Molecular Mechanism In Cytoplasmic Male Sterile Line Zhong41A Of G.trilobum

Cytoplasmic male sterility is a kind of maternal genetic phenomenon that is unable to produce normal viable pollen and it is controlled by nucleus gene and cytoplasm gene.It is not only broadly used in hybrid seed production for improving cotton quality and yield,but also an important material to researchers for studying the interaction between cytoplasm gene and nucleus gene.In addition,compared with rice,rape and other crops,the research on the CMS mechanism of cotton is far behind.Therefore,it has important significance for researching the mechanism of cotton CMS.In this paper,cotton CMS Zhong41 A had indehiscent anthers that was unable to shed pollen,and its stamen filament was significantly shorter than corresponding maintenance line.In order to study the molecular mechanism of sterility,cross sections were firstly applied to confirm the cytological defects and abortive stage of Zhong41 A.Then through the NGS high throughput sequencing platform,the anthers of stage 5,stage 6 and stage 7 in Zhong41 A and Zhong41 B were sampled for RNA-seq in order to observe the dynamic changes of gene expression level before and after the abortion.Furthermore,the sterility mechanism of Zhong41 A was researched by submicrostructure observation and biochemical methods.The main results were described as follows: 1.The confirmation of abortive stage in Zhong41 A through cross sectionsCytological observation of Zhong41 AB anther showed that the beginning of the abortion was occurred in stage 6 of Zhong41 A.In this stage,the microspore mother cells began to appear abnormally,with shrinking and irregular shape.And at stage 7,the peak of abortion was reached,the microspore mother cells were gradually degraded and no tetrad formed.In the late stage of anther development,the anther locules were gradually filled with tapetum cells that were abnormally vacuolated,and unable to produce pollen grain,thereby resulting in the formation of sterility.2.RNA-seq sequencing and data analysisA total of 2,335 differentially expressed genes were found in three stages of anther development by RNA-seq sequencing.In these genes,many DEGs involved in the reactive oxygen metabolism pathway were significantly down-regulated,including the genes related to enzymatic mechanism of scavenging ROS,glutathione metabolic pathway related genes and ascorbate metabolism related genes.In addition,multiple significant transcription factors associated with the development of tapetum,especially the transformation to secretory tapetum,including MS2,AMS,TDF1,DYT1 and AtMYB103,were also seriously down-regulated in the Zhong41 A.3.Discussions on the sterility mechanism of Zhong41AThe results of qRT-PCR confirmed the reliability of transcriptome data.Combined with both results,the expression of genes encoding ROS scavenger enzyme,were significantly down-regulated in Zhong41 A,and the measure of enzyme activity and hydrogen peroxide content showed that the POD activity was decreased and H₂O₂ content was increased in the anther of Zhong41 A,thus casuing the accumulation of ROS.At the same time,the results of the observation from transmission electron microscopy also showed that the tapetum cells were unable to transform in time in the tetrad stage,which underwent abnormal vacuolization till the anther locules were filled gradually,then leading to the formation of CMS.