發(fā)布時間：2018-06-04 13:12

  本文選題：中國美利奴羊 + 內(nèi)皮型一氧化氮合酶　； 參考：《石河子大學》2015年碩士論文

【摘要】：目的:本研究通過對中國美利奴羊e NOS基因編碼區(qū)SNP位點利用SSCP的方法進行檢測,并對有多態(tài)性位點的外顯子進行克隆,找出與布病易感性相關(guān)的SNP位點。用布魯氏菌M5侵染小鼠后對小鼠e NOS基因的濃度、酶活力進行測定并對肝、脾組織中e NOS基因表達量進行檢測,探討了中國美利奴羊e NOS基因與布魯氏菌病易感性相關(guān)性及布魯氏菌侵染過程中e NOS的作用,為選育綿羊抗病新品種及布病預防尋求新途徑提供一定依據(jù)和理論支持。方法:1、將人、綿羊e NOS基因m RNA序列(登錄號:NM_000603.4、NM_001129901.1)利用BLAST比對分析找出人、綿羊e NOS基因的外顯子序列,再通過Gen Bank中的SNP數(shù)據(jù)庫查找人和綿羊的SNP位點。利用DNAMAN6.0軟件對人e NOS基因外顯子上的SNP位點進行分析,獲得多態(tài)性較為豐富的人e NOS基因外顯子序列,再利用Gene Doc軟件比對找出與這些外顯子相似性較高的綿羊e NOS基因外顯子序列。2、本實驗利用布魯氏菌虎紅平板凝集診斷試劑盒對162只中國美利奴羊血液樣本進行布魯氏菌血清檢測,然后利用PCR-SSCP方法對e NOS基因外顯子的SNP位點進行檢測,后對含有SNP位點的外顯子進行克隆測序。通過SHEsis在線分析軟件對檢測出的SNP位點進行布病易感性分析。3、用布魯氏菌M5侵染小鼠,分別在侵染后第0、3、7、14、28天檢測小鼠e NOS的含量和活力,將測得的數(shù)據(jù)導入EXCEL中,利用SPSS17.0對侵染組和對照組的含量和活力進行統(tǒng)計分析。4、用熒光實時定量PCR技術(shù)對侵染第28天小鼠和對照組小鼠e NOS基因進行擴增,通過2-△△Ct法分析和修正數(shù)據(jù),最后將所得結(jié)果導入EXCEL中,得到肝、脾組織相對表達量,對生成的各個樣本的相對表達量采用SPSS17.0軟件進行單因素方差分析。結(jié)果:1、通過生物信息學分析,找到與人同源性高的中國美利奴羊e NOS基因的10個外顯子,這10個外顯子分別是exon2、exon7、exon8、exon13、exon14、exon16、exon17、exon18、exon19、exon20而在人的這10個外顯子中多態(tài)性較為豐富。2、對162只中國美利奴羊進行虎紅平板檢測,共檢測發(fā)現(xiàn)陰性101只,陽性個體為61只,并對出現(xiàn)的陽性樣本進行復檢,布魯氏菌檢出率為37.65%。利用PCR-SSCP的方法對這162只中國美利奴羊e NOS基因10個外顯子進行檢測,發(fā)現(xiàn)中國美利奴羊e NOS基因exon8序列第142位發(fā)生了堿基突變,由G變?yōu)锳,氨基酸由Ala變?yōu)門hr,該突變位點也是一個新的突變位點(ss974768653:A142G)。利用SHEsis在線統(tǒng)計分析軟件和SPSS17.0統(tǒng)計分析軟件,結(jié)果表明該多態(tài)性位點的基因型及等位基因頻率分布均符合Hardy-Weiberg平衡定律(P0.05),具有群體代表性,但該位點的基因型及等位基因頻率分別在侵染組和對照組之間的差異無統(tǒng)計學意義(P0.05)。3、用布魯氏菌M5侵染小鼠后,發(fā)現(xiàn)在侵染后第14天出現(xiàn)了肝、脾腫大。在第0、3、7、14、28天分別測定侵染組和對照組e NOS含量和活力,通過統(tǒng)計分析發(fā)現(xiàn)侵染組和對照組e NOS含量沒有發(fā)生變化,而e NOS活力在侵染后的第14天開始升高。4、利用實時熒光定量對侵染第28天和對照組中小鼠肝、脾e NOS基因進行表達分析發(fā)現(xiàn)侵染組中肝和對照組中肝的相對表達量差異不顯著,P值為0.685(P0.05);在侵染組和對照組中脾的相對表達量差異不顯著,P值為0.512(P0.05)。結(jié)論:1、經(jīng)過PCR-SSCP方法對中國美利奴羊e NOS基因的10個外顯子進行多態(tài)性分析,最終在exon8上發(fā)現(xiàn)1個多態(tài)性位點,但是該多態(tài)性位點與綿羊布病易感性無關(guān)。2、對侵染后的小鼠e NOS含量和活力進行檢測后發(fā)現(xiàn),e NOS含量在侵染后沒有發(fā)生明顯變化,而e NOS活力在侵染后第14天出現(xiàn)升高,說明布魯氏菌侵染小鼠后,并沒有引起e NOS含量的變化,而在第14天以后引起e NOS活力升高,這一過程也可能導致NO釋放量升高。3、利用實時熒光定量PCR對侵染組和對照組小鼠肝、脾進行相對表達量分析后發(fā)現(xiàn)侵染組中肝和對照組肝中e NOS的表達量沒有發(fā)生明顯變化,侵染組中脾和對照組脾中e NOS的表達量沒有發(fā)生明顯變化,說明布魯氏桿菌M5對小鼠肝、脾組織中e NOS的表達沒有影響。
[Abstract]:Objective: to detect the use of SSCP in the SNP loci of the e NOS gene coding region of Chinese Merino sheep, and to clone the exons of the polymorphic loci and find the SNP loci related to the susceptibility to the disease. The concentration of E NOS gene, the activity of the enzyme and the liver and spleen tissue of the mice were detected by the infection of Brucella M5. The expression of E NOS gene was detected, and the correlation between the e NOS gene of Chinese Merino and brucellosis and the role of E NOS in the process of Brucella infection were discussed. It provided a certain basis and theoretical support for the breeding of new varieties of sheep disease resistance and the new way of disease prevention. Method: 1, the m RNA sequence of human and sheep e NOS gene ( Record number: NM_000603.4, NM_001129901.1) using BLAST comparison to find out the exons of human and sheep e NOS gene, and then find the SNP loci of human and sheep through SNP database in Gen Bank. Using DNAMAN6.0 software, the polymorphism of human e NOS gene exons is analyzed by DNAMAN6.0 software, and the polymorphism exons are obtained. Sequence, and then using Gene Doc software to find out the exons of E NOS gene of sheep with high similarity with these exons, this experiment uses Brucella tiger red plate agglutination diagnostic kit to detect Brucella sera in 162 Chinese Merino blood samples, and then PCR-SSCP method is used to detect SNP of the exons of E NOS gene. The exons containing SNP loci were cloned and sequenced. The susceptibility analysis of the detected SNP site was analyzed by SHEsis online analysis software and.3 was used to detect the SNP site. The content and vitality of the mice e NOS were detected on 0,3,7,14,28 days after the infection, and the measured data were introduced into EXCEL, and SPSS was used for SPSS. 17 the content and vitality of the infection group and the control group were statistically analyzed.4. The e NOS gene of the infected mice and the control group was amplified by real time fluorescence quantitative PCR technique. The data were analyzed and modified by 2- Delta Delta Ct method. Finally, the results were introduced into EXCEL to obtain the relative expression of the liver and spleen tissue, and the samples were produced. The relative expression was analyzed by SPSS17.0 software. Results: 1, through bioinformatics analysis, we found 10 exons of the e NOS gene of Chinese Merino sheep with high homology. These 10 exons were Exon2, exon7, exon8, exon13, exon14, exon16, EXON17, exon18, Exon19, and 10 exons. The polymorphism of.2 was more abundant, and 162 Chinese Merino sheep were detected by Tiger red plate, 101 were detected negative, 61 positive individuals were detected, and the positive samples were rechecked. The detection rate of Brucella was 37.65%. using the PCR-SSCP method to detect 10 exons of the 162 Chinese Merino sheep NOS gene, and found that the 10 exons of the 162 Chinese Merino sheep were detected. The 142nd bits of the exon8 sequence of the e NOS gene of the Gome were changed from G to A, the amino acid changed from Ala to Thr, and the mutation site was also a new mutation site (ss974768653:A142G). The results showed that the genotype and allele frequency of the polymorphic loci were used in the SHEsis Online statistical analysis software and SPSS17.0 software. The distribution accorded with the Hardy-Weiberg equilibrium law (P0.05), which had group representation, but there was no significant difference between the genotype and allele frequency between the infection group and the control group (P0.05).3 respectively. After the infection of the mice with Brucella M5, the liver and splenomegaly were found fourteenth days after the infection. The results were measured on the day 0,3,7,14,28 respectively. The content and vitality of E NOS in the infection group and the control group were determined. By statistical analysis, the content of E NOS in the infection group and the control group did not change, while the e NOS activity began to increase in the.4 after the fourteenth day of infection. The expression analysis of the liver and the NOS gene of the spleen E in the infected twenty-eighth days and the control group, and the NOS gene of the spleen E were detected by the real-time fluorescence quantitative analysis. The relative expression of liver in the irradiated group was not significant, the P value was 0.685 (P0.05), the relative expression of spleen in the infection group and the control group was not significant, and the P value was 0.512 (P0.05). Conclusion: 1, 10 exons of the Chinese Merino e NOS gene were analyzed by PCR-SSCP method, and 1 polymorphic loci were found on exon8, but the 1 polymorphic loci were found in exon8. The polymorphic loci were not related to the susceptibility to the susceptibility of sheep brucellosis to.2. After detection of the e NOS content and activity after infection, the content of E NOS did not change obviously after the infection, and the activity of E NOS increased at fourteenth days after infection. It showed that after Brucella infected mice, the NOS content of E was not changed, but after fourteenth days. The activity of E NOS increased, and this process may also lead to the increase of.3 in the release of NO. The expression of E NOS in the liver and the control group of the infected group and the control group was not significantly changed after the analysis of the relative expression of the spleen in the infection group and the control group. The expression of E NOS in the spleen and the control group of the infected group was no more than that of the spleen and the control group. Obvious changes showed that Brucella M5 had no effect on the expression of E NOS in liver and spleen tissues of mice.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.26

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本文編號：1977491