【作者】 王西平；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2004， 博士

【摘要】 我国的野生葡萄不仅抗病,而且和欧洲葡萄杂交亲和性好,可以有效地将抗病基因与欧洲葡萄品质性状相结合,因而将中国野生葡萄抗病基因与欧洲葡萄品质基因有效结合,培育抗病优良葡萄新品种和创造新型葡萄种质是育种理论和生产实践中急需解决的实际问题。本研究在葡萄白粉病发病盛期,对中国葡萄属野生种华东葡萄株系白河-35-1用病叶压片法人工接种葡萄白粉病病原菌,诱导抗病性表达。利用mRNA 差异显示技术结合cDNA 末端快速扩增技术(RACE)获得了中国葡萄属野生种抗白粉病相关基因全长序列,并进行序列分析;采用抗病基因同源序列方法,进行了中国葡萄属野生种抗病基因同源类似物克隆与分析的研究,取得的主要研究结果如下。1.用T11GG、T11AG、T11AC、T11CC、T11CG 等5 个锚定引物与Operon 公司生产的S421～S429 及B0301～B0326 共46 个随机引物组成了引物对230 个,对供试华东葡萄白河-35-1进行了反转录PCR扩增,筛选出了适于葡萄DDRT-PCR的引物组合186个。应用mRNA差异显示技术研究中国葡萄属野生种华东葡萄株系白河-35-1 在白粉病病原菌侵染诱导下,抗白粉病基因的表达,获得了抗白粉病基因差异表达的cDNA 片段10个: T11AC/B0314-323 、T11AC/B0319-456 、T11AC/S429-704 、T11GG/S438-245 、T11GG/B0316-309 、T11GG/B0324-292 、T11AC/B0313-307 、T11AC/B0320-723 、T11GG/B0322-379 、T11CG/S438-449 。其中T11AC/B0319-456 、T11AC/B0320-723 、T11AC/S429-704、T11CG/S438-449、T11GG/S438-245、T11GG/B0316-309、T11GG/B0324-292、T11AC/B0313-307、T11GG/B0322-379 等9 个片段为白粉病病原菌诱导后表达量增加,另外1 个片段为白粉病病原菌诱导后表达量降低。回收这10 个cDNA 片段并进行克隆、测序及同源性比较分析。将T11AC/B0319-456、T11AC/B0314-323、T11GG/S438-245 等3个片段在GenBank 中登录,登录号分别为:CD662167、CD662168、CD662169。2.首次采用5’RACE 与3’RACE 克隆了中国葡萄属野生种抗白粉病防卫基因-芪合成酶基因家族成员7 个,对其测序分析得出其大小分别为1288 bp、1343 bp、1411 bp、1468 bp、1492 bp、1506 bp 与1556 bp。将这7 个cDNA 序列进行开放阅读框架分析,它们都有完整的开放阅读框架,编码392 个氨基酸,分别命名为VpSTS1、VpSTS2、VpSTS3、VpSTS4、VpSTS5、VpSTS6、VpSTS7。氨基酸序列同源性分析表明,它们与其它植物13种STS 具有很高的同源性,介于94%-99%之间。其中VpSTS7 与河岸葡萄VrSTS2,更多还原

【Abstract】 This paper presents that the disease-resistant gene of Chinese Wild Vitis can be effectively combined with the quality gene of Vitis growing in Europe to breed the new fine variety and the new germplasm of Vitis because Chinese Wild Vitis can not only resist diseases but also hybridize with Vitis growing in Europe very well, and because Chinese Wild Vitis can combine its own disease-resistant gene with the quality gene of Vitis growing in Europe. This paper deals with the research in the following two aspects: Firstly, during the period of powdery mildew(Uncinula necator), the disease resistance of Chinese Wild Vitis strain “Baihe-35-1”growing in East China appeared by the way this clone is artificially inoculated with powdery mildew ; and through the combination of mRNA differential display and rapid amplification of cDNA ends, the whole powdery mildew resistant gene sequence of Chinese Wild Vitis is obtained to analyze this sequence. Secondly, the homology sequence of disease resistant gene has been used to study the clone of disease-resistant gene analogs of Chinese Wild Vitis. The novel findings are as follows: 1. Five Anchor Primers (T11GG, T11AG, T11AC, T11CC, T11CG) and 46 random primers(S421～S429 and B0301～B0326)produced by Operon Company are used to form 230 pairs of primers from which 186 DDRT-PRC primer combinations suitable for grape are singled out through the reverse amplification of Chinese Wild Vitis clone “Baihe-35-1”growing in East China. Through the mRNA differential display, powdery mildew resistant gene emerged with the germina source of powdery mildew infecting Chinese Wild Vitis clone “Baihe-35-1”growing in East China. Consequently, 10 powdery mildew resistant gene cDNAs have been found : T11AC/B0314～323、T11AC/B0319～456、T11AC/S429～704、T11GG/S438～245、T11GG/B0316～309 、T11AC/B0313～307、T11AC/B0320～723、T11GG/B0316～309、T11GG/B0322～379 and T11CG/S438～449. These ten cDNAs are cloned, sequenced and homologically analyzed, three of which are submitted to GenBank. The GenBank accession numbers: are CD662167, CD662168 and CD662169 respectively. 2. 5’RACE and 3’RACE(Rapid Amplification of cDNA Ends) have been used to clone anti-powdery mildew(Uncinula necator) defense genes of Chinese Wild Vitis, which are called Stilbene Synthesis(STS) , seven of whose size are 1288 bp,1343 bp,1411 bp,1468 bp,1492 bp,1506 bp and 1556 bp respectively. When these seven sequences of cDNA are analyzed by the way of open reading frame, it has been found out that they have the complete open reading frame and are classified as 392 amino acids with codes called VpSTS7, VpSTS6, VpSTS5, VpSTS4, VpSTS3, VpSTS2, and VpSTS1 respectively. Amino acid multigene alignment analysis showed that these amino acids have the higher identity with other thirteen plants’, the highest percentage of which is 99%; among them, the percentage of sequence identity of VpSTS7 amounts to 99% with Vitis ripariaVrSTS2, and Vitis vinifera VvSTS,VVoSTS, the percentage of sequence identity of VpSTS,VpSTS3,VpSTS5 and VpSTS7 amounts to 98% with VaSTS2 , and 97% with PhSTS, PqSTS and CrSTS , 96% with VvvSTS, VvRS1, VaSTS1,V1STS and VrSTS; and the identity between VpSTS2 and VpSTS6 is 99%, and these two have 95% of identity with VpSTS1; VpSTS1,VpSTS2, and VpSTS6 shows 94% of identity with STS of other plants . 3. According to the design primer of T11AC/B0313-307 cDNA fragment,RACE has been used to clone 5’complete sequence;then ,after splicing their overlapping part, 5’cDNA is combined with T11AC/B0313-307 to form a whole cDNA sequence whose size is 1708 bp.When this cDNA fragment is analyzed by open reading frame(ORF),the results indicated: T11AC/B0313-1708 has a complete ORF sequencing 523 amino acids with code;the start codon of coded protein is ih 17bp; there are a termiator codon—ATG ,a Poly(A) sequence and 120 base pairs in the 3’end.All suggest that a whole cDNA sequence was obtained.Blast shows T11AC/B0313-1708 has no homology sequence in GenBank.The findings show that vitis pseudoreticulata has a new special gene against Uncinula necator.which will play an important role in resisting Uncinula necator. 4. 5’RACE and 3’RACE have been used to clone one interrelated anti-powdery mildew(Uncinula necator) gene of Chinese Wild Vitis----ascorbate peroxidase .The whole length of its VpAPX 1077bp, which is classified as 250 amino acids with codon . Its start code ATG is in 71bp; its 3’RACE end has multiple terminator codes , the terminator sequence AATAAA and the sequence poly(A); its 3’RACE end in the noncoded area contains 254 bases. Compared with multiple sequences, Blast shows that VpAPX has 86% of identity with Solanum tuberosum, Fragaria x ananassa, Nicotiana tabacum and Z.mays; Compared with Brassica oleracea, Hevea brasiliensis, Spinacia oleracea and Arabidopsis thaliana, it shows that the percentage of their identity amounts to 82%; the percentage of VpAPX identity is 84% with Glycine max, 88% with Zantedeschia aethiopica, 80% with Pimpinella brachycarpa and Suaeda maritima and 81% with Oryza sativa. 5. 5’RACE and 3’RACE have been used to clone the interrelated anti-powdery mildew gene of Chinese Wild Vitis---aldehyde dehydrogenase (ALDH) including the whole cDNA sequences of 5’end and 3’end. There are four cDNA sequences in the 5’end, whose sizes are 760bp, 796bp, 865bp and 870bp respectively; there is one cDNA sequence in the 3’end, whose size is 1498bp. The cDNA sequences in the 5’end and 3’end can combine to form four whole cDNA sequences, whose sizes are 1851bp, 1887bp, 1956bp and 1961bp. Through analyzing these four cDNA sequences, the results indicate:Each cDNA sequence contains a whole open reading frame for coded Vitis aldehyde dehydrogenase, whose sizes of coded amino acids are 537, 537, 524 and 477 respectively; and among these cDNA sequences, amino acids coded 1887bp and 1851bp are absolutely same, therefore, in fact three whole cDNA sequences of Vitis aldehyde dehydrogenase have been formed, that is , 1851bp or 1887bp , 1956bp and 1961bp ,which are named VpALDH2a, VpALDH2b and VpALDH1a respectively. The percentages of their coded amino acid identity highly amount to 83%, 77%, 77%, 77%, 79% and 61% respectively with the identity of Lotus corniculatus, Z.mays, Oryza sativa, Nicotiana tabacum, Arabidopsis thaliana and Human aldehyde dehydrogenase. There is a Catalytic and Active Site in the middle part of coded protein , and the “N”end of coded protein VpALDH2a and VpALDH2b is characterized with the expected line Putative Mitochondrial Targeting Sequence , while VpALDH1a doesn’t have this characteristic. All have shown that VpALDH1a, VpALDH2a and VpALDH2b are certainly the coded Vitis aldehyde dehydrogenase. Coded VpALDH1a gene is Cytosolic, while, Coded genes of VpALDH2a and VpALDH2b are Mitochondrial aldehyde dehydrogenase. 6. According to two conserved motifs of R gene, five primers have been designed to form six pairs of primers, and these six pairs of primers have been used to amplify Vitis Dividii. The result has shown that only primer F1 has amplified R3 to form a special DNA sequence. Then, through the PCR amplification, primer F1 has been used to amplify forty-five Vitis, including Thirty-six individual varieties of twelve Chinese Wild Vitis, V. labrusca kyoho, Vinifera, Chenin Blonc, Pinot noir, Muscat Hamburg, V.Vinifera×V.amurensts Beichun, etc, to form about 500bp DNA sequence. Finally, this 500bp DNA sequence band has been recovered, cloned and sequenced to form forty-seven resistant gene analogs, twenty-eight of which has the open reading frame and the rest has contained at least terminator code. 7. When DNASTAR software is used to analyze forty-seven RGAs nucleotide sequences of Vitis Genera, the result shows: the percentages of their identity varies from 46% to 100%; the 5’end of twenty-eight RGAs amino acid sequence with the open reading frame have a disease-resistant gene conserved motif GVGKTT, and the 3’end has a conserved motif GLPLAL, and percentages of their identity varies from 22% to 100%.更多还原