家蝇AMP17基因的克隆及其分子特性和表达模式研究

发布时间：2018-05-03 02:20

  本文选题：家蝇 + AMP基因　； 参考：《中国病原生物学杂志》2017年11期

【摘要】：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(P0.01);雄成虫上调了10 936.37倍(P0.01)。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(P0.05);其次为唾液腺,比体壁上调了1.31倍(P0.01)。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。
[Abstract]:Objective to clone and sequence the AMP17 gene of Musca domestica. Methods the differentially expressed salivary adenoprotein AMP17 gene was screened from the microorganism induced transcription database of Musca domestica. The cDNA library plasmid of the gene was used as template for PCR amplification, and the structure and function of the encoded protein were analyzed by bioinformatics. The specimens of Musca domestica (eggs, larva, pupa, male female adults) and different tissues of 3rd instar larvae (body wall, trachea, salivary gland, fat body, Markov duct and midgut) were collected. Real-time fluorescence quantitative PCR was used to detect the expression of AMP17 gene. Results the specific AMP17 gene fragment of about 495bp. AMP17 gene ORF was 495bp in length, encoding 164 amino acids. The theoretical molecular weight unit was 17.4 脳 10 ~ 3, the isoelectric point was 6.09, and the whole polypeptide chain was hydrophilic. This protein belongs to secretory protein and is mainly distributed outside the cell (including cell wall). There are 5 serine, 3 threonine and 1 tryptophan as protein kinase phosphorylation sites. In the secondary structure, 伪-helix, irregular crimp and extension chain are the main structural elements of protein. Spatio-temporal expression profiles showed that the expression of AMP17 gene was high in the 3rd instar and male adult stage of Musca domestica at different developmental stages, and the egg stage was used as a reference for the expression of AMP17 gene. The sequence of expression level was 20 320.98 times higher than that in egg stage of the 2nd instar larva of female adult larva and 10 936.37 times of P0.01T of male adult larvae in the pupa stage of female adult larva of the third instar larva and male adult larva respectively in the order of the expression level of P0.01and that of the male adult larvae were increased by 20 320.98 times and 10 936.37 times, respectively. The expression of AMP17 gene in different tissues of the third instar larvae of Musca domestica was the highest in different tissues of the third instar larvae of Musca domestica, which was 2.40 times higher than that in the body wall, followed by the salivary gland, which was 1.31 times higher than that in the body wall. Conclusion the AMP17 gene of housefly was cloned successfully and its spatiotemporal expression pattern was preliminarily explored, which laid a foundation for further study of its function.
【作者单位】： 贵州医科大学第二附属医院检验科;贵州医科大学基础医学院;
【基金】：国家自然科学基金项目(No.81760647,81560337)
【分类号】：R384.2
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本文编号：1836542