疏叶骆驼刺杆状DNA病毒及其ORF4编码蛋白与启动子的功能研究

发布时间：2017-12-28 09:41

本文关键词：疏叶骆驼刺杆状DNA病毒及其ORF4编码蛋白与启动子的功能研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：杆状DNA病毒属(Badnavirus)隶属于花椰菜花叶病毒科(Caulimoviridae),是一种类反转录病毒(pararetrovirus),其基因组为不完全环化的双链DNA,大小在7.2 kb~9.2 kb,包含基因间隔区(intergenic region,IR)及三个典型ORFs(ORF1、ORF2、ORF3),其中ORF3最大,编码一个~216 kDa多聚蛋白,包含天冬氨酸蛋白酶(aspartic protease,AP)、外壳蛋白(coat protein,CP)、逆转录酶(reverse transcriptase,RT)和RNA酶H(ribonuclease H,RNase H)等保守功能区。目前,已经在热带、亚热带和温带植物中发现了杆状DNA病毒,但尚未在高寒地区的植物中发现此类病毒。疏叶骆驼刺(Alhagi sparsifolia)为豆科(Leguminosae sp.)多年生植物,主要分布于中亚高寒地区。在前期研究中,本实验室前期以塔克拉玛干地区的疏叶骆驼刺为实验材料,构建了疏叶骆驼刺的转录组数据库。Blast分析发现其中2个转录本与已知杆状DNA病毒基因组序列具有一定的同源性,且其丰度与干旱胁迫正相关。这些数据暗示,疏叶骆驼刺中可能含有一种杆状DNA病毒,因而暂时命名为疏叶骆驼刺杆状病毒(Alhagi bacilliform virus,ABV)。在此基础上,本论文继续开展了ABV基因组克隆、序列分析、进化树分析,以及ABV编码基因及调控序列功能的研究,具体如下:1)ABV基因组克隆与序列分析以塔克拉玛干疏叶骆驼刺总DNA为模板,利用PCR分段克隆,获得了长度为7068 nt的ABV核苷酸序列;序列分析没有发现完整的IR及ORF1序列,但推测其含有对应杆状DNA病毒ORF2、ORF3、ORF4的遗传信息,且ORF3编码蛋白包含典型的AP、RT-RNaseH及CP等保守功能区;利用保守功能区氨基酸序列与已知杆状DNA病毒属成员的相应序列进行比对,结果表明ABV与杆状DNA病毒具有较高同源性;根据系统进化树分析及ICTV规定的杆状DNA病毒分类标准,证明ABV隶属于杆状DNA病毒属,为该病毒属的一个新的种。利用Southern杂交和反向PCR证明了ABV基因组已经整合进入疏叶骆驼刺基因组,以内源类逆转录病毒(endogenous pararetroviruses,EPRV)的形式存在,且整合的ABV元件可能经历了复杂的DNA重组。分析了我国西北12个不同地区(包括塔克拉玛干)的疏叶骆驼刺总DNA,结果仅在阿克苏温宿县、阿克苏乌什县和阿拉尔市等三个邻近地区的样本中没有扩增得到ABV RT-RNaseH保守区,表明ABV在我国西北地区的疏叶骆驼刺中广泛存在。2)ABV ORF4启动子及编码蛋白功能的初步分析利用qRT-PCR证实了PEG处理能够诱导疏叶骆驼刺幼苗ORF4 mRNA的表达,由此推测内源ABV元件可能与疏叶骆驼刺抗旱特性正相关。克隆了ABV ORF4启动子(ORF4-P),构建植物表达载体pORF4-P-GUS,转化获得转基因拟南芥,并通过组织化学染色与GUS定量分析,证明ORF4-P具有干旱诱导表达的特征。克隆了ABV ORF4基因,瞬时表达分析显示ORF4编码蛋白定位于细胞质和细胞核;构建植物表达载体pCAM-ORF4,转化拟南芥,结果显示,与野生型拟南芥相比,转ORF4植株表现莲座叶增多,抽薹推迟等特征,且在甘露醇处理条件下,其根系明显长于野生型。综上所述,本研究在典型高寒荒漠植物——疏叶骆驼刺中发现了一种新的杆状DNA病毒——ABV,并揭示ABV以内源病毒形式存在于疏叶骆驼刺;根据ABV ORF4启动子及编码蛋白功能的初步分析,疏叶骆驼刺可能利用内源ABV元件,调控基因表达或表达蛋白,从而提高其抵御非生物胁迫比如干旱的能力。
[Abstract]:Bacilliform virus DNA (Badnavirus) belonging to the caulimoviridae (Caulimoviridae), is a kind of reverse transcription virus (pararetrovirus) genome, its incomplete ring double stranded DNA the size at 7.2 kb~9.2 KB, contains the intergenic region (intergenic region, IR) and three typical ORFs (ORF1, ORF2 ORF3, ORF3), the largest, ~216 kDa encoding a polyprotein, comprising an aspartic protease (aspartic, protease, AP), (coat protein, CP capsid protein), reverse transcriptase (reverse transcriptase, RT) and RNA H (ribonuclease H, RNase enzyme H) conservative functional areas. At present, rod-shaped DNA virus has been found in tropical, subtropical and temperate plants, but the virus has not been found in plants in the high and cold regions. Alhagi sparsifolia is a perennial of leguminous (Leguminosae sp.), which is mainly distributed in the alpine region of Central Asia. In the previous study, a transcriptional database of camel spines was constructed in the early stage of the laboratory using camel spines in Taklimakan area as the experimental material. Blast analysis found that 2 of the transcripts were homologous to the genome sequences of the known baculovirus DNA viruses, and their abundance was positively correlated with drought stress. These data indicate that the thinning a.pseudoalhagi may contain a rod-shaped DNA virus, thus temporarily named sparsifolia baculovirus (Alhagi bacilliform virus, ABV). Based on the analysis carried out ABV genome cloning, sequence analysis and phylogenetic tree to this thesis, and research the ABV encoding gene and regulatory sequence functions as follows: 1) cloning and sequence analysis of genome ABV Taklimgan sparsifolia total DNA as template, using PCR sub cloning, nucleotide sequence length was ABV 7068 NT; sequence analysis found no IR and complete ORF1 sequence, but that genetic information which contains the corresponding rod-shaped DNA virus ORF2, ORF3, ORF4, and ORF3 encoding protein contains a typical AP, RT-RNaseH and CP were compared using the conserved region; the corresponding sequence of conserved amino acid sequences with known baculovirus DNA region virus is a member, the results showed that ABV and DNA have high homology baculovirus virus; baculovirus DNA virus according to the classification criteria of phylogenetic tree analysis and ICTV rules, that ABV belongs to the bar The genus DNA, a new species of the genus, is a genus of the genus. Using Southern hybridization and reverse PCR, ABV genome has been integrated into the genome of the camel thorn, which exists in the form of endogenous pararetroviruses (EPRV), and the integrated ABV elements may have undergone complex DNA recombination. Analysis of the 12 different regions of Northwest China (including Taklimakan) of Alhagi sparsifolia DNA, results in only three areas adjacent to Akesu Wensu County, Akesu county and Wushi city as sample without alar and amplified ABV conserved region of RT-RNaseH, showed that ABV in Northwest China Alhagi in widely there. 2) a preliminary analysis of the function of ABV ORF4 promoter and coding protein. QRT-PCR confirmed that PEG treatment could induce the expression of ORF4 mRNA in the seedlings of camel thorn, suggesting that the endogenous ABV elements may be positively correlated with the drought resistance characteristics of the camel thorn. The ABV ORF4 promoter (ORF4-P) was cloned, and plant expression vector pORF4-P-GUS was constructed. Transgenic Arabidopsis was transformed into transgenic Arabidopsis thaliana. Histochemical staining and GUS quantitative analysis showed that ORF4-P had the characteristics of drought induced expression. Cloning of ABV ORF4 gene, transient expression analysis showed that ORF4 encoding protein in cytoplasm and nucleus; construct the plant expression vector pCAM-ORF4 was transformed into Arabidopsis thaliana, results show that, compared with wild type Arabidopsis, transgenic ORF4 plants showed increased rosette leaves, delayed bolting features, and in the mannitol treatment conditions, the root was longer than the wild type. In summary, this study in a Typical Alpine desert plant Alhagi in the discovery of a new DNA - ABV and ABV to reveal the rod-shaped virus, endogenous virus exists in the form of Alhagi sparsifolia; according to the preliminary analysis of the ABV promoter and ORF4 encoding protein function, and thinning the camel thorn may use the endogenous ABV element. The regulation of gene expression or protein expression, so as to improve the ability to resist abiotic stress such as drought.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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