家蝇GNBP3基因克隆及感染白色念珠菌后的表达

Cloning and expression pattern of GNBP3 gene under the fungal infection in Musca domestica

对家蝇GNBP3基因进行克隆及生物信息学分析,并对该基因在感染白色念珠菌Candida albicans后的表达情况进行研究。从构建的家蝇Musca domestica幼虫c DNA质粒文库中筛选到GNBP3基因,克隆并运用生物信息学方法对该基因及其编码蛋白进行预测和分析。白色念珠菌注射感染家蝇幼虫并收集标本,逆转录,通过荧光定量PCR检测感染样本中GNBP3基因的表达情况,结果进行统计学分析。研究结果表明,GNBP3基因ORF全长1473 bp,编码490个氨基酸,理论分子量为55.8 k Da,等电点为6.85;感染后不同时间点,感染组与对照组比较,在3 h、12 h、36 h、48 h GNBP3 mRNA的表达明显增高,两组比较有显著性差异(P<0.05),而24 h表达差异性最小;感染后不同组织中感染组与对照组比较,3 h和12 h时,在体壁、血淋巴、脂肪体中的表达量升高,具有显著性差异(P<0.05);24 h时,在血淋巴中的表达量较对照组高,而在唾液腺和脂肪体中的表达量呈下降趋势,均具有显著性差异(P<0.05);48 h,在血淋巴和脂肪体中表达量较对照组高,在中肠的表达量较对照组低,均具有显著性差异(P<0.01)。成功克隆GNBP3基因且在家蝇感染白色念珠菌后GNBP3的表达水平随时间的推移呈现先升高后降低再升高的趋势,在各组织中以在免疫组织(血淋巴、脂肪体)中的表达显著增高,推测其在真菌识别过程中发挥潜在作用。

To cloning and research the expression pattern of GNBP3 Gene in Musca domestica larvae infected by Candida albicans. The GNBP3 gene which was isolated from M. domestica c DNA library was analyzed by the bioinformatics methods. M. domestica larvae infection C. albicans and collect specimens,total RNA was extracted from these samples and reverse transcription for c DNA,the expressions of GNBP3 were detected by real-time fluorescent quantitative PCR. Laboratory data were then analysed statistically.The results indicated that the full-length of GNBP3 gene was 1473 bp, encoding 490 amino acid. The molecular weight 55. 8 k Da and p I of 6. 85. Different time points after infection,infection group GNBP3 mRNA expression increased obviously in 3 h,12 h and 36 h,48 h compared with control group,comparing the two groups have a significant difference（ P〈0. 05）,and 24 h express different minima;Infection group different tissue compared with control group,express in the body wall,hemolymph and blood fat body volume significantly increased in 12 h,3 h（ P〈0. 05）; Higher expression in the haemolymph,but low amount of expression in the salivary glands and the fat body than the control group of24 h,have significant difference（ P〈0. 05）; Expressed in the hemolymph and blood fat body quantity higher,but low expression of intestinal amount than those of the control group in 48 h,all have significant difference（ P〈0. 01）. The M. domestica GNBP3 gene was successfully purified and analyzed by the bioinformatics methods. M. domestica larvae infection C. albicans,GNBP3 expression levels decrease with the passage of time after the first increase and then the trend of rise,the expression significantly increased in immunohistochemical（ hemolymph and blood fat body）,presumably play in the process of identifying the potential role of fungus.