Acta Entomologica Sinica ›› 2017, Vol. 60 ›› Issue (2): 127-135.doi: 10.16380/j.kcxb.2017.02.001

• RESEARCH PAPERS •     Next Articles

Cloning and expression analysis of lysozyme gene AgLYS in the cotton-melon aphid,Aphis gossypii (Hemiptera: Aphididae)

ZHANG Yuan-Chen, LIU Xiang-Dong*   

  1. (College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China)
  • Online:2017-02-20 Published:2017-02-20

Abstract: 【Aim】 This study aims to clone the lysozyme gene of the cotton-melon aphid Aphis gossypii Glover, to construct the expression system of this gene in Escherichia coli, and to analyze its expression profiles in aphids of different day-old and reared on different host plants, so as to further research the biological function of this gene and to illustrate the relationship between lysozyme and symbionts in aphids. 【Methods】 The total RNA was extracted from apterous adults of A. gossypii and used as the template to clone lysozyme gene and to obtain the full-length cDNA sequence using RT-PCR and RACE techniques. The gene fragment of lysozyme gene in A. gossypii without signal peptide sequence was linked to the expression vector pET-30a and then transformed into E. coli to express. The qRT-PCR method was used to examine the relative expression levels of the lysozyme gene in A. gossypii of different day-old (1-19-day-old) and reared on different host plants (zucchini, cucumber and cowpea). 【Results】 A lysozyme gene was cloned from apterous aphids of A. gossypii and named AgLYS with the GenBank accession no. KY575341. The full-length cDNA of AgLYS is 680 bp, and its ORF is 516 bp which encodes 172 amino acid residues. A signal peptide sequence with 36 amino acid residues was found at the N-terminus. The phylogenetic analysis showed that the lysozyme of A. gossypii belongs to i-type and has 90% amino acid sequence identity with that of the pea aphid Acyrthosiphon pisum. AgLYS was expressed in E. coli, and the expressed protein has the molecular weight of approximate 20 kD. AgLYS could be expressed in 1-19-day-old A. gossypii aphids. Its expression levels were lower in 1-9-day-old aphids, but increased extremely significantly in 11-19-day-old aphids (P<0.01). The expression levels of AgLYS were extremely significantly higher in A. gossypii aphids reared on zucchini than in those reared on cucumber and cowpea (P<0.01). 【Conclusion】 Lysozyme i-type was found in A. gossypii, and AgLYS was cloned and expressed in E. coli. The expression of AgLYS is influenced by developmental stages of A.gossypii and host plants. It is inferred that AgLYS might be involved in the regulation of population density of symbionts in A.gossypii.

Key words: Aphis gossypii, lysozyme, gene cloning, gene expression, prokaryotic expression, host plant