Regulating Functions Of Trehalase In The Pathway Of Energy Metabolism And Chitin Biosynthesis In Tribolium Castaneum Revealed By RNA Interference

Trehalase is the first key enzyme in chitin synthesis pathway, and mainly hydrolyzes trehalose as blood sugar in insect into glucose to provide energy for the insect life activities. Two forms of trehalase had been found in insect, namly souble trehalase (Trel) and membrane-bound trehalase (Tre2). Tre2has one or more transmembrane protein structures. Most insect only have two trehalases and a few species have more than3above, but five trehalases had been found in Tribolium castaneum (T. castaneum). Some previous research have reported the role of two types of trehalase played in chitin synthesis pathway, while there weren’t reports about the functions of two trehalases in T. castaneum, including in the chitin synthesis pathways and energy metabolism pathways. T. castaneum is a serious storage pest and model insect, and apply to research genes’ functions. As a pest control target, depth study of the functions of different trehalase genes will be beneficial to study the underlying molecular mechanism, based on this, the development of more effective trehalase inhibitors and low toxicity bio-pesticides, used in biological control.Five T. castaneum trehalase genes (TcTres) namely TcTrel-1, TcTrel-2, TcTrel-3, TcTrel-4and TcTre2had been cloned successfully and have an open reading frame of1626bp,1692bp,1647bp and1662bp, respectively. TcTrel-1cDNA encoded a protein of541amino acids with a predicted mass of approximately61.94kD and a pI of4.7, TcTrel-2cDNA encodsed a protein of563amino acids with a predicted mass of approximately66.16kD and a pI of8.8, TcTrel-3cDNA encoded a protein of548amino acids with a predicted mass of approximately63.98kD and pI of6.8, TcTrel-4cDNA encoded a protein of553amino acids with a predicted mass of approximately63.34kD and a pI of4.8. TcTre-2cDNA had an open reading frame of1929nucleotides. which encoded a protein of642amino acids with a predicted mass of approximately73.91kD and a pI of5.93.Real-time fluorescence quantitative PCR (qRT-PCR) method was used to find developmental profiles of five TcTres mRNA transcripts during the larva-pupa-adult metamorphoses, the results found four soluble trehalase mRNA transcripts were low in the larval stage and reached the maximum before prepupae, while a high expression amount of the TcTre-2gene in the larval stage was found. Semi-quantitative PCR method detected five TcTres gene mRNA were all expressed in the epidermis, fat body, midgut and Malpighian tubules, and differed in the expression levels in different tissues, which TcTrel-4gene mRNA transcripts was higher in the four tissues.Five TcTre genes’ respective characteristics and functions were studied by RNA interference (RNAi).8L and7L larvae as the targets in the study, conducted the single TcTre gene interference, and observed11abnormal phenotypes after RNAi. We find different death rates in five TcTres. RNA interference groups (TcTrel-1-1:35%. TcTrel-2:17%, TcTrel-3:23%, TcTrel-4:42%and TcTre2:39%). Specific results are as follows:In TcTrel-2and TcTrel-4RNAi groups, the proportion of dead insects from lack of energy was84.6%and93.75%, respectively; In all interfering groups, qRT-PCR detected TcTrel-1gene expression increased significantly prior the pre-pupal molt stage, and many abnormal phenotypes from disorder of chitin metabolic pathways were observed in the group of TcTrel-1RNAi; In the group of TcTrel-3RNAi, the number of dead insects due to lack of energy and the number of dead insects due to chitin synthesis disorders were almost the same; Almost all TcTre transcripts went down after TcTre2RNAi. Above results declared5TcTres work more or less in two pathways, but more reflects their specific functions:(1) TcTrel-1mainly took effect in the chitin metabolic pathway in the entire development history of T. castaneum;(2) TcTrel-2mainly hydrolyzes intracellular trehalose to offer the growth and development of T castaneum energy;(3) TcTrel-3mainly hydrolyzes intracellular trehalose to offer energy during whole life of T. castaneum and regulates the expression of CHS1b;(4) TcTrel-4mainly hydrolyzes intracellular trehalose to offer energy during whole life of T. castaneum.(5) TcTre2is a membrane-bound trehalase hydrolyzing extracellular trehalose into glucose to offer energy during whole life of T. castaneum, and regulates the expression of CHS2gene; TcTrel-2. TcTrel-3, TcTrel-4and TcTre2take more effect in energy-offer and TcTrel-1take more effect in chitin metabolism, while they all play the key roles in the growth and development of T. castaneum.