Gene Function Of BdJHAMT In Larval Development Of Oriental Fruit Fly,Bactrocera Dorsalis(Hendel)

The oriental fruit fly,Bactrocera dorsalis(Hendel)(Dipttera: Tephritidae)is common in tropical Asia and subtropical regions.It is a fruit and vegetable pest that can harm a variety of fruits and vegetables.It has great harm,complex food habits,and strong adaptability.It has been isolated by many regions and countries as quarantine pests.Trade creates many restrictions.At present,the chemical control of fruit fly is mainly based on chemical agents.However,once the larvae penetrate into the fruit,the spraying of the agent will be difficult to achieve,and at the same time,it will cause serious environmental pollution and food safety problems.Based on the current genome-wide sequencing data of various types of pests,the discovery of green and safe targets for pharmaceutical agents has continued.Juvenile hormones play an important role in physiological development of insects such as larval growth,pupal metamorphosis,and adult reproduction.Juvenile hormone acid O-methyl-transferase(JHAMT)is a process of juvenile hormone synthesis Important regulatory enzymes.Based on the transcriptome and genomic data of Bactrocera dorsalis,RT-PCR was used to clone the ORF sequences of BdJHAMT and BdJHAMT-like genes and clarified the phylogenetic relationship between BdJHAMT and BdJHAMT-like and other insect homologous sequences;the spatiotemporal expression pattern of the gene in B.dorsalis was analyzed using real-time quantitative PCR and tissue localization of BdJHAMT was done based on immunohistochemistry;The titer of JH ? in the development stages of B.dorsalis was measured by high performance liquid chromatography(HPLC).Finally,the RNA interference function of BdJHAMT in the growth and development of B.dorsalis larvae was explored by feeding dsRNA.The specific research results are as follows:1 Cloning and sequence analysis of JHAMT and JHAMT-like in B.dorsalisThe ORFs of JHAMT and JHAMT-like of JH synthesis pathway genes were cloned from B.dorsalis.Bioinformatics software such as Clustal W and TMHMM found that BdJHAMT and BdJHAMT-like encoded proteins all have conserved SAM binding sites.Phylogenetic tree constructed using software MEGA 5.0 found that BdJHAMT is closer to Diptera insects,while BdJHAMT-like is closer to Hymenoptera.2 Spatiotemporal expression patterns of JHAMT and JHAMT-like genes in B.dorsalisThe expression patterns of BdJHAMT and BdJHAMT-like in different developmental stages of B.dorsalis were analyzed by qRT-PCR.The expression abundance of BdJHAMT was higher in the pre-pupal,early emergence and 9-day-old adults of B.dorsalis,while the expression of BdJHAMT-like in male adults did not fluctuate,and the expression was higher in 9-day-old females.The expression levels of both were low in larval stage.Tissue expression pattern analysis revealed that BdJHAMT-like transcript levels were higher in the larval midgut and corpora allata of adult,while BdJHAMT-like was higher expressed in female adult fat body and midgut.By immunohistochemistry,it was verified at the protein level that BdJHAMT has a specific expression signal in the adult corpora allata,and fluorescent signals were detected in the intestinal and intestinal wall cells of the early larvae.3 JH ? titer test in B.dorsalisIn order to explore the relationship between the titer of JH ? and the expression pattern of BdJHAMT in space and time,a titer detection system for JH ? was established based on HPLC.The results showed that JH ? titers fluctuated regularly during different development stages of B.dorsalis.Among them,the JH ? titer in adult stage was much higher than that in larval and pupal stage,and the JH ? titer in pupal stage was extremely low.The increase of JH ? titers in the early pupae and early emergence of B.dorsalis was consistent with the BdJHAMT mRNA expression pattern.4 Physiological function of BdJHAMT in reproduction and larvae growth of B.dorsalisBy comparing injection and feeding RNAi,it was found that feeding RNAi alone could effectively silence BdJHAMT and it caused a significant lag in the growth and development of the first instar larvae of B.dorsalis,and at the same time,the JH ? titer decreased significantly.High concentration of methoprene treatment could rescue the larval phenotypic defects caused by BdJHAMT silencing,and thus restore body length development.