南方水稻黑条矮缩病毒一步双重RT-PCR 检测技术及其应用

 An one-step dual RT-PCR method was developed for Southern rice black-streaked dwarf virus (SRBSDV) detection from host plants and insect vector white-backed planthopper (Sogatella furcifera Horvath,Hemiptera: Delphacidae), from which two cDNA fragments of the viral genome S5 and S10 were amplified
simultaneously. Two primer pairs, S5-F1 / S5-R2 (5′-ttacaactggagaagcattaacacg-3′ / 5′-atgaggtattgcgtaactgagcc -3′) and S10-oF / S10-oR(5cgcgtcatctcaaactacag -3′ / 5′-tttgtcagcatctaaagcgc -3′), were selectedfrom 40 primer pairs based on SRBSDV genome sequences, and amplified viral S5 ORF1 fragment (819 bp) and cp gene fragment ( 682 bp), respectively. Using total RNA extracts from infected plant leaf tissue or individual planthopper adult as templates, one step RT-PCR reaction in the conditions of annealing temperature of 53℃ with the final concentrations of 240 nmol / L S5-F1 / S5-R2 and 120 nmol / L S10-oF / S10-oR generated a similar yield of two amplicons in argrose electrophoresis analysis. Sequence analysis confirmed the correct
result of the amplification. Additionally, several commercal RNA extraction kits was proven to be fit for the template preparation, and the protocol for total RNA extraction from plant leaf tissue and individual planthopper was simplified by sample grinding direct in eppendorf tube. This method can be used for SRBSDV detection of dried or 70% alcochol soaked planthopper samples stored over one month in room temprature. Key words: Southern rice black-streaked dwarf virus; Sogatella furcifera Horvath     

引起玉米粗缩病的水稻黑条矮缩病毒山东分离物的分子特性

 Four isolates of Rice black-streaked dwarf virus (RBSDV) were collected from the maize plants showing rough dwarf symptom in Linyi and Tai'an,Shandong province.The S10 genomic sequences of these isolates were determined and compared with those of 14 other RBSDV isolates.All of the four sequences were 1 801 base pairs (bp) long including the 5'-UTR of 21 bp and the 3'-UTR of 103 bp.They all contained an open reading frame of 1 677 bp (22-1698),encoding the coat protein (CP) of 558 amino acids.The sequences of these four RBSDV isolates and those of the major cp gene of 14 other isolates available in the GenBank were divided into two groups in the phylogenetic tree.Recombination analysis indicated that the isolate Lym2 was likely a recombinant of isolates Lym1 and Zhjs.     

水稻黑条矮缩病毒S6片段的序列多样性与进化分析

<正>0 引言植物RNA病毒在进化中具有快速变异的能力，有利于病毒扩大寄主范围甚至进化为新病毒[1]。突变和重组是植物RNA病毒变异和遗传多样性的共同进化因素，是群体基因变异的最初来源[2]。由灰飞虱传播的水稻黑条矮缩病毒(Rice black streaked dwarf virus, RBSDV) 属于呼肠孤病毒科(Reoviridae)斐济病毒属(Fijivirus),能引发玉米粗缩病和水稻黑条矮缩病。     

我国水稻黑条矮缩病和玉米粗缩病研究进展

上世纪60年代初我国华东和华北地区分别发现了水稻黑条矮缩病和玉米粗缩病,后来都分别发生了2次大流行,同时都以病害防治为目标分别开展了研究,探明了当地病害发生规律,提出了相应的防治方法,获得了不同程度的防病效果.80年代以来,水稻黑条矮缩病的发生报道仅限于华东地区局部地市,玉米粗缩病的发生涉及华北、东北、西北、西南和华中地区13个省市.根据各地对两病病原形态、寄主及症状、介体昆虫及传病特性等方面相似性的报道,提出了我国玉米粗缩病与水稻黑条矮缩病病原异同性问题.经近10年来对两病用生物学和分子生物学方法进行比较鉴定,证明我国玉米粗缩病和水稻黑条矮缩病病原同属水稻黑条矮缩病毒(RBSDV).同时基本探明了水稻黑条矮缩病在浙江杂交稻区和华北玉米区的再次流行成灾的原因,提出了相应的防治措施,并有效地控制了病害的流行危害.     

控制玉米粗缩病和水稻黑条矮缩病的化学防治方法

玉米粗缩病和水稻黑条矮缩病都是由灰飞虱传播的毁灭性病害,而且目前还没有防治病毒病的有效农药,所以,防治两种病毒病的关键是防治灰飞虱.     

湖南省南方水稻黑条矮缩病毒分离物组分S10编码的ORF序列克隆与分析

采用RT-PCR方法扩增并克隆了2013年湖南省水稻上发生的南方水稻黑条矮缩病毒(Southern rice blackstreaked dwarf virus,SRBSDV)S10片段的近全长序列,采用生物信息学软件Mega、RDP和DnaSP分析其编码的开放阅读框(open reading fragment,ORF)遗传多样性。结果表明:2013年湖南省15个SRBSDV分离物S10编码的ORF序列同源性在99.5%以上,不同分离物中存在3~30个突变位点,绝大部分的核酸突变为无义突变,没有发现可能的重组。在系统发育树上,15个分离物聚类为2个分支,其中湖南汉寿的10个分离物和永州的2个分离物(HuNyz12和HuNyz29)与我国浙江分离物聚为1个亚组、湖南永州的其他3个分离物与我国南部的云南、海南等及越南的分离物聚为1个亚组。系统发育结果表明,SRBSDV随传毒介体白背飞虱迁飞从我国南部向北部扩散。     

南方水稻黑条矮缩病毒湖南分离物S9片段ORF序列分析

本文克隆了湖南省2011-2013年水稻的15个SRBSDV分离物S9两个基因,并测定了其核苷酸序列,序列分析与比较表明,S9两个ORF序列同源性均大于99.5%。突变位点分析表明,S9编码的两个ORF有3~9个突变位点,但大部分的核酸突变为无义突变。系统发育分析表明,S9编码的两个ORF高度同源,处于相同的的系统发育分支。     

玉米粗缩病和水稻黑条矮缩病的发生及防治对策

本文根据玉米粗缩病和水稻黑条矮缩病近几年来的发生特点和为害情况 ,具体阐述这两种病害的发病症状、发生规律、病原生物学、发生影响因素、合理的防治措施 ,为今后更好地识别和防治这两种病害提供了理论基础     

水稻黑条矮缩病毒江苏玉米分离物全基因组序列及进化分析

 利用RT-PCR从江苏玉米上克隆水稻黑条矮缩病毒(RBSDV)的全基因组,其基因组10个片段核苷酸数目和蛋白编码情况与已报道的RBSDV分离物基本一致。 基因组的序列一致性分析和系统进化树表明,RBSDV基因组片段间存在频繁的RNA重排现象;江苏玉米分离物(JS)与湖北玉米分离物(HuB)和河北小麦分离物(HeB)的整体亲缘关系较近,而与安徽分离物(AH-MX8)的整体亲缘关系最远。综合本研究及前人对S8 和S10的研究,RBSDV基因组不同片段的进化中RNA重组的作用不同: S1、S2和S4中没有RNA重组;S3、S5、S6、S8和S10中存在低频率重组;S7 和S9存在较高频率重组,其中S7的高频率重组尤为显著。     

水稻黑条矮缩病和玉米粗缩病的发生特点及防治策略

水稻黑条矮缩病和玉米粗缩病都是病毒病害。“两病”的重要感病期平均为营养生长至生殖生长的过渡期,危害盛期也都在生殖生长旺期。受害作物感病愈早,危害愈重。“两病”都能致受害作物矮化,生长畸形,阻碍正常生长,严重影响产量和品质。     

高效、准确的玉米粗缩病人工接种鉴定技术

为建立高效、准确的玉米粗缩病人工接种鉴定技术,通过将玉米人工接种水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV),研究了温度对RBSDV在灰飞虱体内循回期的影响以及玉米接种苗龄、接种强度及接种时间对人工接种鉴定效果的影响。结果表明:病毒在灰飞虱体内的循回期随温度升高而缩短,在平均温度分别为18.50、23.50、27.50℃时,循回期分别为36.27、21.76、16.38 d;在玉米芽鞘期至2叶1心期接种,接种强度每苗为7~10头虫,接种时间72~96 h条件下,鉴定效果优于其它处理;感病对照郑单958、掖107、掖478发病充分,病情指数为97.70%~100.00%。用已知抗性水平的玉米自交系和杂交种进行验证,鉴定结果表明该人工接种鉴定技术可客观反映供试玉米材料的实际抗性水平。     

玉米抗粗缩病遗传及分子标记研究

Maize rough dwarf disease caused by Rice black-streaked dwarf virus (RBSDV) is transmitted by planthopper in China. Identification and development of resistant hybrids are complicated because of the inconsistencies in viral disease pressure every year. Marker-assisted selection can provide means for main-taining virus resistance alleles even in the absence of disease. In this paper a F₂ segregation population was constructed to identity the molecular markers linked to the resistance gene using a cross between a resistant and a susceptible parents (Qi319×Ye107). Fifteen-day-old seedlings of F₂ population were exposed to small brown planthoppers carrying RBSDV for 3 days in specific inoculation chamber. The inoculated plants were transplanted to screenhouse after removing the insects completely. In plant maturity stage the disease resistance of all the individuals were visually assessed. The results showed that 17, 8, 11, 51 and 122 plants were scaled from 0-4 respectively, in which 0 means no symptoms and 4 represents highly susceptible. Chi-square test demonstrated that the segregation ratio of phenotype was 1∶15 (resistant: susceptible) or 1∶6∶9 (resistant∶moderate∶susceptible) in the F₂ population, indicating RBSDV resistance of maize was controlled by two recessive genes. The F₂ individuals DNA were extracted and 261 SSR (simple sequence repeat) primers derived from maize genome ten chromosomes were selected from maize GDB database to construct genetic linkage map. The linkage map consisted of 71 polymorphic SSR markers, spanning a genetic distance of 996.6 cM with an average interval of 14.0 cM between adjacent markers. The resistant and susceptible gene pools were set up for BSA (bulked segregant analysis) and 6 polymorphism markers were obtained with BSA-SSR method between the two pools. The F₂individuals were further analyzed with 6 polymorphism markers. Chi-square test showed that phi 051, umc1407 and umc1432, mapped on chromosome 7 and 10, exhibited segregation distortion significantly and very significantly in susceptible individuals. These three SSR markers were identified as potential markers linked to the resistant loci.     

水稻黑条矮缩病毒在灰飞虱消化系统的侵染和扩散过程

 水稻黑条矮缩病毒 (Rice black streaked dwarf virus, RBSDV) 由介体灰飞虱(Laodelphax striatellus Fallén)以持久增殖型方式传播, 其编码的P9 1蛋白是形成病毒复制和子代病毒粒体装配的场所—病毒原质(viroplasm)的组分之一。为了明确RBSDV在介体昆虫体内的侵染循回过程, 本研究通过原核表达的P9 1蛋白免疫注射兔子制备P9 1抗体, 应用免疫荧光标记技术研究P9 1在饲毒后不同时期的介体灰飞虱体内的定位。共聚焦显微镜观察到饲毒后3 d, P9 1出现在介体中肠的少数上皮细胞内;饲毒后6 d, 在中肠外表的肌肉细胞分布有P9 1;饲毒后10 d, P9 1分布于中肠和后肠表面的肌肉, 同时在唾液腺也能观察到P9 1的存在。结果表明RBSDV在介体灰飞虱体内首先侵染中肠上皮细胞并复制, 随后扩散到中肠表面的肌肉细胞, 并通过环肌和纵肌扩散到中肠和后肠, 最后扩散到唾液腺。本研究首次直观地阐述了RBSDV在灰飞虱消化系统的侵染和扩散过程, 为有效阻断灰飞虱携带并传播病毒奠定基础。     

南方水稻黑条矮缩病毒检测方法的比较

为明确南方水稻黑条矮缩病毒（Southern rice black-streaked dwarf virus, SRBSDV）检测方法的最佳适用范围,对其现有检测方法Real time RT-PCR、RT-LAMP、RT-PCR的灵敏性及特异性进行了比较,并分析了依据SRBSDV单克隆抗体3F1建立的斑点免疫结合印迹（dot immunobinding assay, DIBA）方法对检测植物寄主和白背飞虱Sogatella furcifera Horvth的特异性。结果表明,灵敏性以Real time RT-PCR方法最高,其次为RT-LAMP方法,而普通RT-PCR方法相对较低。这3种方法均可特异性检测SRBSDV植物寄主和白背飞虱;DIBA方法可以满足SRBSDV和水稻黑条矮缩病毒（Rice black-streaked dwarf virus, RBSDV）植物寄主和白背飞虱大量样品的检测,但不能区分SRBSDV和RBSDV。Real time RT-PCR方法实现了短时间内对SRBSDV RNA拷贝数的相对定量;RT-LAMP方法全程恒温反应,无需热循环仪。     

检测南方水稻黑条矮缩病毒胶体金免疫试纸条的建立

由白背飞虱Sogatella furcifera(Horváth)传播的南方水稻黑条矮缩病毒(Southern rice blackstreaked dwarf virus,SRBSDV)是目前我国南方水稻上危害最严重的病毒,为开发简便、快速、准确的SRBSDV病毒检测技术和检测试剂,以感染SRBSDV的植物粗提液为免疫原,利用杂交瘤技术制备了2株抗SRBSDV的单抗(14A8和15G6),并利用制备的单抗建立了可快速、特异、灵敏地检测SRBSDV的胶体金免疫试纸条。结果表明,2株制备单抗的抗体类型及亚类均为Ig G1、kappa链,单抗腹水的间接ELISA效价均达到10~(-7);Western blot分析表明,2株单抗均与SRBSDV的外壳蛋白亚基有特异反应,而不与水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)反应。以制备14A8和15G6单抗分别为捕获抗体和胶体金标记抗体,开发成能在5 min内准确、特异地检测水稻植物和白背飞虱传毒介体体内SRBSDV的胶体金免疫试纸条;灵敏度分析表明,该检测试纸条的检测水稻病叶的灵敏度达到1∶6 400倍(g/m L),检测单头携毒白背飞虱的灵敏度达到1∶51 200倍(单头/μL)。田间样品检测结果表明,该试纸条的检测结果与RT-PCR的符合率达到100%。建立的SRBSDV胶体金免疫试纸条可对南方水稻黑条矮缩病毒进行快速、特异、灵敏的诊断和检测。     

Improved method for assaying maize plant resistance to maize rough dwarf disease by artificial inoculation and real-time RT-PCR

The study of maize rough dwarf disease (MRDD) and breeding for resistance requires inoculation of maize plants by means of planthoppers. The plant age, insect density and inoculation duration are main factors in the success of maize rough dwarf disease inoculation. These parameters were tested using a susceptible maize inbred line Ye478. Using one or two-leaf plants, 15 planthoppers per plant and a five day inoculation duration, the line Ye478 was the most susceptible with 100% diseased plants; F112132 was moderately susceptible with 60% diseased plants and 90110 and F022411 were resistant without any disease. The results were consistent with those from six years of field studies. Using enzyme-linked immunosorbent assay (ELISA) and real-time quantitative RT-PCR, rice black-streaked dwarf virus was detected in severely diseased plants. The plants were rated from 0 to 3 according to their symptoms at the time of flowering. Plants scoring 0, 1 and 2 could not be distinguished by ELISA, only by real-time quantitative RT-PCR. All of the plants with a score of 3 were positive by ELISA and real-time quantitative RT-PCR. The significant differences in the average viral contents in plants with different symptom ratings could be distinguished by using real-time RT-PCR.     

南方水稻黑条矮缩病防控药剂的创制与应用

南方水稻黑条矮缩病是我国及越南等东南亚国家稻区水稻的重要病毒病。该病害潜隐性强、危害重、防控难度大,对水稻生成构成极大威胁。本文综述了南方水稻黑条矮缩病毒的基因组结构及功能、分子进化、病毒与植物寄主或媒介昆虫间的互作、测报技术、药剂创制和田间综合防控的研究进展,并对未来工作进行了展望与讨论。     

水稻黑条矮缩病毒外壳蛋白基因的克隆、原核表达及抗体制备

为探讨水稻黑条矮缩病毒（Rice black-streaked dwarf virus,RBSDV）的种群变异,采用RT-PCR方法从感染RBSDV山东分离物RBSDV-JN1的水稻中克隆该病毒的S10片段,并进行序列分析,进而构建包含RBSDV-JN1的CP基因的原核表达载体,导入大肠杆菌Escherichia coli BL21（DE3）诱导表达;并以表达的融合蛋白为抗原,制备病毒的多克隆抗体。结果显示：S10片段全长为1 801 bp,包含1个1 677 bp编码框,编码558个氨基酸的外壳蛋白（CP）;与GenBank已注册的19个RBSDV的CP基因序列相比较发现,病毒各分离物间核苷酸的序列相似性为90.5%~99.8%,氨基酸的序列相似性为95.9%~100.0%;地理位置较近的分离物间序列相似性较高,RBSDV种群分布呈现区域性差异。原核表达载体pET-RBSDV-CP经IPTG诱导,获得了分子量约为63 kD带有His标签的目的蛋白。用该蛋白制备的多克隆抗体经ELISA、Western blot和Dot-blot ELISA检测显示,效价为1：81 000,且具有良好的特异性。