發(fā)布時間：2018-03-09 06:32

  本文選題：山楂　切入點：ACLSV　出處：《沈陽農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：蘋果褪綠葉斑病毒(Apple chlorotic leaf spot virus,ACLSV)是危害極大的果樹潛隱性病毒之一,該病毒感染果樹初期大多沒有明顯癥狀,較難及早發(fā)現(xiàn),但是隨著樹體的生長,病毒會逐年積累,造成果園減產(chǎn)。ACLSV侵染的植物種類眾多,不同的ACLSV分離物在基因組序列上存在較大的變異。因此,本研究基于轉錄組高通量測序解析的山楂屬植物ACLSV基因組序列設計病毒檢測引物,建立了優(yōu)化的病毒檢測體系,并探討基于山楂組培苗的ACLSV脫除技術,為揭示ACLSV在山楂屬植物中的傳播及培育山楂無病毒苗奠定基礎。主要研究如下:1.根據(jù)山楂屬植物ACLSV基因組序列設計病毒RT-PCR檢測引物,比較cDNA模板濃度、Taq DNA聚合酶種類及病毒檢測采樣部位對病毒RT-PCR檢測效果的影響。結果表明,引物對F3/R3適合作為病毒的檢測引物;RT-PCR擴增時,20μ1L的PCR體系中加入1 μL未經(jīng)稀釋的cDNA較為適宜;Promega公司的Taq DNA聚合酶適用于ACLSV的PCR檢測;嫩葉擴增出的譜帶最強,說明其病毒含量最高,適合作為病毒檢測試材的采樣部位。2.根據(jù)ACLSV外殼蛋白基因保守序列設計了特異性引物和TaqMan探針,以構建的PMD18-ACLSV-CP陽性重組質粒為標準品繪制TaqMan探針定量RT-PCR標準曲線,并對該方法的靈敏性、重復性和實用性進行檢測,由此建立了山楂屬植物中基于TaqMan探針實時熒光定量RT-PCR技術的ACLSV快速檢測體系。結果顯示,以重組質粒為標準品建立的標準曲線相關系數(shù)達0.99,擴增效率高,建立的TaqMan探針實時熒光定量RT-PCR病毒檢測體系靈敏度為100 copies·μL-1,比常規(guī)RT-PCR高100倍,批內與批間變異系數(shù)均小于0.91%。3.利用建立的山楂屬植物中ACLSV的檢測體系,對80份山楂屬植物攜帶ACLSV的情況進行檢測,結果18份山楂試材檢測出ACLSV,帶毒率為22.5%,并且這18份試材均屬于山楂(C.pinnatifida)或大果山楂(C.pinnatifida var.major),在供試的阿爾泰山楂(CAltaic.(Loud.)Lange)、綠肉山楂(C.Chlorosarca Maxim)、伏山楂(C.BBrettschnhdeSchneid)及毛山楂(C.Mamioiczii Schneid)中未檢測到帶病毒的植株。4.為了建立山楂屬植物ACLSV脫除體系,比較了熱處理(37 ℃C、30 d)、化學處理(培養(yǎng)基中添加20mg·L-1的三氮唑核苷,處理40d)、熱處理與化學處理結合方法(處理30 d)3種方法對山楂組培苗中ACLSV的脫除效率。首先以感染ACLSV的'新賓軟籽,、'秋紅,的1 a生枝條為試材,通過莖尖培養(yǎng)及增殖繼代獲得生長健壯的山楂組培苗;然后以組培苗為試材,進行不同脫毒處理;最后采用ACLSV檢測體系檢測材料的帶病毒情況。結果顯示,熱處理或熱處理結合化學處理均可完全脫除ACLSV,而單純化學處理的脫毒效率與品種有關,其中'新賓軟籽'和'秋紅'的脫毒率分別為88.9%和57.1%,表明組培苗熱處理是脫除山楂植株中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，

本文編號：1587401