本文選題：中國野生葡萄　切入點：白粉病　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：葡萄(Vitis spp.)是世界上重要的經(jīng)濟水果,葡萄白粉病是由葡萄白粉菌(Uncinula necator[Schw.]Burr.)引起的危害葡萄生產(chǎn)的主要真菌病害之一,給葡萄生產(chǎn)造成巨大的經(jīng)濟損失。中國野生華東葡萄白河-35-1(Chinese wild Vitis pseudoreticulata‘Baihe-35-1’)和毛葡萄商-24(Vitis quinquangularis‘Shang-24’)對白粉病具有很強的抗性,挖掘中國野生葡萄抗白粉病基因和研究其功能對葡萄抗病育種具有重要的意義。本研究在項目組前期研究基礎(chǔ)上,克隆了中國野生葡萄白河-35-1抗白粉病相關(guān)基因VpCN和VpTNL1、毛葡萄商-24 VqTNL2及其啟動子序列,構(gòu)建了過表達載體和瞬時表達載體分別轉(zhuǎn)化擬南芥和葡萄,對其抗病功能和啟動子活性進行了驗證;克隆了中國野生葡萄RPM1類基因,進行了其表達和生物信息學(xué)分析,取得以下主要研究結(jié)果:1、從前期中國野生華東葡萄白河-35-1轉(zhuǎn)錄組數(shù)據(jù)庫中發(fā)現(xiàn)一條抗白粉病候選基因,命名為VpCN,其在接種白粉菌后誘導(dǎo)表達,克隆測序后,其開放閱讀框(ORF)長1,773bp,編碼590個氨基酸。生物信息學(xué)分析顯示其N端含有一個Rx-CC like結(jié)構(gòu)域,C端含有一個保守的NB-ARC結(jié)構(gòu)域,異源表達VpCN的擬南芥植株出現(xiàn)三種表型:正常表型、侏儒型和葉片融合表型。正常表型增強了擬南芥對白粉病菌(Golovinomyces cichoracearum)和Pst DC3000的抗性�？寺∑鋯幼�,并構(gòu)建了啟動子與GUS報告基因融合載體,在歐洲葡萄“紅地球”葉片瞬時表達顯示啟動子有病原菌誘導(dǎo)活性。進一步對啟動子5'端缺失分析并與GUS報告基因融合,在葡萄葉片中瞬時表達顯示起始密碼子(ATG)上游400 bp序列有可能是響應(yīng)白粉菌最短片段,TC rich repeats作用元件有可能參與響應(yīng)葡萄白粉菌。2、從前期中國野生華東葡萄白河-35-1轉(zhuǎn)錄組數(shù)據(jù)庫中發(fā)現(xiàn)一條抗白粉病候選基因,命名為VpTNL1,其在接種白粉菌后誘導(dǎo)表達,克隆測序后其全長3,903 bp,ORF長2,622bp,推導(dǎo)編碼873個氨基酸,3'UTR長1281 bp。其定位在18號染色體上,包含4個外顯子和3個內(nèi)含子。生物信息學(xué)分析顯示其N端預(yù)測有類似動物白介素I受體的TIR結(jié)構(gòu)域和一個NB-ARC結(jié)構(gòu)域,其C端含有4個連續(xù)的LRR結(jié)構(gòu)域。異源表達VpTNL1的擬南芥植株出現(xiàn)兩種表型:侏儒型和正常表型。正常表型增強了對擬南芥白粉菌(G.cichoracearum)和Pst DC3000的抗性。對其啟動子克隆和順式作用元件預(yù)測顯示:其啟動子序列含有與病原菌及非生物脅迫相關(guān)的順式作用元件。將其與GUS報告基因融合,構(gòu)建瞬時表達載體后轉(zhuǎn)化歐洲葡萄“紅地球”葉片,結(jié)果顯示啟動子具有活性。對啟動子5'端缺失分析顯示,TCA element作用元件可能參與響應(yīng)SA,TC rich repeats作用元件可能參與響應(yīng)葡萄白粉菌。3、從前期中國野生毛葡萄商-24株系中的轉(zhuǎn)錄組數(shù)據(jù)庫中發(fā)現(xiàn)一條抗白粉病候選基因,命名為VpTNL2,在接種白粉菌后VpTNL2誘導(dǎo)表達下調(diào),分析了在根、莖、葉、花和果皮中的表達模式,VpTNL2在上述不同組織中存在著表達差異�？寺y序顯示,cDNA全長3,835 bp,ORF長3,783 bp,編碼1,260個氨基酸。生物信息分分析顯示其N端存在著一個TIR結(jié)構(gòu)域和NB-ARC結(jié)構(gòu)域,C端存在著6個連續(xù)LRR結(jié)構(gòu)域。克隆其啟動子并對啟動子元件預(yù)測,啟動子序列含有多個參與生物脅迫和非生物脅迫的作用元件,構(gòu)建了啟動子融合GUS報告基因的瞬時表達載體,采用農(nóng)桿菌介導(dǎo)的瞬時轉(zhuǎn)化法轉(zhuǎn)入葡萄葉片后,證明其啟動子有活性。4、從中國野生華東葡萄白河-35-1克隆了VpRPM1,VpRPM1-4和VpRPM1-5,ORF長度分別為2,794 bp,465 bp和2,556 bp,從中國野生毛葡萄中克隆出VqRPM1-2和VqRPM1-3,ORF長分別為2805和2727 bp,其推導(dǎo)的氨基酸都具有保守的NB-ARC結(jié)構(gòu)域。對其在白粉菌侵染后的表達分析顯示,5個基因都顯著下調(diào)。對歐亞葡萄“黑比諾”RPM1類基因生物信息學(xué)分析表明,在基因組中鑒定出17個RPM1類基因,同線性結(jié)果分析顯示染色體大片段復(fù)制和串聯(lián)重復(fù)有可能是RPM1類基因在染色體擴增的主要方式。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S436.631.1
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本文編號：1659671