中文摘要：

【目的】克隆棉蚜Aphis gossypii Glover溶菌酶基因并进行原核表达,分析其在不同日龄和不同寄主植物上棉蚜体内的表达特征,为进一步研究溶菌酶的功能及其与棉蚜共生菌间的关系奠定基础。【方法】以无翅棉蚜成虫总RNA为模板,采用RT-PCR与RACE方法获得棉蚜溶菌酶基因的cDNA全长序列;将信号肽序列剪切后的棉蚜溶菌酶基因片段连接到原核表达载体pET-30a,并转化到大肠杆菌Eschericha coli中进行诱导表达;采用定量PCR方法测定该基因在不同日龄（1-19日龄）以及西葫芦、黄瓜和豇豆等不同寄主植物上棉蚜体内的相对表达量。【结果】克隆获得棉蚜溶菌酶基因,命名为AgLYS（Gen Bank登录号：KY575341）,cDNA全长为680 bp,开放阅读框为516bp,编码172个氨基酸残基。AgLYS的N-末端含有长度为36个氨基酸残基的信号肽序列。系统发育分析表明,棉蚜溶菌酶属于i型溶菌酶,并且与豌豆蚜Acyrthosiphon pisum i型溶菌酶基因的氨基酸序列一致性达90%。AgLYS可在大肠杆菌中诱导表达,表达蛋白分子量为20 k D左右。AgLYS在1-19日龄的棉蚜体内均有表达,但在1-9日龄棉蚜体内表达量较低,11日龄后的棉蚜体内表达量极显著升高（P〈0.01）。同时,在西葫芦上饲养的棉蚜体内AgLYS的表达量极显著高于在黄瓜和豇豆上饲养的棉蚜体内的表达量（P〈0.01）。【结论】从棉蚜中克隆获得了i型溶菌酶基因AgLYS,并在大肠杆菌中诱导表达。棉蚜AgLYS基因的表达量受蚜虫日龄及所取食寄主种类的影响,推测棉蚜i型溶菌酶AgLYS可能与棉蚜体内共生菌种群密度的调控相关。

英文摘要：

【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 c DNA 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 p ET-30 a 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 Gen Bank accession no. KY575341. The full-length c DNA of AgLYS is 680 bp,and its ORF is 516 bpwhich 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 itype 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 k D.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