Identification And Functional Analysis Of Humidity Receptor Genes In Bactrocera Dorsalis’ S Third Instar Larvae

Insect growth and development, survival and reproductiondirectly are related with Humidity affects, which resulting in the occurrence of pests in different period, the amount of occurrence and degree of harm. For insect, the ability to perceive the environment humidity is an important skill to survive in nature. Little insects is the best resreach model for humidity sensing.Bactrocera dorsalis(Hendel) is a worldwide quarantine pest, its host range is wide, which mainly endanger more than 250 kinds of crops and fruits including 40 families. Current control strategies of B. dorsalis is limited on chemical control and male-killed technology. However, these methods are hard to achieve effective outcome. This fly is seriously restrict in the development of fruits and vegetables industry. Current research showed that the new target behavior modifier could be developed with insect key functional target gene. In this study, based on the pupation behavior of the fruit fly larvae, the function of key gene was defined, which could provide scientific discovery for pupation behavior in molecular level.In this study, we selected B. dorsalis’ s third instar larva as test insect. For one side, the humidity sensing gene(Transient Receptor Potential, TRP) was identified in previous larva transcriptome data. Then, RNAi and behavioral assay were employed to confirm the gene function. Thought the study, the results are carry out as follows: 1) the full-length TRPPainless genes of B. dorsalis’ s coding sequence and bioinformaticsUsing RACE technology and cloning techniques, the full length of two humidity sensing genes related(TRPPainless: Bdor TRP1 and Bdor TRP2) were obtained. The genetic characteristics of Bdor TRP1 and Bdor TRP2 were defined as follows: Bdor TRP1 is 2793 bp nucleic acid sequence and encoding 930 amino acids. Bdor TRP2 is 2796 bp and encoding 931 amino acids. The sequence alignment and homology analysis showed that the protein sequence highest matched with Drosophila melanogaster(56%) 、Musca domestica(67%) and Ceratitis capitata(88%) separately. The sequence Clustal W alignment show that Bdor TRP1 and Bdor TRP2 have six conserved domains, in accord with other insect TRP genes. Phylogenetic analysis showed seven clades which including TRPA, TRPN, TRPV, TRPM, TRPML, TRPP and TRPC assigned, and both Bdor TRP1 and Bdor TRP2 are claded with TRPA. In addition, Bdor TRP1 and Bdor TRP2 are also high similarity(97.1%). The genetic relationship show that Bdor TRP1 and Bdor TRP2 belong to TRPA family in accord with Deml Wtrw humidity sensing genes. Which were closed with TRPV family. Thus, Bdor TRP1 and Bdor TRP2 genes were identified as candidate humidity sensing gene in the B. dorsalis’ s third instar larva. 2) The functional study of B. dorsalis’ s TRPPainless genesRNA interference technology and real-time PCR technology were employed to determine the the humidity sensing feature. The results show that the average relative expression of TRPPainless1 gene by interference is 0.57 times than non-interference. the average relative expression of TRPPainless2 gene by interference is 0.63 times than non-interference. Thus the interference effectiveness is significant and obvious.Throught behavioral assay including the selectivity in the different humidity sand, buried time, jumping height and other related factors, the different behavioral data have been compared in interference and non-interference.The results show that the interference and non-interference larva have significant preference,the selectivity is 40% and 60% humidity sand.The non-interference insects number of 40% and 60% sand selectivity rate is 70%; The interference insects number of 40% and 60% sand selectivity rate is 53%. The time to buried the sand between interference and non-interference were compared, the buried time of interference is 197 s and non-interference is 212 s. The jumping height between interference and non-interference is no significant differences, all is 5-6 cm. In conclusion, TRPPainless is important for Humidity selectivity in B.dorsalis’ s third instar larva.