大白菜细胞核雄性不育相关基因挖掘与鉴定

发布时间：2018-01-13 22:27

本文关键词：大白菜细胞核雄性不育相关基因挖掘与鉴定　出处：《沈阳农业大学》2017年博士论文　论文类型：学位论文

【摘要】：细胞核雄性不育(GMS)是常见于高等植物中的遗传性状。利用雄性不育系配制杂交种,可以免去去雄操作,降低杂交制种成本,提高杂交率。大白菜(Brassicacampestris L.ssp.Pekinensis)是异花授粉植物,其杂种优势十分显著。利用雄性不育系配制杂交种,是大白菜杂种优势利用的重要途径。大白菜细胞核雄性不育两用系'AB01',是本课题组育成的稳定遗传核基因雄性不育材料,已用其配成了具有100%不育株率和100%不育度的核基因雄性不育系,具有极高的利用价值。本研究利用RNA-seq技术和iTRAQ技术对大白菜细胞核雄性不育两用系'AB01'的可育株与不育株花蕾进行转录组和蛋白质组分析,获得了大量差异表达基因(DEGs)和差异表达蛋白(DEPs),并富集到了相关的代谢途径。通过系统分析DEGs和DEPs的生物学功能,挖掘鉴定出一批与雄性不育相关的基因和蛋白,对部分基因和蛋白进行了表达模式验证。上述结果为大白菜核基因雄性不育研究提供良好的数据平台,也为雄性不育分子机理的探索奠定了基础。主要结果如下:1.供试的大白菜细胞核雄性不育两用系'AB01'的不育株雄蕊退化,花药干瘪,无花粉。利用石蜡切片进行解剖观察,发现雄蕊败育起始于四分体时期,花粉母细胞异常聚集,绒粘层细胞异常膨大,挤压小孢子直至降解死亡,导致不育株花粉败育。2.利用RNA-seq技术对雄性不育两用系'AB01'的可育株与不育株花蕾进行高通量测序,获得了 4,795个DEGs,包括699个在不育株花蕾中上调表达的基因和4,096个下调表达的基因,其中,有212个是在可育株或不育株花蕾中特异性表达的基因。参考大白菜基因组注释信息,筛选出8个花药和花粉发育相关基因,52个花粉壁发育相关基因,22个内源激素相关基因,28个转录因子相关基因。3.对DEGs进行GO功能注释和KEGG Pathway富集分析,获得了 30个显著的GO terms和8个显著富集的Pathways,其中,淀粉与蔗糖代谢、戊糖和葡萄糖醛酸酯转化、氨基酸代谢和植物激素信号转导等代谢途径可能与花粉败育有关。选出24个可能与花药和花粉发育相关的DEGs进行了 qRT-PCR分析,获得的结果验证了 RNA-seq数据的准确性。对其中的15个DEGs进行了不同发育时期花药的表达分析,发现Bra002004(BrAMS)基因在小孢子发育的四分体阶段高表达。BrAMS表达模式分析表明,其仅在可育株雄蕊中高表达。测序结果表明,该基因于不育株启动子区域起始密码子ATG上游409-934bp处缺失526bp,这可能是导致其在转录水平差异表达的主要原因。原位杂交实验证明,Bra002004表达的部位是花药绒粘层和花粉。4.利用iTRAQ技术对雄性不育两用系'AB01'的可育株与不育株花蕾进行了差异蛋白质组分析,共鉴定到5,932个蛋白质,其中DEPs数量为1,494个,包括749个在不育花蕾中上调表达的蛋白和745个下调表达的蛋白。根据蛋白质注释信息,筛选出可能与雄蕊发育相关的41个DEPs,包括5个花药或花粉发育相关蛋白;7个转录因子相关蛋白,29个花粉壁发育相关蛋白。选择16个DEPs进行表达模式分析,验证了 iTRAQ数据的准确性。5.对DEPs进行GO功能注释和KEGG Pathway富集分析,获得了 216个GO terms和6个显著富集的Pathways。其中,碳水化合物分解代谢过程、己糖代谢过程、胞外区、细胞壁和氧化还原活性等前30个GO terms被显著性富集;丙酮酸代谢、三羧酸循环、植物激素信号转导、糖酵解/糖异生、淀粉和蔗糖代谢和光合碳循环相关代谢途径可能参与雄性不育花粉败育进程。6.鉴于本实验室前期已将'AB01'的雄性不育基因定位于大白菜A07染色体,对利用RNA-seq技术和iTRAQ技术筛选出的DEGs和DEPs,在A07染色体上的分布情况进行了统计,获得了 46个位于A07染色体上可能与花粉发育相关的DEPs和DEGs。其中,在定位区间6700-6800kb中比对到Bra014993和Bra01495两个未知功能的DEGs。7.对RNA-seq和iTRAQ数据进行关联分析,获得了 132个在转录水平与蛋白水平同时差异表达的基因和蛋白,其中122个下调表达(基因下调-蛋白下调),10个上调表达(基因上调-蛋白上调),两者之间的相关性系数1=0.5485。对这132个DEGs/DEPs的功能进行注释,16个为未知功能的DEGs/DEPs,116个分别被注释到花粉壁发育相关的酶类、细胞色素P450家族蛋白、激酶相关的受体蛋白、过氧化物酶、羧酸脂酶等功能类别。GO功能注释和KEGG Pathway富集分析结果表明,水解酶活性GO term被显著性富集,内含40个DEGs/DEPs;丙酮酸代谢、淀粉和蔗糖代谢、戊糖和葡萄糖醛酸酯转化、植物激素信号转导、类黄酮生物合成和ABC转运蛋白等Pathways及其所富集到的DEGs/DEPs可能与雄蕊发育有关。
[Abstract]:Genic male sterility (GMS) is a common genetic traits in higher plants. Hybrid seed using male sterile line can be removed from the emasculation operation, reduce the cost of hybrid seed production, improve the hybridization rate. Chinese Cabbage (Brassicacampestris L.ssp.Pekinensis) is a cross pollinated plant, its heterosis is very significant. Hybrid seed using male sterile line is an important way to utilize heterosis in Chinese cabbage. Genic male sterile line'AB01', is a male sterile genetic stable nuclear genes bred by our research group, has used its match with 100% sterile rate and 100% of infertile male sterile gene, with high utilization value. This study uses RNA-seq technology and iTRAQ technology of genic male sterile line'AB01' of the fertile and sterile flower buds transcriptome and proteome analysis, get a lot of difference Different expression gene (DEGs) and differential expression protein (DEPs), and to the enrichment of the related metabolic pathway. By systematically analyzing the biological function of DEGs and DEPs, identified a number of mining related with the male sterility gene and protein of gene and protein expression patterns were verified. The results provide a good the data platform of nuclear male sterile gene of Chinese cabbage, it also laid the foundation for exploring the molecular mechanism of male sterility. The main results are as follows: sterile stamens 1. tested genic male sterile line'AB01'degeneration, anther withered, no pollen. Were observed by paraffin, found the stamen starting at four tetrad stage, abnormal accumulation of pollen mother cells, tapetal cells abnormal enlargement, until death leads to degradation of microspore extrusion, sterile pollen abortion of.2. male sterile line'A with RNA-seq Technology The B01'of the fertile and sterile flower buds by high-throughput sequencing, obtained 4795 DEGs, including 699 in sterile buds of up-regulated genes and 4096 downregulated genes, among them, 212 are in fertile or sterile buds in the expression of specific genes in Chinese cabbage. Genome annotation information, and selected 8 anther and pollen development related genes, 52 pollen wall development related genes, 22 endogenous hormone related genes, 28 transcription factor related gene.3. of DEGs GO and KEGG Pathway functional annotation enrichment analysis, obtained 30 significant GO and 8 terms significantly enriched Pathways, the metabolism of starch and sucrose, pentose and glucuronate conversion, amino acid metabolism and plant hormone signal transduction pathway may be related to pollen abortion. Select 24 may be associated with anther and pollen development related DEGs The qRT-PCR analysis results verify the accuracy of RNA-seq data. On one of the 15 DEGs analyzed the expression of anthers at different developmental stages, Bra002004 (BrAMS) gene is highly expressed in four separate stages of microspore development analysis showed that the expression pattern of.BrAMS, the only in the fertile stamens. High expression strains sequenced the results showed that the gene promoter region upstream of the start codon ATG 409-934bp deletion at 526bp in sterile plants, which may be the main cause of the expression at the transcriptional level. The difference demonstrated in situ hybridization, the expression of Bra002004 is part of the anther tapetal and pollen.4. using iTRAQ technology on the male sterile line'AB01'fertile and sterile buds of differential proteome analysis, 5932 proteins were identified, of which DEPs number 1494, including 749 up-regulated in male sterile flower buds and egg white 745 protein down regulated expression. According to the annotation information, and screened 41 stamen development related to DEPs, including 5 anther or pollen development related protein 7; transcription factor related protein 29, pollen wall development related protein. 16 DEPs expression pattern analysis, to verify the iTRAQ data the accuracy of.5. on DEPs GO and KEGG Pathway functional annotation enrichment analysis, obtained 216 GO terms and 6 Pathways. significantly enriched the metabolic process of decomposition of carbohydrates, hexose metabolism, extracellular region, cell wall and redox activity of 30 GO terms were significantly enriched; pyruvate metabolism, three tricarboxylic acid cycle plant hormones, signal transduction, glycolysis / gluconeogenesis, starch and sucrose metabolism and photosynthetic carbon cycle related metabolic pathway may be involved in male sterile pollen abortion process of.6. in the laboratory before the time has been'AB01 'the male sterility gene is located on chromosome A07 of Chinese cabbage, screened by RNA-seq technology and iTRAQ technology of DEGs and DEPs, the statistical distribution of the A07 chromosome were obtained, 46 located on the A07 chromosome may be related to pollen development of DEPs and DEGs. in the interval 6700-6800kb in comparison to Bra014993 positioning Bra01495 and two unknown functions of DEGs.7. on the RNA-seq and iTRAQ data for correlation analysis, obtained 132 at the transcriptional level and protein level differences between gene and protein expression, of which 122 (down - down regulate the expression of gene protein expression), 10 up-regulated expression (mRNA up-regulated protein), the correlation coefficient between 1=0.5485. between the 132 DEGs/DEPs function annotation, 16 unknown function of DEGs/DEPs, 116 were annotated to pollen wall development related enzymes, cytochrome P450 family Protein kinase related receptor protein, peroxidase, carboxylesterase and other functions of class.GO function annotation and KEGG Pathway enrichment analysis showed that term was significantly GO hydrolase activity concentration, containing 40 DEGs/DEPs; pyruvate metabolism, starch and sucrose metabolism, pentose and glucuronate transformation, plant hormone signal transduction, biosynthesis of flavonoids and ABC transporter Pathways and DEGs/DEPs may be related to the enrichment of stamen development.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S634.1

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