本文關(guān)鍵詞：疏葉駱駝刺桿狀DNA病毒及其ORF4編碼蛋白與啟動子的功能研究　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 桿狀DNA病毒 疏葉駱駝刺 內(nèi)源類逆轉(zhuǎn)錄病毒 轉(zhuǎn)基因 干旱脅迫

【摘要】：桿狀DNA病毒屬(Badnavirus)隸屬于花椰菜花葉病毒科(Caulimoviridae),是一種類反轉(zhuǎn)錄病毒(pararetrovirus),其基因組為不完全環(huán)化的雙鏈DNA,大小在7.2 kb~9.2 kb,包含基因間隔區(qū)(intergenic region,IR)及三個典型ORFs(ORF1、ORF2、ORF3),其中ORF3最大,編碼一個~216 kDa多聚蛋白,包含天冬氨酸蛋白酶(aspartic protease,AP)、外殼蛋白(coat protein,CP)、逆轉(zhuǎn)錄酶(reverse transcriptase,RT)和RNA酶H(ribonuclease H,RNase H)等保守功能區(qū)。目前,已經(jīng)在熱帶、亞熱帶和溫帶植物中發(fā)現(xiàn)了桿狀DNA病毒,但尚未在高寒地區(qū)的植物中發(fā)現(xiàn)此類病毒。疏葉駱駝刺(Alhagi sparsifolia)為豆科(Leguminosae sp.)多年生植物,主要分布于中亞高寒地區(qū)。在前期研究中,本實驗室前期以塔克拉瑪干地區(qū)的疏葉駱駝刺為實驗材料,構(gòu)建了疏葉駱駝刺的轉(zhuǎn)錄組數(shù)據(jù)庫。Blast分析發(fā)現(xiàn)其中2個轉(zhuǎn)錄本與已知桿狀DNA病毒基因組序列具有一定的同源性,且其豐度與干旱脅迫正相關(guān)。這些數(shù)據(jù)暗示,疏葉駱駝刺中可能含有一種桿狀DNA病毒,因而暫時命名為疏葉駱駝刺桿狀病毒(Alhagi bacilliform virus,ABV)。在此基礎(chǔ)上,本論文繼續(xù)開展了ABV基因組克隆、序列分析、進(jìn)化樹分析,以及ABV編碼基因及調(diào)控序列功能的研究,具體如下:1)ABV基因組克隆與序列分析以塔克拉瑪干疏葉駱駝刺總DNA為模板,利用PCR分段克隆,獲得了長度為7068 nt的ABV核苷酸序列;序列分析沒有發(fā)現(xiàn)完整的IR及ORF1序列,但推測其含有對應(yīng)桿狀DNA病毒ORF2、ORF3、ORF4的遺傳信息,且ORF3編碼蛋白包含典型的AP、RT-RNaseH及CP等保守功能區(qū);利用保守功能區(qū)氨基酸序列與已知桿狀DNA病毒屬成員的相應(yīng)序列進(jìn)行比對,結(jié)果表明ABV與桿狀DNA病毒具有較高同源性;根據(jù)系統(tǒng)進(jìn)化樹分析及ICTV規(guī)定的桿狀DNA病毒分類標(biāo)準(zhǔn),證明ABV隸屬于桿狀DNA病毒屬,為該病毒屬的一個新的種。利用Southern雜交和反向PCR證明了ABV基因組已經(jīng)整合進(jìn)入疏葉駱駝刺基因組,以內(nèi)源類逆轉(zhuǎn)錄病毒(endogenous pararetroviruses,EPRV)的形式存在,且整合的ABV元件可能經(jīng)歷了復(fù)雜的DNA重組。分析了我國西北12個不同地區(qū)(包括塔克拉瑪干)的疏葉駱駝刺總DNA,結(jié)果僅在阿克蘇溫宿縣、阿克蘇烏什縣和阿拉爾市等三個鄰近地區(qū)的樣本中沒有擴(kuò)增得到ABV RT-RNaseH保守區(qū),表明ABV在我國西北地區(qū)的疏葉駱駝刺中廣泛存在。2)ABV ORF4啟動子及編碼蛋白功能的初步分析利用qRT-PCR證實了PEG處理能夠誘導(dǎo)疏葉駱駝刺幼苗ORF4 mRNA的表達(dá),由此推測內(nèi)源ABV元件可能與疏葉駱駝刺抗旱特性正相關(guān)�？寺×薃BV ORF4啟動子(ORF4-P),構(gòu)建植物表達(dá)載體pORF4-P-GUS,轉(zhuǎn)化獲得轉(zhuǎn)基因擬南芥,并通過組織化學(xué)染色與GUS定量分析,證明ORF4-P具有干旱誘導(dǎo)表達(dá)的特征。克隆了ABV ORF4基因,瞬時表達(dá)分析顯示ORF4編碼蛋白定位于細(xì)胞質(zhì)和細(xì)胞核;構(gòu)建植物表達(dá)載體pCAM-ORF4,轉(zhuǎn)化擬南芥,結(jié)果顯示,與野生型擬南芥相比,轉(zhuǎn)ORF4植株表現(xiàn)蓮座葉增多,抽薹推遲等特征,且在甘露醇處理條件下,其根系明顯長于野生型。綜上所述,本研究在典型高寒荒漠植物——疏葉駱駝刺中發(fā)現(xiàn)了一種新的桿狀DNA病毒——ABV,并揭示ABV以內(nèi)源病毒形式存在于疏葉駱駝刺;根據(jù)ABV ORF4啟動子及編碼蛋白功能的初步分析,疏葉駱駝刺可能利用內(nèi)源ABV元件,調(diào)控基因表達(dá)或表達(dá)蛋白,從而提高其抵御非生物脅迫比如干旱的能力。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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