| There is abundant resistance in Chinese wild Vitis species. Utilizing these excellent germplasm to breed new grape varieties, which have not only good cultivated characters but also disease resistance, is a long-time target o f disease resistance breeding in grape. In order to gain insight into the molecular mechanism of the pathogeny-induced resistance and to investigate the expression of resistant gene related cDNAs, we initiated a study for isolation of the powdery mildew resistance gene or related cDNAs of Vitis using mRNA differential display PCR. This technique provided a molecular foundation for disease resistance breeding and also holded promise for the study for differential gene expression in fruits.In the vineyard, Uncinula necator was inoculated on the leaves of the grape materials: Vitis pseudoreticulata Baihe-35-1, Vitis vinifera Carignane and FI 6-12-3 to induce the expression of disease resistance. With thereinafter steps: total RNA extraction from leaves at different sampling periods, cDNAs reverse transcription, sequencing electrophoresis, silver staining, Northern blot, sequencing and Blast searches alignment in DNA database, we gained these results:1. To gain the high purity and integrity RNA, the modified SDS/phenol, guanidinum thiocyanate and commercial total RNA extraction kit were employed. These methods all can obtain total RNA from young leaf of grapevine, but only the modified SDS/phenol method can extract undegraded total RNA from mature leaf and ripe fruit skin. Following this efficient procedure, we routinely obtained 191.40μg/g ~261.20μg/g total RNA from leaf and 31.50 μg/g from ripe fruit skin. The ratio of A260/A280=1.8-2.0. The RNA is highly stable and sufficiently pure for DDRT-PCR, RACE, Northern hybridization and cDNA library construction.2. By comparing key factors in DDRT-PCR, the DDRT-PCR reaction system for Vitis was established. The optimizing reaction system contained 1.0uL cDNA, 0.25mmol-L-1 dNTPs, 1.5mmol-L-1 MgCb, 2.5mmol-L-1 anchored primer, 2.5mmol-L-1 arbitrary primer, and 1.0 U Taq. This DDRT-PCR system can be a useful tool to study fruits developmental7processes at the RNA level.3. By researching the expression of powdery mildew resistance gene after infection in Baihe-35-1 using DDRT-PCR. Five cDNA fragments related to resistance powdery mildew were identified using this approach. T11CA/B0313-410 (Vptual) is 410bp, T11CA/B0304-474 (Vprdrf 2) is 474bp, T11CA/B0315-360 (VprdrfS ). is 360bp, T11CA/B0322-280 (Vprdrf4) is 280bp, TilGC/S11-428 (Vprdrf7) is 428bp. Vprdrf 7, which has no alignment in DNA database, is a new gene of powdery mildew resistance. The accession numbers of GenBank database are CD662166, CD347662, CD347663, CD347664, CD347666.4. By comparing the differential expression cDNAs of Carignane and 6-12-3, 6 sensitive disease reduced cDNA fragments, T11GC/B0316-220, T11GC/B0316-450, T11CA/B0314-960, T11GC/B0316-440, T11CA/B0304-420 , T11GC/B0316-220 , T11CA/B0320-210 were gained. All of these fragments displayed similarly on Carignane and 6-12-3 after the pathogeny infection, were screened using this technique. Besides, 5 specific expression cDNA fragments of Carignane, T11GC/B0316-410 , T11GC/B0316-250, T11CA/B0304-420, T11CA/B0340-720, T11CA/B0316-1000 and 12 specific expression cDNA fragments of 6-12-3, T11GC/B0316-265 , T11GC/B0316-430, T11CA/B0320-390 , T11CA/B0304-390 , T11CA/B0304-550 , T11CA/B0313-290, Tl 1CA/B0313-340 , Tl 1CA/B0313-380 , Tl 1CA/B0313-450 , T11CA/B0313-650 , T11CA/B0313-780, T11CA/B0304-470, were also obtained.5. Sequencing and alignment analysis shows: Vptual, which is 410 bp, encodes the leaf a-tubulin gene of Vitis. The transcription of Vptual is depressed in the process of inducing. It is a down-regulated cDNA. Vprdrf4,partial sequence of leaf ribosomal protein of Vitis, the length of Vprdrf4 is 280 bp. By reason of the expression of it is enhancing during the course after fungi infected, Vprdrf4 is up-regulated cDNA.
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