Molecular Cloning And Functional Validation Of Candidate Effectors From Cotton Bollworm

Cotton bollworm(Helicoverpa armigera)belongs to Lepidoptera noctuidae,which is one of the important pests harming cotton and harms the quality and yield of cotton all year round.With the long-term use of chemical insecticides,the environmental pollution is aggravated,and the large number of transgenic Bt cotton plants lead to the resistance of cotton bollworm to Bt cotton.Therefore,it is necessary to seek more insect resistant means to effectively control pests.The interaction between plants and herbivorous insects makes the proteins secreted in insect saliva affect the plant defense system.For example,elicitors in insect saliva can induce the plant defense response,while effectors in insect saliva can inhibit the plant defense response.By studying how the effectors in the saliva of cotton bollworm affect the defense mechanism of cotton itself,and finding the target of interaction between effectors and cotton,we can provide new research ideas for creating new insect resistant cotton.Starting from the effectors of the cotton bollworm,six candidate effectors were screened from the EST database of the salivary glands of the cotton bollworm through bioinformatics methods,and the target genes were amplified to further construct a super expression vector.The effectors were transiently expressed in tobacco to observe whether they would cause necrosis of leaf cells.At the same time,the cotton target genes interacting with the effectors were screened using yeast double hybrid technology,Knock out and overexpress the corresponding cotton endogenous target genes,obtain cotton transgenic materials,and then conduct insect resistance identification,providing new resistant materials for cotton insect resistance research.The results are as follows:1.The potential gene functions of six candidate effectors were annotated through bioinformatics analysis: neutral lipase(Lipn013),trypsin 0445(Ha OG200445)and 0369(Ha OG200369),proteins containing peptidase S1 domain,are a new type of trypsin,protein1282(Ha OG211282)with serine endopeptidase activity,aminopeptidase N(A3KLP1),and salivary secretory peptide 1277(Ha OG21277).2.By observing whether the transient expression of tobacco will cause cell necrosis,it was found that A3KLP1 candidate effector will cause cell necrosis,and the other candidate effectors did not observe this phenomenon.It suggests that A3KLP1 may inhibit plant defense response.It was observed that Lip013 candidate effectors were located in the nucleus and cell membrane of plants through subcellular localization.The yeast two hybrid experiment was conducted on A3KLP1 candidate effectors,and the target gene 0488(Gh＿D07G0488)and 8960(Ghir＿D12G008960.1)genes interacting with cotton were screened,and the point-to-point verification was conducted.The observation results showed that the interaction was successful.Further,CRISPR/Cas9 and overexpression techniques were used in cotton to verify the function of the interacting target genes.3.The six candidate effectors were used to construct a super expression vector and stably transform cotton.The T0 transgenic cotton materials were obtained.Nine and 11 positive plants of 0445 and Lipn013,10 and 13 positive plants of 1277 and 1282,and 10 and 16 T0 positive plants of 0369 and A3KLP1 were obtained through PCR detection.At the same time,the transcription level of positive plants was detected.The results of q RT-PCR showed that the expression of 0445-3 and 0445-4 was high,the expression of 1277-2 and 1277-9 was high,and the expression of 1282-1 and 1282-20 was high;The expression levels of 0369-7 and 0369-11 were high,and those of A3KLP1-9,A3KLP1-12 and A3KLP1-13 were high.In order to explore whether effectors would affect the feeding of cotton bollworms,the leaf feeding experiment of T0 generation transgenic cotton was carried out.The results showed that the body weight of cotton bollworms fed on the leaves of 0455-3,1277-9 and A3KLP1-12 transgenic cotton increased most obviously,which increased by 0.09 g,0.13 g and 0.1g compared with the control group.After feeding on cotton leaves,the individual size of cotton bollworm was significantly different from that of control group.The length of bollworm feeding on transgenic cotton leaves of 0445-3,1277-9 and A3KLP1-12 increased significantly by 2 mm to 3 mm compared with the control.To further verify this result,the preferential feeding experiment was carried out,and the results showed that the feeding area of the leaves of 0445-3,1277-9 and A3KLP1-12 was significantly larger than that of Jin668 control.These results indicated that the expression of 0445,1277 and A3KLP1 in cotton inhibited the defense response of cotton to some extent.This study preliminarily analyzed the potential functions of insect oral secretions(effectors)through molecular biology,which is of great significance for understanding the complex interaction between plants and herbivorous insects and developing cotton breeding materials with broad-spectrum resistance.