奶牛瘤胃原虫引物设计优化及其在日粮效应评价中的应用

发布时间：2018-04-05 09:05

  本文选题：瘤胃原虫　切入点：多样性　出处：《中国农业科学院》2015年硕士论文

【摘要】：瘤胃原虫在反刍动物日粮营养成分的降解、瘤胃内环境的维持和反刍动物甲烷排放方面具有重要作用,因此近年来受到研究者的广泛关注。基于未培养技术的研究方法越来越多的被应用在瘤胃原虫多样性研究之中,18S r RNA基因发育分析和定量PCR是瘤胃原虫研究中较为常见的方法。但是,现有的瘤胃原虫引物设计较早,难以覆盖新发现的原虫。因此,本研究旨在设计与优化瘤胃原虫18S r RNA全长基因扩增引物,并应用于评价日粮效应对奶牛瘤胃原虫群落多样性和数量的影响。为此,本研究主要分为三个部分:1、从Silva数据库中下载137条纤毛亚门原虫的基因序列,利用保守区域设计4条引物,结合已知2对原虫引物,组合形成5对瘤胃原虫18S r RNA全长基因扩增引物。对这5对引物进行碱基覆盖度和特异性的验证后,发现引物P.324f和P.1747r_2碱基覆盖度高(大于98%),可有效区分瘤胃原虫和其他真核微生物。该引物扩增片段为1423 bp更适用于瘤胃原虫群落多样性和系统发育分析,作为最优引物用于原虫多样性研究。为了优化原虫多样性分析中OTU划分阈值,利用0.010-0.041阈值对原虫18S r RNA序列进行OTU划分,发现在0.015阈值下相同序列被分到不同OTU中的概率和同一个OTU包含两个不同原虫种属的概率最低。2、分别以苜蓿干草与玉米青贮(MF)和玉米秸秆作(CSA)为日粮主要纤维来源,以豆粕和杂粕(CSB)作为日粮主要蛋白质来源,进行奶牛饲喂试验。在试验的第91天晨饲前(0 h)和后(2 h)采集瘤胃液,提取微生物DNA,对瘤胃原虫18S r RNA基因测序并进行系统发育分析。结果表明,不同纤维来源日粮处理后文库差异显著(Libshuff,P0.005),但是不同蛋白质来源之间差异不显著。不同日粮处理对瘤胃原虫丰富度没有显著影响,以苜蓿干草和玉米青贮为纤维来源日粮原虫多样性显著高于玉米秸秆组,但是不同蛋白质来源日粮间原虫多样性差异不显著。系统发育分析表明,瘤胃中优势原虫主要来源于Dasytricha(54%)和Entodinium(35%)。3、在不同纤维和蛋白质来源日粮影响瘤胃原虫群落结构的情况下,探究瘤胃原虫数量的变化。试验材料和方法同第二部分内容,但是试验进行到91天后,所有处理组统一回归的MF日粮,再继续试验16天。在试验的第31(只有晨饲后)、61、91和107天的晨饲前和后采集瘤胃液。通过显微镜观察对上述样品的瘤胃原虫进行计数,结果表明不同处理间瘤胃原虫数量没有显著差异。通过对91天样品微生物DNA的定量PCR分析也出现上述相似结果,表明不同纤维和蛋白质日粮对瘤胃总原虫数量没有显著影响。为了对原虫18S r RNA基因克隆文库结果进行验证,又对瘤胃内主要的原虫种属Dasytricha和Entodinium进行了相对定量PCR分析,结果发现不同纤维和蛋白质来源日粮对Entodinium数量没有显著影响;但是与饲喂苜蓿干草和玉米青贮组相比,以玉米秸秆为纤维来源日粮能使Dasytricha数量显著增加。综上所述,本试验获得了一对碱基覆盖度高、特异性好并且扩增片段较长的瘤胃原虫18S r RNA全长基因扩增引物;优化了原虫OTU划分的阈值;以苜蓿干草和玉米青贮为纤维来源日粮组原虫多样性高于玉米秸秆饲喂组,系统发育分析表明瘤胃原虫主要归属于Dasytricha和Entodinium,玉米秸秆为纤维来源饲喂后瘤胃Dasytricha相对丰度显著增加。
[Abstract]:In the degradation of rumen protozoa on ruminant animal nutrition food, plays an important role in maintaining and ruminant animal methane in the rumen environment, so in recent years has attracted the attention of researchers. The research method is not based on the culture technology more and more are used in the study of rumen protozoa diversity, 18S R RNA gene phylogenetic analysis and PCR is a common method for quantitative study of rumen protozoa in the rumen protozoa. However, primers were designed to cover the existing earlier, the newly discovered protozoan. Therefore, this study aims to design and optimization of 18S r of rumen protozoa RNA gene amplification primers, and to evaluate the effect of dietary diversity and quantity of dairy cow rumen protozoa community effect. Therefore, this research is mainly divided into three parts: 1, 137 gene sequences downloaded from the Silva database Ciliophora protozoa, 4 primers were designed by the conserved region, With 2 of protozoa known primers, combined to form a total length of 5 18S r on rumen protozoa RNA gene primers. Base verification coverage and specificity of the 5 pairs of primers, primers P.324f and P.1747r_2 found that the base coverage is high (more than 98%), which can effectively distinguish the protozoa and other eukaryotic microorganisms. The primer the amplified fragment was 1423 BP for rumen protozoa community diversity and phylogenetic analysis, as the optimal primer for the protozoa diversity research. In order to optimize the threshold OTU partition analysis of the diversity of protozoa, the OTU division of 18S R RNA protozoa sequences by using 0.010-0.041 threshold, found in 0.015 under the same threshold sequence was divided into different probability OTU the same OTU contains two different kinds of protozoa of the genus.2 were the lowest probability, to alfalfa hay and corn silage (MF) and corn straw (CSA) as dietary fiber in soybean meal and the main source. Miscellaneous meal diets (CSB) as the main source of protein, dairy cow feeding test. In the test ninety-first days before the morning feeding (0 h) and after (2 h) acquisition of rumen microbial DNA extraction, 18S R, on rumen protozoa RNA gene sequencing and phylogenetic analysis. The results showed that the difference of different fiber library the source of dietary treatment significantly (Libshuff, P0.005), but no significant difference between different protein sources. Different dietary treatments have no significant effect on rumen protozoa abundance, on alfalfa hay and corn silage as the dietary fiber source protozoan diversity was significantly higher than that of maize straw group, but not significantly different protein source diets between protozoan diversity difference. Phylogenetic analysis showed that the rumen protozoa advantage comes mainly from Dasytricha (54%) and Entodinium (35%).3, with different fiber and protein source diets affect the community structure of the original insect rumen, exploration The changes in the number of rumen protozoa. Materials and methods with second parts, but the test was carried out after 91 days, all treatment groups unified regression of the MF diet, and then continue to test 16 days. In the test thirty-first (only after morning feeding), 61,91 and 107 days before and after the morning feeding collecting rumen fluid. Count by microscope on the samples of rumen protozoa, results showed that there was no significant difference between treatments. The number of rumen protozoa by quantitative PCR analysis of 91 day samples of microbial DNA also appeared similar results show that different fiber and protein diets had no significant effect on the total number of rumen protozoa. In order to verify the 18S r protozoa RNA gene clone library results on ruminal protozoa and the main species of the genera Dasytricha and Entodinium are analyzed and relative quantitative PCR, the results showed that different sources of dietary fiber and protein on Entodinium The amount of no significant impact; but compared with those fed alfalfa hay and corn silage, corn straw was the dietary fiber source can cause a significant increase in the number of Dasytricha. In conclusion, this experiment was on a base with high coverage, primer 18S r of rumen protozoa RNA gene specific fragment and the longer the protozoa; OTU partition threshold optimization; on alfalfa hay and corn silage for dietary fiber source protozoa diversity is higher than that of corn straw feeding group, phylogenetic analysis showed that rumen protozoa mainly attributable to Dasytricha and Entodinium, corn straw fiber source after feeding rumen Dasytricha relative abundance increased significantly.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816
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本文编号：1714094