A Preliminary Study On The Abortion Mechanism Of Cytoplasmic Male Sterile Line SaNa-1A ​​in Brassica Napus L. And The Mining Of Fertility-related MicroRNAs

Cytoplasmic male sterility(CMS)is one of the most effective ways to use heterosis in Brassica napus.In recent years,the researches related to CMS are focuse on origins and structure characteristics of sterility genes,the mechanism of sterility gene and the restoration of fertility.The new type CMS line in this research was selected from the progenies of somatic hybrid,which was obtained by fusing B.napus and S.alba protoplast.This research had a comparative study mainly about:cytological study of anther and microspore;investigating the molecular mechanism of abortion.This study also used small RNA sequencing to investigate the role of miRNAs regulatory network in male sterility.The main results are as follows:(1)When comparing the differences from the morphlolgy of floral organs between CMS and the maintainer lines,we have found the sterile flowers were usually smaller than that of fertile flowers of maintainer lines.The stamens of CMS were poorly developed with no mature pollen.When observing the microstructure of anther,we found there were no other inclusions except cell nucleus in the microspores of sterile line during the uninucleate stage.Moreover,the tapetum cells were severely vacuolated and separation from endothecium,microspores were adhered together and surrounded by the tapetum cells.Finally,they all dissolved in the non-dehiscent anther.The ultrastructure observation of anther showed that PMCs were observed contain many abnormalities organelles structure in sterility line.During the late period of anther development,the microspores were intense vacuolation and organelles were all vanished,because the tapetum didn’t secrete nutrient substance to microspores in time.Besides,the result of H2O2 subcellular locatization showed the accumulation of great amounts of H2O2 in the cell wall and intercellular space of CMS line anther during the PMC stage.Therefore,these results suggested that abortion happened since the beginning of anther development in CMS line.(2)By using TUNEL technique to identify anther tapetum cell PCD,we identified that the normal anther’s tapetum cells underwent PCD during the tetrad stages,and the PCD phenomenon was strongest in the uninucleate stage.PCD singnals was not obvious in tapetum cells during tetrad period,However,a little signals were found in tetrads of sterile lines.Above results showed that sterile anther tapetum degradation was delayed.(3)sRNA sequencing was used to identificate miRNAs and targets in B.napus CMS line and maintainer line.As a result,25 differential expression miRNAs were identified.Several target genes for CMS occurrence-related miRNAs encoded important transcription factors and functional proteins,such as ARF transcription factors,MYB transcription factors,etc.Two target genes were verified with 5’RACE.Besides,the expression patterns of 9 CMS occurrence-related miRNAs and their targets during two stages of anther development were validated by qRT-PCR and the analysis of target genes indicated that most targets were negatively regulated by correspoding miRNA.In addition,a hypothetical miRNA-mediated regulatory model of CMS during anther development was proposed.