优秀单板U型场地滑雪运动员外周血淋巴细胞cDNA文库构建及ESTs分析.doc

优秀单板U型场地滑雪运动员外周血淋巴细胞cDNA文库构建及ESTs分析 　　摘 要：??建一个优秀单板U型场地滑雪运动员血液的cDNA文库，经测序从中获取大量的表达序列标签（Expressed Sequence Tags）ESTs，用于基因筛选和功能预测。方法：通过对多名优秀冰雪运动员血液样品的总RNA进行提取、混合总RNA，合成双链cDNA、蛋白酶K消化、Sfi1酶切并连接载体、λ噬菌体的包装，经扩增最终获得优秀冰雪运动员cDNA文库；随后从文库中随机挑选500个克隆提交至北京诺赛基因组研究中心有限公司进行测序，测序结果利用Chromas软件去低质量样品，使用GenBank中的BLAST功能对所获序列进行比对获取相关信息，将获得的ESTs信息提交PANTHR和GO（Gene Ontology）生物信息数据库进行基本归类分析，从中获取运动员cDNA文库中所涵盖的重要遗传信息。结果：得到滴度分别为1.32×106 pfu/mL和1.65×1010 pfu/mL的未扩增文库和扩增后文库，扩增后文库的重组率为90.6%；得到长度在300～1 100 bp序列360个，平均长度606 bp；通过BLAST比对得出201条具有注释的基因信息，利用PANTHER数据库基本分析发现这些基因分布在27个pathway上，其中TXN、MDH1、ARL1、ARPC3、ACTG1等基因分别分布在参与新陈代谢、氧化应激反应、心血管系统机能等相关联的信号通路上，推测其可能与运动能力存在关联。通过GO分析对所测序列进行归类得到在该数据库中具有功能注释的基因143个，参与31项不同功能，主要包括细胞组分、分子功能和生物过程。结论：构建了一个全长的运动员cDNA文库，其质量符合构建cDNA文库的标准 关键词：单板U型场地滑雪；外周血淋巴细胞；全长cDNA文库；表达序列标签 中图分类号：G 804.2 文章编号：1009-783X（2017）03-0254-05 文献标识码：A Abstract： Objective： The aim of this study is to construct a cDNA library of blood from elite half-pipe snowboarders. And we can get a large number of ESTs （Expressed Sequence Tags） from it by sequencing the genes. We can also use it for gene screening and function prediction. Methods： The cDNA library of blood from elite half-pipe snowboarders was constructed by a series of experiments extracting and mixing Total RNA of several elite ice-snow athletes， synthesizing double-stranded DNA， proteinase K digestion， Sfi 1 enzyme digestion， connecting with vector， packaging phage λ and amplification. Then 500 clones from the library were randomly selected to be sequenced. Low-quality samples in the results of sequence were removed by Chromas software. The relevant information of ESTs was captured by matching gene sequences with BLAST of GenBank. Then the information of ESTs was presented to PANTHR and GO（Gene Ontology）bioinformatics database to make a basic classification and analysis and to get some important genetic information in the cDNA library of athletes. Results： The titer of cDNA library unamplified was 1.32×106pfu/ml， and the titer of