Analysis Of Expression And Aphid Resistance Synthetic Anti-aphid Gene (ASGNA) In Arabidopsis

Cotton is the main material in the textile industry with an important economic value. Cotton plant is esaily subjected to damage by pests and diseases, which decrease yield and quality, even cause economic losses. Aphid is one of the most important pests of cotton. In recent years, aphidis cause damage to cotton is becoming more and more serious with the improvement of irrigation conditions and ecological environment in agriculture. For a Long-term, the prevention and controlling of aphis mainly rely on chemical pesticides, which leads to serious predators of aphids destruction and the pollution of environment. Therefore, cultivating the high resistant quality cotton has been the goal of breeders.With the rapid development of molecular biology and recombinant DNA technology, one means of controlling pests is using gene-engineering technology to transfers exogenous insecticidal gene into cotton and obtain transgenic insect-resistant cotton. Snowdrop lectin (Galanthus navalis agglutinin, GNA) is from the snowdrop, which has good resistance to Homoptera sap-sucking pests (such as rice planthopper and aphids). GNA can effectively inhibit the growth and reproduction of pests, but harmless to human beings、animals and the natural enemies of the aphids. As a result, GNA has a good application prospect in the research of transgenic insect-resistant cotton.In this research, on the base of electronic cloning snowdrop lectin (GNA), we optimize and exchange its codons, artificial synthesis a new gene that express in dicot plants through over-lap PCR. Two expression vector35S-ASGNA and D7-ASGNA containing a synthesized gene encoding snowdrop lectin (Galanthus nivalis agglutinin,GNA) driven by35S constitute promoter and D-7inducible promoter were constructed and then transferred into Arabidopsis thaliana by Agrobacterium-mediated transformation method. Five transformed pure plants were derived. PCR analysis was performed and the exogenous gene integration was confirmed.The aphid resistance bioassay was carried out under artificial condition; and some transgenic lines demonstrated strong against aphid resistance capability. Based on these researches, we tried to transfer ASGNA into cotton using Pollen-Tube Pathway. The main results are as follows:1. By using bioinformatics softwares, LECGNA2was obtained (gene sequence GenBank:M55556.1).On the basis of not changing the sequence of amino acids,122bit A changed to T,290bit G changed to T,299bit G changed to A,350A changed to T,365T changed to C,434G changed to T,470A changed to T,482G changed to T, making it more suitable express in dicotyledonous plants, this new snowdrop lectin gene named ASGNA and12primers were designed and synthesised according to over-lap PCR.2. A new snowdrop lectin gene (ASGNA) synthesized through over-lap PCR. Full-length of512bp,17nucleotides in the5’untranslated region,21nucleotides in the3’untranslated region and a length of474bp ORF, encoding157amino acids.3. Using vectors of pGEM-T easy, pEASYTM-T5Zero、pKannibal and pART27, two expression vector35S-ASGNA and D7ASGNA containing a synthesized gene encoding snowdrop lectin (Galanthus nivalis agglutinin,GNA), which driven by35S constitute promoter and D-7inducible promoter,espectively were constructed.4. Two expression vector35S-ASGNA and D7-ASGNA transferred into Arabidopsis thaliana by Agrobacterium-mediated transformation method. Under sterile conditions kanamycin screening transformed ASGNA pure plants were derived.5. PCR analysis was performed in the level of DNA and RNA, a new snowdrop lectin (ASGNA) gene was successfully transferred into Arabidopsis thaliana.6. The aphid resistance bioassay were carried out under artificial condition; the aphid reduction rate in different transgene lines ranged from12%to75%based on statistic data and some transgenic lines demonstrated strong against aphid resistance capability. Compared to wild Arabidopsis, transgenic Arabidopsis can decrease the reproduction of aphid greatly. As a result, transgenic Arabidopsis have an inhibitory effect on aphid.7. Explore the methods using pollen-tube pathway transform the new ASGNA gene into cotton.