Expression characteristics of trehalose-6-phosphate synthase genes and their roles in the regulation of carbohydrate metabolism in Sogatella furcifera (Hemiptera: Delphacidae) ZHANG Dao-Wei, QIU Ling-Yu, KANG Kui, YU Ya-Ya, ZENG Bo-Ping, CHEN Jing, TANG Bin
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【Aim】 Trehalose in insects is mainly synthesized in the fat body by trehalose-6-phosphate synthase (TPS), which can induce trehalose accumulation and thus plays a protective role when insects are under extreme environmental stress. This study aims to analyze the developmental and tissue expression patterns of two TPS genes in Sogatella furcifera, and their roles in the regulation of carbohydrate metabolism, so as to explore the specific roles of TPS genes in the growth and development of S. furcifera. 【Methods】 Based on the partial sequences of two trehalose synthase genes, SfTPS1 and SfTPS2,obtained in the early stage of the laboratory, gene cloning and sequencing were also carried out in this experiment. The two TPS gene sequences of the S. furcifera were determined by comparison. The phylogenetic tree based on the amino acid sequences of SfTPS and TPS proteins from other insect species was constructed by MEGA 7.0 software. The expression profiles of these two TPS genes in different developmental stages (from the day 1 4th instar nymph to 3 day-old adult), and different adult tissues (head, leg, wing, midgut, fat body, epidermis and Malpighian tubules) of S. furcifera were detected by qRT-PCR. The double-stranded RNA (dsRNA) of the two genes were synthesized and injected into the day 1 5th instar nymphs of S. furcifera for the RNAi. At 48 and 72 h after RNAi, the changes in expression levels of trehalase genes TRE1-1, TRE1-2 and TRE2, the contents of trehalose, glucose and total glycogen, and the trehalase activity were determined. 【Results】 The ORFs of SfTPS1 and SfTPS2 are 2 424 and 2 115 bp in length, respectively, and the numbers of encoded amino acids are 807 and 704, respectively. The predicted protein molecular weights are 90.37 and 80.56 kD, respectively, and the isoelectric points are 6.08 and 6.10, respectively. Moreover, the two TPS amino acid sequences of S. furcifera show the highest identities with Nilaparvata lugens TPS1 and TPS2. Developmental expression profiles revealed that SfTPS1 and SfTPS2 were expressed from the 4th instar nymphal stage to the adult stage, and tissue expression profiles revealed that the two genes showed significantly higher expression levels in Malpighian tubules, midgut and epidermis ofa S. furcifer adults. After RNAi of SfTPS1, the expression levels of TRE11 and TRE2 in the RNAi group increased slightly and significantly, respectively, as compared with those of the control group (dsGFP injection group), and the rel ative expression level of TRE1-2 increased significantly at 48 h and decreased significantly at 72 h after RNAi. The soluble trehalase activity did not change significantly, and the membrane-bound trehalase activity increased significantly, while the contents of trehalose, glucose and total glycogen in the 5th instar nymphs of S. furcifera increased significantly. After RNAi of SfTPS2, the expression levels of TRE1-2 and TRE2 in the RNAi group increased significantly at 48 h, while declined significantly at 72 h. And the expression level of TRE1-1 increased significantly at 48 and 72 h as compared to those of the control group. The soluble trehalase activity decreased significantly at 48 h and increased significantly at 72 h as compared to those of the control group after RNAi. The membrane-bound trehalase activity increased significantly at 72 h after RNAi of SfTPS2. In addition, the glucose content reduced significantly at 48 h after RNAi of SfTPS2, but the contents of trehalose, glucose and total glycogen in the 5th instar nymphs of S. furcifera increased significantly at 72 h after RNAi. 【Conclusion】 The expression levels of TRE1-1, TRE1-2 and TRE2 are affected and thus the trehalose content is regulated by regulating the expression of TPS genes in vivo in S. furcifera. The results provide a theoretical basis for later application of TPS as the target gene for pest control.