Molecular cloning and functional characterization of N-β-alanyl-dopamine hydroxylase gene in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)
DU Xiao-Yan1,2, FU Kai-Yun2,*, XU Qing-Yu3, Tursun AHMAT2, DING Xin-Hua2, HE Jiang2, GUO Wen-Chao4,*
（1. College of Plant Science,Tarim University, Alar, Xinjiang 843300, China; 2. Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis, Ministry of Agriculture, Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China; 3. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; 4. Institute of Microorganism Application, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China）
Abstract 【Aim】 This study aims to clarify the function of N-β-alanyldopamine (NBAD) hydrolase gene important in melanin biosythesis in the Colorado potato beetle, Leptinotarsa decemlineata by RNA interference. 【Methods】 The NBAD hydrolase gene in L. decemlineata was characterized by data mining based on its transcriptome, its cDNA was cloned by RT-PCR, and the gene completeness and phylogeny were determined by multiple alignment and phylogenetic analysis, respectively. The expression levels of NBAD hydrolase gene in different developmental stages, tissues of the 4th instar larvae and male gonad and ovary of adults of L. decemlineata were detected by qPCR. The color change during larval growth was observed after RNAi, and the mechanism how the expression of NBAD hydrolase gene was influenced by juvenile hormone (JH) and molting hormone (MH) was assayed .【Results】 An NBAD hydroxylase gene was cloned from L. decemlineata and named Ldtan (GenBank accession no.: KY221866). Its encoded protein shows the highest amino acid sequence identity with the homologous proteins from Tribolium castaneum and Dendroctonus ponderosae and clustered into the same clade with them. The spatial expression profiles showed that Ldtan were highly expressed in ventral nerve cord, hindgut and cuticle of L. decemlineata, with the relative expression levels of 99.36±0.95, 17.79±3.11 and 9.21±0.12, respectively, while the temporal expression profiles showed that its expression level increased along with larval growth and reached the peak at the adult stage. Knockdown of Ldtan gene by feeding dsLdtan to the 2nd instar larvae not only led to tanned color, but also a degree of lethal effect. Knocking down the expression of JH synthesis and signal-related genes by RNAi downregulated the expression of Ldtan, while knocking down the expression of MH synthesis and signal-related genes by RNAi upregulated the expression of Ldtan. 【Conclusion】 The results suggest that Ldtan is involved in melanin synthesis in L. decemlineata, and JH and MH probably regulate its expression.
DU Xiao-Yan,FU Kai-Bin,XU Qing-Yu et al. Molecular cloning and functional characterization of N-β-alanyl-dopamine hydroxylase gene in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)[J]. ACTA ENTOMOLOGICA SINICA, 2018, 61(6): 637-645.