發(fā)布時(shí)間：2018-06-29 16:03

  本文選題：柑橘潰瘍病 + 生長素��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：柑橘潰瘍病被列為世界性的檢疫性病害,其病原菌為地毯草黃單胞桿菌致病變種Xanthomonas axonopodis pv.citri(Xac),該病引起落葉、枯枝和落果,導(dǎo)致果品質(zhì)量降低,嚴(yán)重影響柑橘的經(jīng)濟(jì)價(jià)值。生長素是調(diào)控植物生長發(fā)育必不可少的激素,與植物的感病性相關(guān)。本研究表明,病原菌可以通過調(diào)控生長素信號(hào)轉(zhuǎn)導(dǎo)途徑來增強(qiáng)其致病性。對(duì)柑桔潰瘍病高感品種紐荷爾臍橙(Citrus sinensis Osbeck)和高抗品種四季桔(C.madurensis)受潰瘍病侵染后的轉(zhuǎn)錄組進(jìn)行表達(dá)分析,發(fā)現(xiàn)生長素代謝途徑相關(guān)基因GH3.1、GH3.6和GH3.1L在高感品種紐荷爾臍橙中的表達(dá)水平顯著高于其在高抗品種四季桔中的表達(dá)水平,暗示GH3.1、GH3.6和GH3.1L可能與柑橘潰瘍病抗(感)性緊密相關(guān)。GH3蛋白酶具有酰胺合成酶活性,能夠催化游離態(tài)IAA向氨基酸結(jié)合態(tài)IAA轉(zhuǎn)移,使IAA失去活性,進(jìn)而調(diào)控生長素信號(hào)途徑。因此,研究這些基因在柑桔潰瘍病侵染過程中的作用,將為闡明生長素信號(hào)途徑參與調(diào)控柑桔潰瘍病發(fā)展的分子機(jī)制提供一定的證據(jù),同時(shí)也為提出新型、有效控制潰瘍病的措施提供理論依據(jù)。本研究從紐荷爾臍橙中克隆了CsGH3.1、CsGH3.6和CsGH3.1L基因,并分析了它們?cè)诩~荷爾臍橙和四季桔中受潰瘍病誘導(dǎo)的表達(dá)情況。在此基礎(chǔ)上,通過在高感品種晚錦橙(C.sinensis Osbeck)中對(duì)這些基因進(jìn)行超量和抑制表達(dá),評(píng)價(jià)了轉(zhuǎn)基因晚錦橙的潰瘍病抗性變化及其可能的機(jī)制。主要研究如下:(1)序列分析顯示紐荷爾臍橙中CsGH3.1、CsGH3.6和CsGH3.1L的編碼序列長度分別為1797bp、1887bp和1836bp,分別編碼599、629和612個(gè)氨基酸。同源性分析表明紐荷爾臍橙中的CsGH3.1、CsGH3.6和CsGH3.1L均與克里曼丁(C.clementine)的同源性達(dá)到100%。系統(tǒng)進(jìn)化樹分析表明紐荷爾臍橙中CsGH3.1、CsGH3.6和CsGH3.1L分別與AtGH3.1、OsGH3.6和AtGH3.1的親緣關(guān)系較近,且都有相同的保守結(jié)構(gòu)域。GH3.1和GH3.6的啟動(dòng)子序列在紐荷爾臍橙和四季桔中的同源性都超過90%,紐荷爾臍橙和四季桔中的GH3.1啟動(dòng)子均有生長素響應(yīng)元件TGA-box。(2)將紐荷爾臍橙和四季桔葉片接種潰瘍病后,實(shí)時(shí)熒光定量PCR(qPCR)檢測CsGH3.1、CsGH3.6和CsGH3.1L基因的表達(dá)水平。結(jié)果顯示,與誘導(dǎo)0天相比,紐荷爾臍橙和四季桔中CsGH3.1、CsGH3.6和CsGH3.1L都受潰瘍病誘導(dǎo)表達(dá)。誘導(dǎo)5天后,高感品種紐荷爾臍橙中CsGH3.1、CsGH3.6和CsGH3.1L的表達(dá)水平顯著高于四季桔。這些結(jié)果說明CsGH3.1、CsGH3.6和CsGH3.1L基因在柑橘潰瘍病的侵染過程中具有重要作用。(3)用濃度為10μmol·L-1的不同激素及抑制劑處理紐荷爾臍橙葉片3天后,接種潰瘍病,觀察統(tǒng)計(jì)其在感病2天,3天和5天時(shí)的病斑面積。結(jié)果顯示經(jīng)過生長素(IAA),生長素類似物(IBA和NAA)處理過的葉片在潰瘍病誘導(dǎo)后,其病斑面積增大但是并不顯著;水楊酸(SA)及其生物合成抑制劑多效唑(PP333)處理過的葉片顯著抑制了潰瘍病病斑的發(fā)展。值得注意的是,生長素運(yùn)輸抑制劑NPA顯著抑制葉片病斑的發(fā)展,暗示抑制生長素信號(hào)傳導(dǎo)能增強(qiáng)柑橘潰瘍病抗性。(4)用濃度為10μmol·L-1的不同激素及抑制劑處理紐荷爾臍橙葉片,分別在激素誘導(dǎo)0,6,12,24,72小時(shí)后檢測CsGH3.1、CsGH3.6和CsGH3.1L基因的表達(dá)水平。結(jié)果顯示外施IAA,IBA,NAA明顯誘導(dǎo)CsGH3.1和CsGH3.1L的表達(dá),而SA、PP333和NPA沒有顯著誘導(dǎo)CsGH3.1和CsGH3.1L的表達(dá)。CsGH3.6的表達(dá)在上述激素和抑制劑處理前后均無明顯差異。這些結(jié)果表明CsGH3.1和CsGH3.1L可能參與柑橘生長素信號(hào)途徑的調(diào)控。(5)超量表達(dá)CsGH3.1、CsGH3.6和CsGH3.1L基因的轉(zhuǎn)基因晚錦橙形態(tài)發(fā)生了改變,主要表現(xiàn)為葉片向上卷曲,葉片幾乎都較野生型葉片小,顏色淺,整片葉子較野生型的葉片下垂,植株呈枯萎狀態(tài)。但是RNAi抑制表達(dá)CsGH3.1、CsGH3.6和CsGH3.1L的轉(zhuǎn)基因晚錦橙表型沒有變化。(6)抗病性評(píng)價(jià)結(jié)果顯示,與野生型植株相比,超量表達(dá)CsGH3.1、CsGH3.6和CsGH3.1L的轉(zhuǎn)基因晚錦橙葉片對(duì)柑橘潰瘍病的抗病性顯著增強(qiáng),其生長素IAA的水平顯著降低,而抑制表達(dá)的轉(zhuǎn)基因晚錦橙對(duì)柑橘潰瘍病的抗病性沒有變化。以上結(jié)果表明CsGH3.1、CsGH3.6和CsGH3.1L基因參與柑橘潰瘍病的抗病性過程。超量表CsGH3.1、CsGH3.6和CsGH3.1L基因可能是通過抑制活性生長素的積累來增強(qiáng)柑橘對(duì)潰瘍病的抗性。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S436.66

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