Cloning and Bioinformatics Analysis of Trehalase Genes in Conopomorpha sinensis Bradley

Abstract

【Objective】Trehalase (Tre) is a key enzyme in trehalose metabolism of Conopomorpha sinensis Bradley, which plays an important role in energy metabolism and growth development by specifically hydrolyzing trehalose into glucose. The study aims to clone two trehalase genes (CsTre1 and CsTre2) from Conopomorpha sinensis Bradley, to clarify its expression patterns in different developmental stages and tissues, and to analyze the molecular characteristics of the two genes and their enzyme proteins.【Method】Based on the transcriptome data of C. sinensis, the full-length cDNA sequences of CsTre1 and CsTre2 were cloned with the rapid amplification of cDNA ends (RACE)-PCR. Bioinformatics analysis was performed with software such as ORF Finder, ProtParam, SignalP 4.1, ProtScale, NetPhos2.0 Server and IQ TREE. The mRNA expression patterns of CsTre1 and CsTre2 in different developmental stages and tissues of C. sinensis were detected by using real-time quantitative PCR (RT-qPCR).【Result】The open reading frame of CsTre1 was 1 701 bp, encoding 566 amino acids, and the protein molecular weight was 64.53 kD. The open reading frame length of CsTre2 was 1 821 bp, encoding 606 amino acids, and the protein molecular weight was 69.08 kD. Signal peptide prediction analysis showed that both front-ends of CsTre1 and CsTre2 had a signal peptide, with positions of 1-16 and 1-17, respectively. The analysis on the secondary structure of the sequence showed that both CsTre1 and CsTre2 were mainly composed of α-helix and random coil, CsTre1 had 24 Sers, 15 Tyrs, and 10 Thrs that may serve as binding sites for protein kinases, while CsTre2 had 27 Sers, 10 Tyrs, and 13 Thrs that may serve as binding sites for protein kinases. The RT-qPCR results revealed that CsTre was expressed throughout all developmental stages of C. sinensis. In the expression pattern of adult stage, the expression level of CsTre1 was much higher than that of CsTre2, and the expression level of male adults of CsTre1 dropped sharply after the fourth day.【Conclusion】The study successfully cloned two trehalose genes of Conopomorpha sinensis Bradley. According to the results of analysis on their molecular characteristics and expression patterns, CsTre1 may be the main gene regulating trehalose metabolism in Conopomorpha sinensis Bradley. The research results provide important clues for elucidating the function of trehalase genes, which lay a solid foundation for the development pest control strategies.

Keywords

• trehalase
• gene cloning
• bioinformatics analysis
• conopomorpha sinensis bradley
• expression pattern