猪蛋白编码基因内SINE转座子插入多态位点鉴定及其对基因活性和表型的影响

The biological roles of transposon in the evolution of genome and gene have attracted more and more attention, and molecular markers based on the transposon insertion polymorphism (TIP) also play an important role in the studies of plant genome mapping, and the origin and evolution of human and genetic disease association. We have obtained some initial results in the transposon annotation in pig, however, the number of transposon insertion locus within the protein coding genes in pig genome, and the impacts of these insertions on the gene activity and phenotypes remain totally unknown, which are worth for further investigation. Based on the previous annotation results of SINE in our laboratory, this project intend to use the different resources of wild boars, native pig breeds, commercial pig breeds, cultured pig breeds (lines) as the research material to screen TIPs on the whole-genome level, and obtain the data of the SINE-TIPs within protein coding genes. Then the association analysis of these SINE-TIPs with the phenotypic variations were conducted to identify the potentially functional TIPs. Finally, two to three SINE-TIPs will be selected to investigate the molecular mechisims of their effects on the gene activity and phenotypes. This study will facilitate the understanding of the role of transposon in the evolution of pig genome and genes, and promote the studies of pig functional genomics and molecular breeding. Two to three papers are expected to be published in the international reputation journals, at least two patents will be applied, and 3-5 graduates will be trained.

转座子在基因组和基因进化中的作用越来越受到关注，转座子插入多态（TIP）分子标记在植物基因组作图、人类起源进化和遗传疾病关联等研究领域中发挥了重要作用。项目组前期在对猪基因组转座子注释中取得了初步成果，但猪基因组上蛋白编码基因内转座子插入位点数量以及这些转座插入对基因活性和表型的影响尚不清楚。本项目拟基于SINE转座子的注释结果，在不同来源的野猪、家猪原始品种、商业化品种、培育品种（系）中开展全基因组水平蛋白编码基因内SINE转座子插入多态位点（SINE-TIP）筛选；获取蛋白编码基因内SINE-TIP位点数据，并进行表型关联分析，鉴定与表型关联性较强的蛋白编码基因内SINE-TIP，并系统研究2-3个SINE-TIP对基因活性的影响及其引起表型变异的分子机制。预期成果将推动猪功能基因组学和分子育种研究进程；预期在国际威权杂志上发表研究论文2-3篇；申请专利2项以上；培养研究生3-5名。

本项目借助多品种猪基因组数据进行了逆转座子插入多态鉴定工作，对SINE插入年龄分析发现SINEA1最年轻且呈现出最高的插入多态性率，其次是SINEA2和SINEA3，是结构变异的重要来源。进而构建了全基因组SINE-RIP挖掘方案，获得了36284个SINE-RIPs，均匀分布在染色体中，平均密度为14.5/Mb，超过65%的SINE-RIP分布在基因中，对应约四分之一的基因，其中260个SINE RIPs位于蛋白编码基因外显子区，且多为非编码区。进一步在GH-IGFs、ZNFs等基因家族中鉴定到34个有效SINE-RIPs；在LEPTIN等基因中鉴定到3个有效SINE-RIPs；在毛色相关基因和繁殖相关基因中鉴定到55个有效SINE-RIPs。进一步注释发现GFBP3-1-RIP、GHR-RIP-10、LEPROT-SINE-RIP和ZNF2-SINE-RIP在各靶基因的核心启动子附近，且实验证实，GFBP3-1-RIP位点中的SINE插入对靶基因启动子有促进作用，而其余三个位点的SINE插入对靶基因启动子有抑制作用。基因表达分析结果显示，GFBP3-1-RIP位点SINE插入个体的GFBP3基因在肝脏和背膘中均显著增强表达（P<0.01）；GHR-RIP-10位点SINE插入个体的GHR基因在腿肌（P<0.05）和背最长肌（P<0.01）中显著抑制基因表达。进一步将上述位点与大白猪群体中与生长和繁殖性能进行关联分析，结果显示IGFBP3-1-RIP位点与校正背膘厚（P<0.01）和眼肌厚（P<0.05）显著相关；ZNF2-SINE-RIP位点与校正背膘显著相关（P<0.05）；LEPROT-SINE-RIP位点（P<0.01）与百公斤日龄和宰前体重显著相关；ESR1-SINE-RIP1位点与大白猪校正背膘厚和百公斤日龄显著相关（P<0.05）；Ktn1-STIP位点与断奶窝重显著相关（P<0.05）；CWH43-9位点与大白猪产活仔数显著相关（P<0.05）。同时我们利用部分SINE-RIPs标记成功进行了小型猪、苏山猪、地方猪的遗传多样性分析，其结果准确反映出群体遗传状态或家系关系，证实了其有效性。项目执行期间论文20篇，其中SCI论文14篇；授权专利8项，申请专利2项；培养硕士生8名，博士生2名，博士后1名。