马铃薯卷叶病毒(PLRV)检测及系统进化关系的研究进展

马铃薯卷叶病毒（PLRV）是导致马铃薯严重减产的世界性病毒病害，本文介绍了PLRV的研究状况，检测方法，不同分离物CP序列同源性进行了系统进化树比较并对这些研究进行了总结。     

马铃薯卷叶病毒(PLRV)内蒙古分离物基因间隔区(IS)的克隆与序列分析

根据Genbank已报道马铃薯卷叶病毒(PLRV)基因组序列,分析其基因间隔区(IS)两端的保守区,自行设计、合成一对特异性引物,以PLRV内蒙古分离物总RNA为模板,经RT-PCR扩增得到含PLRVIS的一段369 bp的cDNA,克隆于载体pBS-T中。重组质粒经PCR鉴定、酶切分析和核苷酸序列测定,并进一步与PLRV其他分离物的同源序列作比对。结果表明:克隆的PLRV内蒙古分离物的IS序列与其他全部已发表的13个全基因组中的IS核苷酸序列有很高的同源性,最高达到100%,平均为97.90%,高于这13个PLRV全基因组序列96.81%的同源性,说明IS序列不仅在PLRV的不同株系间比较保守,而且在PLRV的全基因组序列中也是相对保守的。研究结果预示,将IS构建成RNA干扰型结构导入马铃薯,将有可能获得抗PLRV多种株系且抗性更高的转基因植株。     

重组CP多克隆抗体在马铃薯卷叶病毒DAS-ELISA检测中的应用

先经过饱和硫酸铵沉淀分离,再用Protein G亲和层析柱纯化,从马铃薯卷叶病毒(PLRV)重组CP作抗原制备的抗血清中获得了高纯度多克隆抗体( IgG),并用戊二醛法一步法进行碱性磷酸酶的标记获得了酶标抗体(IgGAP).将纯化的抗体与酶标抗体用于感染PLRV病叶的检测,DAS-ELISA反应呈阳性,结果表明,用重组...     

四倍体马铃薯抗卷叶病毒的分子标记开发及性状关联分析

病毒病是马铃薯生产中的主要病害之一,侵染马铃薯的病毒中,影响较为严重和发生较为普遍是卷叶病毒(potato leaf roll virus,PLRV)引起的卷叶病。本研究通过田间性状调查得到50株马铃薯‘合作88’自交F2代抗PLRV性状分离群体,首先利用DAS-ELISA明确侵染病毒的类型,随机选取5株抗病群体和5株感病群体,通过Illumina 2X覆盖度重测序,随后以马铃薯双单倍体DM为参考基因组进行单倍体组装和注释。通过基于Perl语言下的MISA程序进行全基因组SSR挖掘和比对,共筛选出277个与抗卷叶病相关的特异性SSR标记;接下来随机选取180个SSR标记并设计引物,PCR扩增后通过LabChip GX Touch检测得到232个等位位点,其中多态性位点152个,基于Minimum-Evolution的聚类分析发现,在遗传相似性系数为0.80时,能把抗PLRV两个性状分离群体区分。利用JoinMap 4.0构建了‘合作88’抗PLRV连锁的遗传图谱;遗传图谱共包含8个连锁群,总长度为2684.3 cM,标记间平均距离11.57 cM。最后用TetraploidMap检测到5个与抗卷叶病相关的QTL,其遗传贡献率分别为4.31%、8.70%、10.89%、13.52%、7.82%。基于mapping的单倍体测序数据,5个QTL分别被定位于第Ⅹ号、Ⅲ号、Ⅰ号、Ⅷ号、Ⅸ号染色体。本研究可为高通量开发马铃薯抗卷叶病分子标记提供技术参考,也可为精细定位马铃薯优良性状相关基因提供理论依据。     

双重RT-PCR同步检测马铃薯A病毒和马铃薯卷叶病毒

根据马铃薯A病毒全基因序列和马铃薯卷叶病毒衣壳蛋白区序列分别设计PVA和PLRV的特异性引物,应用双重RT-PCR同步检测马铃薯A病毒和马铃薯卷叶病毒,分别得到300bp和222bp大小的扩增片段.试验从反转录、PCR等方面对双重RT-PCR同步检测2种病毒进行了探索和优化.结果表明反转录反应中dNTPs浓度、2种病毒下游引物浓度比例以及PCR反应中Mg2+浓度对双重RT-PCR同时检测PVA和PLRV有较大的影响.     

蚕豆萎蔫病毒 2 号（BBWV2）基因组 RNA2的序列测定

以表现顶枯症状的11个辣椒病株的dsRNA为模板，用蚕豆病毒属（Fabavirus）的兼并引物Fab5_R1F和Fab5～R1R进行PCR扩增，其中10个病株扩增到长约400bp的预期大小的片段．选取其中代表病株XJP1—1的PCR产物进行克隆、测序，得到389nts的序列片段。基因库检索表明，该片段为蚕豆萎蔫病毒2号（BBWV2）基因组RNA的片段。说明经PCR检测，具有400bp片段的10个田间病株均有BBWV2侵染．将基因库中已登陆的BBWV2的RNA，核苷酸序列进行序列比对，然后根据RNA，的序列保守区设计引物R2F-1F和R2F-1R，以病株的总RNA为模板．对分离物XJP1—1进行RNA，组分的克隆、测序，获得1302nts的RNA，序列，序列比较显示．该序列与新疆侵染加工番茄的分离物XJ14—1具有最高的序列同源性，为88．9％。     

脱毒马铃薯试管苗快繁及微型薯诱导技术

马铃薯是无性繁殖作物,在生产繁殖过程中极易感染病毒,并世代传递,病毒为害逐年加重,产量逐年下降。侵染马铃薯的主要病毒有马铃薯卷叶病毒（PLRV）、马铃薯Y病毒（PVY）、马铃薯X病毒（PVX）、马铃薯A病毒（PVA）、马铃薯S病毒（PVS）、     

河北省番茄黄化卷叶病毒的分子鉴定及序列分析

本研究旨在对河北省发生的番茄黄化卷叶病毒病的病原进行分子鉴定,并对其病原分子的变异和进化情况进行分析。根据已知的番茄黄化卷叶病毒（Tomato yellow leaf curl virus, TYLCV）基因组序列设计特异引物进行PCR扩增,对获得的特异片段进行回收、克隆和测序,而后根据测定序列重新设计引物扩增病毒样品DNA-A的近全长序列,并进行同源性比较及分子系统进化分析。结果显示利用PA1-F/R引物从采集的4个病毒样品中均扩增得到大小约为645bp的特异片段,DNA-A全长序列测定和分析发现4个病毒样品的全长为2781-2782个核苷酸,共编码6个ORFs。基因组比较发现,4个样品DNA-A与山东、上海、浙江、安徽以及日本、澳大利亚报道的TYLCV的同源性最高,均达99.0%以上,并与美洲、非洲及欧洲报道的TYLCV同属一个大的分支。研究结果表明在河北省采集的4个病株样本均为感染了番茄黄化卷叶病毒所致,而且极有可能同属于TYLCV-Israel株系的分离物。     

山西运城番茄黄化曲叶病毒的初步鉴定

近年来,番茄黄化曲叶病毒(tomato yellow leaf curl virus,TYLCV)对山西运城的番茄生产造成严重危害。利用烟粉虱传双生病毒简并引物PA/PB、PA/P2对病株进行检测,分别扩增出约500 bp和约1 200 bp的特异性片段,表明病株感染了双生病毒。进一步利用番茄黄化曲叶病毒特异性引物AF1/AR1进行扩增,对获得的特异性片段测序、序列比对及系统进化分析,结果表明,从样品中获得2个分离物,长度均为654 bp,与TYLCV分离物的相似性高达99%~100%,两分离物间的相似性为99.8%;系统进化分析表明,2个分离物与除TYLCVSeychelles之外的TYLCV聚在一个大的分支,与所有国内的及墨西哥的(TYLCV-Mexico)分离物聚在一个小的分支,与其他病毒分离物的系统进化关系较远。初步判断这2个分离物应属于番茄黄化曲叶病毒,且可能是由中国东南沿海地区和日本传入的。     

马铃薯卷叶病毒与S病毒重组双CP多克隆抗体的制备

为了制备出马铃薯卷叶病毒(PLRV)与S病毒(PVS)重组双CP多克隆抗体并用于PLRV与PVS这2种病毒的间接与DAS-ELISA检测,构建了PLRV与PVS融合双CP基因的原核表达载体pET22b-LRCP/SCP,用超声破碎法代替溶菌酶法裂解重组菌BL21(pET22b-LRCP/SCP)并提取菌体蛋白,用镍离子亲和层析纯化获得了分子质量为51.2 ku的高纯度的重组双CP。用该重组双CP作抗原免疫家兔获得了高效价(1∶128 k)抗血清。纯化的重组双CP多克隆抗体与PLRV或PVS阳性标准物特异性结合,与另外4种马铃薯主要病毒(PVX、PVY、PVA与PVM)无交叉反应。间接ELISA反应中,稀释度为1∶3 200的重组双CP多克隆抗体与PLRV或PVS阳性物都呈现阳性反应;在DAS-ELISA反应中,稀释度均为1∶100的重组双CP多克隆抗体及其碱性磷酸酶标记物与PLRV或PVS的阳性标准物也分别呈现阳性反应。结果表明,制备的重组双CP抗体既可用于PLRV与PVS这2种病毒的间接ELISA检测,也可用于DAS-ELISA检测。     

Molecular characterization of the progeny of <Emphasis Type="Italic">Solanum etuberosum</Emphasis> identifies a genomic region associated with resistance to potato leafroll virus

Potato leafroll virus (PLRV; Genus Polerovirus; Family Luteoviridae) is one of the most important virus pathogens of potato worldwide and breeders are looking for new sources of resistance. Solanum etuberosum Lindl., a wild potato species native to Chile, was identified as having resistances to PLRV, potato virus Y, potato virus X, and green peach aphid. Barriers to sexual hybridization between S. etuberosum and cultivated potato were overcome through somatic hybridization. Resistance to PLRV has been identified in the BC₁, BC₂ and BC₃ progeny of the somatic hybrids of S. etuberosum (+) S. tuberosum haploid × S. berthaultii Hawkes. In this study, RFLP markers previously mapped in potato, tomato or populations derived from S. palustre (syn S. brevidens) × S. etuberosum and simple sequence repeat (SSR) markers developed from tomato and potato EST sequences were used to characterize S. etuberosum genomic regions associated with resistance to PLRV. The RFLP marker TG443 from tomato linkage group 4 was found to segregate with PLRV resistance. This chromosome region has not previously been associated with PLRV resistance and therefore suggests a unique source of resistance. Synteny groups of molecular markers were constructed using information from published genetic linkage maps of potato, tomato and S. palustre (syn. S. brevidens) × S. etuberosum. Analysis of synteny group transmission over generations confirmed the sequential loss of S. etuberosum chromosomes with each backcross to potato. Marker analyses provided evidence of recombination between the potato and S. etuberosum genomes and/or fragmentation of the S. etuberosum chromosomes.     

马铃薯上PVY、PVS和PLRV的三重RT-PCR检测

根据PVY、PVS和PLRV外壳蛋白(Coat protein,CP)基因序列的保守区域设计各自的引物,利用三重RTPCR技术实现了在同一反应体系中同时扩增出3种病毒产物.PVY、PVS和PLRV扩增产物测序长度均与目的片段的长度相符,分别为781,181,364 bp;各种病毒产物的测序结果同GeneBank中的序列...     

<Emphasis Type="Italic">In vitro</Emphasis> production of PLRV and PSTVd-free plants of potato using electrotherapy

Potato leafroll virus (PLRV) and Potato spindle tuber viroid (PSTVd) were responsible for major economic losses in potato production. Effect of electrotherapy on the regeneration response and production of PLRV and PSTVd-free plants from potato explants is reported. DAS-ELISA and RT-PCR were used for indexing of infected and in vitro-regenerated potato plants. Shoot tips, nodal segments, and tuber sprouts of infected potato plants were exposing to electric current intensities of 5- 25 mA in separate lots for a time period of 5-25 min and cultured on medium. Regeneration response was not significantly changed by exposing explants to 5-10 mA for 5-10 min. Electrotherapy at 15, 20, and 25 mA for 15-25 min had slightly decreased regeneration response of explants and current treatment at 20 and 25 mA for 20 min gave an appreciable number of PLRV and PSTVd-free plants. In a comparative study, electrotherapy of shoot tips and sprouts at 20 mA/20 min proved effective for knocking out virus and viroid without any adverse effecta on regeneration response and production of plantlets. Shoot tips gave 46.72% PLRV and 45.90% PSTVd-free plants with regeneration response of 84.71% and sprouts gave 42.85% PLRV and 43.60% PSTVd-free plants with regeneration response of 92.35% by using 20 mA/20 min treatment. Nodal segments showed less regeneration response (78.47%) and produced a low frequency of PLRV (34.51%) and PSTVd (33.62%)-free plants after this treatment. The protocol is simple and effective for eradicating PLRV and PSTVd from infected potato germplasm.     

Resistance to the Potato Leafroll Virus (PLRV) in Diploid Potatoes

A high level of PLRV resistance has been found in four diploid genotypes originating from resistant ancestors widely utilized in European potato breeding. Plants of these genotypes were difficult to infect not only with aphids, but also with graft inoculation. Their resistance is associated with limited virus spread, but not with intolerance. The level of PLRV resistance in these genotypes appears to be comparable to a high level of resistance detected recently in some wild potato species. Evaluation of virus concentration after graft inoculation with PLRV was found useful in the selection of potato genotypes highly resistant to PLRV.     

双重检测马铃薯X和Y病毒试纸条制备技术研究

为了实现在马铃薯种薯生产田现场同时快速检测马铃薯X（PVX）和Y病毒（PVY）,利用胶体金标记和免疫层析技术研制了PVX-PVY双重检测试纸条。采用柠檬酸三钠还原法制备胶体金溶液,在波长526nm处有最大吸收值,金颗粒直径约25nm。同时标记PVX和PVY兔多克隆抗体,将抗体标记物喷射于玻璃纤维纸上,作为金标垫。将PVX和PVY抗体分别划线于硝酸纤维素膜上作为检测线,将羊抗兔抗体划线于硝酸纤维素膜上作为质控线,制备胶体金试纸条。结果表明,研制的双重试纸条能够在2min之内同时检出PVX和PVY。PVX病汁液检测稀释限度可达10⁶倍（W/V）,PVY可达10⁵倍（W/V）,其中对PVX检测更灵敏。用其他4种常见病毒（PVS、PLRV、PVA和PVM）检测,未出现交叉反应。在田间采集马铃薯叶片,分别利用试纸条与DAS-ELISA检测,结果一致性非常高。由此可知,该试纸条具有操作简便、反应快捷的特点,特别适用于田间及口岸一线对于马铃薯X和Y病毒的同时检测。     

一步法RT-PCR和两步法RT-PCR对马铃薯病毒诊断研究的比较

本研究采用试剂盒和Total试剂两种方法从马铃薯叶片和茎秆中提取总RNA,同时设计了一步法RT-PCR和两步法RT-PCR的反应体系,依据马铃薯X病毒、马铃薯Y病毒、马铃薯S病毒、马铃薯A病毒以及马铃薯卷叶病毒的基因组RNA保守序列设计特异性引物对,并运用两种RT-PCR反应体系对染病的马铃薯进行病毒检测,结果与报道的这五种马铃薯病毒核苷酸序列进行BLAST比对, 表明扩增的五种马铃薯病毒靶带的序列与靶序列完全一致。两种诊断方法对马铃薯病毒核酸进行浓度检测,结果证明,两步法RT-PCR具有较高灵敏性。但这两种方法均可快速、简便、有效地诊断马铃薯病毒。     

Heritility of field resistance to potato leafroU virus in cultivated potato

Potato progenies in a line x tester mating design and the clonal parents were screened for field resistance to potato leafroll virus (PLRV) to determine the heritability of this trait. Twelve advanced potato clones or varieties were crossed as pistillate parents to two pollen testers. The seedling progenies and clonal parents were exposed to aphid-transmitted potato leafroll virus for two growing seasons. Cumulative infection by potato leafroll virus was determined by post-season sero-logical indexing of foliage grown from sprouted tubers after 2 years of exposure. Narrow-sense heritability was estimated from regression of mid-parent on progeny as h = 0.72. This estimate indicates a high level of useabie genetic variance for PLRV resistance in advanced breeding materials. Although variation in resistance to PLRV appears to be a quantitative trait in susceptible and moderately resistant clones, per-formance of the most resistant parents suggests that genes with major effects may be present. These results are similar to the conclusions of other researchers who found one or two genes controlling the pheno-types of extreme resistance, resistance to infection, or suppression of virus titre.     

抗PVY马铃薯品种遗传多样性的RAPD分析

采用RAPD技术分析了国内外37种抗PVY马铃薯资源。提取马铃薯(Solanumtuberosum)新鲜叶片的总DNA,用17种RAPD引物进行随机多态性扩增,利用遗传多样性信息,进行亲源关系的分子鉴定。试验表明:平均每10个碱基引物扩增出6￣21条谱带,共获得164条特异性谱带,平均每个引物扩增获得9.8条多态性谱带,多态性比率平均为76.3%。RAPD分析表明37种抗PVY马铃薯资源之间的遗传距离介于0.06￣0.68之间,平均值为0.35,平均遗传距离介于0.27￣0.50之间,聚类分析将37种抗PVY材料划分为三个类,聚类结果同材料的血缘关系密切,并且表现出一定的地域特性。根据扩增的特异性谱带可进行抗PVY马铃薯资源的分子鉴定,为抗PVY育种亲本选配提供了依据。