Molecular cloning, sequence analysis and expression profiles ofLdeSid-1a and LdeSid-1c genes in Leptinotarsa decemlineata (Say)

Author of the article:YANG Shuai1** FU Kai-Yun2 WU Jia-He3 LI Guo-Qing4Tuerxun2 GUO Wen-Chao2***

Author's Workplace：1. College of Agronomy, Shihezhi University, Shihezi 832003, China; 2. Research Institute of Plant Protection, Xinjiang Academy ofAgricultural Sciences, Urumqi 830091, China; 3. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Science; 4. Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

Key Words：Leptinotarsa decemlineata, Sid-1, spatial and temporal expression pattern, real-time quantitive PCR, mRNA transcrpt level

Abstract：

      [Objectives]  To clone and analyze two Sid-1 full coding sequences and expression profiles in Leptinotarsa decemlineata. [Methods]  Full coding sequences of two Sid-1s were amplified by RT-PCR and RACE-PCR, conserved motifs and origin of Sid-1s were analyzed by multiple alignment and construction of phylogenetic trees, and temporal and spatial expression levels detected by qPCR. [Results]  A full Open Reading Frame (ORF) of LdeSid-1a and LdeSid-1c were cloned by RT-PCR and 5¢/3¢ RACE from 4^th instar larvae of the Colorado Potato Beetle, Leptinotarsa decemlineata. The respective mRNA fragments were 2 887 and 3 733 base pairs in length, encoding proteins comprised of 759 and 782 amino acids with a predicted molecular mass of approximately 86.19 and 90.27 ku. Similarity of these two proteins was 59%. Alignment of LdeSid-1a and LdeSid-1a with Sid-1s of other insects indicates that LdeSid-1a and LdeSid-1c are members of Coleptera Sid-1s family. LdeSid-1a and LdeSid-1c possess 11 transmembrane structures and 4 highly conserved motifs at the N-terminal. The temporal expression pattern of LdeSid-1a and LdeSid-1c determined by qPCR shows that expression of these two genes gradually increases as larvae grow; LdeSid-1c is highly expressed in the egg stage. Spatial expression patterns indicate that these two genes were expressed in all tested tissues; moderately expressed in the digestive system, including the foregut, midgut and hindgut, and predominantly expressed in the nervous system. The LdeSid-1a and LdeSid-1c sequences have been deposited with GenBank under Accession Numbers KR153284 and KR153285. [Conclusion]  LdeSid-1a and LdeSid-1c have typical Sid-1 structure and expression patterns, and phylogenetic analysis indicate these two genes may play key roles in larval development.