Study On The Differential Transcriptomics And Proteomics Analysis Between Ja-cms And Its Maintaince Of Cotton

Cotton(Gossypium L.) is one of the most important economic crops in the world. Male sterility is a simple and efficient pollination control system that has been widely used in hybrid cotton breeding.(CMS) is not only the basis in crop heterosis utilization, but also is one of the hot topics in the study of biology, and has important significance to understand the genetic mechanism of plant life activities. However, only a small number of genes are known to be specifically involved in this developmental process and the molecular mechanism of the cytoplasmic male sterility(CMS) is still poorly understand. Therefore, understanding of male sterility mechanism has important theoretical significance and application value. The exploitation of plant male sterility is enhanced by combining transcriptome with proteome analysis of developing pollen.Using a new generation of sequencing technology(NGS) transcriptome combined with proteome using two-dimensional gel electrophoresis and mass spectrometry, we studied(DE) genes and protein of flower buds at the microspore abortion stage between CMS and its maintainer. By integrating both transcriptome and proteome data, we gained a more comprehensive assessment of cotton gene expression. The main results are as follows:1. Here we present, for the first time, extensive sequence and transcript abundance data for the buds transcriptome of obtained between the two lines were captured at both sporogenous cells and microsporocyte stage stages.using the Illumina Tru Seq? sequencing platform. Our further goal was to identify patterns of gene expression during bamboo flower development. Approximately 28 million sequencing reads were generated and assembled de novo, yielding 86093 high quality unigenes with an average length of 755 bp.2. Bioinformatics analysis were performed on the obtained unigene. The result are as follows:(1) Gene function was annotated based on the following databases: Nr(NCBI non-redundant protein sequences);Nt(NCBI non-redundant nucleotide sequences);Pfam(Protein family);KOG/COG(Clusters of Orthologous Groups of proteins);Swiss-Prot(A manually annotated and reviewed protein sequence database);KO(KEGG Ortholog database);GO(Gene Ontology).(2) Open reading frame of 36257 unigene were identified according to the best matched results, and sequence of CDS and amino acid were identified according to standard codon table. Using estscan(3.0.3) software to predict the sequence of CDS for no matched unigene, and there were 28323 genes predicted.(3) Picard- tools v1.41 and samtools v0.1.18 were used to sort, remove duplicated reads and merge the bam alignment results of each sample. GATK2 software was used to perform SNP calling. There were 518108 locis of SNP and 38345 locis of Indel were detacted. SSR of the transcriptome were identified using MISA. we detected 17460 locis of SSR, and 16496 pairs of PCR primers for the development of marker.(4) Based on a rigorous algorithm, significant differential expression genes of the buds at two abortive stages from sterile line and its maintainer line(B2, B3, K2 and K3) were respectively detected 5457, 3178, 3608 and 3999. Compared with the maintainer line, 709 differentially expressed genes(DEGs) were detected at sporogenous cells stage in JA-CMS, including 293 up-regulated genes and 416 genes down regulated expression; There were 644 DEGs were detected at microsporocyte stage in JA-CMS, including 263 up-regulated genes and 381 genes down regulated expression; There were 17 DEGs were detecte in floral buds of JA-CMS between sporogenous cells stage and microsporocyte stage, including 8 up-regulated genes and 9 genes down regulated expression; There were 38 DEGs were detecte in floral buds of JB between sporogenous cells stage and microsporocyte stage, including 29 up-regulated genes and 9 genes down regulated expression.(5) Cluster analysis of the DEGs was performed Using hierarchical clustering, K-means clustering and SOM clustering. The statistical analysis data demonstrated that DEGs identified in this experiment were likely to be involved in energy metabolism, carbohydrate metabolism, transcription factor, amino acid metabolism and signal transduction related genes, and that most of the observed genes might contribute to abnormal floral development in JA.(6) To gain insight into the functional categories and biological pathways active which were altered between JA and JB, groups of up- and down- regulated genes were annotated according to function annotation convention. GO and KEGG analyses were performed to determine and further to understand the biological functions of those differentially expressed genes(DEGs).Using JB as reference, oxidoreductase and the dioxygenase activity decreased of JA between sporogenous cells stage and microsporocyte stage,while the genes associated with photosynthesis and flavonoids are up-regulated.3. Seven genes were randomly selected and their expression levels were confirmed by quantitative RT-PCR, and these genes showed consistent expression patterns with the digital gene expression(DGE) data.4. We optimized the protein extraction method and the two-dimensional electrophoresis conditions, and obtained one serial of two-dimensional electrophoresis system which be suitable to cotton buds. In this system, the protein expression profiles with high definition, high resolution, and p H3-10 NL were got when the total protein of buds in cotton sterile line and maintainer line was extracted with improved phenol method, and the immobilized p H gradient(IPG) strips with 18 cm, p H 3-10 NL were used, and 800 ug protein sample were added in electrophoresis. This 2-DE condition can be effectively applied to analysis the cotton anther proteome.5. The different-display proteomics analysis in the cytoplasmic male sterility lines and its maintainer by technologies of the optimized two-dimensional electrophoresis, mass spectrometry(MS) and bioinformatics and so on. The two-dimensional electrophoresis map of the buds at two abortive critical stage from sterile line and its maintainer line(B2, B3, K3 and K3) were respectively detected 1505, 1540, 1554 and 1540 protein spots which were analyzed by PDQuest8.0.1 software.6. There were 15 protein spots differentially expressed more than 2 folds(p<0.05) were selected to further analysis by MALDI-TOF/TOF-MS/MS and 15 protein spots were successfully identified with a success rate of 100.0%. These fifteen proteins were identified as significant results by using MASCOT search NCBInr green plant protein database. They were involved in energy metabolism(3), carbohydrate metabolism(3), Carbon metabolism(3), amino acid metabolism(1), Lipid metabolism(1), fatty acid metabolism(1), pollen development(1), stress response(1) and unknown function(1), and these genes located in the mitochondria, chloroplasts, nucleus and membrane.7. The cotton anther development is subject to a complex regulatory networks, multiple genes differentially expressed in multiple metabolic pathways affect pollen fertility. This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of cotton gene expression that could not have been attained using each methodology independently. Combining candidate DE proteins with candidate DE transcripts correlates more directly with the actual abundance and function of these DE products, and provides insights into their biological importance in CMS. Detection of DE proteins in combination with DE transcripts is a potentially powerful method for comprehensive analysis of CMS.8. This study compared RNA editing events in eight mitochondrial genes(atp 4, 6, 8, 9, and cox1) among three lines(maintainer JB, CMS- JA, and restorer JR). These events were quantified by sequencing of cloned versions of these genes as c DNAs. Using five pairs of specific primers of atp4, atp6, atp8, atp9 and cox I was amplified by PCR among JA-CMS, its maintainer and restorer line, and DNA sequence alignment of these genes were finished. The number of RNA editing sites in atp4 and atp9 mt DNA genes reported in three other dicot plants was compared with the number of editing sites in cotton detected in this study. The number of RNA editing sites was relatively conserved in atp4 and atp9. In cotton, cox Ⅰ appeared to have more RNA editing sites than this found in the gene of other plant. The putative trans-membrane structures of the coxⅠproteins were different, and their trans-membrane directions were opposite. The results demonstrated the important role of RNA editing in the production of the functional COXⅠsubunit and suggested the RNA editing be likely associated with cytoplasmic male sterility.