Transcriptional Regulatory Mechanism Of HaSCP-2 In The Cotton Bollworm,Helicoverpa Armigera

Helicoverpa armigera is a highly resistant omnivorous agricultural pest belonging to the Lepidoptera Noctuidae family.There are many important economic crops,including cotton and vegetables which has been harmed by H.armigera.With the long term use of pesticides,H.armigera produced resistance to many pesticides and even the Bacillus thuringiensis(Bt).Therefore,in order to promote the sustainable green development of agriculture,the new,safe and efficient biological pesticide is urgent to be developed.Studies have shown that cholesterol is essential for the growth and development of cotton bollworm.However,the cotton bollworm can not use its own substances to synthesize cholesterol,so it has to obtain cholesterol from the outside world to meet the needs of its growth and development.It was proven that sterol carrier protein-2(SCP-2)play a crucial role in the absorption and transportation of the cholesterol in insects.The H.armigera SCP-2(HaSCP-2)gene was cloned and identified in our laboratory.The structure of this protein was studied by NMR.However,the regulation mechanism of HaSCP-2 gene expression in H.armigera is still unclear.The studies were carried out in two aspects.On the one hand,by using mutation,cell transfection,protein expression,EMSA,real-time fluorescence quantitative PCR,RNA interference and other experimental techniques,the regulatory functions of cis-acting element E-box and transcription factor HaAP-4 on the promoter of HaSCP-2 gene were investigated.The following results were obtained:(1)A pGL3-400 bp recombinant plasmid was constructed,an E-box element located in the region of-400 bp in the HaSCP-2 promoter was mutated,and the luciferase activities were measured by transfecting H.armigera cells.Results showed that the activity of SCP-2 promoter was significantly down-regulated by 70%after mutating E-box elements;(2)OpIE2-AP-4--his-N1 recombinant plasmid was constructed and transfection,measured enzyme activity,the data showed that over-expression of HaAP-4 positively regulates the SCP-2 promoter;(3)HaAP-4 siRNA is transfected in Ha cells.HaAP-4 and HaSCP-2 mRNA transcription levels were detected by real-time fluorescence quantitative PCR,and results showed that knockdown the expression of HaAP-4 caused a significant decrease of HaSCP-2 mRNA transcription;(4)The HaAP-4 proteins were successfully expressed using the prokaryotic expression vector of pET-28a.A large amount of the protein was purified and the rabbit was immunized to prepare the antibody;(5)The exploration of interaction between HaAP-4 Protein and E-box Sequence by gel electrophoresis migration assay(EMSA),and the results showed that the transcription factor HaAP-4 can specifically bind with the E-box sequence of the HaSCP-2 promoter to regulate the transcription of the HaSCP-2.On the other hand,we studied the function of HaAP-4 and HaSCP-2 in H.armigera(in vivo).The main results were as follows:(1)HaAP-4 siRNA was injected into the larva of H.armigera and the midgut RNA is extracted.The expression of HaAP-4 and HaSCP-2 mRNA were detected by real-time fluorescence quantitative PCR.The results showed that HaAP-4 siRNA inhibited the mRNA transcription of HaAP-4,and further led to the significantly decrease of SCP-2 expression,indicating that HaAP-4 promotes the expression of SCP-2 protein;(2)The fat body of the cotton bollworm was also collected to detect the cholesterol level in the HaAP-4 RNAi experiments.Results showed that the cholesterol content in the HaAP-4 siRNA injected group was significantly lower than that of the control group injected with DEPC water.This result suggested that the expression of HaSCP-2 was inhibited after interfering with the transcription factor HaAP-4,which led to the decrease of cholesterol absorption in the cotton bollworm.This is consistent with the fact that SCP-2 plays a role in insects'absorption of exogenous cholesterol,also further proved that HaAP-4 affected the absorption of cholesterol by the regulation of HaSCP-2 expression;(3)The growth and development of the cotton bollworm,including the body weight and growth rate of the bollworm,were recorded,counted,and analyzed after the expression of HaAP-4 were interfered.The results showed that after the HaAP-4 was interfered,the growth and development of the cotton bollworm have produced some significant changes.It is speculated that the transcription factor HaAP-4 can affect the absorption and utilization of cholesterol by controlling the expression of HaSCP-2,and further affect the growth and development of the cotton bollworm.In this paper,the function of the E-box element in the promoter region of HaSCP-2 and the regulation of HaSCP-2 gene by the transcription factor HaAP-4 were studied.Furthermore,the effects of HaAP-4 on the regulation of HaSCP-2 and its effects on the absorption of cholesterol,growth and development of H.armigera were further validated in vivo.All these results are not only important for the research of SCP-2 synthesis and transcriptional regulation in insects,but also provided important theoretical data for the development of new effective insecticides.