中国野生葡萄编码NB-ARC结构的抗白粉病基因克隆及功能分析

发布时间：2018-03-24 19:18

  本文选题：中国野生葡萄　切入点：白粉病　出处：《西北农林科技大学》2016年博士论文

【摘要】：葡萄(Vitis spp.)是世界上重要的经济水果,葡萄白粉病是由葡萄白粉菌(Uncinula necator[Schw.]Burr.)引起的危害葡萄生产的主要真菌病害之一,给葡萄生产造成巨大的经济损失。中国野生华东葡萄白河-35-1(Chinese wild Vitis pseudoreticulata‘Baihe-35-1’)和毛葡萄商-24(Vitis quinquangularis‘Shang-24’)对白粉病具有很强的抗性,挖掘中国野生葡萄抗白粉病基因和研究其功能对葡萄抗病育种具有重要的意义。本研究在项目组前期研究基础上,克隆了中国野生葡萄白河-35-1抗白粉病相关基因VpCN和VpTNL1、毛葡萄商-24 VqTNL2及其启动子序列,构建了过表达载体和瞬时表达载体分别转化拟南芥和葡萄,对其抗病功能和启动子活性进行了验证;克隆了中国野生葡萄RPM1类基因,进行了其表达和生物信息学分析,取得以下主要研究结果:1、从前期中国野生华东葡萄白河-35-1转录组数据库中发现一条抗白粉病候选基因,命名为VpCN,其在接种白粉菌后诱导表达,克隆测序后,其开放阅读框(ORF)长1,773bp,编码590个氨基酸。生物信息学分析显示其N端含有一个Rx-CC like结构域,C端含有一个保守的NB-ARC结构域,异源表达VpCN的拟南芥植株出现三种表型:正常表型、侏儒型和叶片融合表型。正常表型增强了拟南芥对白粉病菌(Golovinomyces cichoracearum)和Pst DC3000的抗性。克隆其启动子,并构建了启动子与GUS报告基因融合载体,在欧洲葡萄“红地球”叶片瞬时表达显示启动子有病原菌诱导活性。进一步对启动子5'端缺失分析并与GUS报告基因融合,在葡萄叶片中瞬时表达显示起始密码子(ATG)上游400 bp序列有可能是响应白粉菌最短片段,TC rich repeats作用元件有可能参与响应葡萄白粉菌。2、从前期中国野生华东葡萄白河-35-1转录组数据库中发现一条抗白粉病候选基因,命名为VpTNL1,其在接种白粉菌后诱导表达,克隆测序后其全长3,903 bp,ORF长2,622bp,推导编码873个氨基酸,3'UTR长1281 bp。其定位在18号染色体上,包含4个外显子和3个内含子。生物信息学分析显示其N端预测有类似动物白介素I受体的TIR结构域和一个NB-ARC结构域,其C端含有4个连续的LRR结构域。异源表达VpTNL1的拟南芥植株出现两种表型:侏儒型和正常表型。正常表型增强了对拟南芥白粉菌(G.cichoracearum)和Pst DC3000的抗性。对其启动子克隆和顺式作用元件预测显示:其启动子序列含有与病原菌及非生物胁迫相关的顺式作用元件。将其与GUS报告基因融合,构建瞬时表达载体后转化欧洲葡萄“红地球”叶片,结果显示启动子具有活性。对启动子5'端缺失分析显示,TCA element作用元件可能参与响应SA,TC rich repeats作用元件可能参与响应葡萄白粉菌。3、从前期中国野生毛葡萄商-24株系中的转录组数据库中发现一条抗白粉病候选基因,命名为VpTNL2,在接种白粉菌后VpTNL2诱导表达下调,分析了在根、茎、叶、花和果皮中的表达模式,VpTNL2在上述不同组织中存在着表达差异。克隆测序显示,cDNA全长3,835 bp,ORF长3,783 bp,编码1,260个氨基酸。生物信息分分析显示其N端存在着一个TIR结构域和NB-ARC结构域,C端存在着6个连续LRR结构域。克隆其启动子并对启动子元件预测,启动子序列含有多个参与生物胁迫和非生物胁迫的作用元件,构建了启动子融合GUS报告基因的瞬时表达载体,采用农杆菌介导的瞬时转化法转入葡萄叶片后,证明其启动子有活性。4、从中国野生华东葡萄白河-35-1克隆了VpRPM1,VpRPM1-4和VpRPM1-5,ORF长度分别为2,794 bp,465 bp和2,556 bp,从中国野生毛葡萄中克隆出VqRPM1-2和VqRPM1-3,ORF长分别为2805和2727 bp,其推导的氨基酸都具有保守的NB-ARC结构域。对其在白粉菌侵染后的表达分析显示,5个基因都显著下调。对欧亚葡萄“黑比诺”RPM1类基因生物信息学分析表明,在基因组中鉴定出17个RPM1类基因,同线性结果分析显示染色体大片段复制和串联重复有可能是RPM1类基因在染色体扩增的主要方式。
[Abstract]:Grape (Vitis spp.) is the world's most important economic fruit, grape powdery mildew is caused by powdery mildew (Uncinula necator[Schw.]Burr.) is one of the main hazards caused by fungal diseases in grape production, causing huge economic losses to the production of grape. Chinese wild grape white East River -35-1 (Chinese wild Vitis pseudoreticulata "Baihe-35-1") and grape -24 (Vitis quinquangularis Shang-24) has a strong resistance to powdery mildew, mining China wild grape powdery mildew resistance gene and study its function has important significance for grape breeding. In this study, the project team based on the previous research of cloning Chinese Baihe wild grape powdery mildew resistance related gene VpCN and -35-1 VpTNL1, taking quinquangularis -24 VqTNL2 and its promoter sequence, constructed over expression vector and transient expression vectors were transformed into Arabidopsis and its resistance to grapes. The function and the promoter activity was verified; cloned Chinese wild grape RPM1 gene for its expression, and bioinformatics analysis, the main results are as following: 1, former period China wild v.pseudoreticulata Baihe -35-1 transcriptome database found an anti white candidate gene, named VpCN, its expression after inoculation with powdery mildew, cloned and sequenced. The open reading frame (ORF) length of 1773bp, encoding 590 amino acids. Bioinformatics analysis showed that the N contains a Rx-CC like domain, C contains a conserved NB-ARC domain, heterologous expression of Arabidopsis VpCN has three phenotypes: normal the dwarf phenotype, and leaf fusion phenotype. Normal phenotype in Arabidopsis enhanced the powdery mildew resistance (Golovinomyces cichoracearum) and Pst DC3000. The cloned promoter and construct the promoter fused with GUS gene The carrier, show promoter bacteria induced expression in European grape "Red Globe". Further analysis of leaf instantaneous start and end deletion of 5'fused with GUS gene in grape leaves, transient expression shows the start codon (ATG) 400 bp upstream sequences may be the shortest response to powdery mildew fragments, TC rich repeats effect of components may be involved in the response of grape powdery mildew.2, formerly Chinese wild v.pseudoreticulata -35-1 period of Baihe transcriptome found a powdery mildew resistance gene, named VpTNL1, was induced in after inoculation with powdery mildew, cloning and sequencing of the full-length 3903 BP, ORF is 2622bp long, encoding 873 amino acids, 3'UTR 1281 the bp. is located on the 18 chromosome, including 4 exons and 3 introns. Bioinformatics analysis showed that the N terminal domain of TIR prediction with similar animal interleukin I receptor and a N The B-ARC domain, the C terminal contains 4 consecutive LRR domain. Heterologous expression of Arabidopsis plants VpTNL1 two: dwarf phenotype and normal phenotype. Normal phenotype in Arabidopsis enhanced powdery mildew resistant and Pst (G.cichoracearum) DC3000. The Promoter Cloning and cis acting elements of the promoter prediction showed that: with the sequence of pathogens and abiotic stress related cis elements. The fused with GUS gene, transformation of European grape Redglobe leaves construct transient expression vector, results showed that the promoter activity of the promoter. With 5'deletion analysis showed that TCA element elements may be involved in the response of SA, TC rich the effect of repeats components may be involved in the response of grape powdery mildew.3 former period transcriptome database China wild grape -24 strains found in a powdery mildew resistance gene, named VpTNL2 And in the expression of VpTNL2 induced by inoculation with powdery mildew, analyzed in root, stem, leaf, flower and fruit in the expression pattern of VpTNL2, there are differences in expression in the different tissues. Cloning and sequencing showed that cDNA was 3835 BP, ORF 3783 BP, encoding 1260 amino acids. The biological information analysis display N there is a TIR domain and NB-ARC domain, C end there are 6 consecutive LRR domain. The cloned promoter and the promoter element predicted promoter sequence containing multiple involved in biotic stress and abiotic stress acting elements, construct the instantaneous promoter fused with GUS reporter gene expression vector transformation method into the grape leaves using Agrobacterium mediated transient, prove that the promoter activity of.4, from Chinese wild v.pseudoreticulata Baihe -35-1 clone VpRPM1, VpRPM1-4 and VpRPM1-5, ORF length were 2794 BP, 465 BP and 2556 BP from Cloning of the VqRPM1-2 and VqRPM1-3 Kunino Momo grape, ORF were 2805 and 2727 BP, and the deduced amino acid has a conserved NB-ARC domain. The analysis shows that the expression of powdery mildew after infection, 5 genes were down regulated. The Eurasian grape Pinot Noir "RPM1 gene bioinformatics analysis showed that identified 17 RPM1 genes in the genome, with the results of linear analysis showed that the large fragment of chromosome replication and tandem repeats may be RPM1 gene amplification of chromosome in a major way.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.631.1
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本文编号：1659671