Study On Cry1Ca Receptors Of The Spodoptera Litura

The popularization and application of transgenic Bt cotton has effectively controlled the damage of various lepidopteran pests to agricultural crops,and as a biological control method,it has significant ecological,economic and social benefits.Currently,most of Bt cotton planted in China are expressing Cry1 Ac gene.Although they can effectively control the main target insect pests,such as Helicoverpa armigera,their control effects on Spodoptera litura are still minor.In order to continuously applicating Bt cotton,it is necessary to look for other Bt proteins which can expand the insecticidal spectrum of existing Bt cotton.In this paper,we find that Cry1Ca7 protein(China has its independent intellectual property rights)has a significant better control effect on Spodoptera litura than Cry1 Ac protein through bioassay experiments,so further research on the insecticidal mechanism of Cry1 Ca protein against Spodoptera litura was carried out.The following experiments were performed:1.The results of bioassay showed that Cry2 Ab had high insecticidal efficiency against all the six species of lepidopteran pests;Cry2Ah had good lethal effect on H.armigera,S.exigua,S.litura and had certain efficiency against O.furnacalis and C.suppressalis;meanwhile it had significant weight inhibition effect on M.separate rather than lethal effect.Cry1 Ca had obvious high control efficiency against S.litura,and also good effects on S.exigua,H.armigera and C.suppressalis.Vip3 Aa had good efficiency against S.exigua and S.litura,and adequate effects on O.furnacalis,H.armigera and C.suppressalis.2.Transmission electron microscope was used to observe the pathological changes in the midgut tissues of Spodoptera litura after feeding on Cry1 Ca protein.The results showed that both the columnar and goblet cells in the control group were neat and orderly,slender and dense,the nucleus was round or oval,the nuclear membrane was clear,the chromatin was evenly distributed,the inner ridge of mitochondria was clearly visible.The endoplasmic reticulum was linearly arranged in the cytoplasm.After feeding the diet containing Cry1 Ca toxin,the microvilli of columnar cells and goblet cells were swollen and distorted,the shape of the nucleus was elongated or deformed,there was no obvious nuclear membrane and the chromatin distribution was uneven,and the mitochondria were deformed.The endoplasmic reticulum becomes disorganized,swollen and deformed,and the number is reduced.3.By establishing a receptor pull-down platform,the Cry protein was first coupled with the pre-activated medium,BBMV of Spodoptera litura was then passed through the medium mentioned above to wash away non-specific binding proteins and contamination,and the specific binding protein was detected by SDS-PAGE and samples were sent to the company for LC-MS-MS(Q-TOF)analysis to classify them.The results showed that Cry1 Ca can bind with APN,ACTIN,Polycalin,ABCC2 and some other proteins.4.According to the result of receptor pull-down,the ABCC2 gene was selected for further study.RT-q PCR was used to detect the different expression of ABCC2 gene in different tissues and different instars of Spodoptera litura.It was found that the relative expression of ABCC2 was increased with the development of Spodoptera litura larvae.The expression level in the midgut and Malpighian tubules were significantly higher than other tissues,which is in accordance with reports indicating ABCC2 transporter is involved in metabolism and transport functions.The ABCC2 extracellular membrane fragment was expressed by prokaryotic expression and the protein was extracted and purified(about 47 k Da).Ligand blot experiments demonstrated that it can bind specially with Cry1 Ca in vitro.The p Ac5.1B-EGFP-ABCC2 plasmid was constructed and transfected into Sf9 cells for function identification.The results showed that the sensitivity to Cry1 Ca and Cry1 Ac in Sf9 cells transfected with ABCC2 gene was significantly increased(P<0.05).In conclusion,the toxicity of Cry1 Ca against Spodoptera litura was measured and some binding receptors were identified.Their functions were also verified.Our results provide a theoretical basis for the prevention and control of Spodoptera litura and give suggestions for better application fo the transgenic cotton.