Clonging Of Two Bt Genes And Construction Of Plant Expression Vector In Which Fused The Bt Genes

In recent years, plant insect-resistant genetic engineering has been deeply studiedand applied. Ecological safety and rational use of transgenic plants have also been widelyconcerned around. In the study and evaluation of ecological security of the transgenicplants, The effective ways and reasonable use methods are being explored by which toavoid the adversely ecological environment caused by the transgenic plants. However,the ecological security evaluation and rational utilization of the transgenic plants must befounded on the fully understanding of the resistant selectivity of the transgenic plants.Currently, a lot of work has been carried out in genetically engineered insect-resistantpoplar breeding worldwide, by which many different insect-resistant genes have beentransferred into poplar successfully. Bacillus thuringiensis (Bacillus thuringiensis, calledBt) was the most deeply researched and utilized widely as insect-resistant genes update.According to the insecticidal characteristic encoded by Bt insecticidal protein, The Btgenes could be divided into six major categories. However, anti Btcryâ… lepidopteran pestsand pest resistance Coleoptera BtCryâ…¢gene were the most widely used in poplarinsect-resistant genetic engineering at present.So far, Transfer-Bt gene has been achieved initial results on non-target insects,edaphon, mammalian and human impact of such research. Research showed that thetransgenic insect-resistant poplars with different type genes of Bt had certain selectedresistances on insects, and had not toxic on non-target insects. It showed that transfer-Btgene in poplar had few insecticidal spectrum and well-targeted features. On the otherhand, it also showed that it had good safety, and had not harm to non-target insects andother biological hazards. In order to expand insecticidal spectrum and improve resistanceeffect, transferred different types of Bt hybrid trees were mix-planted or the differenttypes of Bt gene were constructed in one expression vector. By this way, transgenicpoplar trees with multiple Bt genes were generated by gene transformation. Theinsect-resistant capacity of transgenic trees could be greatly enhanced by complementaryinsect-resistant characteristic of a variety of insect-resistant genes in productionapplicationsIn the experiment, the pCAMBIA1305-Bt1 plant expression vector fused Bt1 gene, the pCAMBIA1305-Bt3 plant expression vector fused Bt3 gene, and thepCAMBIA1305-Bt1-Bt3 plant expression vector fused all Bt1 and Bt3 gone wereseparaterly constructedThe main results were summarized as follows:1. Bt1 gene was cloned from pBin438 by PCR. The result of sequence analysis showedthat length of the gene was 1836 bp. It was 100% identity to the original sequence of Bt1(reconstructed sequence).2. Bt3 gene was cloned from pBCC3 by PCR. The result of sequence analysis showedthat length of the gene was 1794 bp. It was same as the Bt3 gene released in NCBIGenBank(accession No. M84650).3. In the experiment, The Bt1 gene was inserted into the position between CaMV35Spromoter and NOS terminator of expression vector pCAMBIA1305, based on DNArecombinant technology. The binary expression vector pCAMBIA1305-Bt1 has beenconstructed.4. In this experiment, the Bt3 gene was inserted into the position between CaMV35Spromoter and NOS terminator of the expression vector pCAMBIA1305 by DNArecombinant technology. The pCAMBIA1305-Bt3 plant expression vectors has beencreated.5. 35S-Bt3-NOS gene segment was cloned from pCAMBIA1305-Bt3 by PCR. The35S-Bt3-NOS gene segment was inserted into the position between Smaâ… and Hindâ…¢of media expression vector pCAMBIA1305-Bt1. The pCAMBIA1305-Bt1-Bt3 plantexpression vectors fused two genes Bt1 and Bt3has been achieved.