| Tomato was high level of consumption and one of the most widely cultivated vegetable crops around the world. The gray mold(Botrytis cinerea), it was one of the most destructive diseases of the tomato, which caused the production loss and low quality and had become one of the main factors to limit high production and high quality of tomato. Chitinase hydrolyzed chitin, which was the main component of great majority of fungal cell wall, and there was a synergetic effect against fungal diseases. Currently, chitinase genes were employed in plant genetic engineering for cultivatuing new cultivars of resistance to plant fungal diseases.In this research, we constructed a plant expression vector p CAMBIA1301-35S-Eu CHIT1, which contained the E. ulmoides chitinase gene Eu CHIT1 driven by the Ca MV35 S promoter. The Eu CHIT1 was introduced into tomato via Agrobacterium-mediated callus transformation and shoot apex transformation. The regeneration plants were acquired by screening and identification. We obtained transgenic plants with resistance to Botrytis cinerea. The main results were summarized as follows: 1 Constructing the plant expression vector contained the Eu CHIT1 gene and genetic transfomation of tomatoConstructing the plant expression vector p CAMBIA1301-35S-Eu CHIT1 by one-step cloning. The p CAMBIA1301-35S-Eu CHIT1 carryed a E. ulmoides chitinase gene Eu CHIT1 driven by the Ca MV35 S promoter and a selectable maker gene Hyg. Gain of regenerated p CAMBIA1301-35S-Eu CHIT1 was transformed by freeze-melt method into Agrobacterium tumefaciens strain LBA4404. We obtained 38 transgenic plants by Agrobacterium-mediated callus transformation and gained 12 transgenic plants by Agrobacterium-mediated shoot apex transformation. 2 Effect of Eu CHIT1 gene expression on tomato chitinase activitiesThe chitinase activities in wild type and transgenic plants were mensurated by snail enzyme digestion method. The results showed that the chitinase activities in transgenic plants was 2059.48 U·g-1, 62.14% higher than that in wild type. This indicated that expression of the transgene Eu CHIT1 in tomato plants increased chitinase activities. 3 Effect of Eu CHIT1 gene expression on tomato resistance to Botrytis cinereaThe effect of transgene expression on fungal resistance was conducted by determining onset time and lesion size of infection by Botrytis cinerea on tomato leaf explants. The results showed that the lesions of the transgenic and wild type leaf explants appeared for 2 days and 4 days after inoculation respectively. The transgenic plants occur Botrytis cinerea disease lately 3 d compared to wild type, and the mean diameter of necrotic lesion in transgenic leaf explants was 26.28% smaller than in wild type, reached to 6.93 mm for 4 days post-inoculation. This suggested that expression of the transgene Eu CHIT1 in tomato plants conferred resistance to Botrytis cinerea infection. 4 Effect of Eu CHIT1 gene expression on tomato protective enzyme activitiesThe SOD, POD, CAT activities and the MDA content in wild type and transgenic plants were determined before and after 6 days inoculation with Botrytis cinerea. Before inoculating pathogen, the POD activities in transgenic plants were 48.63% higher than that in wild type, reached to 2825.85 U·g-1(FW); MDA contents in transgenic plants were 28.28% lower than that in wild type, reached to 21.84 nmol·g-1(FW); SOD and CAT activities had not significant difference between wild type and transgenic plants. However, after inoculating to Botrytis cinerea, SOD, POD and CAT activities in transgenic plants were 19.48%, 116.08% and 53.80% higher than that in wild type, respectively, reached to 510.44 U·g-1(FW), 5423.92 U·g-1(FW) and 603.59 U·g-1(FW), and the MDA contents in transgenic plants were 37.65% lower than that in wild type, reached to 26.49 nmol·g-1(FW). This indicated that expression of the transgene Eu CHIT1 in tomato plants enhanced the antioxidant capacity, and reduced damage to Botrytis cinerea. 5 Effect of Eu CHIT1 gene expression on tomato pathogenesis-related protein genesThe transcript levels of tomato pathogenesis-related protein genes in wild type and transgenic plants were analyzed before and after 6 days inoculation with Botrytis cinerea. The results showed that the relative expression level of pathogenesis-related protein genes PR-1a?PR-2 and PR-5 in transgenic plants were respectively 2.23, 11.69 and 1.80 folds than in wild type. The relative expression level of PR-NP24 in transgenic plants had no significant difference compared to wild type. But after inoculating to Botrytis cinerea, expression of PR-1a, PR-2, PR-NP24 and PR-5 in transgenic plants were higher significantly than in wild type, an increase of 4.35, 10.37, 16.58 and 5.02 folds, respectively. This indicated that expression of the transgene Eu CHIT1 in tomato plants increased relative expression level of pathogenesis-related protein genes and enhanced disease resistance of transgenic plants. |
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