柑橘黄化脉明病毒诱导尤力克柠檬细胞程序性死亡研究

发布时间：2018-04-18 03:39

本文选题：柑橘黄化脉明病毒 + 尤力克柠檬　；参考：《西南大学》2017年硕士论文

【摘要】：柑橘黄脉病是柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)引起的一种新型柑橘病害,严重威胁柠檬和酸橙产业,主要造成叶片脉明和黄化。目前该病在我国重庆、四川等地相继爆发,尤其以四川安岳柠檬产区为害最为严重。研究发现细胞程序性死亡(programmed cell death,PCD)与植物抗病过程密切相关。植物抵抗病原物入侵时产生的过敏性反应(hypersensitive reaction,HR)是植物抗病的的重要手段之一,也是PCD的一种表现。本研究以健康尤力克柠檬为试材,采用嫁接传毒的方法,通过对CYVCV侵染后不同时期尤力克柠檬病毒含量、PCD相关基因、代谢酶活性进行测定,并结合TUNEL原位末端标记、电子显微镜、琼脂糖凝胶电泳等方法对PCD进行检测,从而了解CYVCV诱导柑橘细胞程序性死亡机制。主要研究结果如下:1、通过DTBIA与免疫荧光技术确定CYVCV在尤力克柠檬叶片主脉的韧皮部、分泌腔周围以及薄壁细胞中发生。2、TUNEL技术检测结果表明:在感病初期,随着感病时间的增加,尤力克柠檬叶片出现PCD阳性反应,并随着CYVCV含量的增加而不断加强;而在感病后期CYVCV含量降低,PCD阳性反应减少。因此推测CYVCV可以诱导尤力克柠檬PCD的产生,同时PCD阳性反应随CYVCV含量的增加不断增多。3、CYVCV胁迫下,尤力克柠檬根、茎、叶都出现不同程度的PCD阳性反应,由强到弱依次为根、茎、叶,与CYVCV含量分布相一致。因此推测CYVCV含量的增加可以促进尤力克柠檬叶片PCD的产生。4、CYVCV诱导尤力克柠檬叶片超微结构变化:(1)叶绿体淀粉粒增多,嗜锇颗粒增多且体积变大,叶绿体肿胀、畸形,病变严重的叶绿体出现解体现象。(2)少量线粒体出现外膜溶解、嵴突模糊、解体等现象。(3)细胞核出现染色质凝聚且边缘化,核膜溶解,核质高度浓缩等典型PCD现象。进一步证明PCD可以诱导柑橘细胞程序性死亡。5、通过实时荧光定量PCR检测CYVCV胁迫下尤力克柠檬不同时期PCD相关基因CsCysp、Cit-Dad1-1和MCA1的表达,结果显示,CsCysp、Cit-Dad1-1基因表达量下调,MCA1基因表达量上调,从而促进PCD的产生。6、CYVCV侵染后,感病组织caspase-3蛋白酶活性随感病时间的增加而增加;丙二醛含量呈先下降后上升趋势,且高于对照组,表明细胞膜脂过氧化作用增加;感病前期细胞内SOD、CAT、POD酶活性总体呈下降趋势,细胞抗氧化能力减弱,引起ROS积累,从而诱导PCD的产生;感病后期抗氧化酶活性升高,加快活性氧的清除,从而抑制PCD的产生。
[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.
【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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