本文選題：家蠅 + AMP基因　； 參考：《中國(guó)病原生物學(xué)雜志》2017年11期

【摘要】：目的克隆家蠅AMP17基因并進(jìn)行、序列分析,對(duì)其時(shí)空表達(dá)模式進(jìn)行初步探索。方法從微生物誘導(dǎo)的家蠅轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中篩選差異高表達(dá)唾液腺蛋白AMP17基因。以該基因的cDNA文庫(kù)質(zhì)粒為模板進(jìn)行PCR擴(kuò)增,運(yùn)用生物信息學(xué)方法對(duì)其編碼蛋白進(jìn)行結(jié)構(gòu)與功能分析。取家蠅不同生活史時(shí)期標(biāo)本(卵,各齡幼蟲(chóng),蛹,雄雌成蟲(chóng))及3齡幼蟲(chóng)不同組織部位(體壁、氣管、唾液腺、脂肪體、馬氏管及中腸)標(biāo)本,采用實(shí)時(shí)熒光定量PCR檢測(cè)AMP17基因表達(dá)情況。結(jié)果 PCR擴(kuò)增得到約495bp的特異性AMP17基因片段。AMP17基因ORF全長(zhǎng)495bp,編碼164個(gè)氨基酸,理論分子質(zhì)量單位17.4×103,等電點(diǎn)為6.09,整個(gè)多肽鏈表現(xiàn)為親水性。該蛋白屬于分泌蛋白,主要分布在細(xì)胞外(包括細(xì)胞壁)。磷酸化位點(diǎn)分析該蛋白,有5個(gè)絲氨酸、3個(gè)蘇氨酸、1個(gè)色氨酸可能成為蛋白激酶磷酸化位點(diǎn)。其二級(jí)結(jié)構(gòu)中主要以α-螺旋,不規(guī)則卷曲和延伸鏈為蛋白最大量的結(jié)構(gòu)元件。時(shí)空表達(dá)譜顯示,家蠅不同發(fā)育時(shí)期中,以卵期作為參照,AMP17基因在3齡及雄成蟲(chóng)時(shí)期表達(dá)量高,表達(dá)水平排列順序?yàn)?齡幼蟲(chóng)雄成蟲(chóng)1齡幼蟲(chóng)雌成蟲(chóng)蛹期2齡幼蟲(chóng)卵,3齡幼蟲(chóng)時(shí)期表達(dá)量比卵期上調(diào)了20 320.98倍(P0.01);雄成蟲(chóng)上調(diào)了10 936.37倍(P0.01)。以體壁作為參照,AMP17基因在家蠅3齡幼蟲(chóng)不同組織的表達(dá)以馬氏管表達(dá)最高,比體壁上調(diào)了2.40倍(P0.05);其次為唾液腺,比體壁上調(diào)了1.31倍(P0.01)。結(jié)論成功克隆了家蠅AMP17基因并初步探索了其時(shí)空表達(dá)模式,為進(jìn)一步研究其功能奠定了基礎(chǔ)。
[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, 偽-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.
【作者單位】： 貴州醫(yī)科大學(xué)第二附屬醫(yī)院檢驗(yàn)科;貴州醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(No.81760647,81560337)
【分類(lèi)號(hào)】：R384.2
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本文編號(hào)：1836542