| Helicoverpa armigera belongs to the Noctuidae in Lepidoptera and is a major agricultural pest.It is polyphagous and distributed all over the world.It harms many fields such as agriculture,horticulture and floristry.There are more than 20 parasitic plants.Its main host plant is cotton,which has caused great economic losses to the cotton areas in North China,Yunnan and Xinjiang province.The control of cotton bollworm mainly experienced chemical pesticides and transgenic Bt cotton,but the misuse of chemical pesticides was harmful to the ecological environment and led to strong resistance.Under long-term selective pressure,this pest also developed strong resistance to Bt toxins,which limited the application of transgenic Bt cotton.It is urgent to develop new and efficient insecticides.Since insects cannot synthesize cholesterol by themselves,their growth and development depend on the absorption and transport of exogenous cholesterol.In the uptake of cholesterol from the outside,sterol carrier protein-2(SCP-2)plays an important role,which provides an excellent potential target for new pesticides.The Helicoverpa armigera SCP-2 gene was cloned and its function was clarified in our previous studies,but the regulation mechanism of HaSCP-2 gene was still unclear.In this study,Alibaba2.1 and TFD online tools were used to predict the transcription factor and its binding site in the 1.8 kb sequence of the HaSCP-2 promoter.It was found that the transcription factor of Fork head could bind to the 1.8kb promoter sequence.There are two potential binding sites for the Fork head transcription factor,which are located-742～-758 bp and-764～-780 bp upstream of the HaSCP-2 promoter.In order to explore the regulation role of the Ha-Fork head on the HaSCP-2,this thesis conducts the research from the following four aspects.Firstly,by using immunofluorescence,mutation and overexpression experiments,the effects of Ha-Fork head and its binding sites on the HaSCP-2 promoter activity were studied.The results were as follows:(1)Immunofluorescence experiment showed that Ha-Fork head was successfully expressed in the Ha cells and localized in the nucleus.(2)two deletion mutant plasmids of potential binding sites,mpB10 and mpB20 were constructed and transfected into Ha cells to detect the activity of the HaSCP-2 promoter.The results showed that a single deletion mutation of the binding elements significantly reduced the promoter activity by about 40%,indicating that these two binding sites may be important to the HaSCP-2 promoter activity.(3)the overexpression of the Ha-Fork head made the HaSCP-2 promoter activity was significantly up-regulated by about 4 times,indicating that Ha-Fork head plays a positive regulatory role in the transcription of HaSCP-2.Secondly,the pET-28a-Ha-Fork head recombinant plasmid was constructed and the Ha-Fork head protein was expressed in Escherichia coli.The expressed protein was partially soluble.After purification by nickel column affinity chromatography,about 10 mg of pure recombinant protein was obtained,which provided high-quality antigens for antibody preparation and other further research.Then,the temporal and spatial expression profiles of the Ha-Fork head gene in cotton bollworm were determined by RT-qPCR.The expression of this gene is very low in the egg stage and the young larval stage.The expression level gradually increases from the 4th instar,and reached to the highest in the 3rd day of 5th instar.Spatially,the expression levels of Ha-Fork head in different tissues are different at the 3rd day of 5th instar and pre-pupa stage.Ha-Fork head is expressed at the highest level in the midgut.The above results suggested that the Ha-Fork head transcription factor could be involved in the transcriptional regulation of HaSCP-2 in the midgut tissue.Finally,through injection and nanomaterial-mediated feeding,the Ha-Fork head gene was interfered in vivo.The results showed that both the injection method and the feeding method could successfully interfere with the expression of Ha-Fork head.When Ha-Fork head was down-regulated,HaSCP-2 was also down-regulated,indicating that Ha-Fork head had an positive effect on HaSCP-2 transcription.At the same time,after Ha-Fork head was knocked down,the cholesterol content in the larval fat body tissue decreased significantly by 55%and 47%compared to the wild-type control group,respectively.In a series of bioassays on the growth and development of the cotton bollworm,after the Ha-Fork head expression was disturbed,the growth of cotton bollworm delayed,larvae weight loss,larvae died,pupation rate and emergence rate decreased.Both the egg rate and hatching rate decreased.In particular,the pupation of the cotton bollworm was inhibited,resulting in abnormal pupation or failure of the pupation,causing its growth to stagnate in the prepupal period of the life stage.This study proved that Ha-Fork head had a positive regulatory role in the transcription of HaSCP-2 in vitro and in vivo.The bioassay further verified that after Ha-Fork head was interfered,HaSCP-2 was down-regulated,the accumulation of cholesterol nutrient in insect reduced,which finally had impact on the growth and reproduction of the cotton bollworm.This study the foundation for further research and elucidation of the mechanism of HaSCP-2 expression regulation,and also provide a theoretical support for the research and development of new green insecticides. |
PDF Full Text Request