【作者】 杨柳；

【导师】 曹孟籍；

【作者基本信息】 西南大学 ， 植物病理学， 2022， 硕士

【摘要】 柑橘是中国及世界上重要的经济水果之一,迄今已有139个国家种植。中国作为主要的柑橘生产国,种植面积和产量均居世界首位,然而柑橘各种各样的病害阻碍着我国柑橘产业的发展。作为多年生木本植物,柑橘主要通过嫁接进行无性繁殖,以致于柑橘植株在生长发育中容易受到多种病毒的侵染并造成病毒的累积,导致植物代谢紊乱,从而引起病症与危害,如感病树体树势减弱、叶片花叶、黄化、皱缩、品种退化等,最终造成柑橘果实产量和品质下降,降低经济效益。病毒的鉴定与检测是病毒病害防控的前提和基础。高通量测序技术因其巨大的优势,目前已成为植物病毒鉴定和检测的强有力工具。因此,本研究借助高通量测序技术,在表现为褪绿、黄斑症状的台北柚叶片中发现了一种新的柑橘病毒,进一步通过构建该新病毒全长cDNA侵染性克隆以探究其生物学特性。同时建立了该新病毒的q RT-PCR常规检测技术体系,为该病毒性病害的监测及制定科学合理的防控策略奠定了基础。本文主要研究内容及结果如下:1.通过高通量测序,在表现为褪绿、黄色斑点症状的台北柚叶片中发现并鉴定出一种正单链RNA新病毒,暂将其命名为柑橘黄斑病毒(Citrus yellow spot virus,CiYSV)。CiYSV是一条由8 061个核苷酸(Nucleotide,nt)组成的正单链RNA病毒。其基因组包含3个开放阅读框(Open reading frame,ORF)和两个非编码区(Untranslated region,UTR)。5’端和3’端UTR的长度分别为75 nt和204 nt。ORF1编码分子量约233 k Da的复制相关蛋白,其包含5个保守结构域,分别编码甲基转移酶,烷烃羟化酶,蛋白水解酶,解旋酶以及依赖于RNA的RNA聚合酶。该复制相关蛋白RP氨基酸序列与乙型线形病毒科(Betaflexiviridae)下柑橘病毒属(Citrivrus)病毒RP氨基酸序列相似性最高(40.84%–43.62%)。ORF2具有病毒运动蛋白相关的保守结构域,编码运动蛋白(Movement protein,MP),该蛋白氨基酸序列与细胞质弹状病毒属(Cytorhabdovirus)病毒MP序列相似度(28.0%–33.2%)比Betaflexiviridae科病毒更高(13.00%–13.68%)。ORF3编码28 k Da外壳蛋白,其上有Betaflexiviridae科病毒外壳蛋白相似的保守结构域。基于核苷酸、氨基酸序列相似性与遗传进化分析,可将CiYSV划分到Betaflexiviridae科Trivirinae亚科下,并且根据ICTV第九次分类报告中Betaflexiviridae科内分属标准,CiYSV与科内各属病毒基因组结构、基因序列均存在明显差异。因此我们认为该病毒可能代表了Betaflexiviridae科Trivirinae亚科内一个暂定的新属级分类单元。2.本研究构建了CiYSV的全长cDNA克隆并初步验证了其侵染性和致病性。克隆获得CiYSV全长cDNA,连入目标载体p Cass4-Rz,成功构建重组载体p CiYSV。通过农杆菌介导注射接种了本氏烟、心叶烟,15 d后,RT-PCR检测和斑点杂交确定了CiYSV能侵染心叶烟,但未引起明显症状,不能侵染本氏烟。另外,通过农杆菌介导真空浸润接种强德勒柚黄化幼苗、尤力克柠檬、凤凰柚等多个柑橘品种,接种30 d后,利用RT-PCR结合斑点杂交方法明确CiYSV能够侵染强德勒柚,但未引起明显症状;在RT-PCR检测阳性的尤力克柠檬、凤凰柚的叶片上发现黄斑症状,说明这两个品种对CiYSV比较敏感。以上结果表明本研究构建的CiYSV全长cDNA克隆具有侵染性,也初步验证了其克隆的致病性。3.本研究建立了CiYSV的q RT-PCR检测体系。以CiYSV的CP基因为靶标基因设计了特异性引物,通过筛选确定了建立检测体系最佳的引物。通过对引物浓度以及退火温度优化建立了CiYSV的实时定量检测体系,明确引物浓度为100nmol/L,退火温度为60℃时,熔解曲线呈单峰,反应Ct值最小。经样品验证,该检测体系特异性良好,灵敏度较普通RT-PCR提高了10倍。更多还原

【Abstract】 Citrus is one of the important economic fruits in China and the world.It is planted in 139 countries so far.As a major producer of citrus,China ranks first in both area and yield in the world.However,various citrus diseases hinder the development of citrus industry in China.As a perennial woody plant,citrus is asexually propagated mainly through grafting,so that citrus plants are vulnerable to virus infection and cause the accumulation of virus during their growth and development,resulting in plant metabolic disorders,and causing diseases and hazards,Such as the weakened tree vigor of susceptible trees,leaf mosaic,yellowing,shrinkage,and variety degradation,etc.,which will eventually lead to a decline in the yield and quality of citrus fruits and reduce economic benefits.The identification and detection of virus is the premise and foundation of virus disease prevention and control.Due to its huge advantages,high-throughput sequencing technology has become a powerful tool for plant virus identification and detection.Therefore,in this study,with the help of high-throughput sequencing technology,a new citrus virus was identified in the leaves of Taipei-pomelo showing chlorosis and yellow spots symptoms.Then,the full-length cDNA infectious clone of the virus was constructed to explore biological characteristics of the new virus,and q RT-PCR detection system of the new virus was also established,It laid the foundation for the monitoring of the viral disease and the formulation of scientific and reasonable prevention and control strategies.The main research results of this paper are as follows:1.A novel positive single-stranded RNA virus,tentatively named Citrus yellow spot virus according to the symptoms,was identified by high-throughput sequencing in leaves of Taipei pomelo with chlorosis and yellow spot symptoms in this study.Citrus yellow spot virus is a plus-single stranded RNA virus.The genome of CiYSV contains 8061 nucleotides(nt),including three open reading frames(ORFs).The length of its5’-terminal untranslated region(UTR)is 75 nt,and of its 3’UTR is 204 nt.ORF1 encodes a replicase with a molecular weight of about 233 k Da.In this putative protein,there are five conserved domains,namely,methyltransferase,alkane hydroxylase,peptidase,helicase,and RNA-dependent RNA polymerase.The amino acid sequence of the replicase was most similar to that of citriviruses(40.84%-43.62%).ORF2 encodes movement protein whose amino acid sequence is more similar to that of cytorhabdoviruses(28.0%-33.2%)than that of betaflexivirids(13.00%-13.68%).ORF3 encodes a 28-k Da coat protein,which contains a conserved betafleviral coat protein domain.Based on analyses of nucleotide and amino acid sequences,and genetic evolution,CiYSV can be classfied into the subfamily Trivirinae(family Betaflexiviridae),and according to the demarcation criteria,members of different genera within the family generally share < 45% nt sequence identity in each gene,and CiYSV was significantly different from viruses of other genera in this family.Therefore,we considered that CiYSV may represent the first member of a tentative new genus of the Trivirinae subfamily of Betaflexiviridae.2.In this study,the full-length cDNA clones of Citrus yellow spot virus were constructed and its infectivity and pathogenicity were preliminary verified.The full-length cDNA of CiYSV was amplified by PCR and inserted into the target vector p Cass4-Rz and the recombinant vector p CiYSV was successfully constructed.Then,the infectious clones were inoculated into Nicotiana.benthamiana and N.glutinosa via agrobacterium-mediated method.After 15 days postinoculation(dpi),RT-PCR detection and dot-blot hybridization verified that CiYSV could infect N.benthamiana,but did not cause obvious symptoms and could not infect N.glutinosa.In addition,several citrus varieties such as Qiangdeleyou pomelo,Eureka lemon,and Fenghuangyou pomelo were inoculated by Agrobacterium-mediated vacuum infiltration.It was confirmed that CiYSV could infect Qiandeleyou pemolo by RT-PCR and dot-blot hybridization detection,at 30 dpi,but did not cause obvious symptoms;Yellow spot symptoms were found on the leaves of Eureka lemon and Fenghuangyou pomelo,which were positive by RT-PCR detection,indicating that these two varieties are more sensitive to CiYSV.The above results indicated that the full-length cDNA clone of CiYSV constructed in this study was infectious,and also preliminarily verified the pathogenicity of the clone.3.In this study,q RT-PCR detection system of Citrus yellow spot virus was established.Specific primers were designed using CiYSV CP gene as target region,the real-time quantitative detection system of Citrus yellow spot virus was established by screening primer,and optimizing primer concentration and annealing temperature.The results showed that when the primer concentration was 100 nmol/L and the annealing temperature was 60℃,the reaction Ct value was the lowest,the melting curve was a single peak,and the amplification efficiency reached 99%.Moreover,the established q RT-PCR detection system has the characteristics of strong specificity and good repeatability,and its sensitivity is 10 times higher than that of ordinary RT-PCR.更多还原