Functional Verification Of Six Effectors Of Cotton Bollworm And Creation Of New Insect Resistant Cotton Flower Materials

The oral secretion(OS)of insects,also known as regurgitation,is a mixture of saliva secreted by the salivary glands of insects and intestinal secretions.In the process of longterm coevolution between insects and plants,insects have evolved effectors to inhibit plant defense.During insect feeding on host plants,OS is secreted into plants and affects plant defense response.The identified OS effectors mainly come from stinging sucking mouthparts insects.There are few reports about chewing mouthparts insects.Helicoverpa armigera is a lepidopteran insect,which exists widely in nature and seriously affects the production of crops.The oral secretion of Helicoverpa armigera contains a variety of proteins that regulate plant hormone response,and one of the effector proteins can stimulate plants to produce direct and indirect defense.Therefore,the study of the role and interaction mechanism of the effectors of the oral secretion of cotton bollworm in regulating cotton’s defense response to pests will provide an important theoretical basis for the research and development of new cotton resistant to pests.In this study,six candidate effectors were screened from the salivary gland EST database of Helicoverpa armigera using bioinformatics methods,and they were transiently expressed in tobacco to observe whether these candidate effectors could cause phenotypic necrosis of tobacco;Then,through prokaryotic expression experiment and Western Blotting,it was tested whether the effectors could be expressed in vitro,and then transgenic cotton materials were obtained through stable genetic transformation.Through positive detection of transgenic cotton at the molecular level,the expression of q RT-PCR gene was identified to detect whether the effectors were highly expressed in transgenic cotton.The transgenic cotton lines with high expression were selected for insect resistance identification,Detect whether the effector is highly expressed in the transgenic cotton,and conduct insect resistance identification.Find the target gene that the key effector interacts with cotton through yeast double hybrid experiment.Knock out and overexpress the target gene in cotton to obtain cotton materials with insect resistance.It provides a new theoretical basis and germplasm materials for cotton insect resistance research.The results are as follows:1.Through bioinformatics analysis,it was found that the six candidate effectors were all known functional proteins,and four of them belonged to serine trypsin(XM＿021344467,XM＿021340592,EU32555,XM＿021327997),which played an important role in insect digestion and metabolism,signal transduction and immune defense.A trypsin class(DQ847154).One belongs to the coactivation factor(XM＿021341986).Observe whether the candidate effector will cause cell necrosis by injecting tobacco for transient expression.It is found that 7997(XM＿021327997)will cause cell necrosis of tobacco,while the other candidate effectors will not cause cell necrosis of tobacco.2.The candidate effectors were constructed into prokaryotic expression vector p GEX-4T-1 and transformed into E.coli BL21(DE3).Through prokaryotic expression induction,SDS-PAGE and Western blotting were used to detect the purification effect and identify the expression product.The relative molecular weight of the target protein was about 12.19 k D(XM＿03127997),27.01 k D(XM＿021344467),15.16 k D(XM＿021341986),17.72 k D(EU325555),18.96 k D(DQ847154),respectively.The detection results were consistent with expectations,and soluble protein products were obtained.The target genes for interaction between key effector(XM＿021327997)and cotton were found through yeast double hybrid experiment,respectively Ghir＿D09G022580,Ghir＿A13G002860,Ghir＿D10G023950,knockout and overexpression of these three endogenous target genes in cotton are currently in the differentiation and induction stage.3.The six effectors were stably expressed in cotton,and six candidate effector transgenic cotton materials were successfully obtained.Twenty 4467,six 1986,five 0592,23 325,23 8471,and 19 7997 positive plants were harvested,respectively.Later,the expression amount of transgenic positive cotton plants was detected.The detection results showed that the expression amount of foreign genes in 4467-4,4467-10,4467-11,1986-17,1986-14,1986-18,0592-20,0592-22,0592-23,3255-25,3255-26,3255-28,8471-37,8471-38,8471-39,7997-46,7992-45,7997-48 plants was high.The transgenic cotton lines with high expression were selected for the experiment of insect reception(feeding experiment).In order to confirm whether the candidate effectors will affect the feeding of the cotton bollworm,we conducted a preference feeding identification experiment.The results confirmed that the weight of the cotton bollworm that feeds on 1986-18,7997-46 transgenic cotton increased significantly,0.15 g and 0.11 g more than the control group.The results showed that the shape and size of the cotton bollworm that feeds on cotton also changed significantly compared with the control group,The length of the cotton bollworm fed on 1986-18,7997-46 transgenic cotton was significantly increased by 2 mm to 3 mm compared with the control,and it was more plump and vigorous.This result was further verified,and a preference feeding experiment was conducted.The results were similar to the above weighing results.It was found that the leaf area of 1986-18,7997-46 was significantly larger than that of Jin668 control.It was confirmed that the expression of1986-18,7997-46 in cotton inhibited the defense response of cotton.This study identified the key effector in oral secretions of cotton bollworm,which is specific to the control of the cotton bollworm,and will not affect the natural enemies of the cotton bollworm,solve the problem of the resistance of the cotton bollworm to pesticides and Bt cotton,and have a strong use value in the biological control of the cotton bollworm.