| Sugarcane is an important sugar crop,but it has been a great loss of crop yield due to insect pests.Bt gene has broad spectrum insecticidal property,and the cultivation of transgenic insect-resistant varieties provides a new idea for pest control of sugarcane.Cry1Aa,CrylAb and Cry1Ac are the most widely used in transgenic insect-resistant breeding in Bt insecticidal proteins.They’re highly homologous and they have the same binding site on cadherin-like protein which do not overlap completely.Insects have strong interactive resistance to these three toxins.So CrylA toxin protein is difficult to adapt to the need of resistance management.As the second generation of insecticidal genes,Cry2A gene has a low homology with Cry1A gene.And it has different binding sites on the insect brush border membrane which is helpful to improve the resistance of target pests to Bt insect-resistant genes.In order to identify the control effect of Cry2A gene on sugarcane pests,genetic transformation of Cry2A gene was carried out on sugarcane.The main research results of this topic are as follows:(1)We successfully constructed the Cry2A gene efficient plant expression vector Cry2A-3300 driven by the corn Ubi promoter,and conducted the identification of recombinant plasmids.The result shows that recombinant plasmids can be used for subsequent genetic transformation.(2)Using Agrobacterium-mediated technology to genetically transform sugarcane embryogenic callus materials,a total of 46 resistant plants were obtained through screening and regeneration culture.(3)We conducted PCR amplification test on resistant plants,25 plants were positive,and the positive rate reached 54%.(4)Southern Blot test was carried out on the positive plants.The detected transgenic strains showed hybridization signals,and the exogenous genes were successfully integrated into the sugarcane genome with 1-4 different copy numbers(5)The positive plants were detected by RT-PCR,and all positive plants were effectively expressed at the transcriptional level.(6)Fluorescence quantitative PCR analysis of the obtained transgenic strains revealed that the expression of Cry2A gene was different among different transgenic strains.The expression of Cry2A gene was the highest in Cry2A-2,which was 12.35 times higher than the strains with low expression.(7)We conducted ELISA analysis on the obtained transgenic plants and found that different strains were also different of the protein expression.The expression of Cry2A protein was the highest in Cry2A-2,which was 12.72 times higher than the strains with low expression.(8)Insect resistance identification of transgenic plants had been done with cotton bollworm larvae which grew in the same state.We fed insects with feed containing transgenic and non-transgenic sugarcane leaves.It was found that the Cry2A gene can effectively inhibit the growth of pests,which will be beneficial to field control later.By comparing the results of fluorescence quantitative PCR,ELISA and indoor anti-insect identification,it was found that plants with different expression levels of gene Cry2A and protein expression levels showed different insect resistance.The higher the expression in some plants,the stronger the insect resistance of the strain was. |
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