棉蚜溶菌酶基因AgLYS的克隆与表达分析

发布时间：2018-06-07 23:16

  本文选题：棉蚜 + 溶菌酶　； 参考：《昆虫学报》2017年02期

【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(P0.01)。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(P0.01)。【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。
[Abstract]:[objective] to clone and express the lysozyme gene of Aphis gossypii Glover in cotton aphid, and analyze its expression characteristics in different days and host plants. In order to further study the function of lysozyme and the relationship between lysozyme and symbiotic bacteria, the full-length cDNA sequence of lysozyme gene of cotton aphid was obtained by RT-PCR and race. The lysozyme gene fragment of APHIS gossypii was ligated into the prokaryotic expression vector pET-30a and transformed into Escherichia coli for induction expression. Relative expression of cotton aphid on cucumber and cowpea. [results] lysozyme gene of cotton aphid was cloned. The full length of the cDNA is 680bp.The open reading frame (ORAF) is 516bp.The N- terminal of AgLYYS encodes 172 amino acid residues. The N-terminal of AgLYS contains 36 amino acid residues. Phylogenetic analysis showed that the lysozyme of cotton aphid belongs to type I lysozyme, and the amino acid sequence of the lysozyme gene of aphid Acyrthosiphon pisum i is 90. AgLYS can be induced and expressed in Escherichia coli. The molecular weight of the expressed protein was about 20kD. AgLYS was expressed in cotton aphids of 1-19 days old, but the expression of Ag-LYS in cotton aphids at 1-9 days old was significantly higher than that in cotton aphids after 11 days of age. At the same time, the expression of AgLYS in cotton aphid raised on zucchini was significantly higher than that in cotton aphid raised on cucumber and cowpea. [conclusion] I type lysozyme gene AgLYS was cloned from cotton aphid and expressed in Escherichia coli. The expression of AgLYS gene in cotton aphids was affected by the aphids' age and host species. It was suggested that the type I lysozyme AgLYS might be related to the density of symbiotic species in cotton aphids.
【作者单位】： 南京农业大学植物保护学院;
【基金】：国家自然科学基金项目(31672034) 江苏省自然科学基金项目(BK20141368)
【分类号】：S433
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本文编号：1993236