鉴别影响猪脊椎数主效基因VRTN的因果突变位点

The porcine vertebrae consist of cervical, thoracic, lumbar, sacral and caudal vertebrae. The numbers of cervical and sacral vertebrae are always fixed whereas the number of thoracic and lumbar vertebrae varies considerably in pigs. The number of vertebrae is significantly associated with carcass length, and it has been estimated that one extra vertebra can expand the carcass length of about 15 mm in adult pigs. In our previous study, we characterize the VRTN gene that underlies phenotypic variation in thoracic vertebrae based on a series of genetic analyses. Moreover, we identified two most likely causative mutations in this gene. However, the real causative mutation is still unclear. And the temporal and spatial expression and molecular mechanisms of this gene remains unexplored. In this study, we plan to implement RT-PCR and Western blot to investigate the expression characteristics of VRTN in early embryonic body segment in pigs. Using the Chip-seq analysis, we attempt to dissect the roles of VRTN binding sites in regulation network of vertebra formation. In addition, the applicants intend to adopt Luciferase reporter assay and VRTN-LacZ transgenic mice to identify the association between these two mutations and VRTN transcriptional control, thus preliminarily distinguishing the causative mutation. Finally, we plan to introduce single and both mutations into mouse VRTN by site-directed integration using the Crispr/cas9 system, with the aim to finally determine the causative mutation affecting thoracic vertebrae on SSC7 that is expected to increase the number of thoracic vertebrae in genome-editing mice.

猪的脊椎包括颈、胸、腰、荐和尾椎，其中颈、荐和尾椎相对固定而胸椎和腰椎存在变化。脊椎数与胴体长明显相关，每增加一根脊椎数可使成年猪体长增加15 mm。申请人及所在课题组通过一系列遗传学分析鉴别到了影响猪胸椎数的主效基因VRTN及其两个候选主效突变位点，但并未甄别出哪一个是真正的因果突变，有关该基因的时空表达特性、分子作用机理也仍属空白。本课题拟采用RT-PCR和Western blot研究VRTN基因在猪早期胚胎体节中的时空表达特性，通过Chip-seq分析其蛋白结合位点在胸椎形成调控网络中的作用；利用Luciferase报告基因表达体系和VRTN-LacZ转基因小鼠解析两个突变位点与基因转录调控的关联性并初步鉴别出因果突变位点；通过Crispr/cas9基因编辑技术将两个突变位点定点整合入小鼠VRTN基因中，通过基因编辑小鼠胸椎数是否增加以最终确定因果突变位点。

本课题首先针对杜长大群体（609头）开展胸椎数的全基因组关联（GWAS）分析；结果表明，与本实验室之前在杜洛克×二花脸、苏太和二花脸×通城群体中的GWAS结果相吻合。针对小鼠5.5-12.5天（dpc）胚胎的数字PCR检测表明，VRTN在胚胎体节发育时期（7.5-9.5 dpc）高量表达。上述结果进一步支持VRTN是影响胸椎数的因果基因的结论。针对4个候选因果突变的荧光素酶报告检测发现，g.19034A>C和g.20311_20312ins291突变等位基因可上调靶基因的表达且具有加性效应。LacZ转基因小鼠研究表明，双拷贝291 bp片段可特异性地在9.5 dpc鼠胚的头部、心脏、体轴后端、新形成的体节及前体节中胚层前端上调LacZ基因的表达。此外，VRTN在猪胸部体节形成时期沿前后体轴的表达从头到尾逐渐减弱，且17.5 dpc猪胚中，g.19034A>C和g.20311_20312ins291两位点纯合突变型（QQ）个体的VRTN mRNA及蛋白表达量均显著高于野生型（qq）个体。上述结果支持该两个位点是调控胸椎数的因果突变位点。随后开展的细胞核定位实验、免疫共沉淀测序等实验，证明了VRTN定位于细胞核中，是一种新的转录因子。VRTN纯合敲除鼠胚在9.5 dpc和10.5 dpc时体轴翻转失败，体节数少于13个，在11.5 dpc消化殆尽；一半以上VRTN杂合敲除成年小鼠脊椎发育异常，其肋骨数减少一对或单侧减少一根。针对17.5 dpc的QQ和qq型猪胚开展RNA-seq和通路分析，表明NOTCH2是VRTN的靶基因之一，VRTN调控NOTCH信号通路内体节发育重要基因HES1的表达。此外，课题组还搭建了高效的家猪CRISPR/Cas9基因组编辑平台，以SSODN为修复模板的编辑技术，点编辑效率达到20%；实现了13.4 kb外源基因的定点整合，编辑效率达5.76%。利用搭建的编辑平台，课题组将巴马香猪胎儿成纤维细胞中VRTN的qq基因型编辑成QQ，为下一步研制VRTN基因编辑猪奠定了基础。总之，本课题的研究首次从生物学功能层面对影响猪胸椎数的因果基因VRTN进行了验证，揭示了VRTN是一种对胸椎发育至关重要的转录因子，为基于VRTN-QTNs的猪胸椎数（肋骨数）分子育种提供了坚实有力的证据，同时为人类先天性脊柱畸形等相关疾病的临床研究提供了重要的借鉴。