Discovery And Expression Analysis Of Genetic Male Sterility Related Genes In Chinese Cabbage

Genic male sterility is a genetic trait that commonly exists in higher plants.There are many benefits of using hybrid seeds from the male sterility line.On the one hand,we can avoid the hand emasculate operation.On the other hand,we can also reduce the cost of hybrid seed production and improve hybrid rate.Chinese cabbage is a typical allogamous plant with bisexual flowers and significant heterosis.Therefore,using male sterility line is a good way to produce its hybrid seeds.The GMS AB line 'AB01' is a genetically stable male sterile plant that we have incubated in our laboratory.We have utilized the sterile line to successfully prepare male sterile materials with 100%sterile plant rate and 100%sterile degree.In this research,by studying the fertile buds and sterile buds of GMS AB line 'AB01' in Chinese cabbage at transcription and protein level,using RNA-seq and iTRAQ methods,we obtained a large number of differentially expressed genes/proteins(DEGs/DEPs)and their significant enrichment pathways.Through systematic analysis of the biological functions of DEGs and DEPs,a series of sterility related genes and proteins were discovered and identified.We further selected some of them for expression pattern verification.The above findings could provide reliable information for the analysis on male sterility line and lay the foundation for exploring the sterility molecular mechanism in Chinese cabbage.The main results are summarized in the following paragraphs:1.The test materials of fertile stamen of GMS AB line 'AB01' in Chinese cabbage was degraded,and its anther was shriveled,with no pollen was left.By dissecting and observing paraffin sections of fertile and sterile anthers that were at different stages of the development,we could presume that the anthers began to show abnormalities at the tetrad stage.The microspore mother cells(MMCs)clustered together in the center of the locule,the tapetal cells continued to expand,filling in the space where the microspores had degraded and collapsed,and finally leading to pollen sterility in sterile plants.2.We performed transcriptome analysis of the mRNA from fertile and sterile buds of GMS AB line 'AB01' in Chinese cabbage,using RNA-seq,and obtained a total number of 4,795 DEGs.These include 699 genes that were significantly up-regulated and 4,096 genes that were down-regulated in sterile buds.Among them,we obtained 212 specifically expressed genes(SEGs)in fertile or sterile plants.Refering to the genome annotation information in Chinese cabbage,we found 8 anther and pollen-related genes,52 pollen cell wall-related genes,22 phytohormone-related genes,and 28 transcription factor genes.3.After analyzing the DEGs with its GO functional annotation and KEGG Pathway,we obtained 30 significant GO terms and 8 significant Pathways.Among the Pathways,starch and sucrose metabolism,pentose and glucuronate interconversions,amino acid metabolism and plant hormone signal transduction,etc.,were found to be potentially related to pollen sterility in Chinese cabbage.24 possibly anther and pollen related genes were also selected for qRT-PCR analysis,and the results verified the accuracy of the RNA-seq data.Meanwhile,we selected 15 DEGs/DEPs from the above analysis for further verification of anther development periods,and found that Bra002004(BrAMS)was highly expressed in tetrad stage.PCR products from the two samples were sequenced,and we found that there was an absence of 526 bp in the promoter regions in sterile plants,from 409 to 934 bp upstream of start codon ATG.The promoter region deletion may be an important factor that leads to the gene Bra002004's differential expression at the transcription level.qRT-PCR verification showed that Bra002004 was only expressed in the stamens of fertile buds.Also,this gene was detected in tapetum and pollen grain by in-situ hybridization experiment.4.We performed proteormics analysis of fertile and sterile buds of male sterile AB line 'AB01' with iTRAQ technology,and a total of 5,932 proteins were identified.Among them,we obtained 1494 DEPs,including 749 proteins that were significantly up-regulated and 745 proteins that were down-regulated in sterile buds.Base on the annotation of DEPs,41 DEPs that may related to stamen development were selected,including 5 anther and pollen related proteins,7 transcription factor related proteins,and 29 pollen cell wall-related proteins.Then,we selected 16 DEPs from above for expression pattern verification.5.We obtained 216 GO terms and 6 significant Pathways,after analyzing the DEPs with its GO functional annotation and KEGG Pathway.Among them,carbohydrate catabolic process,hexose metabolic process,extracellular region,cell wall,oxidoreductase activity,etc.,were the top 30 significant GO terms.The Pathways,such as pyruvate metabolism,TCA cycle,plant hormone signal transduction,glycolysis/gluconeogenesis,starch and sucrose metabolism,and carbon fixation in photosynthetic organisms may be part of the pollen sterility process of male sterile plants.6.Since the male sterility gene of 'AB01' material has been located on linkage A07 in the previous study in our laboratory,we summarized the distribution of DEGs and DEPs in this locus which was identified by using RNA-seq and iTRAQ technology.We obtained 46 DEGs and DEPs that were located on linkage A07,which were possibly related to pollen development.Among them,Bra014993 and Bra014995 were found in the interval"6700-6800kb".7.After performing correlation analysis of the data obtained from RNA-seq and iTRAQ technology,we found a total of 132 DEGs/DEPs that expressed both up-regulation or down-regulation at the level of transcription and protein.Among them,122 were down-regulated(gene down-regulated and protein down-regulated)and the other 10 were up-regulated(gene up-regulated and protein up-regulated)in sterile buds.The correlation coefficient is r=0.5485 between them.We further annotated the functionality of the 132 DEGs/DEPs,and we found that 116 DEGs/DEPs were related to pollen wall related enzymes,cytochrome P450 proteins,receptor proteins kinase-related,carboxylesterase,peroxidase,etc.,and 16 of the rest were unknown in functionality.The result of GO functional annotation showed that hydrolase activity was significantly enriched,and there were 40 DEGs/DEPs in this GO term.Several important Pathways that may related to stamen development process were pyruvate metabolism,starch and sucrose metabolism,pentose and glucuronate interconversions,plant hormone signal transduction,and flavonoid biosynthesis and ABC transporters.