牛microRNA的分离、鉴定及其表达谱研究

发布时间：2018-03-25 02:01

本文选题：牛　切入点：microRNA　出处：《河北农业大学》2015年硕士论文

【摘要】：牛不仅具有重要的经济价值更有很高的生物价值。越来越多的证据表明,非编码RNA在转录后水平对动物的生长发育起了很重要的调节作用。Micro RNAs(mi RNAs)是一类内源性的长度大约在22nt左右的非编码单链RNA分子,广泛地分布在动、植物及病毒中。它可以通过与靶基因m RNA的3’UTR区域碱基互补配对的方式,来使靶m RNA发生降解或者抑制其翻译。牛mi RNA研究相对滞后,本研究构建了由3月龄胎儿、6月龄胎儿、12月龄青年牛3个不同生长时期的多个组织混合的mi RNA的c DNA文库,然后对3个文库进行高通量测序,结合生物信息学方法对牛的mi RNA进行深度挖掘,并通过茎环RT-q PCR的方法检测了部分mi RNA在3个不同生长时期的组织表达谱,取得的主要成果如下:构建了3个由处于3种不同生长时期的多个组织mi RNA混合的c DNA文库,通过对3个文库的高通量测序,分别获得了24665837,19963158,20286502的原始读段(raw reads)。经过质量控制后,得到高质量的reads(mapped reads)的数量分别为23619093条,18746297条,18455215条。3个文库中得到的mi RNA序列长度均主要分布在20~24nt,且在22nt处达到峰值。并且3个文库中的Unique Reads在牛的所有染色体上均有分布。采用mi RDeep程序对测序结果进行分析,3个文库中分别获得了696,646,605个mi RNAs,其中已知mi RNA的数量分别为449,435,430个;未知mi RNA的数量分别为247,211,175个。采用茎环法RT-q PCR对9个mi RNAs进行了组织表达谱研究,发现bta-mi R-1、bta-mi R-133、bta-mi R-206在背最长肌和心肌中特异性表达,bta-mi R-378在背最长肌中特异性表达,bat-mi R-338、bta-mi R-421、bta-mi R-2453、Novel_mi R-381、Novel_mi R-86在3月龄胎儿的各个组织中均有不同程度的表达,在6月龄胎儿和青年时期时仅在个别组织中表达。本研究通过对3个c DNA文库的高通量测序并结合生物信息学分析,发现了大量的牛mi RNA序列,同时鉴定了部分mi RNA的表达谱,为后续牛的基因组学相关研究奠定了理论基础。
[Abstract]:Cattle have not only important economic value, but also high biological value. There is growing evidence that, Non-coding RNA plays an important role in regulating the growth and development of animals at post-transcriptional level. Micro RNAs(mi RNs are a class of non-coding single-stranded RNA molecules with endogenous length about about 22nt, which are widely distributed in animals. In plants and viruses, it can degrade or inhibit the translation of target m RNA by complementing the 3'UTR region of the target gene m RNA. In this study, we constructed the c DNA library of mi RNA mixed from three tissues of 3-month-old fetal / 6-month-old young cattle at 12 months of age, and then carried out high-throughput sequencing of the three libraries. In combination with bioinformatics method, the deep mining of bovine mi RNA was carried out, and the tissue expression profiles of partial mi RNA in three different growth stages were detected by means of RT-q PCR of stem ring. The main results are as follows: three c DNA libraries mixed with mi RNA in three different growth stages were constructed. By high-throughput sequencing of the three libraries, the original reading segment of 24665837 / 1996315820286502 was obtained. The number of high quality reads(mapped was 23619093. The length of mi RNA sequence from the three libraries was mainly distributed in 20 ~ 24nt, and reached a peak value at 22nt. The Unique Reads of the three libraries was found on all chromosomes of cattle. The results of sequencing were analyzed by using mi RDeep program. 696646605 mi RNAss were obtained from 3 libraries, among which the number of known mi RNA was 449435430; The number of unknown mi RNA was 247211175. The tissue expression profiles of 9 mi RNAs were studied by RT-q PCR with stem ring method. It was found that bta-mi R-1 bta-mi R-133n bta-mi R-206 was specifically expressed in the longissimus dorsi muscle and myocardium and bta-mi R-378 was expressed specifically in the longissimus dorsi muscle. In this study, a large number of bovine mi RNA sequences were identified by high-throughput sequencing of three c DNA libraries and bioinformatics analysis, and partial expression profiles of mi RNA were also identified. It lays a theoretical foundation for further studies on bovine genomics.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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