九香虫i型溶菌酶基因的克隆与细菌诱导表达分析

Cloning and induced expression analysis of the i-type lysozyme genes in Coridius chinensis

溶菌酶是一组进化上保守的酶,在昆虫中主要分为c型和i型两类。为明确i型溶菌酶在九香虫Coridius chinensis体内的表达模式与功能,我们对九香虫的两个i型溶菌酶基因CcLys-i1和CcLys-i2进行了克隆,这两个基因的编码区分别为504 bp和432 bp,分别编码167和143个氨基酸。序列分析显示,CcLys-i1和CcLys-i2缺乏与催化活性相关的谷氨酸(E)和丝氨酸(S)。同源性和聚类分析显示,CcLys-i1和CcLys-i2与斯氏珀蝽Plautia stali的i型溶菌酶相似性最高,分别为80.36%和48.95%。采用实时荧光定量PCR(RT-qPCR)解析CcLys-i1和CcLys-i2基因的时空表达谱,结果表明它们在九香虫不同发育阶段都有表达,CcLys-i1在成虫中表达水平最高,而CcLys-i2在5龄若虫中表达水平最高;它们在所检测的成虫不同组织中都有表达,都在脂肪体中表达水平最高。九香虫被注射细菌后24 h,CcLys-i1和CcLys-i2的表达显著上调;九香虫被饲喂细菌后,这两种基因的表达水平没有显著变化。九香虫的这两种i型溶菌酶基因的表达受到自身免疫机制的调控,可以被诱导而参与免疫应答,但不具有参与消化的功能。该研究为进一步明确九香虫i型溶菌酶基因的功能奠定基础。

Lysozymes are a group of evolutionarily conserved enzymes and can be divided into c-type and i-type in insects.In order to clarify the expression pattern and function of the i-type lysozyme in Coridius chinensis,we cloned two i-type lysozyme genes CcLys-i1 and CcLys-i2.The coding regions of these two genes were 504 bp and 432 bp,encoding 167 and 143 amino acids,respectively.Sequence analysis showed that CcLys-i1 and CcLys-i2 lacked glutamic acid(E)and serine(S)related to catalytic activity.Homology and cluster analyses showed that CcLys-i1 and CcLys-i2 shared the similarity of 80.36%and 48.95%,respectively,with the i-type lysozyme from Plautia stali.The spatiotemporal expression profiles of CcLys-i1 and CcLys-i2 were analyzed by using real-time quantitative PCR(RT-qPCR).The results indicated that these two genes were expressed at various developmental stages of C.chinensis,with the highest expression levels for CcLys-i1 in the adults and for CcLys-i2 in the fifth-instar nymphs;they were expressed in different tissues of adults and had the highest expression level in the fat bodies.The expression levels of CcLys-i1 and CcLys-i2 were significantly upregulated within 24 h post bacterial injection,but there were no significant changes in the expression levels of these two genes post bacterial feeding.The expression of these two i-type lysozyme genes in C.chinensis was regulated by its own immune mechanism and could be induced to participate in immune response,but they were not involved in digestion.This study laid a foundation for further elucidating the functions of the i-type lysozyme genes in C.chinensis.