【摘要】：從家蠅EST測(cè)序數(shù)據(jù)庫(kù)中篩選獲得家蠅幾丁質(zhì)酶基因MDCII,對(duì)該基因進(jìn)行克隆及分子特性分析,探討其在家蠅不同組織、不同發(fā)育時(shí)期及經(jīng)不同微生物誘導(dǎo)后的時(shí)空表達(dá)模式。利用EST測(cè)序技術(shù)從已構(gòu)建的家蠅幼蟲(chóng)c DNA質(zhì)粒文庫(kù)篩選出MDCII基因,運(yùn)用生物信息學(xué)方法分析該基因序列及其編碼蛋白的理化特性,采用鄰接法構(gòu)建系統(tǒng)進(jìn)化樹(shù);PCR技術(shù)擴(kuò)增目的基因,構(gòu)建p EASY-E1-MDCII重組質(zhì)粒,轉(zhuǎn)化到Trans1-T1克隆感受態(tài)細(xì)胞中;采用實(shí)時(shí)熒光定量PCR(Real Time PCR)技術(shù),檢測(cè)MDCII基因在不同發(fā)育時(shí)期和不同組織部位的表達(dá)差異;采用注射法將不同微生物導(dǎo)入到家蠅3齡幼蟲(chóng)體內(nèi),Real Time PCR檢測(cè)誘導(dǎo)后不同時(shí)間點(diǎn)MDCII基因表達(dá)水平的變化。結(jié)果顯示,MDCII基因的ORF框全長(zhǎng)1 374 bp,編碼457個(gè)氨基酸,理論分子量51.6 k D,進(jìn)化樹(shù)分析比對(duì)與果蠅成蟲(chóng)盤(pán)生長(zhǎng)因子的遺傳距離較近。構(gòu)建了具有正確基因序列的p EASY-E1-MDCII重組質(zhì)粒。MDCII基因在家蠅不同發(fā)育階段中均有不同程度的表達(dá),在3齡幼蟲(chóng)中,以唾液腺和脂肪體中的表達(dá)水平較高;在白色念珠菌、金黃色葡萄球菌、大腸埃希菌誘導(dǎo)后3 h,MDCII基因均出現(xiàn)明顯的表達(dá)上調(diào)。MDCII基因?qū)儆趲锥≠|(zhì)酶中成蟲(chóng)盤(pán)生長(zhǎng)因子,參與了家蠅的生長(zhǎng)發(fā)育,在免疫防御過(guò)程中也發(fā)揮了一定作用。
[Abstract]:The chitinase gene MDCII, was selected from the EST sequencing database of housefly to clone the gene and analyze its molecular characteristics. The expression patterns of chitinase gene in different tissues, different developmental stages and induced by different microorganisms were discussed. The MDCII gene was screened by EST sequencing from the constructed cDNA library of housefly larva c DNA, and the physicochemical properties of the gene and its encoded protein were analyzed by bioinformatics. The recombinant plasmid of p EASY-E1-MDCII was constructed and transformed into the competent Trans1-T1 cells by using the method of contiguous construction of phylogenetic tree-PCR technique, and real-time fluorescence quantitative PCR (Real Time PCR) technique was used to amplify the target gene, and the recombinant plasmid was transformed into the competent cells of Trans1-T1. The expression of MDCII gene was detected in different developmental stages and different tissues, and the changes of MDCII gene expression at different time points after induction were detected by injecting different microorganisms into the third instar larvae of Musca domestica. The results showed that the ORF frame of MDCII gene encoded 457 amino acids with theoretical molecular weight of 51.6 kD, and the genetic distance between phylogenetic tree analysis and adult disk growth factor of Drosophila melanogaster was close. The recombinant plasmid of p EASY-E1-MDCII. MDCII with correct gene sequence was constructed and expressed in different developmental stages of Musca domestica. In the third instar larva, the expression level was higher in salivary gland and fat body, while in Candida albicans, the expression level was higher in the third instar larva, and in Candida albicans. The expression of MDCII gene in Staphylococcus aureus and Escherichia coli were obviously up-regulated. MDCII gene belonged to adult disk growth factor in chitinase, which participated in the growth and development of housefly and played a certain role in immune defense.
【作者單位】： 貴州醫(yī)科大學(xué);貴州醫(yī)科大學(xué)附屬醫(yī)院腫瘤生物治療中心;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(81560337,81160204)
【分類(lèi)號(hào)】：Q78;Q966
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本文編號(hào)：2214045