山楂属植物中苹果褪绿叶斑病毒检测及病毒脱除方法研究

发布时间：2018-03-09 06:32

  本文选题：山楂　切入点：ACLSV　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)是危害极大的果树潜隐性病毒之一,该病毒感染果树初期大多没有明显症状,较难及早发现,但是随着树体的生长,病毒会逐年积累,造成果园减产。ACLSV侵染的植物种类众多,不同的ACLSV分离物在基因组序列上存在较大的变异。因此,本研究基于转录组高通量测序解析的山楂属植物ACLSV基因组序列设计病毒检测引物,建立了优化的病毒检测体系,并探讨基于山楂组培苗的ACLSV脱除技术,为揭示ACLSV在山楂属植物中的传播及培育山楂无病毒苗奠定基础。主要研究如下:1.根据山楂属植物ACLSV基因组序列设计病毒RT-PCR检测引物,比较cDNA模板浓度、Taq DNA聚合酶种类及病毒检测采样部位对病毒RT-PCR检测效果的影响。结果表明,引物对F3/R3适合作为病毒的检测引物;RT-PCR扩增时,20μ1L的PCR体系中加入1 μL未经稀释的cDNA较为适宜;Promega公司的Taq DNA聚合酶适用于ACLSV的PCR检测;嫩叶扩增出的谱带最强,说明其病毒含量最高,适合作为病毒检测试材的采样部位。2.根据ACLSV外壳蛋白基因保守序列设计了特异性引物和TaqMan探针,以构建的PMD18-ACLSV-CP阳性重组质粒为标准品绘制TaqMan探针定量RT-PCR标准曲线,并对该方法的灵敏性、重复性和实用性进行检测,由此建立了山楂属植物中基于TaqMan探针实时荧光定量RT-PCR技术的ACLSV快速检测体系。结果显示,以重组质粒为标准品建立的标准曲线相关系数达0.99,扩增效率高,建立的TaqMan探针实时荧光定量RT-PCR病毒检测体系灵敏度为100 copies·μL-1,比常规RT-PCR高100倍,批内与批间变异系数均小于0.91%。3.利用建立的山楂属植物中ACLSV的检测体系,对80份山楂属植物携带ACLSV的情况进行检测,结果18份山楂试材检测出ACLSV,带毒率为22.5%,并且这18份试材均属于山楂(C.pinnatifida)或大果山楂(C.pinnatifida var.major),在供试的阿尔泰山楂(CAltaic.(Loud.)Lange)、绿肉山楂(C.Chlorosarca Maxim)、伏山楂(C.BBrettschnhdeSchneid)及毛山楂(C.Mamioiczii Schneid)中未检测到带病毒的植株。4.为了建立山楂属植物ACLSV脱除体系,比较了热处理(37 ℃C、30 d)、化学处理(培养基中添加20mg·L-1的三氮唑核苷,处理40d)、热处理与化学处理结合方法(处理30 d)3种方法对山楂组培苗中ACLSV的脱除效率。首先以感染ACLSV的'新宾软籽,、'秋红,的1 a生枝条为试材,通过茎尖培养及增殖继代获得生长健壮的山楂组培苗;然后以组培苗为试材,进行不同脱毒处理;最后采用ACLSV检测体系检测材料的带病毒情况。结果显示,热处理或热处理结合化学处理均可完全脱除ACLSV,而单纯化学处理的脱毒效率与品种有关,其中'新宾软籽'和'秋红'的脱毒率分别为88.9%和57.1%,表明组培苗热处理是脱除山楂植株中ACLSV的有效方法。
[Abstract]:Apple chlorotic leaf spot virus (ACLSVV) is one of the most harmful latent virus in fruit trees. Most of the virus infected fruit trees have no obvious symptoms at the initial stage, so it is difficult to detect it early. However, as the tree grows, the virus accumulates year by year. There are many kinds of plants infected with reduced yield of Orchard. ACLSV, different ACLSV isolates have great variation in genome sequence. In this study, we designed primers for virus detection based on the ACLSV genome sequence of Hawthorn plants with high throughput sequencing, established an optimized virus detection system, and discussed the ACLSV removal technology based on Hawthorn tissue culture seedlings. In order to reveal the spread of ACLSV in Hawthorn plants and to cultivate virus-free Hawthorn seedlings, the main studies are as follows: 1. According to the ACLSV genome sequence of Hawthorn plants, primers for the detection of virus RT-PCR were designed. The effects of the concentration of cDNA template and the type of Taq DNA polymerase and the sampling site of virus detection on the detection of virus RT-PCR were compared. Primer pair F3 / R3 was suitable for RT-PCR amplification of virus. Adding 1 渭 L undiluted cDNA into the PCR system of 20 渭 L was more suitable for PCR detection of ACLSV by using Taq DNA polymerase of Promega Company, and the spectrum bands amplified from young leaves were the strongest. The results showed that the virus content was the highest, and it was suitable to be used as the sampling site for virus detection. 2. According to the conserved sequence of ACLSV coat protein gene, specific primers and TaqMan probes were designed. Using the constructed PMD18-ACLSV-CP positive recombinant plasmid as the standard sample, the quantitative RT-PCR curve of TaqMan probe was drawn, and the sensitivity, repeatability and practicability of the method were detected. The rapid detection system of ACLSV in Hawthorn plants based on real-time fluorescence quantitative RT-PCR technique of TaqMan probe was established. The results showed that the correlation coefficient of standard curve established with recombinant plasmid was 0.99, and the amplification efficiency was high. The sensitivity of the real-time fluorescent quantitative RT-PCR virus detection system with TaqMan probe was 100 copies 路渭 L ~ (-1), which was 100 times higher than that of conventional RT-PCR, and the coefficient of variation within and between batches was less than 0.91%. 3. The detection system of ACLSV in Hawthorn plants was established. ACLSV was detected in 80 Hawthorn plants. Results ACLSVV was detected in 18 Hawthorn samples, with a virus rate of 22.5m, and all the 18 samples belonged to C. pinnatifida) or C. pinnatifida var. majorus. No disease was detected in CAltaic.Loud.Langee, C. Chlorarca Maxima, C. BBrettschnhde Schneididi and C. Mamioiczii Schneididi. In order to establish the ACLSV removal system of Hawthorn, The chemical treatment (20 mg 路L ~ (-1)) of nucleoside triazolium at 37 鈩，

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