| Locusta migratoria manilensis(Meyen)is a major economic pest in the world.The methods of controlling locusts are mainly biological control of Metarhizium anisopliae and chemical control of organic pesticides.Biological control is widely used in agricultural production with the advantages of harmless to humans and animals,no residue,and no drug resistance.However,it can not overcome the disadvantages such as low lethal efficiency and long lethal time.One of the main reasons for this problem is that locusts have a strong immune defense response.If we can directly inhibit the immune response of locusts,we can improve the lethal efficiency of M.anisopliae.The study of FK506 binding protein is helpful to understand the immune mechanism of insects and provide new ideas for the development of new biological pesticides.In this paper,the FKBP52 gene of L.migratoria manilensis was cloned,and the effect of FKBP52 protein on the infection of L.migratoria manilensis by M.anisopliae was determined by purified protein treatment and RNAi,respectively.The main results of this study are as follows:1.Combined with the previous laboratory data analysis of L.migratoria manilensis midgut transcriptome,FKBP52 gene was successfully cloned.The results of sequencing showed that the open reading frame of the gene was 1242bp,encoding 413 amino acids.The protein had two conserved domains of FKBP_C superfamily between amino acids 38～130 and amino acid 158～247,which belongs to one of the members of FKBP_C superfamily.The FKBP52 gene was introduced into the expression vector to express and purify the target protein,and the specific antibody was prepared.2.qRT-PCR and Western Blot techniques were used to detect the expression of FKBP52 gene in different tissues of adult and in the midgut of nymph with different instars.It was found that FKBP52 gene was expressed in the integument,hemolymph,fat body,leg,midgut,testis and ovary of both male and female adults,and the expression was the highest in the midgut of male adults.Taking the expression of FKBP52 gene in the integument of male adults as the standard parameter,the expression of FKBP52 gene in the midgut of male adults was 4.13 times higher than that in the integument,and FKBP52 gene was expressed in egg,the 1st instar nymph body,2nd-5th instar nymph,female and male adults midgut,among which the expression level was the highest in egg.Taking the expression of FKBP52 gene in the 1st instar nymph as the standard parameter,the expression of FKBP52 gene in egg was 3.89 times higher than that of 1st instar nymph,which was significantly higher than that of other stages.3.The effect of purified protein on the infection of L.migratoria manilensis by M.anisopliae was detected by qRT-PCR and enzyme-linked immunosorbent assay(ELISA).The results showed that when M.anisopliae and FKBP52 protein were used to treat locusts at the same time,the cumulative mortality of increased rapidly,reaching 93.33%on the 10th day,which was significantly higher than that of M.anisopliae alone(60.00%),and the activities of peroxidase(POD),superoxide dismutase(SOD)and calcineurin(CaN)decreased significantly.The expressions of MyD88,Dorsal,Cactus and Defensin genes downstream of humoral immune Toll pathway were significantly decreased at the transcriptional level.4.In order to further verify the function of FKBP52 gene,we used RNA interference technique to detect the effect of interference FKBP52 gene on the infection of L.migratoria manilensis by M.anisopliae.The results of RNA interference showed that FKBP52 interference could significantly enhance the immune ability of locusts to inhibit M.anisopliae infection(the cumulative mortality rate of 10 days was only 35.56%);the activities of calcineurin and protective enzymes of locusts increased significantly after FKBP52 interference;the expressions of MyD88,Dorsal,Cactus and Defensin downstream genes of humoral immune Toll pathway were significantly increased at the transcriptional level after FKBP52 interference. |
PDF Full Text Request