| Cytoplasmic male sterility (Cytoplasmic male sterility, CMS) exist widely in plant kingdom and is the important way of crop heterosis utilization. The molecular mechanism of CMS in research provides the theoretical foundation for the construction of artificial sterile lines, therefore, to explore, discover genes associated with CMS function is of great significance. This study conducting high-throughput transcriptome sequencing on the RNA of kenaf sterile line and maintainer line anther, Through bioinformatics analysis, development the gene expression profile of anther, study regulatory network of the development of kenaf anther gene. On the base of DEG(Digital Gene Expression) analysis combine with the result of RT-PCR,finality select SF3 gene,through cloning, carrier construction and verification the function of genetically modified, results following:1.By analyzing the transcriptome we obtained 29,656,489 and 30,712,685 raw paired-end reads from the CMS and maintainer lines, respectively. These reads were eventually assembled into 54,563 unigenes with a mean size of 1,015bp. As a result, 45,930(84%)sequences were annotated against the nr protein database. 15,977(29%)sequences were assigned to 286 kyoto encyclopedia of genes and genomes (KEGG) pathways,20,289(37%)sequences have Clusters of Orthologous Groups classifications, and 38,611 unigenes(71%)have at least one gene ontology (GO) term assigned and could be categorized into 50 functional groups. By using the digital gene expression (DGE) method,4,584 transcripts were detected with at least twofold differences between CMS and maintainer lines. We performed GO and KEGG pathway enrichment analysis of deferentially expressed genes (DEGs). The DEGs were assigned to 155 GO terms and enriched to 74 KEGG pathways.There are 67 transcript only exists in the sterile lines genome, 78 transcript only exist in maintainer lines2..According to the results of sequencing by homo-logy cloning method, comparing the sequencing results of SF3 gene with transcriptome sequencing sequences, select high homologous gene sequences, select the high homology gene sequences.Find the longest open reading frame with ORF in finder, design the primers before the beginning and after the termination codes of the ORF,then amplification over the gene, through the comparison to find the full-length of SF3 gene.3 Using the technology of RT-PCR,The cDNAs are from kenaf ((P3A and P3B))anthers and roots which are in different periods,a clear gel electrophoresis atlas, shows that the expression are obvious difference in mono-nuclear and bi-nuclear period of SF3 gene, the results of this are the same with A.Eliasson’s view:SF3 gene is the cumulative consistency in pollen development; The same happen between the same period of Sterile and Maintain,The express content of male sterile line is lower than Maintain line system, but there is not obvious deference in roots of two-line, which might explain the amount of gene expression have significant differences in P3A and P3B, this difference has temporal and spatial specificity.4.According to the sequence of SF3 gene design its expression vector, and choose the specific sequence as interference zone, building SF3 RNAi expression vector and over expression vector.laying the theoretical foundation for the further study of this gene.ompare SF3 gene Sequence with tobacco’the homologous is greater than 75%. Using root carcinoma agrobacterium mediated transfer methods putting the RNAi vector into tobacco, and research its gene function. |
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