Cloning And Analysing For The Gene Sequences Of Resistance To Uncinula Necator In Chinese Wild Vitis Species

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%.