Expression Analysis And Functional Study Of NPC1 Gene In Bemisia Tabaci

Cholesterol is an important substance for the metabolism,growth and hormone synthesis of insect cells,but insects themselves cannot synthesize this kind of substance,and need to obtain it from plants or other foods.Controlling the absorption and transformation of cholesterol in insects may be a new method for pest control way.Insect body involved in the absorption,transport and metabolism of plant sterol gene is relatively conservative,NPC1 is one of the key genes,the vast majority of the insects have two NPC1 genes,and a few insects such as whiteflies only 1 NPC1 gene in the genome,and analyze the gene sequence and function could be used to a new theory reference for the important agricultural pest control.In this paper,Bemisia tabaci was taken as the research object.According to the sequence of NPC1 gene in its genome data,the full length of NPC1 gene was amplified by PCR,and its expression was analyzed.The function of this gene was studied by means of RNA interference（RNAi）and inhibitors of ds RNA mediated by plant leaves.The main results are as follows:（1）According to the gene sequences obtained from NCBI database and Bemisia tabaci genome database,Primer5.0 primer design software was used to design amplification primers.The multi-fragment c DNA sequence of the gene was amplified by PCR,and the complete c DNA sequence was obtained after splicing.It was inferred that the three conserved domains of the protein sequence were complete.Sequence alignment and evolutionary analysis showed that it was highly consistent with other NPC1 genes in insects.（2）NPC1 gene was differentially expressed in Bemisia tabaci.Different hidden species of Bemisia tabaci（Bemisia tabaci type B,Bemisia tabaci type Q,Bemisia tabaci type ZHJ-1）,different instars（egg,1st to 2nd instar nymph,3rd instar nymph,4th instar nymph,female adult,male adult）and different parts（head,chest and abdomen）were selected for RNA reverse transcription and fluorescence quantitative PCR.The results showed that NPC1 gene was expressed in Bemisia tabaci types B,Q and ZHJ-1,but there was no difference in expression level.NPC1 gene was expressed in all developmental stages of Bemisia tabaci.The expression level of NPC1 gene was higher in male and female adults,but lower in the 1st to 2nd,3rd and 4th instars.NPC1 gene was expressed in all parts of Bemisia tabaci,with the highest expression level in the abdomen.（3）RNA interference experiment was conducted by plant leaf mediated ds RNA.The results showed that the NPC1 gene of Bemisia tabaci adults treated with ds NPC1 was down-regulated by 66%compared with that of ds EGFP control,and the oviposition quantity and survival rate of Bemisia tabaci adults treated with ds NPC1 were significantly reduced compared with that of control.Compared with the control group,the NPC1 gene was down-regulated by 39% after ds NPC1 treatment,and the survival rate of the nymphs was significantly reduced.（4）The effects of cholesterol and cholesterol inhibitors on Bemisia tabaci were studied by double membrane method.The expression of NPC1 gene was affected by cholesterol concentration.The expression of NPC1 gene in Bemisia tabaci adults fed 1 mg/m L cholesterol was significantly higher than that of 0.25 mg/m L,0.5 mg/m L and 2 mg/m L cholesterol.Moreover,the expression level of cholesterol NPC1 increased with the increase of cholesterol concentration in 0.25 mg/m L,0.5 mg/m L and 1 mg/m L.Cholesterol concentration affected the survival rate of Bemisia tabaci.The survival rate of Bemisia tabaci increased with the increase of cholesterol concentration after feeding 0.25 mg/m L,0.5 mg/m L,1 mg/m L and 2 mg/m L cholesterol.Cholesterol inhibitors inhibited the expression of NPC1 gene in Bemisia tabaci.The gene expression level decreased with the increase of cholesterol inhibitor concentration,and the expression level of U18666 A treatment was lower than that of ezetimibe treatment.The survival rate of Bemisia tabaci decreased significantly with the increase of cholesterol inhibitor concentration,and the survival rate of Bemisia tabaci treated with U18666 A was significantly higher than that treated with ezetimibe.