Transcriptomic And Proteomic Analysis On Recessive Genic Male Sterile Line 1355A Of Upland Cotton

Cotton is one of the most important economic crops, how to improve the cotton yield and quality more effectively is one of the important issues for the cotton breeding. Thus,cotton heterosis utilization is one strategy to solve this proplem. The genic male sterility system has been considered as the main way in heterosis utilization of cotton as well as artificial emasculation system,because of the widely existed restorers, no cytoplasmic negative influence and stable sterility. Therefore, it is significant to detect the molecular mechanism of genic male sterility. The male sterile line 1355 A of Gossypium hirsutum is the single-gene recessive GMS system, and is an ideal material to study the sterile molecular mechanism of genic male sterility for its stable sterility and obvious phenotype. In order to understand the sterile molecular mechanism of 1355 A, the RNA-seq technology and 2-DE technology were performed to exploit the differentially expressed genes(DEG) at transcription and translation levels. Furthermore, the DEGs of 1355 A were verified by cytological and molecular biological methods. The chief results were shown as follows: 1. Classification fourteen stages of anther development in the male fertile line 1355BFourteen stages of anther development and the relationship between anther development stages and bud length(from the nectary to the top of the bud) were identified based on the results of cross sections on 1355 B anther. The bud length of 0.5-1 mm, 1-2 mm, 2-3 mm, 3-4 mm, 4-5 mm, 5-6 mm, 6-7 mm, 7-8 mm, 8-9 mm, 9-14 mm, 14-24 mm, >24 mm but not flowering, anther dehiscence(the anther in early stage of flowering) and anther senescent(the anther in later stage of flowering) was corresponded to stage 1 to stage 14 respectively. One important difference between anther development in cotton and Arabidopsis thaliana is that the tapetal cell degradation commences earlier in cotton than in Arabidopsis thaliana. 2. The defective pollen wall of 1355AThe cross sections on 1355 AB anther indicated that the first detectable sign of male sterility occurs during the early uninucleate microspore stage(stage 8) in the 1355 A plants. In the 1355 A male sterile plant, the exine spine is not produced at stage 8, consistent with the observations from the SEM(scan electron microscope). Meanwhile 1355 A pollen cytoplasm was empty, and only residual defective pollen walls were present in the anther locules. Furthermore, observed by TEM(transmission electron microscope), the pollen nexine of 1355 A is thicker than that of 1355 B, and the 1355 A pollen intine was not formed. There results suggested the defective in the 1355 A male sterile plant were the thicker nexine, laking of intine, exine without spine and degraded pollen cytoplasm. The thicker nexine in the 1355 A male sterile plant may lead to the other phenotype and finally to male sterility. 3. RNA-seq analysisThe bioinformatics analysis using RNA-seq indicates that a total of 2446 differentially expressed genes were identified between the anthers of 1355 AB lines. Several subsets of genes, including fatty acid biosynthesis-related genes, fatty acid metabolism-related genes and glucose- and phenylpropanoid pathway- related genes are differentially expressed during 1355 B and 1355 A anther development associated with male sterility. The results of the lipid stain Sudan black B also suggests that abnormal lipid metabolism is associated with abnormal pollen wall development at stage 8 in the 1355 A anther. 4. Two-dimensional electrophoresis analysisThere are 47 differentially expressed proteins at stage 7 and stage 8 by using two-dimensional electrophoresis technology. Several subsets of differentially expressed proteins involved in the metabolismof fatty acids and glucose, which were consistent with the results from the RNA-seq analysis. 5. Expression analysis of recessive male sterility related genes in 1355AThe expression of homologous genes from Arabidopsis thaliana, associated with pollen wall development, were analysed by qRT-PCR in both upland cotton lines 1355 AB. The expressions of MS188 and TEK for sexine and nexine formation, respectively, were significantly up-regulated at stage 7 between 1355 B and 1355 A. Meanwhile, the sporopollenin synthesis-related genes, incuding CYP704B1, ACOS5 and MS2, were differently expressed. Therefore, the expression of homologous genes associated with pollen wall development were differently expressed, which lead to forming the abnormal thicker nexine, and finally to male sterility in male sterile line 1355 A.