烟草NtWRKY40在植物应答病毒侵染过程中的作用

WRKY转录因子家族在植物的抗病、抗逆反应中具有重要功能。已有研究表明,烟草花叶病毒(TMV)的侵染显著地诱导烟草Nt WRKY的表达,有必要进一步探明该基因在植物应答病毒侵染过程中的作用。采用PCR的方法克隆获得Nt WRKY cDNA,生物信息学分析结果显示,该基因属于WRKYⅡa亚族成员,与绒毛状烟草NtoWRKY40高度同源,命名为NtWRKY40。以此建立了过表达该基因的转基因烟草,并以TMV为毒源进行了转基因烟草和野生烟草的侵染实验,以观察NtWRKY40在烟草应答病毒侵染过程中的作用。实验结果表明,野生烟草在TMV侵染后9 d,NtWRKY40的表达量显著升高,而NtWRKY40过表达转基因烟草在病毒侵染后,病毒相关基因的表达高于野生型对照,与染病程度成正相关,说明过表达NtWRKY40增加了植株对病毒的敏感性,该基因为负调控因子。此外,为探索应用人工miRNA的抗病毒技术,以烟草天然miR167前体为骨架、马铃薯Y病毒(PVY)外壳蛋白基因的一段反向互补序列为成熟序列,构建了amiR167-PVY植物表达载体并转化烟草,以抑制PVY。对amiRNA转基因植株进行抗病毒实验的结果显示,amiR167-PVY能够部分抑制病毒基因的表达,转基因植株具有一定的抗病毒能力。     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系(PVY-C)HC-Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K₃₂₆(Nicotina tabacum cv.K₃₂₆)。PCR和Southern blot分析证明了HCPro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HCPro中心区域介导转基因烟草中PVY-C和黄瓜花叶病毒(CMV)、PVYC和马铃薯X病毒(PVX)之间的协生作用,从而明确了PVY-C HC-Pro中心区域为病毒协生作用的功能区域。     

TMV和PVY外壳蛋白双基因融合干涉及烟草转化

为了降低烟草花叶病毒(fobacco mosaic virus,TMV)和马铃薯Y病毒(potato virus Y,PVY)复合侵染对烟草带来的危害,本实验找到TMV-CP和PVY-CP基因部分保守序列,将保守序列进行双基因融合,此双基因即为RNAi的靶序列,用限制性内切酶将双基因从pMD18-T载体上切下,正反向连接到pUCCRNAi载体后,经酶切鉴定后定向连接到含超强启动子的pC2300-35S-OCS表达载体上,利用冻融法将此表达载体导入只含辅助质粒的根癌农杆菌中,构建含靶序列反向重复结构的RNAi双元载体系统,提取转化质粒,经酶切验证鉴定表明TMV和PVY外壳蛋白基因植物表达双元载体构建成功.并转化烟草,获得了3株对TMV和PVY抗性显著提高的转基因烟草.     

利用病毒载体在烟草中瞬时表达融合HBsAg基因

利用马铃薯PVX病毒载体构建了外源人工融合乙肝表面抗原HBsAg基因的表达载体,在烟草中利用农杆菌介导进行瞬时表达,以快速鉴定外源基因瞬时表达的状况以及重组蛋白的免疫活性。利用PCR技术从含有人工融合HBsAg基因的表达载体中分别扩增出LP PreS1 PreS2 S、PreS1 PreS2 S、PreS2 S序列,将其分别与PVX病毒载体pgR106连接,构建成PVX-LP、PVX-S1和PVX-S2等3个转化载体,并将此载体导入农杆菌菌株GV3101中用于侵染烟草植株叶片。感染植株经RT-PCR、RNA Dot blotting和HBsAg蛋白的ELISA检测显示,3个人工融合的HBsAg基因均可在植物体内得到转录,翻译成具有活性的蛋白。结果表明,外源融合HB-sAg基因经过植物病毒载体瞬时表达系统可以在植物系统中正常转录和翻译。     

利用FLP/frt重组系统产生无选择标记的转基因烟草植株

在植物转基因植株产生过程中,对转化细胞进行抗性筛选是通用程序,转化细胞的抗性一般是抗生素抗性或除草剂抗性,将赋予转化细胞抗性的选择标记基因删除是提高转基因植物生物安全性的重要措施。来自于啤酒酵母的FLP/frt位点特异性重组系统可有效删除同向定点重组位点frt之间的基因。通过多步骤重组,建立了可在植物中广泛应用的FLP/frt位点特异性重组系统。该系统包括含有frt位点的植物表达载体pCAMBIA1300-betA-frt-als-frt和含有由热诱导启动子hsp启动的FLP重组酶基因的植物表达载体pCAMBIA1300-hsp-FLP-hpt。利用二次转化的方式将二者先后转入烟草植株,热激处理后,热诱导型启动子hsp调控的重组酶FLP基因的表达催化位于选择标记基因als两侧同向frt位点间的重组反应,有效地删除了选择标记基因als。41%的经热激处理的二次转化植株发生了选择标记基因的删除,表明该系统在获得无选择标记基因的转基因植株中有很好的应用价值。     

高效Bt抗虫基因表达结构的构建及其在转基因烟草中表达行为的研究

构建了高效植物表达载体pBinMoBc,其携带有超强表达复合启动子OM及Ω因子控制下的CryIA?基因,作为对照,本实验构建了含有CaMV35S启动子控制下的CryIA?基因的植物表达载体pBinoBc。分别使用两个植物表达载体转化烟草,ELISA检测表明,在pBinMoBc转基因烟草中CryIA?基因的平均表达水平是pBinoBc的2.44倍,最高可达可溶蛋白的0.255%。抗虫检测结果表明,pBinMoBc转基因烟草与pBinoBc转基因烟草相比,具有更强的抗棉铃虫效果。上述结果表明,OM启动子比CaMV 35S启动子更具有实际应用价值,此结果在植物抗虫基因工程研究中具有重要意义。     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系（PVY－C）HC－Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K326(Nicotina tabacum cv.k326)。PCR和Southern blot分析证明了HC－Pro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HC－Pro中心区域介导转基因烟草中PVC－C和黄瓜花叶病毒（CMV）、PVY－C和马铃薯X病毒（PVX）之间的协生作用,从而明确了PVY－C HC－Pro中心区域为病毒协生作用的功能区域。     

棉花半乳糖苷酶基因启动子的分离及其在转基因烟草中的表达

β-半乳糖苷酶(β-galactosidase, EC 3.2.1.23)由植物中广泛分布的一类糖基水解酶组成, 被认为与细胞壁多糖的代谢相关. 棉花(Gossypium hirsutum) β-半乳糖苷酶基因已被成功分离, 被命名为GhGal1. RNA杂交实验显示该基因在棉花纤维发育的伸长期优势表达. 为了分析GhGal1基因的时空表达调控, 本研究构建了GhGal1启动子区域(1770 bp)与β-葡糖醛酸糖苷酶(glucuronidase, GUS)基因融合的双元载体, 通过农杆菌转化烟草植株. 对转基因植株分析的结果表明: 此转基因果实中的GUS活性比阴性和阳性对照的活性高. GUS组织定位分析表明: β-半乳糖苷酶基因能在根组织的分生区、子叶、维管束组织、果实和表皮毛中表达. 此外, 调控区域的序列分析揭示该序列含有一些果实/种子特异表达以及与表皮毛表达相关的保守元件. 这些结果显示了GhGal1启动子在转基因烟草植株中的时空表达特征, 并提供了GhGal1基因参与棉花纤维发育的一些重要线索.     

绿色荧光蛋白基因(GFP)在抗虫转基因植物研究中的应用(英文)

用合成的cry1Ac基因与绿色荧光蛋白基因 (GFP)构成融合蛋白基因 ,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg ,经根癌农杆菌介导转化了烟草。在紫外灯照射下 ,观察到转基因植株叶片中有较强的绿色荧光 ;经抗虫试验、PCR、Southernblot和Westernblot等检测 ,表明该重组植物表达载体能够在转基因植物中有效表达外源基因 ,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统 ,简化了抗虫转基因植物筛选程序 ,有助于快速获得双价抗虫转基因植株。     

表达Harpin蛋白的转基因马铃薯降低晚疫病斑生长率

以苹果火疫病菌 (Erwiniaamylovora)的Harpin蛋白基因和马铃薯 prp1 1基因启动子为主要元件 ,探索了以利用病原侵染诱导植物过敏性反应为目标的抗病基因工程新策略 .通过构建Harpin蛋白基因的 3个植物表达载体和遗传转化 ,获得68个转基因马铃薯植株 .Southern ,Northern和Westernblot分析证明 ,Harpin蛋白基因实现了在转基因植株中的插入、转录和蛋白表达 ,用Phytophthorainfestans复合生理小种测定表明 ,Harpin蛋白在转基因植株中的组成型表达和病原侵染诱导表达均能降低病斑的扩展速率 ;在病原侵染诱导Harpin蛋白基因表达的转基因植株中 ,发现有 2个植株共 3 0个接种叶片上无菌丝形成 ,病斑局限于接种点内 ,表明过敏性反应的遗传操作在植物抗真菌病基因工程中具有广阔的应用前景 .     

改造的马铃薯Y病毒复制酶基因介导高度抗病性

提取马铃薯Y病毒中国分离株(PVY—c)的mRNA作为模板,随机六聚脱氧核苷酸和寡聚dT为引物合成了单链cDNA。通过聚合酶链式反应(PcR)获得了PVY—C的核内含体b(Nib)全长cDNA克隆。在对其进行全序列分析的基础上,构建了PVY—CNIb基因全长．5’端缺失381个碱基和Nib反义RNA三种不同形式高等植物表达载体。在土壤农杆菌LBA4404的介导下,转化烟草生产品种NC89,获得了所有三种表达载体的转基因植株。通过分子生物学检测和抗性分析发现不同形式的Nib基因序列的转基因植株对马铃薯Y病毒表现不同程度的抗性。其中,以5’端缺失的Nlb的基因转化植株表现最好,从总共20个这类转化株系中筛选到4个株系至少在100μg／m1 PVY—C接种浓度下,表现完全的抗病效果。从总共39个全长Nib基因转化株系中,仅有一个株系,在100μg／ml PVY—c的攻毒接种下具有完全的抗病性。所有33个Nib基因反义RNA的转化植株中,无一株系表现完全的抗病效果,但是有部分株系能不同程度地延缓或减轻发病程度,并有部分植株在发病后50d左右有恢复健康的趋势。虽然能够在上述3种形式的Nib基因序列的转基因植物中检测到相应的RNA的转录产物,但是均未能检测到其相应的蛋白表达产物。     

Potato Y Potyvirus Helper Component Proteinase Enhances Long-distance Movement of Potato X Potexvirus

To mutagenize two conserved CCCT and PTK motifs in the central domain of Chinese strain of potato Y potyvirus (PVY-C) helper component proteinase (HC-Pro), four mutants of HC-Pro gene were obtained by PCR and site-directed mutagenesis, and then were inserted into the constitutive expression vector pBin438. Leaves from tobacco ( Nicotiana tabacum L. cv. K326) were transformed with these four plant expression plasmids by Agrobacterium -mediated transformation, respectively. Southern and Western blotting analyses showed that these four mutants were integrated into tobacco genomic DNA and could express the corresponding proteins in most of the transgenic plants. The challenge of transgenic plants with potato X potexvirus (PVX) revealed that the expression products of PVY-C HC-Pro mutants in transgenic plants greatly abolished functions of HC-Pro in enhancing the accumulation and pathogenicity of PVX, indicating that CCCT and PTK motifs of HC-Pro were required for PVX/PVY synergism. Meanwhile, the results demonstrated that PVY-C HC-Pro had a function in accelerating the long-distance movement of PVX in these transgenic plants for the first time.     

马铃薯Y病毒蚜传辅助因子促进马铃薯X病毒长距离运输

采用PCR和定点突变法,对马铃薯Y病毒中国株系（Chyinese strain of potato Ypotyvirus,PVY-C)蚜传辅助成分（helper component proteinase,HC-Pro)基因中心区域的CCCT基序和PTK基序进行定点改造,获得了4种突变体。然后将突变体砍降到植物表达载体pBin438中,所得到的重组体通过根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)Conn）介导法转了烟草（Nicotiana tabacum L.cv.K326).Southern blotting和Western blotting分析表明4种突变体已经成功整合到烟草的基因组中,并在蛋白水平上得到了表达。马铃薯X病毒（potato X potexvirus,PVX)对转基因烟草的攻毒实验表明,4种突变体均使PVY－C HYC－Prog严重丧失了促进PVX病毒粒子在寄主体内积累和提高PVX致病性的功能,说明CCCT、PTK基序为PVY－C HYC－Pro介导PVX／PVY协生作用所必需。同时证明了HC-Pro具有增强PVX在寄主体内长距离运输的功能。     

Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance

Changes in glucose-6-phosphate dehydrogenase (G6P DH; EC 1.1.1.49) activity caused by infection of tobacco ( Nicotiana tabacum L.) leaves with potato virus Y (PVY), cucumber mosaic virus, potato virus X, tobacco rattle virus and turnip mosaic virus, the subcellular localisation of G6P DH isozymes in mesophyll protoplasts derived from healthy and PVY-infected tobacco leaves, as well as G6P DH control and the relationship of its isozymes with the degree of tobacco resistance to PVY multiplication, were studied. The activities of G6P DH were markedly increased in locally and systemically infected leaves and the time courses of the activity linearly correlated with those of virus multiplication. In leaves infected with PVY, the activity time courses of the crude and the partially purified G6P DH were coincident. This probably indicates the involvement of coarse regulation of the enzyme. PVY content linearly correlated with enhanced G6P DH activity in leaf discs derived from susceptible, tolerant and resistant cultivars of tobacco. The increased activity of the enzyme in infected protoplasts and plant tissues was predominantly caused by the increased activity of chloroplastic isozymes. This was confirmed by the specific staining of isozymes after electrophoretic separation of chloroplastic proteins of tobacco leaves. These findings enable the degree of resistance to virus multiplication to be quantified for the use of gene manipulation and breeding.     

Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

Expression of recombinant trichosanthin,a ribosome-inactivating protein,in transgenic tobacco

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.     

Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection.

Tobacco genes encoding the PR-1a protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-1a gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. No such induction was found with upstream sequences of 643 base pairs or shorter of the PR-1a gene. When the PR-1a upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35S core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic virus- and salicylate-responsive elements between positions -643 and -689 in the PR-1a promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.