我国马铃薯抗病毒基因工程研究进展

10年来我国已合成克隆了PVX、PVY、PLRV的外壳蛋白基因 ,复制酶基因 ,蛋白酶基因 ,基因调控序列 ,核酶cDNA及其他各种基因并进行了序列分析。建立了完善的致瘤农杆菌介导的马铃薯转化技术。通过外壳蛋白基因介导 ,复制酶基因介导 ,表达基因调控序列和核酶等基因工程途径 ,获得了一批不同程度上抗PVX、PVY、PVX和PVY ,PVY和PLRV ,PLRY及高抗PSTVd的转基因马铃薯栽培种。这些转基因马铃薯多数已进入田间试验。不久一批抗病毒的优质高产的马铃薯栽培种 ,经过进一步发育 ,必将在生产中推广应用。抗病毒转基因马铃薯培育成功 ,为我国马铃薯病毒病害防治开辟了一条崭新的途径 ,也必将有力地促进我国马铃薯生产的发展     

马铃薯抗晚疫病和病毒病转基因研究现状与展望

从Harpin蛋白基因表达诱导抗性、Osmotin蛋白诱导抗性、病原诱导葡萄糖氧化酶(GO)基因表达生成H2O2获得抗性途径对抗马铃薯晚疫病抗性转基因研究现状进行了综述;从病毒外壳蛋白(CP)基因介导抗病性、复制酶基因介导抗病性、用核酶切割介导抗病性、病毒基因调控序列介导抗性途径对马铃薯病毒病抗性转基因研究现状进行了综述。     

通过农杆菌介导把Harpin_(Ea)基因导入马铃薯的初步研究

应用农杆菌介导的叶盘法进行马铃薯遗传转化研究。通过D3、D4培养基筛选法 ,把HarpinEa蛋白基因导入马铃薯四倍体栽培种大西洋 (Atlantic) ,获得 10 7个转化株系。并经过卡那霉素的抗性鉴定和PCR分子检测 ,有 5 9株PCR检测呈阳性 ,占所有转化植株的 5 5 14%。结果表明HarpinEa基因已整合到马铃薯的基因组中。转化植株的抗病性鉴定正在进行中。     

抑制马铃薯无性系植株病毒的积累减少马铃薯卷叶病毒病的传播

在培育抗病毒品种的工作中,抗侵染是最常选择的特性。事实上,马铃薯抗卷叶病毒(PLRV)病的育种,这一仔细考虑的选择可能是抗性的唯一类型(Davison,1973),而其他类型大多被忽视。近年的研究表明,一些马铃薯无性系和栽培品种对PLRV的抗性至少有8个组成部分(Barker等,1985,1986;     

湖南省马铃薯主产区马铃薯病毒种类及流行分析

马铃薯是世界第四大粮食作物,其病毒病危害严重。2010年对湖南马铃薯主产区采集的66个病毒标样进行了RT-PCR检测,结果表明,检测出的马铃薯病毒有马铃薯Y病毒(PVY)、马铃薯卷叶病毒(PLRV)、马铃薯X病毒(PVX)、马铃薯S病毒(PVS)、马铃薯A病毒(PVA)和马铃薯纺锤块茎类病毒(PSTVd)。其中PVS的检出率最高,为54.5%,其次是PVX,检出率为45.5%,PVY的检出率为39.4%,PSTVd和PVA的检出率均为21.2%,PLRV的检出率为18.2%。2~4种病毒的复合侵染现象较为普遍。PVY中重组型PVY占85.7%。     

几丁质酶基因转入马铃薯品种东农303的研究

以马铃薯极早熟品种东农 30 3的试管薯为外植体 ,通过农杆菌介导法成功地将几丁质酶(PBch)基因导入马铃薯中。薯块培养基MS +1mg LNAA +2mg LZT ,共培养 3d后 ,转到含卡那霉素 5 0mg L ,头孢噻肟钠 2 0 0mg L的相同的培养基 ,待抗性芽长到 1～ 2cm时 ,转入含卡那霉素75mg L的生根培养基进行生根筛选。对获得的 4株转基因植株进行PCR检测及PCR Southern杂交检测 ,有 3株呈阳性 ,转化率为 3 2 %。     

北方晚熟马铃薯病毒抗性鉴定及分子标记检测

病毒病等病害是中国马铃薯产业的重要限制因子。为了马铃薯病毒病等病害的防治,研究通过病毒接种鉴定了15个北方晚熟马铃薯品种(系)对马铃薯Y病毒(Potato virus Y,PVY)、马铃薯X病毒(Potato virus X,PVX)、马铃薯S病毒(Potato virus S,PVS)和马铃薯A病毒(Potato virus A,PVA)四种主要马铃薯病毒的抗性,同时检测了这些品种部分病害的相关分子标记。有10份试验材料至少对一种病毒表现出抗性,其中‘京张薯4号’‘冀张薯8号’‘冀张薯12号’‘2013-19-14’和‘2013-89-2’同时具有3种病毒抗性,表明京张薯和冀张薯系列马铃薯具有较好病毒综合抗性。标记检测结果显示,‘冀张薯8号’‘2013-38-32’和‘2013-89-2’的PVY抗性可能来源分别为Ry_adg、Ry_chc、Ry_sto,而‘冀张薯12号’的PVX抗性可能来源于Rx1。根据标记与抗性表型的符合程度推测,上述马铃薯品种(系)的PVY抗性可能主要来源Ry_sto,PVX抗性可...     

马铃薯早熟品种与晚熟品种对马铃薯Y病毒（PVY）和马铃薯卷叶病毒（PLRV）的生理反应

马铃薯病毒病是导致马铃薯退化的主要原因之一,寻找能够有效降低病毒病对马铃薯生产的影响一直是马铃薯研究和生产者的追求。通过研究马铃薯中早熟品种‘LK99’和晚熟品种‘陇薯3号’对马铃薯Y病毒(PVY)和马铃薯卷叶病毒(PLRV)的生理反应,进一步明确和比较了中早熟品种‘LK99’和晚熟品种‘陇薯3号’在PVY或PLRV胁迫下的一些生理变化。以未感病和分别感染了PVY、PLRV的马铃薯中早熟品种‘LK99’和晚熟品种‘陇薯3号’为研究对象,在马铃薯苗期、块茎形成期、块茎膨大期、淀粉积累期采用紫外分光光度计测定了所取叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性、丙二醛(MDA)含量、叶绿素含量,并进行了分析比较;同时,在块茎膨大期测定株高、茎粗、地上鲜重,成熟后测产。试验结果表明,病毒侵染马铃薯植株后,植株生长受抑制,块茎产量降低、有关保护酶(SOD、POD、CAT)活性增强、丙二醛含量升高、总叶绿素含量减少。植株感染了PVY或PLRV后块茎产量、有关保护酶(SOD、POD、CAT)活性、丙二醛含量、总叶绿素含量的相对变化量表明‘陇薯3号’的受损程度较‘LK99’严重。以块茎产量作为评价标准,则PVY对马铃薯的危害程度较PLRV更为严重。马铃薯植株对PLRV的应激性反应比对PVY强烈,表现为酶活相对增强幅度大、丙二醛含量相对增量高、总叶绿素含量相对减量高。     

利用dsRNA介导的抗病性获得抗马铃薯Y病毒（PVY）的转基因烟草

为了获得抗马铃薯Y病毒(PVY)转基因烟草,分别扩增PVY HC-Pro基因3’端正、反向片段,构建反向重复植物表达载体.利用农杆菌介导法转化普通烟草(Nicotiana tabacum)云烟85,经抗性筛选及抗病性鉴定,获得T0代转基因抗病烟草株系.繁殖T0代抗病株系,抗病性鉴定发现,部分抗病株系的T1代烟株发生了一定比例的抗感分离.Real-time PCR检测发生抗感分离的T1代烟株,发现抗病烟株中PVY HC-Pro基因RNA积累水平显著低于感病烟株,说明抗病烟株中HC-Pro基因发生了RNA沉默.利用dsRNA技术沉默HC-Pro基因,获得了烟草品种云烟85的T1代转基因抗PVY株系.     

利用体细胞杂交获取马铃薯软腐病的抗性

对马铃薯四倍体栽培种和二倍体野生种Solanumbrevidens的体细胞杂种通过叶片离体培养获得的四倍体植株,以及用四倍体栽培种进行回交获得的五倍体植株的块茎对软腐病的抗性进行了测定。结果表明,由体细胞杂种通过叶片组织离体培养再生植株中,有一个株系SC107对软腐病菌Erwiniacarotovora具有较强的抗性。在用不抗软腐病的马铃薯栽培种对体细胞杂种进行回交获得的杂种后代中,大部分株系对软腐病菌具有高水平的抗性,从而说明Solanumbrevidens对软腐病的抗性基因已转移到马铃薯栽培种。     

A Reexamination of the Effectiveness of Ribavirin on Eradication of Viruses in Potato Plantlets in vitro Using ELISA and Quantitative RT-PCR

The potato plantlets singly infected by PVA, PLRV, PVS, PVX and PVY and mix-infected by PVM, PVS and PVY were cultured on MS medium with different concentration of ribavirin. The effects of ribavirin on growth of the plantlets and efficiency of virus elimination were investigated. Results showed that the plant height and fresh weight obviously decreased with increase of ribavirin concentration from 0 mg/L to 150 mg/L, and most of the plantlets could not survive when the concentration reached 200 mg/L. According to the ELISA tests, ribavirin was more efficient for eradicating PVA, PVM, PVS and PVX than PVY and PLRV, and healthy plantlets could be obtained with high frequency (up to 100 %) by culturing with 75?~?150 mg/L ribavirin after 2?~?3 subcultures. Whereas, only 33?~?66 % PVY and PLRV infected plantlets were found to be virus-free after 3 subcultures with 75?~?150 mg/L ribavirin. The results of quantitative RT-PCR (qPCR) indicated that ribavirin could obviously reduce virus content in the plantlets. Except PLRV was detected positive after 3 subcultures with ribavirin, the healthy seedlings were obtained from infected stocks at the first or end of propagation and no viruses could be detected at the post-eradication stage. No apparent difference of genetic variation resulted from ribavirin treatment was found by SSR analysis between the control and the treated plantlets. All of these results above proved that ribavirin treatment in vitro was an effective method to eliminate viruses in the propagation of potato.     

Occurrence and Distribution of Potato Pests and Diseases in Kenya

Potato plays an important role in food security in Kenya but yields are low (<10 t/ha), and this is partly attributed to the lack of healthy planting material. This study is the first wide-scale survey to determine the occurrence and distribution of common potato pests and diseases in Kenyan seed (certified and quality declared) and ware crops. Potato crops growing on 101 farms in 21 districts were examined. Approximately 36% of plants in farmers’ fields sampled both during the long rains (main potato-growing season) and short rains seasons displayed virus-like disease symptoms. Six viruses (potato leafroll virus (PLRV), Potato virus A (PVA), potato virus M (PVM), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY)) were detected using double antibody sandwich enzyme-linked immunosorbent assay in potato samples. Sequencing of polymerase chain reaction products from PVY-infected plants revealed the presence of recombinant strains of PVY (NTN and Wilga). Four aphid species, Macrosiphum euphorbiae, Aphis gossypii, Myzus persicae, and Aphis fabae, colonized potato in all districts, occurring in greater numbers west of the Great Rift Valley than to the east. There was a positive correlation between virus incidence and aphid numbers in the long rains (main) potato-growing season. PLRV, PVM, PVS, PVX, and PVY were detected in solanaceous weeds. Ralstonia solanacearum was detected in soils from 13 farms in 8 of the 18 districts surveyed. Approximately 38% of soil samples were infested with Meloidogyne spp. Phytophthora infestans isolates belonging to the US 1 and 2_A1 genotypes were identified. Although many economically important diseases are present in Kenya, the lower aphid incidence in districts east of the Great Rift Valley may indicate that these districts are more suitable for seed potato production.     

Modification of serological techniques and their evaluation for detection of potato viruses in seed certification related activities

Development of alternative serological techniques to ELISA for detection of potato viruses offers advantages for monitoring virus incidence and for seed potato certification systems. Several trials showed that multiplex tissue print immunoassay (TPIA) and dot blot immunoassay (DBIA) might represent fast, practical, and sensitive alternatives for the detection of: Potato leaf roll virus (PLRV), Potato virus S (PVS), Potato virus X (PVX) and Potato virus Y (PVY), from green and/or tuber tissues. In TPIA, the specific precipitation patterns in infected tissues of leaf petioles or stem cross sections, observed with each virus, allowed identification of the specific virus or mixed infections in a single multiplex assay. For detection of PVY in green tissues, DBIA was shown to be over 50 times more sensitive than ELISA. TPIA and ELISA from the tuber stem end or from eyes might be used for rapid detection of PVY and PVS in seed potato tubers without prior germination. PVS was evenly distributed in potato tuber tissue, while PVY was localized in the vascular tissue beneath the epidermis, with irregular distribution along the periphery of the potato tuber. For laboratories in developing countries lacking time and facilities for tests based on tuber germination, monitoring for PVS and PVY using TPIA in tuber tissue may be a suitable alternative to ELISA.     

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.     

Simultaneous detection of potato viruses Y,leafroll, X and S by DAS-ELISA technique with artificial polyvalent antibodies (APAs)

Summary A modification of the double antibody sandwich-enzyme linked immunosorbent assay (DASELISA) was used to try to detect simultaneously PVY, PLRV, PVX and PVS in both leaf and sprout samples, using artificial polyvalent antibodies (APAs). Each virus, singly or in a mixture, was detected with similar sensitivity using either specific antibodies or APAs. The APA for all four viruses is suitable for routine leaf testing, but only the APA for PVY+PVX+PVS is suitable for routine sprout testing.     

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。