23.5、cmdh and aldh2of the proteins were detected by fluorescence real-time quantitative PCR. The results showed that the mRNA expression levels of atpl, atpβ and hsp23.5gene in conversion line were more higher than that in sterility line, accorded with the result of proteins analysis. But the genes of cMDH and ALDH2were opposite to proteins analysis. Blasting clonal sequences of atpl, atpβ and hsp23.5to highest homology sequences from NCB1, the identities were99%,100%and94%respectively.The results as above showed definitely that in BNS male sterility, a number of differential expressed proteins were seperated, and corresponding genes in transcrtiption level also had significant differences. Especially hsp23.5, atpl and atp β genes, expessed down-regulated significantly in sterility line. So we thoght that they might be the important sterility genes of BNS. Acorrding to the function and characteristics of the proteins and genes, it is suggested that gene hsp23.5most likely be the upstream infertility genes, and genes atp1, atpβ, impotant infertility correlation genes under the regulation of sterility genes. The other differential expressed proteins separated and identified in2-DE and mass spectrum should be the downstream proteins regulated by infertility genes.