马铃薯Y病毒外壳蛋白基因的克隆及全序列测定

自田间采集的马铃薯病叶中提取马铃薯Y病毒 (PVY)总RNA ,人工合成引物P1、P2 ,通过RT PCR扩增合成PVY cp的cDNA ,并将其克隆到 pGEM TEasy载体上。经限制酶谱分析后进行全序列测定 ,结果表明 :该基因由 80 1个核苷酸组成 ,编码 2 67个氨基酸 ,与文献报道的 3个PVY cp基因相比同源性均在 90 %以上 ,且其编码的氨基酸同源性均在 94 4 %以上。说明PVY cp基因具有较高的保守性     

香蕉MaERF-1基因的克隆、序列分析及表达载体构建

从香蕉中克隆了1个乙烯响应因子(ERF)Ma ERF-1。序列分析表明,该基因存在1个完整的开放阅读框(ORF)729 bp,编码243个氨基酸。多序列比对和进化树分析表明,Ma ERF-1所编码的蛋白与其他植物中ERF编码的蛋白具有较高的一致性。其中与马来西亚野生香蕉同源性最高达98%,与油棕、菠萝、海枣、葡萄、荷花、烟草的Ma ERF编码的氨基酸序列的同源性分别为65%、60%、59%、54%、53%、51%。Ma ERF-1编码的蛋白质分子量为26 139.03 u,理论等电点p I为7.81,其亲水性氨基酸均匀分布在整个肽链中,多于疏水性氨基酸。通过PCR和酶切反应鉴定成功构建该基因的表达载体。     

苏云金芽孢杆菌chi36基因的克隆、表达和酶活性分析

通过PCR方法从苏云金芽孢杆菌010菌株克隆出几丁质酶基因chi36,该基因开放阅读框(ORF)的长度为1 083bp,编码360个氨基酸。氨基酸序列分析表明:Bt 010的Chi36与Bt15A3、Bc28-9、Bc6E1的Chi36相似性分别为99%、99%和96%,其N-端具有27个氨基酸的信号肽序列。该蛋白归类为18家族几丁质酶,属于一种胞外酶。将chi36基因插入到PGEX-KG原核表达载体的表达框中,构建成原核表达载体并转化到大肠杆菌E.coli BL21(DE3)进行蛋白表达,酶活性测定结果表明,表达产物对几丁质有降解作用,在pH值为5.0、温度为55℃时酶活性最高。     

大豆rbcL基因克隆、序列分析及原核表达

利用叶绿体基因组保守性的特征,根据菜豆、豌豆、烟草的rbcL基因序列设计引物,从大豆叶绿体DNA中克隆rbcL基因,全长序列为1488bp,包括1449bp的开放阅读框,编码482个氨基酸。相似性比较显示,此序列与其它10个物种rbcL基因核苷酸的同源性为85.37%～95.31%,氨基酸的同源性为90.87%～96.47%。将该基因与表达载体pET-30a（+）连接,转化大肠杆菌Rosseta感受态细胞,PCR和酶切鉴定筛选阳性克隆,阳性菌液IPTG诱导后经10%SDS-PAGE分析,结果显示：诱导表达出分子量约为60kD的特异融合蛋白。     

HARDY基因的克隆、原核表达及其植物表达载体的构建

为了给转HARDY(HRD)基因小麦研究奠定基础,利用PCR技术从拟南芥中克隆HRD基因,进行生物信息学分析,预测其编码的蛋白质结构与功能,构建原核表达载体pET28a(+)-HRD,转化大肠杆菌BL21(DE3),IPTG诱导蛋白的表达,同时构建pBin-HRD植物表达载体。测序分析结果表明,克隆的HRD与NCBI发布的拟南芥核苷酸序列的同源性为99%,其开放阅读框长555bp,可编码184个氨基酸,具有许多重要功能位点;成功构建了原核表达载体pET28a(+)-HRD和植物表达载体pBin-HRD,诱导表达出大小约为23.3kD的蛋白,与理论值相近。     

花生条纹病毒（红安分离物）cp基因的克隆和序列分析

从湖北省红安县花生种传苗中得到花生条纹病毒（Peanut stripe virus, PStV）分离物（PStV-Hongan），提取感病叶片的总RNA，RT-PCR扩增了其外壳蛋白基因（cp），克隆至pGEM-Teasy载体，转化大肠杆菌并鉴定。阳性质粒序列测定结果表明，该cp含有861个核苷酸，编码分子量为32.4kDa的蛋白。该株系cp与PStV其它株系核苷酸序列同源性为94.6%~98.8%，氨基酸序列同源性为96.2%~99.3%。株系间cp核苷酸序列系统进化树结果表明，Hongan株系与中国其它株系亲缘关系较近，而与东南亚其它株系关系较远。     

大豆主要过敏原Gly m Bd 30K基因的克隆及其原核表达载体的构建

利用RT-PCR克隆大豆主要过敏原Gly m Bd 30K的全长基因,根据序列设计带有酶切位点的特异性引物,扩增大豆GlY m Bd 30K的完整开放阅读框,与pET一28a载体连接,构建原核表达载体.结果表明:克隆了大豆主要过敏原Gly m Bd 30K基因,且构建了其原核表达载体.该基因含有长度为1 140 bp的开放阅读框,编码379个氨基酸.该蛋白质的相对分子质量为42 758,等电点为5.08.序列同源性分析发现其与数据库中已知的Gly m Bd30K基因同源性很高,因此认为其是大豆的过敏原基因,在GenBank数据库中的登录号为EU883600.克隆的大豆主要过敏原Gly m Bd 30K基因及构建的原核表达载体,为大豆主要过敏原Gly m Bd 30K的重组表达和免疫活性鉴定等奠定基础.     

芝麻黄花叶病病原的分子鉴定

从芝麻黄花叶病株上分离得到花生条纹病毒2个分离物（Peanut stripe virus sesame isolate, PStV-se1和PStV-se2）,提取感病叶片的总RNA,RT-PCR扩增外壳蛋白基因（cp）片段。序列测定结果显示,cp基因含有861个核苷酸,编码分子量为32.4kDa的蛋白。2个株系cp基因与PStV其它株系核苷酸序列同源性为94.4%～99.9%,氨基酸序列同源性为93.7%～100%。株系间CP氨基酸序列系统进化树结果表明,芝麻分离物与中国其它寄主来源的PStV株系处于同一进化分支。     

大豆胞囊线虫热激蛋白基因Hsp70的克隆与原核表达

采用RT-PCR技术结合基因组步移技术,从大豆胞囊线虫(Heterodera glycines)中克隆了热激蛋白70基因(Hsp70)的全长cDNA序列(Hg-Hsp-70),全长1 953 bp,GenBank登录号为FJ816100.1。碱基序列分析结果表明,该序列含有9个外显子与8个内含子,与其它线虫具有较高的同源性。氨基酸序列分析表明,该基因编码650个氨基酸,相对分子量为70.7 kD,具有2个Hsp70基因家族的签名序列,与其它线虫的该基因氨基酸序列具有很高的同源性。构建了原核表达载体Hsp70pEASY-E1,采用IPTG诱导蛋白表达,SDS-PAGE电泳结果表明,当IPTG终浓度为0.2～1.2 mmol.L-1时均能显著诱导表达;以终浓度0.8 mmol.L-1的IPTG诱导5 h蛋白表达量达到最大值。     

茶树谷胱甘肽过氧化物酶编码基因CsGPX1功能分析

为明确茶树[Camellia sinensis(L.)O.Kuntze.]谷胱甘肽过氧化物酶(Glutathione peroxidases,GPX)编码基因CsGPX1的功能,本文利用茶树转录组数据克隆获得CsGPX1的编码区全长序列,进行序列比对与分析,在此基础上,将CsGPX1在烟草中进行过表达获得转CsGPX1基因烟草,并比较野生型烟草与转基因烟草耐旱性的差异,验证CsGPX1功能。序列分析表明,CsGPX1编码序列(CDS)长723 bp,编码240个氨基酸。BLAST比对发现,该基因与桃树(Prunus persica)的GPX基因具有高度同源性(>85%)。CsGPX1编码的氨基酸序列具有GPX蛋白保守特征序列和区别于其他家族成员的特有的结构域——磷脂氢谷胱甘肽过氧化物酶(PHGPX)。干旱胁迫处理结果显示,过表达CsGPX1烟草株系抗旱性强于野生型。GPX酶活性测定结果表明,转基因植株的GPX酶活性高于野生型植株。以上研究结果表明,过表达CsGPX1可提高转基因烟草的耐旱能力,说明CsGPX1可能与茶树的耐旱性能相关。本研究为提高茶树的耐旱性能研究提供了新的理论途径,并对降低茶园管理的成本具有一定的积极意义。     

Potato virus X isolates from potato collected in eastern Canada with different symptoms in tobacco differ in their coat proteins

Three Canadian isolates of potato virus X (PVX) that produced distinctly different symptoms in tobacco (Nicotiana tabacum cv. Samsun) were analyzed for differences in their coat proteins using SDS-PAGE, denaturing isoelectric focusing, peptide analysis and nucleotide sequencing. The amino acid sequence of the coat protein in a mild isolate differed from that of isolates that produced severe or intermediate symptoms at two locations. However, the amino acid sequences of the Canadian isolates that produced severe or intermediate symptoms were identical and were the same as the coat proteins of PVX isolates from Argentina, China, The Netherlands, and United Kingdom. These results suggest that regions of the PVX genome in addition to the coat protein gene may be involved in viral-host interactions.     

马铃薯病毒外壳蛋白融合基因转化马铃薯及其抗病性分析

将多种病毒的有效核酸片断拼接成融合基因转入马铃薯可获得多抗马铃薯材料。针对马铃薯生产中分布广泛、危害严重并经常混合感染的马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯卷叶病毒(PLRV)和马铃薯S病毒(PVS),开展了利用基因工程方法获得兼抗4种马铃薯病毒转基因马铃薯材料的研究。试验在前期获得含4种马铃薯病毒外壳蛋白基因片段的质粒pART27-XSYV-rh的基础上,通过根癌农杆菌(Agrobacterium tumefaciens)介导转化马铃薯(Solanum tuberosum)品种‘陇薯3号’,PCR扩增和PCR-Southern杂交证明,4价融合基因已整合到马铃薯基因组中。qRT-PCR分析表明,该融合基因在转基因植株中能正常表达。3株转基因植株的抗病性鉴定结果表明,2株对4种病毒同时具有抗性;1株对PLRV侵染表现阳性,对另外3种病毒同时具有抗性。     

甜椒脉斑驳病毒（PVMV）在海南的发现与检测

2014年在海南辣椒病毒病调查过程中发现了一种疑似病毒感染的辣椒样品,主要表现为叶片黄化、绿斑驳、叶脆易折断,且在田间发生较多。利用马铃薯Y病毒属的简并引物对其叶片总RNA进行RT-PCR检测,并将约1 700 bp目的片段克隆到pMD18-T载体上进行测序和BLAST分析。结果表明：该条带序列（包含部分NIb和部分cp基因）与已收录的甜椒脉斑驳病毒（Pepper veinal mottle virus,PVMV）（GenBank登录号：FM202327）序列相似性最高,达99％。设计cp基因的特异引物,对上述样品进行cp基因扩增并构建以cp基因序列为基础的系统进化树,发现海南辣椒上的PVMV与台湾的PVMV分离物ns1株同源性最高。田间检测结果表明：海南黄灯笼辣椒上PVMV的检出率高达74.07％,说明PVMV可能成为海南辣椒生产上的潜在威胁。     

Extreme resistance to infection by potato virus X in genotypes of wild tuber-bearingSolanum species

Summary Clones derived from thirty-one different accessions (nineteen of Argentine origin) belonging to eightSolanum species were screened for resistance to infection by potato virus X strain cp (PVX cp) by mechanical inoculation of plantlets that had been micropropagated in vitro. Estimates of PVX multiplication obtained by enzyme linked immunosorbent assay and slot blot nucleic acid hybridization allowed the identification of resistant clones derived from five accessions belonging toS. commersonii S. oplocense, S. sparsipilum andS. tuberosum andigena. Resistant genotypes supported PVX concentrations 5 to 15 times smaller than did the susceptible control cultivar Spunta. Graft inoculation test confirmed the presence of extreme resistance similar to that conferred by the ‘immunity’ gene X¹ (also called RX_act).     

应用RT-PCR技术检测马铃薯A病毒

参考GenBank中马铃薯A病毒(potato virus A,PVA)的保守序列,利用Primer6.0引物设计软件设计并合成了一对特异性引物PVAF、PVAR,以此引物利用RT-PCR方法对PVA保守序列基因进行了特异性扩增。结果表明:引物PVAF、PVAR能从已知的感染PVA病毒的植株中扩增出834bp的cDNA特异性片段;该RT-PCR的检测灵敏度为1pg的病毒核酸,特异性强,重复性好,可用于PVA病毒的快速检测。     

Relationships of defined PVX infection levels to verticillium wilt,yield, and quality of the Russet Burbank potato

Field studies were conducted over a three-year period to investigate effects of potato virus X (PVX) on the Russet Burbank potato cultivar. Seed (PVX-free and PVX-infected) used for this three-year study originated from the same PVX-free source and was grown and stored under similar conditions. PVX-free seed was found to increase yield over PVX-infected plots by 9 to 32%. With these yield benefits, effects of PVX on either the incidence of verticillium wilt or plant nutrition were not significant. There was a trend (P = 0.10) for a reduction of mean tuber weight when the PVX infection level exceeded 19%. Because of increases of undersized potatoes, the yields of U.S. #1 potatoes were reduced as levels of PVX infection were increased. With a PVX infection level of 36%, the yield of U.S. #1 tubers was reduced by 21% when compared with plots having 0% PVX. At an 88% infection level, the yield of U.S. #l’s was reduced still further (36% lower than plots with 0% PVX). Results demonstrate the importance of the level of PVX infection to potato production.     

Volunteer potatoes as a source of potato leafroll virus and potato virus X

Volunteer potatoes were investigated as infection sources for potato leafroll virus (PLRV) and potato virus X (PVX) in a high elevation seed potato growing area of eastern Idaho. Population densities ofMyzus persicae were assessed. Percentage of PLRV and PVX infection of the volunteers and seed potato crops was determined, as well as density of volunteers and certain parameters of volunteer growth and reproduction. Volunteers apparently harbored no more PLRV than the potato crop from which they originated. But they were found to be an important reservoir of PVX with the infection increasing as much as 12.43% in one year. No aphids capable of transmitting PLRV were found although one species that can transmit potato virus Y was recorded. The mean density of volunteers varied from 0 to 84,880 stems/ha. The number of tubers remaining in the field after harvest and winter weather conditions appeared to be the only factors affecting volunteer density. Volunteer plants arising from seed pieces at an average depth of 6.1 cm were found to set an average of 2.1 new tubers per plant at an average depth of 4.0 cm. These results suggest that volunteer potatoes are a significant source of PVX infection in subsequent seed potato crops.     

河北省藁城花生病毒病的RT-PCR检测及外壳蛋白基因序列分析

采用RT-PCR技术对采自河北省农林科学研究院粮油作物研究所藁城堤上试验基地的12份花生叶片样品进行花生病毒病的检测,结果表明:从表现花叶和矮化症状的10份样品中检测到花生条纹病毒(peanut stripe virus,PStV),检出率高达90%,未检测到花生矮化病毒和黄瓜花叶病毒;对其中2个PStV分离物cp基因序列分析的结果表明,该cp全长864 bp。这两个分离物与已报道的PStV其他株系的cp核苷酸序列相似性分别为94.4%~99.2%和94.7%~99.7%,氨基酸相似性分别为95.5%~99.7%和95.8%~100.0%。PStV cp核苷酸序列构建的系统进化树中,这两个分离物与中国大陆其它分离物及美国分离物亲缘关系较近,而与中国台湾、泰国和越南分离物亲缘关系较远。