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。     

野生马铃薯抗PVY材料的鉴定与筛选

通过人工接种的方法对5类野生马铃薯材料进行了马铃薯Y病毒(PVY)的抗性鉴定和筛选。它们对PVY抗性存在明显的差异,其中Solanum stoloniferum(S.A2)×S.stenotomum(104)和S.stoloniferum(S.A5)×S.stenotomum(105)组合抗性最强,属于抗病群体,S.chacoense×S.stenotomum(103)组合属于中抗群体,S.chacoense(102)和S.demissum(101)组合属于感病群体。并从中筛选出一批抗PVY的育种材料:0级抗性材料108份,1级抗病材料56份,3级抗病材料94份。     

武汉地区马铃薯Y病毒株系的类型

马铃薯Y病毒(PVY)是严重危害我国马铃薯生产的主要病毒之一。本研究通过RT-PCR的方法,对依据症状采集的124份马铃薯叶片样品进行了病毒检测,并对其中PVY阳性样品进行了株系类型分析。结果显示,在93份PVY阳性样品中,PVYN/NTN/N??O类型株系占60.2%,而PVYO普通株系占20.4%,还有18个样品(19.4%)显示受到两种类型PVY的混合侵染。进一步分析表明,在PVYN/NTN/N??O类型株系中,PVYNTN、PVYN??O和PVYN三种株系分别占83.8%、12.2%和4.0%;而PVYO普通株系中,PVYO-FL和PVYO-RB两种变异型各占70.3%和29.7%。本研究结果显示,PVYNTN和PVYO-FL是检测样品中主要的PVY株系,该结果为指导马铃薯PVY的防治提供一定的依据。     

NCM-ELISA检测马铃薯Y病毒(PVY)技术的研究及应用

血清学方法是病毒检测的主要手段。本试验通过对马铃薯Y病毒(PVY)的提纯,免疫家兔制备PVY抗血清,并提取PYV免疫球蛋白IgG作为NCM-ELISA反应为一抗,以市售羊抗兔抗血清为二抗,在硝酸纤维素膜(NCM)上进行NCM-ELISA反应检测PVY,建立PVY NCM-ELISA检测反应体系。试验结果显示,NCM-ELISA具有反应特异性强,灵敏度高的优点,检测植物叶片样品的最高稀释度可达到1:250。通过对田间40份样品的NCM-ELISA和DAS-ELISA检测比较,其检测结果吻合率达到100%。由于NCM-ELISA方法可以将样品点在硝酸纤维素膜上,并且可贮存几个星期或将膜送到其他实验室进行检测,因此具有操作简单,使用方便,检测成本低等优点。     

不同品种（系）马铃薯休眠块茎顶端和茎端芽眼组织马铃薯Y病毒含量分析

马铃薯Y病毒（Potato virus Y,PVY）是马铃薯生产中较常见的病毒之一,马铃薯病毒的检测是生产中保障马铃薯品质和产量的重要基础,收获后批量检测是进行种薯质量评价的主要依据之一。研究针对‘56-2’‘Ivory Russet’‘Clearwater Russet‘’龙薯14号‘’龙薯3号‘’龙薯15号‘’龙育201401-70‘’龙薯7号‘’克新23号’和‘克新28号’马铃薯品种（系）休眠块茎样品,采用TRIzol法提取马铃薯休眠块茎顶端和茎端芽眼组织的总RNA,经qRT-PCR和RT-PCR检测分析,比较块茎的顶端与茎端芽眼组织PVY含量分布的情况。结果表明,上述供试材料的休眠块茎茎端芽眼组织PVY浓度均高于顶端芽眼组织,能够精准的检测到病毒的存在;对‘Ivory Russet’品种的休眠块茎选取100个带病样品通过TRIzol法提取顶端与茎端的总RNA,采用4合1的方法合样后进行PVY RT-PCR检测,所有处理的顶端芽眼组织检测条带亮度低于茎端芽眼组织。可见,马铃薯休眠块茎茎端芽眼组织取样能够更加精准的检测出PVY。研究结果为马铃薯种薯收获后PVY的检测及合理取样提供了...     

利用PLDMV/Twin-Strep侵染性克隆纯化HC-Pro病毒蛋白

番木瓜畸形花叶病毒(Papaya leaf distortion mosaic virus,PLDMV)是一种新的潜在威胁番木瓜种植业的病毒,其辅助成分蛋白酶(helper component-proteinase,HC-Pro)是PLDMV编码参与病毒复制、运动、寄主植物症状表现的多功能蛋白,因此纯化获得具有功能活性...     

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     

马铃薯块茎感染烟草脆裂病毒贮藏期条纹症状的研究

马铃薯块茎感染烟草脆裂病毒贮藏期条纹症状的研究ＫｅｒｓｔｉｎＲｙｄｅｎ等烟草脆裂病毒（ＴＲＶ）是一种具广泛寄主范围和地理分布的土壤带病毒。其传染媒体主要是毛刺线虫属（Ｐａｒａｔｒｉｃｈｏｄｏｒｕｓ）和寄生性毛刺线虫属（Ｔｒｉｃｈｏｄｏｒｕｓ）的几个种...     

PVX病毒载体介导2种马铃薯Y病毒科病毒VPg蛋白表达诱导烟草叶片系统坏死的研究

芜菁花叶病毒(Turnip mosaic virus,TuMV)和槟榔坏死环斑病毒(Areca plam necrotic ringspot virus,ANRSV)是马铃薯Y病毒科(Potyviridae)中分别可诱导十字花科作物和槟榔产生坏死病症的2种不同属病毒.VPg(Viral protein genome l...     

Evidence of Recombinant Isolates of Potato Virus Y (PVY) in Argentina

In order to make a first approach in the identification of the genetic diversity of Potato virus Y (PVY) in Argentina, 46 PVY isolates from different potato growing regions of Argentina were characterized both, biological and serologically. Five of them (ST11, RCA5a, RCA6, RCA14b and SSF6) were selected for further genomic analyses. Four genomic fragments containing hot spot regions of recombination (HSR) reported previously were sequenced in each isolate and compared to PVY^N (CS434575.1) and PVY^O (U09509.1) reference genomes looking for genomic recombinations. Isolates with one, two or three recombination points were identified among these, including the two strains considered typical PVY^N (RCA5b) and PVY^O (SSF18) used as controls. This is the first report of the presence of recombined PVY in Argentina using a combination of biological, serological and molecular tools that shed light on the genetic diversity of PVY viruses in this country.     

Cryotherapy of Potato Shoot Tips for Efficient Elimination of Potato Leafroll Virus (PLRV) and Potato Virus Y (PVY)

Viral diseases constitute a major constraint to high yield and high quality production of potato. Potato leafroll virus (PLRV) and Potato virus Y (PVY) are among the most damaging potato viruses and are prevalent in most potato growing areas. In the present study, attempts were made to eliminate PLRV and PVY by three cryogenic protocols, i.e., encapsulation-dehydration, encapsulation-vitrification and droplet. Results showed that both PLRV and PVY could be efficiently eliminated by cryogenic treatments with 83–86% and 91–95% of frequencies of virus-free plantlets obtained for the former and latter, respectively. Frequencies of virus-free plantlets produced by cryogenic treatments were higher than those by meristem culture (56% for PLRV and 62% for PVY) and thermotherapy (50% for PLRV and 65% for PVY), and similar to those by thermotherapy followed by meristem culture (90% for PLRV and 93% for PVY). Survival (75–85%) and regrowth (83–89%) from cryo-treated shoot tips were higher than those from meristem culture (50–55%) and thermotherapy followed by meristem culture (40–50%), but similar to those from thermotherapy (80–87%). The morphology of the plantlets regenerated from cryo-treated shoot tips was similar to that of non-treated plantlets. Thus, cryotherapy would provide an alternative method for efficient elimination of potato viruses, and can be simultaneously used for long-term storage of potato germplasm and for production of virus-free plants.     

Identification of triplex (YYYy) Potato Virus Y (PVY) immune progenitors derived fromSolanum tuberosum ssp.andigena

As part of the International Potato Center’s (CIP) virus resistance breeding strategy, a group of 182 selected clones from intercrosses among duplex Potato Virus Y (PVY) immune progenitors derived fromSolanum tuberosum ssp.andigena (i.e., YYyy × YYyy) was sampled. These clones were test-crossed to the PVY susceptible tester 377964.5 (yyyy) to search for triplex (YYYy) and quadruplex (YYYY) PVY immune potato progenitors. Seedlings of each test-crossed progeny were screened for PVY immunity. Genetic analysis of observed ratios for immunity-susceptibility showed that two of the 182 clones segregated as triplex under the assumption of a random chromatid segregation model. The double reduction coefficient, α, and its standard error were estimated from the experimental data, and had values of 0.1566 and 0.0186 respectively. These results indicated that crossovers take place during meiosis between the locus and the centromere, permitting sister chromatids to migrate to the same pole producing a random chromatid segregation. In the population evaluated, no clone was identified as a quadruplex. The two triplex PVY immune genotypes will produce about 96% of progenies with PVY immunity when mated to PVY susceptible advanced clones or varieties. These progenitors have the potential to provide a durable PVY genetic control and diminish the present impact of this virus on the potato crop. Also, immunity to PVY will significantly simplify the seed production process.     

Foliar Symptoms Caused by Potato mop-top virus on Potato Plants During Vegetative Propagation in Scotland and Their Association With Tuber Yield, Spraing and Tuber Infection

The effect of foliar symptomatic infection by Potato mop-top virus (PMTV) on yield of tubers, spraing and infection in daughter tubers, and foliar symptom development and tuber infection in the following generations of propagation was investigated in commercial seed potato crops in Scotland. Six crops covering cvs Atlantic, Hermes, Nicola and Cara were studied between 2000 and 2006 by labelling paired replicates of plants with foliar symptoms and plants with no symptoms. Tubers from plants with no symptoms rarely produced plants with foliar symptoms in the following generation. Plants with no symptoms produced more infected tubers if they had been derived from plants with foliar symptoms the previous year than from plants with no symptoms. The proportion of daughter plants with foliar symptoms produced by tubers from plants with foliar symptoms in year 1 ranged from 19–41% and seemed to be associated with the severity of foliar symptoms. The detection of PMTV by ELISA in samples of leaves from plants with foliar symptoms ranged from 13% for cv. Cara to 59% for a crop of cv. Atlantic in 2004. The amounts of spraing were generally low but tended to be greater for tubers from plants with foliar symptoms than those from plants with no symptoms. These results indicate that roguing plants with foliar symptoms in seed potato crops could achieve an improvement in crop health but might be impractical when diseased plants are too prevalent.     

转小麦 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥植株的主要抗旱性研究

核氧还蛋白（nucleoredoxin,NRX）可通过还原目标蛋白的二硫键来调控其生物活性,在植物的生长发育和抗逆境胁迫中发挥着重要作用。蛋白质二硫键异构酶（protein disulfide isomerase,PDI）、h型硫氧还蛋白（h-type thioredoxin,TRXh）和蛋白磷酸酶2A催化亚基（protein phosphatase 2A catalytic subunit,PP2Ac）是小麦核氧还蛋白TaNRX1的互作蛋白。为了明确TaNRX1互作蛋白的抗旱性功能,本研究在拟南芥中过表达了小麦 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因,对野生型和转基因拟南芥的表型和抗旱相关生理指标进行了鉴定。结果表明,干旱胁迫处理后,转 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥的根长、存活率、脯氨酸含量均大于野生型,离体叶片失水率、丙二醛（MAD）含量均小于野生型。二氨基联苯胺（diaminobenzidine,DAB）对H₂O₂组织定位染色结果表明,干旱胁迫处理后,转 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D基因拟南芥的H₂O₂含量均低于野生型。上述结果说明,TaNRX1的互作蛋白基因 TaPDI-A、 TaTRXh-A和 TaPP2Ac-D增强了拟南芥对于干旱胁迫的抵抗能力。本研究可为小麦抗旱育种提供候选基因和理论基础。     

转基因抗虫玉米对亚洲玉米螟的抗性评价

采用田间人工接虫鉴定方法,根据玉米抗螟性田间鉴定评价标准,研究转基因玉米Bt799和NC6304YGRR对亚洲玉米螟的抗性.心叶期接虫试验结果表明,以食叶级别作为评价参数,2种转基因玉米对亚洲玉米螟具有良好的抗性,食叶级别均为1级;2种非转基因玉米食叶级别均大于7级.吐丝期接虫试验结果表明,以单株虫孔数、单株活虫数、单株隧道个数及单株隧道长度作为评价参数,2种转基因玉米对亚洲玉米螟抗性均显著高于各自对应的非转基因玉米.转基因抗虫玉米Bt799和NC6304YGRR田间抗虫效果良好,均能保护玉米在整个生育期内不受亚洲玉米螟危害.     

马铃薯植株感染Y病毒后生理指标变化与抗病性的关系

本研究分别以对PVY感病、过敏和极端抗性的品种脱毒试管苗为材料,比较了接种PVY后不同时间内超氧化物歧化酶(SOD)和游离脯氨酸含量2个指标的变化。从整体上看,未接种PVY条件下,感病品种(Superior)植株的SOD活性较极端抗性品种(IVP35)和过敏型品种(Desiree)低。接种后,IVP35的SOD活性下降;Desiree在接种前期0.5～1 d SOD的活性下降,2～7 d后SOD活性上升,再后7～14 d又下降;Superior接种PVY后,SOD活性有上升趋势,且在接种2～3 d变化率出现最大值。另外,IVP35和Desiree在接种PVY后SOD变化率小,变化范围分别为-13.4%～4.6%和-17.8%～8.7%;Superior接种PVY后,SOD活性变幅较大,范围为5.1%～79.2%。被测的3个品种无论接种与否,极端抗性品种和过敏型品种其植株内部游离脯氨酸含量均明显高于感病品种。正常情况下,IVP35和Desiree的游离脯氨酸含量为41.39～48.77μg.g-1干重和33.42～39.11μg.g-1干重,而Superior仅为8.91～10.72μg.g-1干重。接种PVY后,Superior的游离脯氨酸含量明显上升,达到11.23～21.54μg.g-1干重;虽然IVP35和Desiree的游离脯氨酸含量也呈上升趋势,但上升幅度小,含量分别为48.92～72.13μg.g-1干重和42.46～71.34μg.g-1干重。     

乙烯利提高香蕉幼苗抗寒性的生理效应

用200 mg/L乙烯利喷洒香蕉幼苗叶片1 d,再于8℃低温胁迫1 d后,研究乙烯利对香蕉幼苗抗寒性的影响.结果表明:乙烯利预处理提高了香蕉叶片的SOD、POD、CAT活性和香蕉叶片的游离脯氨酸和可溶性蛋白含量;降低了丙二醛含量.表明乙烯利可通过提高抗氧化酶活性和减少膜损伤来增强香蕉幼苗抗寒性.     

Integrated Management of Whitefly, Bemisia tabaci (Gennadius) and Potato Apical Leaf Curl Virus in India

This paper reviews the importance of Potato apical leaf curl virus (PALCV) in India, the biology of its major vector Bemisia tabaci and possible control measures of PALCV and B. tabaci. PALCV has appeared as a new problem in potato growing areas of northwestern plains of India. The first report of PALCV was made from Hissar around 2000. PALCV is a whitefly (B. tabaci) transmitted viral disease and the symptoms are manifested primarily in the form of upward or downward curling of leaves, with 40–75% infection being recorded in cultivars grown in the Indo-Gangetic Plains of India. B. tabaci is a polyphagous pest that feeds on numerous fibre (particularly cotton), food, vegetable and ornamental plants. B. tabaci produces many generations in a year and reaches high population densities. To check the disease incidence, whitefly suppression using imidacloprid either as seed treatment or as foliar application early in the season is recommended. To minimize within-field spread of viruses, removal of virus-infected plants is suggested. Application of mineral and vegetable oils has been found to inhibit virus transmission and possibly can help to avoid difficulties with insecticide resistance in whiteflies.