Identification and expression analysis of chitin synthase and related enzymes in the chitin biosynthetic pathway genes of Cnaphalocrocis medinalis
Author of the article:YU Hai-Zhong1** HUANG Ke-Hui1** WANG Wan-Ling2 LIU Ming-Hui3 YANG Xin1 ZHANG Yan3 XU Jia-Ping1
Author's Workplace：1. School of Life Science, Anhui Agricultural University, Hefei 230036; 2. Rice Research Institute, Anhui Agricultural Sciences, Hefei 230031; 3. Sericultural Research Institute, Hefei 230031
Key Words：Cnaphalocrocis medinalis, CmCHSA, CmCHSB, CmUAP, CmPGM, identify, bioinformatics analysis, chitin biosynthesis
Abstract： [Objectives] The rice leaf folder, Cnaphalocrocis medinalis (Guenee), is one of four rice pest insects that cause serious crop damage. In recent years, chitin synthesis and metabolism has become a focus of pest control research. Cloning and spatio-temporal expression of two chitin synthases, and other two key enzymes in the chitin biosynthetic pathway encoding genes in C. medinalis, were conducted to reveal the function of these genes. [Methods] Based on transcriptome data, we used the PCR and RACE techniques to clone the full length cDNA sequences of 4 key enzymes in the chitin biosynthetic pathway. Prediction of the structure, sequence alignment and phylogenetic analysis of the products of these 4 genes were performed using different bioinformatics software. The relative expression levels of the 4 genes in different developmental stages and larval tissues of C. medinalis were determined with quantitative Real-time PCR. [Results] Two full-length cDNA sequences encoding chitin synthase, and two full-length cDNA sequences encoding other two key enzymes related to the chitin biosynthetic pathway, were obtained. These were; Chitin Synthase A (CHSA), Chitin Synthase B (CHSB), Phosphoacetylglucosamine Mutase (PGM) and UDP-N-acetylglucosamine pyrophosphorylase (UAP) (hereafter CmCHSA, CmCHSB, CmPGM and CmUAP, respectively). Sequence analysis shows that the full length of the CmCHSA gene is 4 868 bp, which encodes a polypeptide of 1 564 amino acids, the full length of the CmCHSB gene is 4 651 bp, which encodes a polypeptide of 1 525 amino acids, the full length of CmPGM gene is 1 934 bp, which encodes a polypeptide of 548 amino acids, and the full length of CmUAP gene is 1 837 bp, which encodes a polypeptide of 487 amino acids. The results of RT-qPCR indicate that CmUAP and CmPGM had higher expression in hemolymph, whereas CmCHSA was more highly expressed in the head and integument than the midgut and CmCHSB was more highly expressed in the midgut than in other tissues. [Conclusion] Four key chitin biosynthetic pathway genes were cloned in C. medinalis. These have significantly different spatio-temporal expression patterns in different developmental and tissues. This work lays a foundation for future research on the function of CmCHS and the chitin biosynthetic pathway.