家蝇AMP17基因的克隆及其分子特性和表达模式研究

Bioinformatic analysis,cloning,and patterns of expression of the Musca domestica AMP17 gene

目的克隆家蝇AMP17基因并进行、序列分析,对其时空表达模式进行初步探索。方法从微生物诱导的家蝇转录组数据库中筛选差异高表达唾液腺蛋白AMP17基因。以该基因的cDNA文库质粒为模板进行PCR扩增,运用生物信息学方法对其编码蛋白进行结构与功能分析。取家蝇不同生活史时期标本(卵,各龄幼虫,蛹,雄雌成虫)及3龄幼虫不同组织部位(体壁、气管、唾液腺、脂肪体、马氏管及中肠)标本,采用实时荧光定量PCR检测AMP17基因表达情况。结果 PCR扩增得到约495bp的特异性AMP17基因片段。AMP17基因ORF全长495bp,编码164个氨基酸,理论分子质量单位17.4×103,等电点为6.09,整个多肽链表现为亲水性。该蛋白属于分泌蛋白,主要分布在细胞外(包括细胞壁)。磷酸化位点分析该蛋白,有5个丝氨酸、3个苏氨酸、1个色氨酸可能成为蛋白激酶磷酸化位点。其二级结构中主要以α-螺旋,不规则卷曲和延伸链为蛋白最大量的结构元件。时空表达谱显示,家蝇不同发育时期中,以卵期作为参照,AMP17基因在3龄及雄成虫时期表达量高,表达水平排列顺序为3龄幼虫>雄成虫>1龄幼虫>雌成虫>蛹期>2龄幼虫>卵,3龄幼虫时期表达量比卵期上调了20 320.98倍(P<0.01);雄成虫上调了10 936.37倍(P<0.01)。以体壁作为参照,AMP17基因在家蝇3龄幼虫不同组织的表达以马氏管表达最高,比体壁上调了2.40倍(P<0.05);其次为唾液腺,比体壁上调了1.31倍(P<0.01)。结论成功克隆了家蝇AMP17基因并初步探索了其时空表达模式,为进一步研究其功能奠定了基础。

Objective To bioinformatically analyze,clone,and determine the patterns of expression of the AMP17 gene of Musca domestica. Methods A transcriptome database was searched for M.domestica highly expressing the AMP17 gene.The gene and the protein it codes for were analyzed bioinformatically.Specimens of M.domestica were collected during different developmental stages（eggs,three instar larvae,pupae,adult females,and adult males）.Specimens were collected from different sites in three instar larvae（the integument,the trachea,salivary glands,the fat body,Malpighian tubules,and the midgut）.The patterns of AMP17 gene expression were detected with real-time quantitative PCR（real-time PCR）. Results A specific AMP17 gene fragment about 495 bp in length was obtained after amplification with PCR.Bioinformatic analysis indicated that full-length ORF of MSP was 495 bp,and the AMP17 gene encoded164 amino acids.AMP17 has a molecular weight of 17.4×10^3 and a pI of 6.09.The polypeptide is hydrophilic and it is a secretory protein found mainly outside cells（but possibly in cell walls）.Protein kinase phosphorylation sites were identified as 5 serines,3 threonines,and 1 tryptophan.The secondary structure of MSP consisted of alpha-helices,random coils,and extended strands.Spatial and temporal patterns of AMP17 expression indicated that expression of the AMP17 gene differed in each stage M.domestica development.With eggs as a reference,the level of AMP17 expression was higher in third instar larvae and males.The level of expression was highest in third instar larvae,followed by adult males,then first instar larvae,adult females,pupa,second instar larvae,and finally eggs.The level of AMP17 expression in third instar larvae was 20 320.98 times that in eggs（P〈0.01）;the level of AMP17 expression in adult males was10 936.37 times（P〈0.01）that in eggs.Levels of AMP17 expression in Malpighian tubules and salivary glands were significantly higher than those in other tissues.The level of AMP17 expression in Malpighian tubules was 2.40 times（P〈0.05）that in the integument;the level of AMP17 expression in salivary glands was 1.31 times（P〈0.01）that in the integument.Spatial and temporal patterns of AMP17 expression indicated that expression of the AMP17 gene differed in each stage of M.domestica development.The level of AMP17 expression was higher in third instar larvae and adult males.The level of AMP17 expression in third instar larvae was significantly higher in Malpighian tubules and salivary glands than that in other tissues. Conclusion This study successfully cloned the AMP17 gene and it preliminarily examined patterns of that gene＇s expression.These findings provide a basis for further study of the function of AMP17.