中国野生毛葡萄钙调蛋白基因克隆及序列分析

于葡萄黑痘病发病盛期,用病叶压片法对高抗黑痘病的中国野生毛葡萄'商-24'接种黑痘病病原菌,采用mRNA差异显示技术进行抗黑痘病基因表达差异的研究.结果显示:(1)获得了T11GG/B0304-400、T11CC/S428-350、T11GG/S424-700、T11AG/S432-350、T11GG/S433-250、T11GG/S433-300、T11AG/S432-300、T11GG/S438-353和T11AG/S424-300等9个基因表达差异cDNA片段.其中T11GG/S438-353 mRNA片段表达在接种后3 d被诱导显著降低,并在之后2 d几乎检测不到.(2)采用RACE技术克隆了T11GG/S438-353 mRNA片段的cDNA全长序列;序列分析表明,该cDNA包含一个607 bp完整的开放阅读框架,编码149个氨基酸;其编码氨基酸序列与拟南芥、欧洲葡萄、柳杉、党参、无梗花栎、欧洲栗及寄生草钙调蛋白的一致性分别为99%、97%、94%、91%、90%、88%和77%.(3)本实验克隆到了负向调控中国野生毛葡萄抗黑痘病的钙调蛋白基因,并命名为VqCaM,其GenBank登录号为EU694099;实时荧光定量PCR结果再次验证VqCaM表达受葡萄黑痘病侵染下调,     

中国野葡萄新基因cDNA全长的克隆及序列分析

根据对中国野生葡萄华东葡萄株系白河-35-1在白粉病诱导下构建的cDNA文库中获得的1条EST序列设计特异引物,以总RNA逆转录产物为模板,进行SMART-RACE扩增。结合生物信息学方法对所获的序列进行开放阅读框、序列同源性分析,并预测了蛋白质的理化性质、信号肽、酶与非酶分析、亚细胞定位以及蛋白质二级结构。结果表明,获得的中国野生葡萄新基因的全长序列为854 bp,其开放阅读框为585 bp,5′非编码区为107 bp,3′非编码区为162 bp;同源性比对表明该新基因推导的氨基酸序列与拟南芥RNA结合蛋白和水稻推定的半乳糖基转移酶的同源性分别为55%和63%,第8~82位氨基酸序列与RNA识别基序相类似,是一个典型的结构功能域;推导该基因表达产物为相对分子质量为21 801.27、等电点为6.86的不稳定蛋白,定位于细胞核,不包含信号肽,二级结构主要是无规则卷曲。     

毛葡萄芪合成酶基因的克隆及序列分析

芪合成酶是白藜芦醇生物合成过程中起关键作用的酶.通过RT-PCR,从高抗葡萄黑痘病的中国野葡萄毛葡萄商-24克隆了芪合成酶基因家族的2个成员VqSTS1和VqSTS2,其cDNA全长均为1 179 bp,编码392个氨基酸.氨基酸序列分析表明,VqSTS1和VqSTS2编码氨基酸247-257位点的IPN（S/F）AGAIAGN ＂是芪合成酶家族固有的氨基酸保守结构域,368-378位点的GVLFGFGPGLT＂是芪合成酶与查尔酮合成酶家族固有的保守序列特征.在编码的392个氨基酸中,VqSTS1和VqSTS2仅有12个氨基酸不同,氨基酸一致性达97%.多重比较分析显示,VqSTS1与VqSTS2与五针松、河岸葡萄、白粉藤、华东葡萄及欧洲葡萄等STS在氨基酸水平的一致性分别为65%、96%、97%和98%.将VqSTS1和VqSTS2登录GenBank,登录号分别为EF158856和EF158857.     

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.     

中国野生毛葡萄芪合成酶基因克隆及表达分析

采用RT-PCR技术克隆中国野生毛葡萄‘丹凤-2’芪合成酶基因,命名为VqDSTS1,并进行序列及表达模式分析.结果表明:VqDSTS1基因cDNA编码区全长为1 179bp,GenBank登录号为JQ342086,编码392个氨基酸;氨基酸序列分析表明,VqDSTS1含有芪合成酶基因家簇的特征识别序列‘IPNSAGAIAGN’和‘GVLFGFG-PGLT’;序列比对显示,VqDSTS1与其他葡萄种质的芪合成酶氨基酸序列一致性在95.2%～98.7%之间;半定量RT-PCR分析表明,VqDSTS1受白粉病诱导表达,呈双峰模式.为进一步研究中国野生毛葡萄‘丹凤-2’芪合成酶基因家族的表达及功能分析提供了基础.     

mRNA差异显示技术克隆中国葡萄属野生种核糖体RNA基因

克隆功能基因是研究植物基因结构及功能的重要途径,利用mRNA差异显示技术,筛选获得了中国葡萄属野生种华东葡萄白河-35-1在人工接种白粉菌前后差异表达基因的cDNA片段Vprdrf4。经Northern杂交验证,该片段为正调控片段,Blaset检索表明与真核生物核糖体RNA基因的同源性高达90%以上,GenBank登录号为CD347664。     

Expression and functional analysis of two genes encoding transcription factors, VpWRKY1 and VpWRKY2, isolated from Chinese wild Vitis pseudoreticulata

In this study, two WRKY genes were isolated from Erysiphe necator-resistant Chinese wild Vitis pseudoreticulata W. T. Wang ‘Baihe-35-1’, and designated as VpWRKY1 (GenBank accession no. GQ884198) and VpWRKY2 (GenBank accession no. GU565706). Nuclear localization of the two proteins was demonstrated in onion epidermal cells, while trans-activation function was confirmed in the leaves of ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was induced rapidly by salicylic acid treatment in ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was also induced rapidly by E. necator infection in 11 grapevine genotypes; the maximum induction of VpWRKY1 was greater in E. necator-resistant grapevine genotypes than in susceptible ones post E. necator inoculation. Furthermore, ectopic expression of VpWRKY1 or VpWRKY2 in Arabidopsis enhanced resistance to powdery mildew Erysiphe cichoracearum, and enhanced salt tolerance of transgenic plants. VpWRKY2 also enhanced cold tolerance of transgenic plants. In addition, the two proteins were shown to regulate the expression of some defense marker genes in Arabidopsis and grapevine. The data suggest that VpWRKY1 and VpWRKY2 may underlie the resistance in transgenic grapevine to E. necator and tolerance to salt and cold stresses.     

Transient expression of glyoxal oxidase from the Chinese wild grape Vitis pseudoreticulata can suppress powdery mildew in a susceptible genotype

Vitis pseudoreticulata glyoxal oxidase (VpGLOX) was previously isolated from the Chinese wild vine V. pseudoreticulata accession “Baihe-35-1” during a screen for genes that are upregulated in response to infection with grapevine powdery mildew (Erysiphe necator, PM). In the present study, a possible function of VpGLOX for defense against PM was investigated using Agrobacterium-mediated transient expression. After optimizing agro-infiltration, VpGLOX was transiently overexpressed in leaves of either PM-susceptible (accession “6-12-2”) or PM-resistant (accession “6-12-6”) plants. The efficiency of transfection was verified using a β-glucuronidase (GUS) reporter and was found to comprise most leaf areas regardless of the initial leaf position. Upon infection with E. necator, clear differences were observed with respect to hyphal development between agro-infiltrated leaves and control groups of both, the susceptible and the resistant, genotypes. The expression of VpGLOX was followed by real-time polymerase chain reaction in both genotypes. Whereas in the susceptible host (“6-12-2”) expression was found to increase only in transfected leaves and remained transient, in the resistant host (“6-12-6”), a second peak appeared later in transfected leaves, probably representing the response of the endogenous VpGLOX. The data support the interpretation that VpGLOX is sufficient to confer resistance to E. necator.     

葡萄VvBAP1基因的克隆及表达特性分析

以抗性葡萄品种‘F-242’组培苗为材料, 利用同源克隆法克隆了葡萄VvBAP1基因。测序结果显示, VvBAP1扩增片段大小为531 bp, 可编码176个氨基酸序列。利用生物信息学分析VvBAP1基因编码的蛋白序列显示, 该蛋白分子量为19.43 kDa, 含有保守的钙离子依赖性的C2结构域; 等电点pI为9.42; 不稳定系数为37.09, 推测为稳定的亲水性蛋白; 含有多个丝氨酸/苏氨酸磷酸化位点。实时荧光定量PCR表明, 该基因在根茎叶中均有表达, 其中在叶片中表达量较高; 盐胁迫、低温等逆境因子及逆境相关的信号物质, 如水杨酸和一氧化氮均可诱导VvBAP1的表达, 其中低温对其表达量影响更为显著, 推测该基因参与了葡萄抵御逆境胁迫的过程, 尤其是与低温相关的过程。     

Physiological Change and Transcriptome Analysis of Chinese Wild Vitis amurensis and Vitis vinifera in Response to Cold Stress

Plant Molecular Biology Reporter - The Chinese wild Vitis amurensis Rupr. is a crucial resource for cold-resistant germplasm, but the molecular mechanism of cold resistance in V. amurensis has not...     

Characterization and Expression Analysis of a Glutathione Reductase Gene from Antarctic Moss Pohlia nutans

Glutathione reductase (GR) is a flavoprotein oxidoreductase and plays an important role in response to oxidative stresses in plants. A cDNA-encoding cytosolic GR [GenBank accession number GACA01029426, designated as Pohlia nutans glutathione reductase gene (PnGR)] was successfully cloned from Antarctic moss P. nutans. The full-length PnGR cDNA has 1,654 bp nucleotides with an open reading frame of 1,494 bp, encoding 497 amino acid residues. The deduced amino acid sequence of PnGR had 87.0 % identity with GR in Physcomitrella patens subsp. patens. The phylogenetic analysis showed that PnGR is clustered together with known cytosolic GR in other plants. In addition, the subcellular localization analysis by observing the transient expression of PnGR–green fluorescent protein fusion protein in Arabidopsis thaliana mesophyll protoplasts also revealed PnGR targeting to cytosol in plant cells. The expression patterns of PnGR under different abiotic stresses were determined by real-time PCR. Compared to the normal condition, the maximal mRNA accumulation of PnGR increased 3.82-fold at 4 °C, 2.92-fold at 10 °C, 4.14-fold with 200 mM NaCl, and 3.17-fold with drought stress, respectively. Together, our results suggested that the inducible PnGR might play an important role in Antarctic moss P. nutans acclimatizing to polar environment.     

山葡萄几丁质酶基因VCH3启动子的分离及鉴定

利用接头PCR技术首次分离了长度为1 216bp的"双优"山葡萄(Vitis amurensis Rupr.)classⅢ几丁质酶基因VCH3上游启动子序列(GenBank登录号AF441123),并用引物延伸方法鉴定了该启动子的转录起始位点,为5'-ATCAAGCAC-31序列中的第二个A.序列分析结果表明,代表真核基因启动子特征的CAAT盒和TATA盒分别位于VCH3启动子转录起始位点上游-122和-29处.另外,在转录起始位点上游-1 181 bp和-293 bp处各有一个水杨酸(SA)响应的顺式作用元件TGACG.为了鉴定该启动子的功能,将该启动子连接到β-葡糖苷酸酶基因(GUS)编码区的上游构建了VCH3启动子-GUS融合基因,并用农杆菌介导叶盘转化法将该融合基因转入烟草栽培品种NC89中.SA处理的转基因烟草根系和叶片GUS酶活性的荧光和组织化学检测结果表明VCH3启动子的驱动作用被SA诱导,因而该启动子在基因工程中将具有潜在的应用价值.