本文選題：柑橘黃化脈明病毒 + 尤力克檸檬　； 參考：《西南大學》2017年碩士論文

【摘要】：柑橘黃脈病是柑橘黃化脈明病毒(Citrus yellow vein clearing virus,CYVCV)引起的一種新型柑橘病害,嚴重威脅檸檬和酸橙產(chǎn)業(yè),主要造成葉片脈明和黃化。目前該病在我國重慶、四川等地相繼爆發(fā),尤其以四川安岳檸檬產(chǎn)區(qū)為害最為嚴重。研究發(fā)現(xiàn)細胞程序性死亡(programmed cell death,PCD)與植物抗病過程密切相關(guān)。植物抵抗病原物入侵時產(chǎn)生的過敏性反應(yīng)(hypersensitive reaction,HR)是植物抗病的的重要手段之一,也是PCD的一種表現(xiàn)。本研究以健康尤力克檸檬為試材,采用嫁接傳毒的方法,通過對CYVCV侵染后不同時期尤力克檸檬病毒含量、PCD相關(guān)基因、代謝酶活性進行測定,并結(jié)合TUNEL原位末端標記、電子顯微鏡、瓊脂糖凝膠電泳等方法對PCD進行檢測,從而了解CYVCV誘導柑橘細胞程序性死亡機制。主要研究結(jié)果如下:1、通過DTBIA與免疫熒光技術(shù)確定CYVCV在尤力克檸檬葉片主脈的韌皮部、分泌腔周圍以及薄壁細胞中發(fā)生。2、TUNEL技術(shù)檢測結(jié)果表明:在感病初期,隨著感病時間的增加,尤力克檸檬葉片出現(xiàn)PCD陽性反應(yīng),并隨著CYVCV含量的增加而不斷加強;而在感病后期CYVCV含量降低,PCD陽性反應(yīng)減少。因此推測CYVCV可以誘導尤力克檸檬PCD的產(chǎn)生,同時PCD陽性反應(yīng)隨CYVCV含量的增加不斷增多。3、CYVCV脅迫下,尤力克檸檬根、莖、葉都出現(xiàn)不同程度的PCD陽性反應(yīng),由強到弱依次為根、莖、葉,與CYVCV含量分布相一致。因此推測CYVCV含量的增加可以促進尤力克檸檬葉片PCD的產(chǎn)生。4、CYVCV誘導尤力克檸檬葉片超微結(jié)構(gòu)變化:(1)葉綠體淀粉粒增多,嗜鋨顆粒增多且體積變大,葉綠體腫脹、畸形,病變嚴重的葉綠體出現(xiàn)解體現(xiàn)象。(2)少量線粒體出現(xiàn)外膜溶解、嵴突模糊、解體等現(xiàn)象。(3)細胞核出現(xiàn)染色質(zhì)凝聚且邊緣化,核膜溶解,核質(zhì)高度濃縮等典型PCD現(xiàn)象。進一步證明PCD可以誘導柑橘細胞程序性死亡。5、通過實時熒光定量PCR檢測CYVCV脅迫下尤力克檸檬不同時期PCD相關(guān)基因CsCysp、Cit-Dad1-1和MCA1的表達,結(jié)果顯示,CsCysp、Cit-Dad1-1基因表達量下調(diào),MCA1基因表達量上調(diào),從而促進PCD的產(chǎn)生。6、CYVCV侵染后,感病組織caspase-3蛋白酶活性隨感病時間的增加而增加;丙二醛含量呈先下降后上升趨勢,且高于對照組,表明細胞膜脂過氧化作用增加;感病前期細胞內(nèi)SOD、CAT、POD酶活性總體呈下降趨勢,細胞抗氧化能力減弱,引起ROS積累,從而誘導PCD的產(chǎn)生;感病后期抗氧化酶活性升高,加快活性氧的清除,從而抑制PCD的產(chǎn)生。
[Abstract]:Citrus yellow vein disease is a new citrus disease caused by Citrus yellow vein clearing virus CYVCV. it is a serious threat to lemon and lime industry, and mainly causes leaf veinlight and yellowing.At present, the disease broke out in Chongqing and Sichuan, especially in Anyue lemon producing area.It was found that programmed cell death was closely related to plant disease resistance.Hypersensitive reactionHR-induced hypersensitive response to pathogen invasion is one of the most important methods of plant disease resistance, and it is also a manifestation of PCD.The aim of this study was to investigate the activity of metabolic enzymes and the activity of metabolic enzymes in different stages of CYVCV infection by grafting and transferring virus from healthy lemons, and combining with the in situ end labeling of TUNEL, the content of Lemon virus and the activity of metabolic enzymes were determined in different stages after CYVCV infection.PCD was detected by electron microscope and agarose gel electrophoresis to understand the mechanism of programmed death induced by CYVCV.The main results were as follows: DTBIA and immunofluorescence techniques were used to determine the occurrence of CYVCV in phloem, perisecretory cavity and parenchyma cells of lemons.The PCD positive reaction appeared in the leaves of Lemon, and increased with the increase of CYVCV content, but decreased in the late stage of the disease.It is speculated that CYVCV can induce the production of PCD of Lemon Ulik, and the positive reaction of PCD was increased with the increase of CYVCV content. Under the stress of CYVCV, the positive reaction of PCD in roots, stems and leaves of Lemon were observed in different degrees, from the strong to the weak, the roots and stems were in turn.The distribution of CYVCV content in leaves was consistent with that in leaves.It is speculated that the increase of CYVCV content can promote the production of PCD in the leaves of Lemon Urex. 4CYVCV induces the ultrastructural changes of the leaves of Lemon Ulik. The chloroplast starch granules increase, osmiophilic granules increase and the volume becomes larger, the chloroplast swelling and deformity.A few mitochondria appeared outer membrane dissolution, cristae blurred and disintegrated. 3) chromatin condensation and marginalization, nuclear membrane dissolution, nuclear cytoplasm concentration and other typical PCD phenomena occurred in the seriously diseased chloroplast.It was further demonstrated that PCD could induce programmed death of citrus cells. The expression of CsCysptr Cit-Dad1-1 and MCA1 in different stages of CYVCV stress was detected by real-time fluorescence quantitative PCR. The results showed that the expression of CsCysptcit-Dad1-1 gene down-regulated the expression of MCA1 gene.In order to promote the production of PCD, the activity of caspase-3 protease in susceptible tissues increased after infection, and the content of malondialdehyde (MDA) decreased first, then increased, and was higher than that of control group, indicating the increase of lipid peroxidation of cell membrane.In the early stage of the disease, the activity of SODCATPOD decreased, and the antioxidant ability of the cells decreased, resulting in the accumulation of ROS, thus inducing the production of PCD. In the late stage of the disease, the activity of antioxidant enzymes increased, and the scavenging of reactive oxygen species was accelerated, thus inhibiting the production of PCD.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S436.66

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