褐黄血蜱HSP70基因克隆与表达分析及中肠微生物构成研究
发布时间：2018-11-07 15:24
【摘要】：本研究以褐黄血蜱作为试验材料,主要研究其中肠内容物的微生物构成、HSP70的基因全长克隆和表达,为寄生虫病的防治提供新的思路。本试验选取三个地区、多个生长阶段的雌蜱和雄蜱,经过鉴定,用于后续试验。1、通过RACE法克隆得到了HSP70基因的cDNA序列全长,并对其进行了多种生物信息学分析。结果显示,褐黄血蜱HSP70的cDNA序列全长2349bp,共编码648个aa,蛋白分子量为71164.46Da,理论等电点pI为5.43。Blast分析显示,褐黄血蜱与彩饰花蜱的HSP70同源性最高。2、利用RT-PCR法对褐黄血蜱中HSP70基因的mRNA相对表达量进行了分析,并以管家基因β-actin为内参。发现HSP70在唾液腺中的表达量高于中肠,在半饱血雌性成蜱组织中的表达量要高于饱血雌性成蜱。此外,随着吸血进程,HSP70的表达量逐渐递增,饱血雌性成蜱的表达量最高。可能预示着HSP70除了在冷热应激中发挥作用外,还与蜱吸血、消化、抵抗病原等生命活动有关。3、采用PCR-DGGE法分析中肠内容物的微生物构成,结果表明,褐黄血蜱中肠内确定存在13种微生物,分别为Rickettsia peacockii-. Candidatus Rickettsia angustus、 Candidatus Rickettsia kingi、肠杆菌、贝纳柯克斯氏体、铜绿假单胞杆菌、肺炎克雷伯菌、蜡样芽孢杆菌、菊欧文氏菌、模仿葡萄球菌、软腐果胶杆菌等。该项研究为蜱传病的研究奠定了基础,具有公共卫生意义。
[Abstract]:In this study, Haemaphysalis brownicolor was used as the experimental material to study the microbial composition of intestinal contents, the full-length cloning and expression of HSP70 gene, and to provide a new idea for the prevention and treatment of parasitic diseases. In this study, female and male ticks from three growing stages were selected for further test. 1. The cDNA sequence of HSP70 gene was cloned by RACE method and analyzed by various bioinformatics. The results showed that the cDNA sequence of Haemaphysalis brownicolor (HSP70) was 2349 BP, encoding 648 aa, proteins with a molecular weight of 71164.46 Da. the theoretical isoelectric point pI was 5.43.Blast analysis. The relative mRNA expression of HSP70 gene in Haemaphysalis brownicolor was analyzed by RT-PCR method, and the housekeeper gene 尾-actin was used as internal control. It was found that the expression of HSP70 in salivary gland was higher than that in midgut, and that in female adult ticks was higher than that in female adult ticks. In addition, the expression of HSP70 increased with the development of bloodsucking, and the expression of HSP70 was the highest in female adult ticks. It may indicate that HSP70 not only plays a role in cold and heat stress, but also is related to the life activities of ticks, such as blood absorption, digestion and resistance to pathogens. 3. The microbial composition of midgut contents was analyzed by PCR-DGGE method. The results showed that, Thirteen microbes were identified as Rickettsia peacockii-. in the midgut of Haemaphysalis brownicolor. Enterobacter Candidatus Rickettsia angustus, Candidatus Rickettsia kingi, Bencox's body, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus cereus, Irving's, Staphylococcus mimicus, Pectinobacterium soft rot, etc. This study lays a foundation for the study of tick-borne disease and has public health significance.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.746
本文编号：2316773
[Abstract]:In this study, Haemaphysalis brownicolor was used as the experimental material to study the microbial composition of intestinal contents, the full-length cloning and expression of HSP70 gene, and to provide a new idea for the prevention and treatment of parasitic diseases. In this study, female and male ticks from three growing stages were selected for further test. 1. The cDNA sequence of HSP70 gene was cloned by RACE method and analyzed by various bioinformatics. The results showed that the cDNA sequence of Haemaphysalis brownicolor (HSP70) was 2349 BP, encoding 648 aa, proteins with a molecular weight of 71164.46 Da. the theoretical isoelectric point pI was 5.43.Blast analysis. The relative mRNA expression of HSP70 gene in Haemaphysalis brownicolor was analyzed by RT-PCR method, and the housekeeper gene 尾-actin was used as internal control. It was found that the expression of HSP70 in salivary gland was higher than that in midgut, and that in female adult ticks was higher than that in female adult ticks. In addition, the expression of HSP70 increased with the development of bloodsucking, and the expression of HSP70 was the highest in female adult ticks. It may indicate that HSP70 not only plays a role in cold and heat stress, but also is related to the life activities of ticks, such as blood absorption, digestion and resistance to pathogens. 3. The microbial composition of midgut contents was analyzed by PCR-DGGE method. The results showed that, Thirteen microbes were identified as Rickettsia peacockii-. in the midgut of Haemaphysalis brownicolor. Enterobacter Candidatus Rickettsia angustus, Candidatus Rickettsia kingi, Bencox's body, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus cereus, Irving's, Staphylococcus mimicus, Pectinobacterium soft rot, etc. This study lays a foundation for the study of tick-borne disease and has public health significance.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.746
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