关键词: 松墨天牛, 肠道, 漆酶, 全长克隆, 原核表达, 表达谱

Abstract: 【Aim】 This study aims to clone and identify the laccase gene MaLac1 from the Japanese pine sawyer, Monochamus alternatus, and to analyze its expression levels in different developmental stages of the beetle, so as to provide some clues for further study of the function of MaLac1. 【Methods】 Based on the transcriptome sequencing data of M. alternatus gut, the full-length cDNA of MaLac1 was cloned from M. alternatus using the RACE method and analyzed by bioinformatic software. MaLac1 sequence was then inserted into the prokaryotic expression vector pET-32a(+) to construct a recombinant plasmid pET-MaLac1, which was then transformed into Escherichia coli Rosetta (DE3) to express MaLac1. The expression patterns of MaLac1 in the gut of M. alternatus at different developmental stages (early instar larva, mature larva, pupa, female adult, and male adult) were assayed by qPCR. 【Results】 The full-length cDNA of MaLac1 from M. alternatus (GenBank accession no.： KY073340) were obtained. Its open reading frame is 2 067 bp in length, encoding a protein of 688 amino acids, with an estimated molecular mass of 78.34 kD and an isoeletric point of 5.30. MaLac1 was predicted to contain an N-terminal signal peptide with 15 amino acids by SignalP 4.1 Server. Sequence alignment analysis showed that MaLac1 shows the typical characteristics of insect laccase genes, with 93% amino acid sequence identity with the laccase gene of Tribolium castaneum. SDS-PAGE results showed that IPTG induced a specific protein band of about 78 kD, which was consistent with the estimated size. The qPCR results showed that MaLac1 was expressed in the gut of M. alternatus at various developmental stages, with the highest expression level in the female adult gut, followed by that in the male adult gut, and the lowest expression level in the larval gut. 【Conclusion】 The expression level of MaLac1 in adults of M. alternatus is significantly higher than that in larvae, and this may be relevant to the feeding differences between adults and larvae. Further study is needed to reveal the exact function of MaLac1 in M. alternatus. 

Key words: Monochamus alternatus, gut, laccase, full-length cloning, prokaryotic expression, expression pattern