›› 2017, Vol. 60 ›› Issue (8): 876-890.doi: 10.16380/j.kcxb.2017.08.004

• RESEARCH PAPERS • Previous Articles     Next Articles

Molecular cloning of the C-type lectin gene PxCTL5 and its mRNA level changes under bacterial stimulation in Plutella xylostella (Lepidoptera: Plutellidae)

YU Jing, XU Xiao-Xia, GAO Yan-Fu, JIN Feng-Liang*   

  1. (Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, College of Agriculture, South China Agricultural University, Guangzhou 510642, China)
  • Online:2017-08-20 Published:2017-08-20

Abstract: 【Aim】 This study aims to clone a C-type lectin gene from Plutella xylostella, to investigate its expression patterns and to elucidate its agglutination on bacteria. 【Methods】 Based on the bioinformatical analysis of genome and transcriptome database of P. xylostella, the full-length cDNA of a C-type lectin gene was cloned from P. xylostella by RT-PCR and rapid amplification of cDNA ends (RACE) techniques. Prokaryotic expression plasmid was constructed and the fusion protein was expressed in E.coli BL21. The polyclonal antibody with high serum titer was prepared using the purified fusion protein to immunize New Zealand white rabbit. Real-time quantitative PCR (RT-qPCR) was employed to analyze the expression profiles of this gene in different tissues (hemocyte, cuticle, fat body, midgut and Malpighiam tubules) of the day-1 4th instar larvae and different developmental stages (egg, 1st-4th instar larva, prepupa, pupa, and adult) of P. xylostella. RT-qPCR and Western blot were used to detect the expression difference of this gene after challenge with E. coli and B. thuringiensis at the mRNA and protein levels, respectively. The agglutination of the recombinant protein to the two bacteria was investigated by microscopic observation. 【Results】 A C-type lectin gene was cloned from P. xylostella and named PxCTL5 (GenBank accession no.: KY807084). Its full-length cDNA is 1 243 bp, with the open reading frame (ORF) of 672 bp, encoding 223 amino acid residues. RT-qPCR analysis revealed that PxCTL5 was expressed in all developmental stages, with the highest expression level in adults of P. xylostella, and mainly in the cuticle, in which the transcript level was up-regulated upon B. thuringiensis or E. coli challenge, with the highest expression level at 18 h and 24 h post infection, respectively. Western blot results also confirmed that the expression level of PxCTL5 in the hemolymph was significantly increased after challenge with B. thuringiensis and E. coli, respectively. Agglutination assay in vitro showed that in the presence of calcium ions, PxCTL5 protein had certain agglutination activity on E. coli and B. thuringiensis with a stronger agglutination effect on B. thuringiensis. 【Conclusion】 The mRNA level of PxCTL5 is significantly induced by bacteria, and PxCTL5 protein has high agglutination activity on B. thuringiensis.

Key words: Plutella xylostella, C-type lectin, gene cloning, microbial challenge, immune response