Fully Motified Bt Insecticidal SCH Gene, Constructed Plant Expression Vectors And Obtained Transgenic Indica Rice

Pests seriously threaten agricultural production. The genetic engineering of insect-resistant plants has been proved very effective in the Integrated Pest Management Program. On the other hand, pests will develop resistance to insect-resistant transgenic plants with the wide planting of these plants. The fully motified gene can not only broaden insecticidal spectrum, it may also delay the process of insects' developing resistance. The study is summarized as following:1. For the purpose of obtaining transgenic rice with mosquitocidal activity, wild-type cryllBal gene (NCBI:X86902, encoding a 81,293Da crystal protein of Bacillus thuringiensis subsp. jegathesan. Purified cryllBal inclusions were highly toxic for mosquito larvae of the species Aedes aegypt, Culex pipiens, and Anopheles stephensi.). The potential processing sites affecting its levels of transcription and translation and conducing mRNA stability were fully motified, such as PPSS sequence, ATTTA sequence, CATTG sequence, and so on. Then, optimizing outer-gene code, we striping Kozak and signal peptides sequences in 5' precursor sequence and striping HDEL and double terminators sequences in 3' UTR section, respectively.2. In this study, Empolying the amino acid insect-specific diptera toxin cryllBal gene was designed according to bias in codon choice of indica rice, and was chemically synthesized by using successive PCR. Modifications and optimization in nucleotide sequence of cryllBal protein. The overall G+C content and G+C contend of bases at third position in codons of the synthesized gene were 49.42% and 65.10%, respectively, while that of the original cryllBal gene are 32.97% and 20.69%, respectively. The synthesized gene was cloned into pBluescript IISK vector and sequenced. Results of sequencing for recombinant plasmid were correct completely. The synthesized gene was named SCH(Sigf\al-CryJlBal-liDEL).3. The newly synthesized SCH gene was cloned into binary vector pCAMBIA-1300 and pCDMAR-Hyg, in which the expression of the SCH gene was under the control of ubiquitin promoter, actin promoter and Nos terminator, respectively. So, Monocotyledones plant expression vector harboring SCH gene and bi-vector harboring SCH & crylA(c) genes were constructed. In pCDMAR-Hyg system, a marker gene and SCH gene are placed on separate transfer DNA (T-DNA), and the T-DNAs are introduced into plant genomes as unlinked fragment. Consequently, the non-selectable genes separate from the marker genes in the progeny of transgenic rice.4. The recombinant plasmid containing the expression cassette was then introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation. Transgenic rice plants were obtained by Agrobacterium-mediated callus transformation, the callus derivated from the immature embryos of indica rice Minhui 86, and their sensitivity to Hygromycin was tested.287 resistant clones were obtained.5. Integration and expression of foreign gene were verified in a number of transgenic indica rice Minhui 86 by PCR and ELISA analysis. In the ELISA analysis of Bt protein content in the leaves of transgenic rice, the average toxin was about 0.243% of total expressed proteins, the result proved that plants with the SCH gene had a 250-fold higher level of toxin protein compared with the wild-type gene.6. Segregation ratio of transgenic rice T0 seeds were detected by Hyg-resistant. The results indicated that segregation ratio of transgenic rice T0 seeds was chaos.