| Disease and pest is an important factor of reducing crops yield,across the globe.For the control of agricultural damage cause by disease and pest,chemical insecticides has extensively used. It more or less can control major pests and thus no great effect have been made to breed varieties resistant to insects.On the other hand, chemical insecticides polluting environment,and it has been posing a threat to the living environment and human health. people always have been looking for a more effective,more friendly environmental, sustainable developmen's way.to control disease and pest.The development of plant genetic engineering opens new prospects for disease and pest control in crop breeding.In last few decades ,several novel genes both from microbial and plant sources have been introduced into plants to control pests. Bacillus thuringiensis endotoxins have been studied extensively and have proved effective against a number of insects falling under the groups of lepidopteran and coleopteran, which feed by chewing plant parts.The insecticidal activity of proteinase inhnhitor also against to lepidopteran .Now it had't been found more gene to be toxic to Homoptera and other major pests.Therefore,developing other pests resistance gene assumes high significance.In the study,the Cplectin gene was transformed into model plant tobacco,transgenic tobacco plants were obtained,and analyzed insect-resistant.On the other hand, the prokaryotic expression vector was constructed. Recombinant plasmid transformed into E.coli and let it expressed reconbinant proteins.The results are follows:1. Using the leaf disc transformation procedure mediated by Agrobacterium tumefacien, the Cplectin gene was transformed into tobacco plants,transgenic tobacco plants were obtained, in which plants were identified by kanamycin-resistant ,histochemistry assay of GUS activity, PCR.2. Investigation of insect-resistance activity of Cplectin transgenic tobacco1) Slug attacked situation of transgenic tobacco and non- transgenic tobacco plantlets in the field were investigated.The results showed non-transgenic tobacco were seriously damaged by slug;and transgenic tobacco were obviously resistant to slug with slightly damage.The average slug attacked indexes were 0.75 and 0.1, respectively.2)Transgenic tobacco and non- transgenic tobacco was planted in greenhouse,and let aphids freely feed between them.Recorded the condition of aphids assemble .The results of field trial showed transgenic tobacco always resistant to aphid.3)The result of aphid bioassay revealed that transgenic plants resisted to aphids and inhibited to the growth of aphid population.The reduction in detached transformed leaf aphid bioassay is 68%,and with 60% reduction in transformed plant aphid bioassay.The result indicated that transgenic plant expressed Cplectin and Cplectin-expressing plant can reduce the population density efficiently.3.Construction of prokaryotic expression vectorThe Cplectin gene was cloned by PCR and insected into PMD18-T vector. The PMD18-T-Cplectin plasmid was digested with Nco I and Xho I to release a fragment containing the coding sequence of Cplectin ,which was inserted into prokaryotic vector PET-28a.The recombinant plasmid had been PCR amplified and enzyme digested,it indicated that the Cplectin gene had successful inserted into the recombinant bacterial expression vector. 4.The recombinant prokaryotic expression vector PET-28a- Cplectin expressed in E.coli.(BL21)The recombinant prokaryotic expression vector Plasmid with the coding sequence of the mature Chimonanthus praecox lectin transformed into E.coli BL21 cell,and which was induced expression through 1mmol/L IPTG.The expressed product was dectected by SDS-PAGE.The result indicated the Cplectin had expressed in E.coli...
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