【摘要】：本研究以褐黃血蜱作為試驗材料,主要研究其中腸內容物的微生物構成、HSP70的基因全長克隆和表達,為寄生蟲病的防治提供新的思路。本試驗選取三個地區(qū)、多個生長階段的雌蜱和雄蜱,經過鑒定,用于后續(xù)試驗。1、通過RACE法克隆得到了HSP70基因的cDNA序列全長,并對其進行了多種生物信息學分析。結果顯示,褐黃血蜱HSP70的cDNA序列全長2349bp,共編碼648個aa,蛋白分子量為71164.46Da,理論等電點pI為5.43。Blast分析顯示,褐黃血蜱與彩飾花蜱的HSP70同源性最高。2、利用RT-PCR法對褐黃血蜱中HSP70基因的mRNA相對表達量進行了分析,并以管家基因β-actin為內參。發(fā)現(xiàn)HSP70在唾液腺中的表達量高于中腸,在半飽血雌性成蜱組織中的表達量要高于飽血雌性成蜱。此外,隨著吸血進程,HSP70的表達量逐漸遞增,飽血雌性成蜱的表達量最高�？赡茴A示著HSP70除了在冷熱應激中發(fā)揮作用外,還與蜱吸血、消化、抵抗病原等生命活動有關。3、采用PCR-DGGE法分析中腸內容物的微生物構成,結果表明,褐黃血蜱中腸內確定存在13種微生物,分別為Rickettsia peacockii-. Candidatus Rickettsia angustus、 Candidatus Rickettsia kingi、腸桿菌、貝納柯克斯氏體、銅綠假單胞桿菌、肺炎克雷伯菌、蠟樣芽孢桿菌、菊歐文氏菌、模仿葡萄球菌、軟腐果膠桿菌等。該項研究為蜱傳病的研究奠定了基礎,具有公共衛(wèi)生意義。
[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.
【學位授予單位】：湖南農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.746

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本文編號：2316773