超量表达生长素早期响应基因CsGH3增强柑橘溃疡病抗性

发布时间：2018-06-29 16:03

本文选题：柑橘溃疡病 + 生长素　；参考：《西南大学》2017年硕士论文

【摘要】：柑橘溃疡病被列为世界性的检疫性病害,其病原菌为地毯草黄单胞杆菌致病变种Xanthomonas axonopodis pv.citri(Xac),该病引起落叶、枯枝和落果,导致果品质量降低,严重影响柑橘的经济价值。生长素是调控植物生长发育必不可少的激素,与植物的感病性相关。本研究表明,病原菌可以通过调控生长素信号转导途径来增强其致病性。对柑桔溃疡病高感品种纽荷尔脐橙(Citrus sinensis Osbeck)和高抗品种四季桔(C.madurensis)受溃疡病侵染后的转录组进行表达分析,发现生长素代谢途径相关基因GH3.1、GH3.6和GH3.1L在高感品种纽荷尔脐橙中的表达水平显著高于其在高抗品种四季桔中的表达水平,暗示GH3.1、GH3.6和GH3.1L可能与柑橘溃疡病抗(感)性紧密相关。GH3蛋白酶具有酰胺合成酶活性,能够催化游离态IAA向氨基酸结合态IAA转移,使IAA失去活性,进而调控生长素信号途径。因此,研究这些基因在柑桔溃疡病侵染过程中的作用,将为阐明生长素信号途径参与调控柑桔溃疡病发展的分子机制提供一定的证据,同时也为提出新型、有效控制溃疡病的措施提供理论依据。本研究从纽荷尔脐橙中克隆了CsGH3.1、CsGH3.6和CsGH3.1L基因,并分析了它们在纽荷尔脐橙和四季桔中受溃疡病诱导的表达情况。在此基础上,通过在高感品种晚锦橙(C.sinensis Osbeck)中对这些基因进行超量和抑制表达,评价了转基因晚锦橙的溃疡病抗性变化及其可能的机制。主要研究如下:(1)序列分析显示纽荷尔脐橙中CsGH3.1、CsGH3.6和CsGH3.1L的编码序列长度分别为1797bp、1887bp和1836bp,分别编码599、629和612个氨基酸。同源性分析表明纽荷尔脐橙中的CsGH3.1、CsGH3.6和CsGH3.1L均与克里曼丁(C.clementine)的同源性达到100%。系统进化树分析表明纽荷尔脐橙中CsGH3.1、CsGH3.6和CsGH3.1L分别与AtGH3.1、OsGH3.6和AtGH3.1的亲缘关系较近,且都有相同的保守结构域。GH3.1和GH3.6的启动子序列在纽荷尔脐橙和四季桔中的同源性都超过90%,纽荷尔脐橙和四季桔中的GH3.1启动子均有生长素响应元件TGA-box。(2)将纽荷尔脐橙和四季桔叶片接种溃疡病后,实时荧光定量PCR(qPCR)检测CsGH3.1、CsGH3.6和CsGH3.1L基因的表达水平。结果显示,与诱导0天相比,纽荷尔脐橙和四季桔中CsGH3.1、CsGH3.6和CsGH3.1L都受溃疡病诱导表达。诱导5天后,高感品种纽荷尔脐橙中CsGH3.1、CsGH3.6和CsGH3.1L的表达水平显著高于四季桔。这些结果说明CsGH3.1、CsGH3.6和CsGH3.1L基因在柑橘溃疡病的侵染过程中具有重要作用。(3)用浓度为10μmol·L-1的不同激素及抑制剂处理纽荷尔脐橙叶片3天后,接种溃疡病,观察统计其在感病2天,3天和5天时的病斑面积。结果显示经过生长素(IAA),生长素类似物(IBA和NAA)处理过的叶片在溃疡病诱导后,其病斑面积增大但是并不显著;水杨酸(SA)及其生物合成抑制剂多效唑(PP333)处理过的叶片显著抑制了溃疡病病斑的发展。值得注意的是,生长素运输抑制剂NPA显著抑制叶片病斑的发展,暗示抑制生长素信号传导能增强柑橘溃疡病抗性。(4)用浓度为10μmol·L-1的不同激素及抑制剂处理纽荷尔脐橙叶片,分别在激素诱导0,6,12,24,72小时后检测CsGH3.1、CsGH3.6和CsGH3.1L基因的表达水平。结果显示外施IAA,IBA,NAA明显诱导CsGH3.1和CsGH3.1L的表达,而SA、PP333和NPA没有显著诱导CsGH3.1和CsGH3.1L的表达。CsGH3.6的表达在上述激素和抑制剂处理前后均无明显差异。这些结果表明CsGH3.1和CsGH3.1L可能参与柑橘生长素信号途径的调控。(5)超量表达CsGH3.1、CsGH3.6和CsGH3.1L基因的转基因晚锦橙形态发生了改变,主要表现为叶片向上卷曲,叶片几乎都较野生型叶片小,颜色浅,整片叶子较野生型的叶片下垂,植株呈枯萎状态。但是RNAi抑制表达CsGH3.1、CsGH3.6和CsGH3.1L的转基因晚锦橙表型没有变化。(6)抗病性评价结果显示,与野生型植株相比,超量表达CsGH3.1、CsGH3.6和CsGH3.1L的转基因晚锦橙叶片对柑橘溃疡病的抗病性显著增强,其生长素IAA的水平显著降低,而抑制表达的转基因晚锦橙对柑橘溃疡病的抗病性没有变化。以上结果表明CsGH3.1、CsGH3.6和CsGH3.1L基因参与柑橘溃疡病的抗病性过程。超量表CsGH3.1、CsGH3.6和CsGH3.1L基因可能是通过抑制活性生长素的积累来增强柑橘对溃疡病的抗性。
[Abstract]:Citrus canker is listed as a worldwide quarantine disease, the pathogen of which is Xanthomonas axonopodis pv.citri (Xac), which causes deciduous, dry and falling fruit, which leads to the decrease of fruit quality and the economic value of citrus. Growth hormone is an essential hormone to control plant growth and growth. This study shows that the pathogenic bacteria can enhance its pathogenicity by regulating the auxin signal transduction pathway. The expression analysis of the transcriptional group after the infection of the citrus ulcers, the Citrus sinensis Osbeck and the high resistance variety, the four seasons orange (C.madurensis), was carried out, and the metabolites of the auxin were found. The expression level of diameter related genes GH3.1, GH3.6 and GH3.1L in high sensitive new navel orange was significantly higher than that in the citrus of high resistance variety, suggesting that GH3.1, GH3.6 and GH3.1L may be closely related to citrus ulcerative disease (sense) resistance (sense), and.GH3 protease has the activity of amide synthesis enzyme, which can catalyze the binding of free IAA to amino acids. The transfer of state IAA makes IAA inactive and then regulates the auxin signaling pathway. Therefore, the study of the role of these genes in the infection process of Citrus ulcers will provide some evidence for elucidating the molecular mechanism of auxin signaling in the regulation of the development of Citrus ulcers, as well as providing a new and effective measure for the control of ulcerative disease. Theoretical basis. This study cloned the CsGH3.1, CsGH3.6 and CsGH3.1L genes from the navel orange, and analyzed the expression of their ulcerative expression in the navel navel orange and four seasons oranges. On this basis, the gene was overexpressed and overexpressed in the highly susceptible variety, late brocade orange (C.sinensis Osbeck), and the transgene was evaluated. The changes and possible mechanisms of resistance to ulcers of late brocade orange were studied as follows: (1) sequence analysis showed that the encoding sequence length of CsGH3.1, CsGH3.6 and CsGH3.1L in navel orange was 1797bp, 1887bp and 1836bp respectively, encoding 599629 and 612 amino acids respectively. Homology analysis showed CsGH3.1, CsGH3.6 and CsGH3.1L in navel orange. The homology of Kleiman Martin (C.clementine) to 100%. phylogenetic tree analysis showed that CsGH3.1, CsGH3.6 and CsGH3.1L in the navel orange were closely related to AtGH3.1, OsGH3.6 and AtGH3.1, and the same conserved domain.GH3.1 and GH3.6 promoter sequences were all over the same homology in the navel navel orange and four four oranges. After 90%, the GH3.1 promoter in the navel orange and four four oranges all had the auxin response element TGA-box. (2) to inoculate the Ulceration Disease of the navel navel orange and the four seasons orange leaves. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression level of CsGH3.1, CsGH3.6 and CsGH3.1L genes. The results showed that the navel orange and the four four orange CsGH3.1 in the four seasons were compared with the 0 days. Both CsGH3.6 and CsGH3.1L were induced by ulceration. 5 days after induction, the expression level of CsGH3.1, CsGH3.6 and CsGH3.1L in the high sensitive breed of new navel orange was significantly higher than that of four seasons. These results showed that CsGH3.1, CsGH3.6 and CsGH3.1L genes had a important role in the infection process of Citrus ulcers. (3) different stimuli with the concentration of 10 u mol. L-1. 3 days after treatment of the leaves of navel orange in 3 days, it was inoculated with ulceration disease and observed the area of the disease in 2 days, 3 days and 5 days. The results showed that the leaf area of the leaves treated by auxin (IAA) and auxin analogs (IBA and NAA) was not significant after the ulcerative disease induced, but the salicylic acid (SA) and its biosynthesis were not significant. The depressant PP333 treated leaves significantly inhibited the development of the lesions of the ulcer disease. It is worth noting that auxin transport inhibitor NPA significantly inhibits the development of leaf spot, suggesting that inhibition of auxin signal transduction can enhance citrus ulceration resistance. (4) different hormones and inhibitors with a concentration of 10 u mol. L-1 treat the navel of the navel The expression level of CsGH3.1, CsGH3.6 and CsGH3.1L genes was detected after 0,6,12,24,72 hours induced by hormone, respectively. The results showed that the expression of CsGH3.1 and CsGH3.1L was obviously induced by IAA, IBA and NAA, while SA, PP333 and NPA did not significantly induce the expression of CsGH3.1 and the expression of the expression before and after the treatment of these hormones and inhibitors. These results showed that CsGH3.1 and CsGH3.1L may be involved in the regulation of Citrus auxin signaling pathway. (5) the morphology of transgenic late brocade orange that overexpressed CsGH3.1, CsGH3.6 and CsGH3.1L genes changed, mainly manifested by the upward curl of leaves, the leaves were almost smaller than the wild type leaves, and the whole leaf was more than the wild type leaf. The plant was drooping and the plant was wilted. But RNAi inhibited the expression of CsGH3.1, CsGH3.6 and CsGH3.1L were not changed. (6) resistance evaluation showed that the resistance of transgenic late brocade orange leaves with CsGH3.1, CsGH3.6 and CsGH3.1L increased significantly to citrus ulcers, and its auxin IAA, compared with wild plants. The resistance of the transgenic late brocade orange to citrus ulcers was not changed. The above results showed that CsGH3.1, CsGH3.6 and CsGH3.1L genes were involved in the disease resistance process of citrus canker. The super scale CsGH3.1, CsGH3.6 and CsGH3.1L genes may enhance citrus ulceration by inhibiting the accumulation of active auxin. Resistance to ulcers.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.66

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