适宜CRISPR/Cas9编辑的生长性状位点预测及其在太湖猪中的应用  

Prediction of Growth Trait Sites Suitable for CRISPR/Cas9 Editing and Its Application in Taihu Pigs(Sus scrofa)

作　　者：赵伟 刘登英 孙浩[1] 岳阳 王起山[1] 潘玉春[1] ZHAO Wei;LIU Deng-Ying;SUN Hao;YUE Yang;WANG Qi-Shan;PAN Yu-Chun(School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学农业与生物学院,上海200240

出　　处：《农业生物技术学报》2018年第11期1880-1889,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31772552);上海市农委项目(沪农科推字(2016)第1-1-4号)

摘　　要：太湖猪(Sus scrofa)作为我国著名的地方猪品种,具有繁殖力高、耐粗饲、抗病性好、肉质佳等诸多特点,是分子育种和基因组选择的重要遗传资源。本研究基于猪数量性状基因座(quantitative trait locus,QTL)数据库、基因本体(gene ontology, GO)、通路分析(ingenuity pathway analysis, IPA)数据库等生物信息学技术对猪生长性状相关基因进行挖掘,同时筛选出其与西方品种存在品种间差异的特异SNPs,通过基因相关性,SNP特异性,SNP位置等信息综合预测可以通过基因编辑改良太湖地方品种的位点,并对猪基因组单导链RNA(single guide RNA, sgRNA)进行脱靶分析,筛选出CRISPR/Cas9编辑的最优sgRNA,旨在为太湖地方猪生长性状研究提供准确真实的基因编辑位点。在猪基因组中,共检测到127 353 502个前间区序列邻近基序(protospacer adjacent motif, PAM)位点,平均每20 bp存在1个PAM位点,共观察到5种PAM类型,包括TGG、AGG、GGG、CGG和NGG,占比分别为34.52%,32.61%,26.43%,6.43%和0.0082%。最终得到猪生长性状候选基因428个,筛选得到太湖流域猪种二花脸、枫泾、嘉兴黑、米猪、中梅山、沙乌头、小梅山的生长相关特异SNPs数量分别为412、282、372、414、393、279、458个,并为每一个位点综合打分,最后依据脱靶分析,为6个品种的每个生长相关SNP位点分别提供4个最优的sg RNA。本研究为后续高效准确的基因编辑工作提供数据基础。As a famous local pig breed in China, Taihu pig （Sus scrofa） has many characteristics such as high fecundity, resistance to rough feeding, good disease resistance and good flesh quality. It has been an important genetic resource for molecular breeding and genome selection. Based on the bioinformatics techniques such as pig quantitative trait locus （QTL） database, gene ontology （GO） database and ingenuity pathway analysis （IPA） database, this study mined the genes related to growth traits in pigs. In order to verify the accuracy of the candidate genes obtained, this study uploaded the candidate genes to the IPA database for online analysis. The results indicated that the candidate genes obtained were mainly enriched in 10 gene networks, and they were mainly enriched in gene expression, body development, cell growth, embryonic development, bone and muscle system development and other functions. Many candidate genes related to pig growth traits have been widely studied and reported. All of the above indicated that the candidate genes of pig growth trait obtained had a certain degree of confidence. This study based on the sequencing data in genotyping by genome reducing and sequencing （GGRS） platform, detected the SNPs of 6 Taihu pig breeds and screened out specific SNPs which had interspecific differences with western breeds. Then through the analyses of genetic correlation, SNP specificity, SNP location and other information, this study predicted the sites where can genetically improve the local breeds of Taihu by gene editing. In order to screen out the best single guide RNA （sgRNA） near the gene editing site and complete the gene editing, this study prepared a script based on Perl language to analyze the whole pig genome, and found out the protospacer adjacent motif （PAM） and sgRNA target sites in pig genome. In the pig genome, a total of 127 353 502 PAM sites were detected, with an average of one PAM site per 20 bp, and these PAM sites were evenly distributed within the chromosome. A

关 键 词：太湖猪 生长性状 CRISPR/Cas9 候选基因 

分 类 号：S813.3[农业科学—畜牧学]