本文關(guān)鍵詞：大白菜細(xì)胞核雄性不育相關(guān)基因挖掘與鑒定　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 大白菜 細(xì)胞核雄性不育 花蕾 RNA-seq iTRAQ

【摘要】：細(xì)胞核雄性不育(GMS)是常見于高等植物中的遺傳性狀。利用雄性不育系配制雜交種,可以免去去雄操作,降低雜交制種成本,提高雜交率。大白菜(Brassicacampestris L.ssp.Pekinensis)是異花授粉植物,其雜種優(yōu)勢十分顯著。利用雄性不育系配制雜交種,是大白菜雜種優(yōu)勢利用的重要途徑。大白菜細(xì)胞核雄性不育兩用系'AB01',是本課題組育成的穩(wěn)定遺傳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,具有極高的利用價值。本研究利用RNA-seq技術(shù)和iTRAQ技術(shù)對大白菜細(xì)胞核雄性不育兩用系'AB01'的可育株與不育株花蕾進(jìn)行轉(zhuǎn)錄組和蛋白質(zhì)組分析,獲得了大量差異表達(dá)基因(DEGs)和差異表達(dá)蛋白(DEPs),并富集到了相關(guān)的代謝途徑。通過系統(tǒng)分析DEGs和DEPs的生物學(xué)功能,挖掘鑒定出一批與雄性不育相關(guān)的基因和蛋白,對部分基因和蛋白進(jìn)行了表達(dá)模式驗證。上述結(jié)果為大白菜核基因雄性不育研究提供良好的數(shù)據(jù)平臺,也為雄性不育分子機(jī)理的探索奠定了基礎(chǔ)。主要結(jié)果如下:1.供試的大白菜細(xì)胞核雄性不育兩用系'AB01'的不育株雄蕊退化,花藥干癟,無花粉。利用石蠟切片進(jìn)行解剖觀察,發(fā)現(xiàn)雄蕊敗育起始于四分體時期,花粉母細(xì)胞異常聚集,絨粘層細(xì)胞異常膨大,擠壓小孢子直至降解死亡,導(dǎo)致不育株花粉敗育。2.利用RNA-seq技術(shù)對雄性不育兩用系'AB01'的可育株與不育株花蕾進(jìn)行高通量測序,獲得了 4,795個DEGs,包括699個在不育株花蕾中上調(diào)表達(dá)的基因和4,096個下調(diào)表達(dá)的基因,其中,有212個是在可育株或不育株花蕾中特異性表達(dá)的基因。參考大白菜基因組注釋信息,篩選出8個花藥和花粉發(fā)育相關(guān)基因,52個花粉壁發(fā)育相關(guān)基因,22個內(nèi)源激素相關(guān)基因,28個轉(zhuǎn)錄因子相關(guān)基因。3.對DEGs進(jìn)行GO功能注釋和KEGG Pathway富集分析,獲得了 30個顯著的GO terms和8個顯著富集的Pathways,其中,淀粉與蔗糖代謝、戊糖和葡萄糖醛酸酯轉(zhuǎn)化、氨基酸代謝和植物激素信號轉(zhuǎn)導(dǎo)等代謝途徑可能與花粉敗育有關(guān)。選出24個可能與花藥和花粉發(fā)育相關(guān)的DEGs進(jìn)行了 qRT-PCR分析,獲得的結(jié)果驗證了 RNA-seq數(shù)據(jù)的準(zhǔn)確性。對其中的15個DEGs進(jìn)行了不同發(fā)育時期花藥的表達(dá)分析,發(fā)現(xiàn)Bra002004(BrAMS)基因在小孢子發(fā)育的四分體階段高表達(dá)。BrAMS表達(dá)模式分析表明,其僅在可育株雄蕊中高表達(dá)。測序結(jié)果表明,該基因于不育株啟動子區(qū)域起始密碼子ATG上游409-934bp處缺失526bp,這可能是導(dǎo)致其在轉(zhuǎn)錄水平差異表達(dá)的主要原因。原位雜交實驗證明,Bra002004表達(dá)的部位是花藥絨粘層和花粉。4.利用iTRAQ技術(shù)對雄性不育兩用系'AB01'的可育株與不育株花蕾進(jìn)行了差異蛋白質(zhì)組分析,共鑒定到5,932個蛋白質(zhì),其中DEPs數(shù)量為1,494個,包括749個在不育花蕾中上調(diào)表達(dá)的蛋白和745個下調(diào)表達(dá)的蛋白。根據(jù)蛋白質(zhì)注釋信息,篩選出可能與雄蕊發(fā)育相關(guān)的41個DEPs,包括5個花藥或花粉發(fā)育相關(guān)蛋白;7個轉(zhuǎn)錄因子相關(guān)蛋白,29個花粉壁發(fā)育相關(guān)蛋白。選擇16個DEPs進(jìn)行表達(dá)模式分析,驗證了 iTRAQ數(shù)據(jù)的準(zhǔn)確性。5.對DEPs進(jìn)行GO功能注釋和KEGG Pathway富集分析,獲得了 216個GO terms和6個顯著富集的Pathways。其中,碳水化合物分解代謝過程、己糖代謝過程、胞外區(qū)、細(xì)胞壁和氧化還原活性等前30個GO terms被顯著性富集;丙酮酸代謝、三羧酸循環(huán)、植物激素信號轉(zhuǎn)導(dǎo)、糖酵解/糖異生、淀粉和蔗糖代謝和光合碳循環(huán)相關(guān)代謝途徑可能參與雄性不育花粉敗育進(jìn)程。6.鑒于本實驗室前期已將'AB01'的雄性不育基因定位于大白菜A07染色體,對利用RNA-seq技術(shù)和iTRAQ技術(shù)篩選出的DEGs和DEPs,在A07染色體上的分布情況進(jìn)行了統(tǒng)計,獲得了 46個位于A07染色體上可能與花粉發(fā)育相關(guān)的DEPs和DEGs。其中,在定位區(qū)間6700-6800kb中比對到Bra014993和Bra01495兩個未知功能的DEGs。7.對RNA-seq和iTRAQ數(shù)據(jù)進(jìn)行關(guān)聯(lián)分析,獲得了 132個在轉(zhuǎn)錄水平與蛋白水平同時差異表達(dá)的基因和蛋白,其中122個下調(diào)表達(dá)(基因下調(diào)-蛋白下調(diào)),10個上調(diào)表達(dá)(基因上調(diào)-蛋白上調(diào)),兩者之間的相關(guān)性系數(shù)1=0.5485。對這132個DEGs/DEPs的功能進(jìn)行注釋,16個為未知功能的DEGs/DEPs,116個分別被注釋到花粉壁發(fā)育相關(guān)的酶類、細(xì)胞色素P450家族蛋白、激酶相關(guān)的受體蛋白、過氧化物酶、羧酸脂酶等功能類別。GO功能注釋和KEGG Pathway富集分析結(jié)果表明,水解酶活性GO term被顯著性富集,內(nèi)含40個DEGs/DEPs;丙酮酸代謝、淀粉和蔗糖代謝、戊糖和葡萄糖醛酸酯轉(zhuǎn)化、植物激素信號轉(zhuǎn)導(dǎo)、類黃酮生物合成和ABC轉(zhuǎn)運蛋白等Pathways及其所富集到的DEGs/DEPs可能與雄蕊發(fā)育有關(guān)。
[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.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S634.1

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本文編號：1420835