关键词: 松褐天牛, 泛素, 基因克隆, 序列分析, 原核表达

Abstract:

The coding sequence of the ubiquitin gene from Monochamus alternatus was cloned with a pair of degenerated primers by using RT-PCR (GenBank Accession No. EU433567). The length of this opening reading-frame (ORF) was 228 bp, encoding a protein of 76 amino acids with molecular mass of 8.49 ku and theoretical isoelectric point of 5.83. Multiple sequence alignment indicated that M. alternatus ubiquitin was very similar to those of the homologous proteins of other eukaryotic species and shared 94%～98% identity with other eukaryotic ubiquitins at amino acid level. Phylogenetic tree based on the nucleotide sequence indicated that M. alternatus had close relationship with Spodoptera litura. The theoretical three-dimensional structure of the ubiquitin gene was generated by homology modeling. Using pET-32a (+) as a fused expressive vector, a recombinant plasmid which contained ubiquitin gene was constructed. Western blotting indicated that the M. alternatus ubiquitin gene was expressed successfully in the BL21 (DE3) strain of E. coli induced by IPTG. This work is a basis for futher studying the function of ubiquitin in M. alternatus.

Key words: Monochamus alternatus, ubiquitin, gene cloning, sequence analysis, prokaryotic expression