Construction Of The Insect-resistant Gene Cry1Ab13 Plant Expression Vector And Transformation In Maize

Maize, the important economic crop in world and the first major food crop in china, has an extensive application in all aspects of social production. The efficiency of maize breeding was significantly improved by the application of transgenic technology, which has played an important role in breeding insect-resistant, disease-resistant, stress-resistant, high-yield and quality improvement. The insect-resistant transgenic maize was most widely applied and most quickly developed among those transgenic maize varieties.The maize production in china has been affected by pests seriously among which the damage caused by the lepidopera Asian corn borer(ACB,O.furnacalis) was the most serious. The maize production has been reduced 10%-30% by corn borer each year in china. The resistance of maize to corn borer can be improved significantly with the introduction of insect-resistant gene into maize by genetic engineering technology.Cry1Ab13 gene fragment was cloned from the cloning vector pUC57-cry1Ab13 and was ligated to plant expression vector pCAMBIA3300-35S-Nos-35S-Bar-Nos by homologous recombinant method, through which the recombinant plant expression vector with Bar gene as a selection marker was obtained. The vector was transfered into maize inbred line H99 by Agrobacterium. The integration and transcription of cry1Ab13 gene were detection and the resistance of transgenic maize lines to corn borer were analyzed indoor and at field.These conclusions were drawn by this study:1. The plant expression vector pCAMBIA3300-35S-cry1Ab13-Nos-35S-Bar-Nos with a Bar gene as selection marker was obtained by cry1Ab13 cloned from cloning vector pUC57-cry1Ab13 inserted into the basic plant expression vector pCAMBIA3300-35S-Nos-35S-Bar-Nos.2. Engineering bacteria was constructed and expression element 35S-cry1Ab13-Nos-35S-Bar-Nos was transfered into maize inbred line H99 by Agrobacterium. 8 T0 generation transgenic plants were obtained by herbicide screening and PCR detection.3. The seeds of the positive transgenic plants after self-pollination were obtained and planted. 200 positive transgenic plants were obtained by generational herb screening and PCR detection.4. 5 positive transgenic plants appeared obvious hybridization bands by southern blotting detection to some T2 generation transgenic plants. There are three copies of cry1Ab13 gene in T2-2-3-1 and single copy in others. Besides, the length of hybridization bands were different among different transgenic event.5. The southern blotting positive plants were detected by Quantitative Real-time PCR to analysis the expression of cry1Ab13. The result show that the cry1Ab13 gene has transcribed in each plant detected except T2-2-3-1. The expression level exists obvious differences not only in the same tissue from different plants but also in different tissues from same plant. The expression level of different tissues arranged in order of relative amount was as below:leaf>bract>filament.6. The resistance identification of transgenic plant to corm borer experiments were conducted indoor and at field. The results show that the resistance to corn borer of transgenic plants were improved significantly and achieved a good insect-resistant effect compared with non-transgenic control.7. The investigation results of agronomic traits show that the hundred-grain weight was significantly improved without the condition of pest control and no great changes in other agronomic traits compared with non-transgenic control.