Study On Brown Planthopper Resistant Transgenic Rice By Transformation Of Cry30Fa1 Gene

In this study, we transferred Brown Planthopper resistance gene Cry30Fa1 into excellent rice restorer line Shuhui 818 (R818) which as receptor material by using Agrobacterium-mediated transformation method to obtain the target gene homozygous transgenic lines through successive inbreeding and DNA detection in T6 generation. By PCR detection and resistance expression identification of target genes, we determined selection marker gene Hyg (R) and insect resistant gene Cry30Fa1 in T5 and T6 generation transgenic materials, finally screening marker-free transgenic rice lines. Transcription of Cry30Fa1 gene expression was detected by RT-PCR method. At the same time, RT-PCR techniques were used to detect the Cry30Fa1 gene copy numbers in the transgenic lines. The Cry30Fa1 gene protein expression was detected by Western Blot method. Insect-resistance of 3 transgenic lines were analyzed with the methods of pest resistance identification in seedling group, individual identification of pest resistance in seeding stage and field identification of pest resistance, respectively, and finally screening the above level of moderate resistance in transgenic rice lines of Brown Planthopper. In addition, we investigated the main agronomic traits of T6 generation transgenic rice lines. The main results were as follows:1. By PCR detection to 184 transgenic lines of T5 generation, we found 150 lines containing Cry30Fa1 genes, and 29 transgenic homozygous lines with target genes have been screened through tracking detection in T6 generation. Using PCR detection and soaking expression detection to identify Hyg (R) gene in 29 transgenic lines, finally obtained 15 marker-free transgenic rice lines.2. By RT-PCR technique, the expressions of Cry30Fa1 genes at transcriptional level were detected in T6 generation of 29 transgenic lines. The results showed that Cry30Fa1 genes in all transgenic lines were expressed at the transcriptional level. However, the gene expression level had great difference, which ranged from 0.19 to 8.53.3. By Western Blot technique, the protein expressions of the Cry30Fa1 genes were detected in T6 generation of 29 transgenic lines. The results showed that protein expressions were not detected in 5 transgenic lines, and the remaining 24 transgenic lines detected the Bt protein expression, but the expression level existed significant difference, which were from between 0.13 to 11.6.4. By analyzing Cry30Fa1 gene copy numbers of transgenic lines, we found that there were great differences among the 29 transgenic lines, however, the copy numbers of the 15 lines belonged to the low copy lines.5. By using pest resistance identification in seedling group and pest resistance identification in field identification, we analyzed insect-resistance of 3 transgenic lines. The results showed that seedling resistance reach to 7 grades which belongs to middle susceptible materials, and field resistance reached to 3-5 grades, which belongs to middle resistance to resistance materials. Meanwhile, we selected individual identification of pest resistance in seeding stage to analysis insecticidal property in 3 transgenic lines. Results showed that Brown planthopper mortality rates in 3 transgenic lines were significantly higher than the parent R818, which showed transgenic lines can improve insecticidal ability.6. Through analyzing 29 transgenic lines main agronomic traits, the results showed that exogenous Bt gene have greatest impact on rice height, and there were significant differences between the 19 lines and their parents. Exogenous Bt gene have a little influences on other agronomic traits, such as heading date, effective tiller number, main panicle length, seed setting rate,1000-grain weight etc. By data analysis, there were more improved lines than deteriorated lines, which meanings as long as we put through a combination of molecular level detection and field breeding work, always selecting the transgenic lines which are similar to their parents or even better than their parents.Currently, the remaining 26 transgenic lines are being conducted a comprehensive resistance analysis, hoping to gain more resistance to Brown planthopper transgenic lines.