Insect-Resistant Identification And Evaluation Of Transgenic Cry1C* Cotton Insect-Resistant Identification And Evaluation Of Transgenic Cry1c* Cotton

Cotton is a combination of fiber and cottonseed,which plays an important role in field crops.With the commercialization and scale of transgenic insect-resistant cotton all over the world,the planting proportion of Bt transgenic insect-resistant cotton is increasing year by year.While bringing economic benefits to farmers,the number of cotton bollworms of main target pests decreases sharply,which is effectively controlled in cotton fields where Bt transgenic insect-resistant cotton is planted.But at the same time,the target pest population has the risk of resistance to Bt protein,and secondary pests such as Spodoptera litura Fabricius have gradually become the main pests harmful to cotton.Using genetic engineering technology to create new varieties of cotton insect-resistant genes is an effective way to solve the current problems.In this paper,using the modified new insect-resistant gene Cry1C*gene,transgenic cotton plants with Cry1C*gene were obtained through a large number of genetic transformation,and their progenies were tested for transgenic copy number,Cry1C*gene expression,Cry1C*protein content,insect-resistance bioassay and so on to evaluate the insect-resistant effect of Cry1C*transgenic cotton lines.The main results are as follows:1.The results of conserved domain prediction show that Cry1C*gene has three conserved domains,which belong to Endotoxin＿N,Endotoxin＿M and Endotoxin＿C families respectively.The amino acid sequence was aligned with Cry1C protein,and the similarity was 84.39%.2.A total of 15 transgenic plants of T0 generation were obtained in the early stage.After protein content determination,6 plants with high Cry1C*protein content and 1transformed plant with low protein content were selected as control.The copy number of T3 generation plants of 7 transgenic plants were identified,of which 4 materials were double copies,1 material was multiple copies,1 material showed multiple copies and single copies,and one material did not detect inserts.3.In the indoor bioassay,gene relative expression and protein content of T₃lines were determined,and the relative gene expression and protein content of T₄lines were detected.The relative gene expression and protein content of stem tissue and root tissue of T₄line were detected.There was a large difference in relative gene expression among different lines.There was also a big difference in leaf protein content among different lines.Among them,the leaves of T₃and T₄generation of BT27 strain were resistant to the second and fourth generation Helicoverpa armigera,and moderately resistant to the third generation Helicoverpa armigera,slightly lower than the existing insect-resistant cotton GK19;to the second and third generation of Spodoptera litura Fabricius,significantly better than the existing insect-resistant cotton GK19;it was resistant to the second and third generations of Spodopter aexigua,which was significantly better than the existing insect-resistant cotton GK19.4.The relative expression of Cry1C*gene and the content of Cry1C*protein in the leaves of the same transgenic line were compared at different stages.The relative expression of Cry1C*gene in the leaves of T₃lines increased at first and then decreased from seedling stage to bud stage and then to boll stage,while the leaf protein content of T₃lines decreased gradually from seedling stage to bud stage and then to boll stage.The relative expression of Cry1C*gene in leaves of T₄line increased at first and then decreased from seedling stage to bud stage and then to boll stage,and the relative expression of leaf Cry1C*gene in seedling stage and boll stage was at the same level.The relative expression of Cry1C*gene in stem tissue and root tissue of T₄line was at the same level at seedling stage and bud stage,and the content of Cry1C*protein in leaf,stem tissue and root tissue of T₄line had no significant difference at different stages.5.In the experiment of boll resistance of T₃strain to the third instar bollworm,only the boll of BT27 strain had not been gnawed into the interior of the boll by the third instar bollworm.6.After 10x deep resequencing of BT27 lines,it is found that the T-DNA region of the vector has been integrated into the genome A01 genome of the receptor.The foreign gene is positively inserted into cotton genome A01 from LB-RB,and the left boundary insertion site was 27401103.