Physiological Function Of Bd Taiman In JH Signaling Pathway Of Bactrocera Dorsalis (Hendel)

Bactrocera dorsalis is a dangerous fruit and vegetable pest,and its host range is wide,causing serious economic losses.Due to its strong environmental adaptability and proliferation intrusion capabilities,B.dorsalis is classified as a quarantine pest by many countries and regions,which has caused many restrictions on international trade.So some entomologists and biologists think that B.dorsalis is one of the most important agricultural pests in the world.The fruit fly lives through four stages of development:eggs,larvae,cockroaches,and adults.The adults lay their eggs inside the fruit,and the larvae invade the interior of the fruit to feed on the food and cause fruit rot or fruit drop.At present,the field control of B.dorsalis is dominated by insecticides,but due to the widespread and irrational use of insecticides,its resistance to insecticides has developed rapidly.Orange fruit fly has become a great threat to the healthy development of China's fruit and vegetable industry.Therefore,it is particularly urgent to find new targets for the prevention and control of B.dorsalis.In this dissertation,the fruit fly was researched.Based on the information of genome and transcriptome database,the Taiman open reading frame of the juvenile hormone signaling pathway gene was cloned by RT-PCR technique,and five alternative splicing of the gene were obtained.The characteristic sequences were annotated;qPCR technique was used to determine the temporal and spatial expression pattern of the five variable cuts of the Taiman of B.dorsalis and the expression pattern of exogenous hormone methoprene on Taiman.On this basis,the use of RNAi technology,using the combination of dsRNA and siRNA to specifically interfere with Taiman expression,explored its physiological functions in the growth and development of B.dorsalis.In addition,we successfully constructed the eukaryotic expression vectors pACT-BdTaiman,pACT-BdMet,pBIND-BdMet,and pBIND-BdTaiman of the BdTaiman and BdMet based on the two-hybrid vectors pACT and pBIND,which laid the foundation for subsequent interactions between transcription factors.The foundation.The main findings are as follows:1.Cloning and sequence analysis of BdTaiman isoforms in B.dorsalisBased on the genome and transcriptome database information of the orange fruit fly,the ORFs of five variable splicing gene sequences of JH signaling pathway genes were obtained from B.dorsalis.The ORFs were named as BdTai-A?GenBank accession number:KY378657?,BdTai-B?GenBank accession number:KY378658?,BdTai-C?GenBank accession number:KY378659?,BdTai-D?GenBank accession number:KY378660?and BdTai-E?GenBank accession number:KY378661?.We analyzed the transmembrane structure and conserved sequences of the five alternative spliceosome encoded proteins of BdTaiman through bioinformatics tools such as TMHMM and SignalP 4.1 Physiological function.2.Expression pattern of BdTaiman isoformsThe qPCR technique was used to analyze the expression pattern of five isoforms in the growth and development stage of B.dorsalis.The results showed that the BdTaiman isoforms were expressed in different degrees in various stages of B.dorsalis larvae.Among them,BdTaiman-A and B had higher expression levels in the early larvae;BdTaiman-D had higher expression levels in later stages of the larvae compared to other variable cuts;in addition,BdTaiman-E and BdTai-core expression levels were basically the same.The differences in the above expression patterns suggest that these variable shearing bodies have different functions in the development of B.dorsalis larvae.Further studies showed that the application of methoprene by spotting resulted in up-regulation of BdTaiman variable-spliced expression.In adults of B.dorsalis,BdTaiman has low expression in fat bodies and ovaries,and high expression in Malpighian tubules and central nervous system.3.Physiological function analysis of the BdTaiman isoforms in the JH signaling pathwayFurther use of RNAi explored the function of BdTaiman in the growth and development of B.dorsalis.Through RNA interference with BdTaiman successfully,the larvae of the treatment group were larvae ahead of time and the downstream gene BdKr-h1 was down-regulated,indicating that the BdTaiman was involved in the transformation of JH signaling pathway in B.dorsalis to prevent larval metamorphosis.After successful silencing of BdTaiman-E by synthesizing specific siRNA,BdKr-h1 was detected in both larvae and“small cockroaches”and the same phenotype as that after injection of dsTai-core was observed.The above phenotypes cannot be interfered with by other shearing bodies,indicating that BdTaiman-E is involved in the transduction of JH signaling pathway as the main factor in the larvae of B.dorsalis.The reason for this phenomenon may be related to the special INDEL-1 region of BdTaiman-E.After successfully interfering with BdTaiman expression of 4-day-old adults of B.dorsalis using RNAi,the ovarian development of B.dorsalis apparently lags behind,and the expression levels of the yolk synthesis genes BdVg1 and BdVg2 decrease,indicating that BdTaiman influences the development of ovary of adults of B.dorsalis through inhibiting the expression of BdVg1 and BdVg2.4.Expression vector construction in luciferase reporter systemTo investigate the interaction between Met and Taiman,the pBIND and pACT vectors provided in the CheckMate^TM mammalian two-hybrid system were used to successfully construct Expression vectors such as pBIND_BdMet,pACT_BdMet,pACT_BdTaiman and pBIND_Taiman using double digestion?Not1 and Mlu1?and recombination techniques.