Corin基因在雪兔季节性毛色变化中的功能研究

Animal seasonal colour changes is important scientific question. Applicants with RNA-Seq and de nove transcriptome, four skin differential expressed genes were obtained between two stages of coat color change, these gene sequences among mountain hare, rabbits and other hares do not lead to differences in protein variations, compared the genome sequences of hare found that Corin gene have a great variations in the promoter region. Corin knockout mouse cause coat color fades, and Corin gene expressed in dermal papilla, so that it have ability for set up relationship between endocrine or nervous-system signals and hair follicle melanocytes, is the only candidate gene. On the basis of previous studies, to identify function of Corin gene in mountain hare, this project intends first get mountain hare Corin gene sequence of variation region, second using CRISPR/Cas9 technology knock out Corin gene of mountain hare, observation its hair color whether variable white; third using CRISPR/Cas9 technology knock mountain hare Corin gene into rabbit genome and replace the different region of rabbit Corin gene, observation hair color whether like mountain hare as appeared seasonal changes. This research has a great theoretical significance for clarifying the regulation of melanogenesis and reception of evironment change.

动物季节性毛色变化是重要的科学问题 。申请者用RNA-Seq和de nove转录组技术获得了雪兔不同毛色变化期间皮肤组织4个差异表达的基因，这些基因的序列和家兔及野兔比较并无导致蛋白质变异的差异；比较变色兔与非变色兔的基因组序列，发现只有Corin基因的启动区发生了很大的变异。Corin基因敲除小鼠毛色变淡，且在毛乳头表达，具备把内分泌或神经系统信号传递到毛囊色素细胞的条件，因此是唯一的候选基因。为了验证雪兔Corin基因的功能，本项目拟首先获得雪兔Corin基因调控区的序列，其次利用CRISPR/Cas9技术将雪兔Corin基因失活，观察其毛色是否变白；第三利用CRISPR/Cas9技术将雪兔Corin基因调控区的变异序列敲入家兔并置换家兔Corin基因相应的区段，观察毛色是否像雪兔一样出现季节性变化。Corin功能的验证对于揭示其调控毛发色素合成及感受环境变化的机理具有重要的理论意义。

动物季节性毛色变化是重要的科学问题。通过转录组技术技术获得了雪兔不同毛色变化期间皮肤组织差异表达基因，这些基因的序列和家兔及野兔比较并无导致蛋白质变异的差异；比较变色兔与非变色兔的基因组序列，发现只有Corin基因的启动区发生了很大的变异。Corin基因敲除小鼠毛色变淡，且在毛乳头表达，具备把内分泌或神经系统信号传递到毛囊色素细胞的条件，是导致雪兔毛色季节性变化的重要候选基因。为了验证雪兔Corin基因的功能，本项目按计划，以家兔基因组为参考，分析了雪兔和欧洲兔的基因组重测序数据。构建了雪兔与獭兔Corin基因的CRISPR/Cas9基因编辑载体，完成了雪兔卵子与受精卵采集、建立了编辑载体转染技术。优化了獭兔卵子与受精卵采集、编辑载体转染技术；建立了獭兔卵子与受精卵培养技术体系，编辑载体的脂质体与电转化技术，胚胎移植技术以及精子载体法转化技术。这些工作都为雪兔和獭兔基因组编辑技术的建立，同时也为进一步揭示Corin及其它基因的功能奠定了技术基础。