本文選題：瘤胃原蟲　切入點：多樣性　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：瘤胃原蟲在反芻動物日糧營養(yǎng)成分的降解、瘤胃內(nèi)環(huán)境的維持和反芻動物甲烷排放方面具有重要作用,因此近年來受到研究者的廣泛關(guān)注�；谖磁囵B(yǎng)技術(shù)的研究方法越來越多的被應(yīng)用在瘤胃原蟲多樣性研究之中,18S r RNA基因發(fā)育分析和定量PCR是瘤胃原蟲研究中較為常見的方法。但是,現(xiàn)有的瘤胃原蟲引物設(shè)計較早,難以覆蓋新發(fā)現(xiàn)的原蟲。因此,本研究旨在設(shè)計與優(yōu)化瘤胃原蟲18S r RNA全長基因擴(kuò)增引物,并應(yīng)用于評價日糧效應(yīng)對奶牛瘤胃原蟲群落多樣性和數(shù)量的影響。為此,本研究主要分為三個部分:1、從Silva數(shù)據(jù)庫中下載137條纖毛亞門原蟲的基因序列,利用保守區(qū)域設(shè)計4條引物,結(jié)合已知2對原蟲引物,組合形成5對瘤胃原蟲18S r RNA全長基因擴(kuò)增引物。對這5對引物進(jìn)行堿基覆蓋度和特異性的驗證后,發(fā)現(xiàn)引物P.324f和P.1747r_2堿基覆蓋度高(大于98%),可有效區(qū)分瘤胃原蟲和其他真核微生物。該引物擴(kuò)增片段為1423 bp更適用于瘤胃原蟲群落多樣性和系統(tǒng)發(fā)育分析,作為最優(yōu)引物用于原蟲多樣性研究。為了優(yōu)化原蟲多樣性分析中OTU劃分閾值,利用0.010-0.041閾值對原蟲18S r RNA序列進(jìn)行OTU劃分,發(fā)現(xiàn)在0.015閾值下相同序列被分到不同OTU中的概率和同一個OTU包含兩個不同原蟲種屬的概率最低。2、分別以苜蓿干草與玉米青貯(MF)和玉米秸稈作(CSA)為日糧主要纖維來源,以豆粕和雜粕(CSB)作為日糧主要蛋白質(zhì)來源,進(jìn)行奶牛飼喂試驗。在試驗的第91天晨飼前(0 h)和后(2 h)采集瘤胃液,提取微生物DNA,對瘤胃原蟲18S r RNA基因測序并進(jìn)行系統(tǒng)發(fā)育分析。結(jié)果表明,不同纖維來源日糧處理后文庫差異顯著(Libshuff,P0.005),但是不同蛋白質(zhì)來源之間差異不顯著。不同日糧處理對瘤胃原蟲豐富度沒有顯著影響,以苜蓿干草和玉米青貯為纖維來源日糧原蟲多樣性顯著高于玉米秸稈組,但是不同蛋白質(zhì)來源日糧間原蟲多樣性差異不顯著。系統(tǒng)發(fā)育分析表明,瘤胃中優(yōu)勢原蟲主要來源于Dasytricha(54%)和Entodinium(35%)。3、在不同纖維和蛋白質(zhì)來源日糧影響瘤胃原蟲群落結(jié)構(gòu)的情況下,探究瘤胃原蟲數(shù)量的變化。試驗材料和方法同第二部分內(nèi)容,但是試驗進(jìn)行到91天后,所有處理組統(tǒng)一回歸的MF日糧,再繼續(xù)試驗16天。在試驗的第31(只有晨飼后)、61、91和107天的晨飼前和后采集瘤胃液。通過顯微鏡觀察對上述樣品的瘤胃原蟲進(jìn)行計數(shù),結(jié)果表明不同處理間瘤胃原蟲數(shù)量沒有顯著差異。通過對91天樣品微生物DNA的定量PCR分析也出現(xiàn)上述相似結(jié)果,表明不同纖維和蛋白質(zhì)日糧對瘤胃總原蟲數(shù)量沒有顯著影響。為了對原蟲18S r RNA基因克隆文庫結(jié)果進(jìn)行驗證,又對瘤胃內(nèi)主要的原蟲種屬Dasytricha和Entodinium進(jìn)行了相對定量PCR分析,結(jié)果發(fā)現(xiàn)不同纖維和蛋白質(zhì)來源日糧對Entodinium數(shù)量沒有顯著影響;但是與飼喂苜蓿干草和玉米青貯組相比,以玉米秸稈為纖維來源日糧能使Dasytricha數(shù)量顯著增加。綜上所述,本試驗獲得了一對堿基覆蓋度高、特異性好并且擴(kuò)增片段較長的瘤胃原蟲18S r RNA全長基因擴(kuò)增引物;優(yōu)化了原蟲OTU劃分的閾值;以苜蓿干草和玉米青貯為纖維來源日糧組原蟲多樣性高于玉米秸稈飼喂組,系統(tǒng)發(fā)育分析表明瘤胃原蟲主要歸屬于Dasytricha和Entodinium,玉米秸稈為纖維來源飼喂后瘤胃Dasytricha相對豐度顯著增加。
[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.

【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S816
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本文編號：1714094