Transcriptional Regulation Of HaSCP-2 By The Transcription Factor Ha-c-Myc In The Cotton Bollworm,Helicoverpa Armigera

Helicoverpa armigera(Lepidoptera:Noctuidae)is an omnivorous and very harmful pest in the world.There are more than 200 host plants endanged by Helicoverpa armigera.They destroyed crops by many different ways.They are seriously harmful in the area of Yangtze and Yellow River Valleys,and Xinjiang province.And they caused serious economic losses to the local people.The abuse of insecticides makes the cotton bollworm to produce strong resistance.And the long-term use of Bt insecticides leads to increased frequency of Bt resistance of the cotton bollworm.It poses a great threat to the sustained and stable development of crops.Therefore,the development of safe and effective insecticides has become a top priority for the control of the cotton bollworm.Studies have shown that the cotton bollworm lacks the key enzymes for the synthesis of cholesterol,so the cotton bollworm must rely on the sterol carrier protein-2(SCP-2)to transport the exogenous cholesterol to the corresponding parts of the body for its growth and development.There is an E-box element for the binding of transcriptional actor c-Myc located in the upstream of the H.armigera SCP-2(HaSCP-2)gene promoter.Mutation of E-box leads to the decrease of HaSCP-2 promtoer activities.However,the regulation mechanism of Ha-c-Myc on HaSCP-2 gene expression is not known at present.This study was carried out in the following four aspects.Firstly,the effects of transcription factor c-Myc on the activity of HaSCP-2 gene promoter were studied by carrying out overexpression experiments and RNA interference.The results were as below:(1)The OpIE2-Ha-c-Myc-his-N1 overexpression recombinant plasmid was constructed,then transfected into Ha cells and the activity of HaSCP-2 promoter was detected.The results showed that overexpression of the Ha-c-Myc significantly increased the activity of the HaSCP-2 gene promoter by approximately 50%.(2)Ha-c-Myc siRNA was designed and transfected into Ha cells.The mRNA transcription levels of Ha-c-Myc gene and HaSCP-2 gene were detected by real-time quantitative PCR.The results showed that the expression of Ha-c-Myc gene was inhibited.The transcription level of HaSCP-2 mRNA was significantly decreased by 50%after the Ha-c-Myc gene was inhibited.Both overexpression and RNA interference experiments indicated that the transcription factor Ha-c-Myc positively regulates the expression of HaSCP-2 gene.Secondly,the recombinant plasmid of pET-32a-c-Myc and pFastBac TMHTA-c-Myc were constructed,and the Ha-c-myc protein was expressed by prokaryotic expression system and Bac-to-Bac insect baculovirus eukaryotic expression system.The prokaryotic and eukaryotic expression of Ha-c-Myc was successfully achieved.In addition,the spatial and temporal expression patterns of Ha-c-Myc gene were detected in the cotton bollworm by fluorescence quantitative RT-PCR.The results showed that the expressions of Ha-c-Myc gene were different in different stages and different tissues of Helicoverpa armigera.Ha-c-Myc had higher expression levels in eggs,5th star larvae and prepupal stage.The expression level of Ha-c-Myc gene is highest in the midgut.And in the other tissues,the order of expression level from high to low is the head,fat body and epidermis.Finally,the role of the transcription factor Ha-c-Myc in the regulation of HaSCP-2 gene expression was further studied in vivo.The RNA interference was performed by injection and feeding methods respectively.The relative mRNA expression of two genes of Ha-c-Myc and HaSCP-2 after interference was detected.Small interfering RNA was introduced into the cotton bollworm by micro injection and nanomaterial-mediated feeding.The results showed that the expression levels of Ha-c-Myc gene and HaSCP-2 gene were significantly down-regulated by the above two methods of interfering.Knockdown of Ha-c-Myc gene caused strong inhibition or even silence of HaSCP-2 gene expression.This indicated that Ha-c-Myc is an important regulator of HaSCP-2 transcription and has a positive regulatory effect on the expression of HaSCP-2 gene.After interferring Ha-c-Myc expression in vivo,the content of cholesterol in the fatbody of cotton bollworm was detected.The growth and development of cotton bollwormwe were observed and recorded.The results showed that after interference,the cholesterol content of the cotton bollworm decreased,the growth of larvae was delayed,the development was abnormal,the pupation and eclosion were inhibited,and the reproduction was weakened.Both the studies in vitro and in vivo demonstrated that the transcription factor Ha-c-Myc plays a positive regulatory role in the expression of HaSCP-2 gene and further validated the effects of Ha-c-Myc on the growth,development and reproduction of cotton bollworm.These studies will help to elucidate the transcriptional regulation mechanism of HaSCP-2 gene and lay a good basis for the studies of potential target of green and effective insecticide.