The Construction Of Recombinant Plasmid Containing Bt Anti-Insect Genes That Is Replicable In Agrobactium Tumenfaciens And Its Genetic Transformation In Rice TP309

Agrobacterium is a Gram-negative and soil-borne phytopathogenic bacterium.It transfers a segment of its Ti(tumor-inducing) palsmid, called T-DNA(transferred DNA) to plants.Finally the bacterial DNA integrates into plant chromosomal DNA in a random fashion.The study of Agrobacterium-plant interactions has been the source of many scientific discoveries including,in the eighties,the development of vectors for plant genetic transformation leading to the production of the first transgenic plants.To date,Agrobacterium is the most widely used system of gene delivery into plants.Virulence(vir) genes of Agrobacterium play the key role in this process.The vir gene system ensures the processing and transfer into the plant nuclear genome of a discrete portion of the Agrobacterium DNA(called T-DNA) flanked by two 25 bp imperfect direct repeats.Rice is one of the most important food crops in the world,and it is also staple food of more than fifty percent population in China.Insect pest which influences the output of rice is a serious problem in provisionment. Traditional approaches like crop-dusting to defend this disease shall make environmental-polluting.At present,cultivating anti-insect rice by genetic engineering is one of the methods to solve this contradiction.The object of this project is to integrate cry1Act gene and vip3A gene which both from Bacillus thuringiensis as the anti-insect genes into the genome of rice through Agrobacterium-meidated methods.Cry1Ac protein has specicif toxicity to lepidopteran insects,while Vip3A protein has specific toxicity to lepidopteran insects.They have different insecticidal spectrum and insecticidal mechanism.Meanwhile, they show synergetic effect to each other.Three pairs of oligos were synthesized,cry1Ac1-S/cry1Ac1-A,cry1Ac2-S/cry1Ac2-A and vip3A-S/vip3A-A.Use these three pairs of oligos and genomic DNA of Bt 4.0718 as the template to amplify upstream of cry1Act gene, downstream of cry1Act gene and vip3A gene.Ligate upstream of cry1Act gene,downstream of cry1Act gene as an intact cry1Act gene.Then put cry1Act gene and vip3A gene under the control of eukaryotic promoter CaMV 35S and Nos terminator.Construct the final binary plamsid pBCAV by cloning these two cassettes into pCAMBIA1300.The plamsid pBACV was transformed into Agrobacterium strain EHA105.Use the recombinant EHA105 strain to infect the calli of TP-309.After selection and generation,some calli that were resistant to hygromycin were generated.Check these calli by PCR,we found cry1Act gene and vip3A gene were successfully integrated into the genome of TP309.Southern blotting and bioassay will be used to test these transformations in the future.