基于CRISPR/Cas9技术研究脊尾白虾类胡萝卜素氧化酶基因的功能及其育种价值评估

Carotenoids, a family of natural pigments, play an important role in animals, which can protect eyesight,enhance immunity and improve the animal reproductive ability..The physiological function based on carotenoids has been used in animal breeding. In this project, Exopalaemon carinicauda is taken as the research animal, and the key genes EcBCMO1 and EcBCO2 on the carotenoids metabolic pathway are selected as the target genes by the candidate gene approach. EcBCMO1 and EcBCO2 will be knockout via CRISPR/Cas9 technology. Then, EcBCMO1- and EcBCO2-knockout mutants are cultivated. The differences of physiological and biochemical characteristic, gene expression and regulation between the mutant and the wild-type prawns will be analyzed. At the same time, EcBCMO1 and EcBCO2 will be recombinantly expressed in Pichia pastoris, and the characteristics of the purified recombinant EcBCMO1 and EcBCO2 will be studied and compared. Based on the above research, the biological functions of EcBCMO1 and EcBCO2 will be clarified. In addition, the differences in resistance to environmental factors between the mutant and the wild-type prawns will be helpful to evaluate the possibility of EcBCMO1 and EcBCO2 in the genetic breeding of crustacean. .In summary, the implementation of this project will be helpful for the study of carotenoid metabolism and physiological function of other crustaceans. In addition, it will provide a model for the cultivation of crustacean varieties with more health, more beautiful appearance, higher nutritional value and stronger resistance.

类胡萝卜素是一类天然色素的总称，在动物体内具有重要作用，可增强机体免疫力，提高动物繁殖能力。本项目拟以脊尾白虾为研究对象，以类胡萝卜素代谢关键基因β-胡萝卜素-15, 15′-单加氧酶（EcBCMO1）与β-胡萝卜素-9′, 10′-单加氧酶（EcBCO2）为目标基因，利用CRISPR/Cas9技术分别构建EcBCMO1和EcBCO2敲除模型，培育稳定遗传的突变品系；对突变品系与野生群体在生理生化、基因表达调控等进行差异分析，结合EcBCMO1和EcBCO2重组蛋白的酶学特征，进而阐释脊尾白虾类胡萝卜素氧化酶的生物学功能；进一步比较EcBCMO1和EcBCO2突变品系与野生群体在不同环境因子胁迫下的抗逆性差异，对类胡萝卜素氧化酶在脊尾白虾遗传育种中的应用价值进行初步评估。通过本项目实施，为后续开展其他甲壳动物类胡萝卜素代谢的生理功能研究及培育体色与肌肉颜色美观、抗逆性强的新品系提供参考。

类胡萝卜素是一类天然色素的总称，在动物体内具有重要作用，可增强机体免疫力，提高动物繁殖能力。本项目以脊尾白虾为研究对象，克隆了其类胡萝卜素氧化酶EcNinaB-X1和EcBCO2基因，利用CRISPR/Cas9技术构建EcNinaB-X1和EcBCO2敲除模型，培育稳定遗传的纯合突变品系；进一步开展了突变品系与野生群体在体色及水生致病菌胁迫下的抗病差异分析，并对其分子机制进行了初步探究。结果发现：（1）获得的EcNinaB-X1和EcBCO2基因突变体的体色（尤其是肝胰脏的颜色）发生了显著性变化，同时，其在病原刺激下的死亡率明显降低。（2）纯合突变体EcBCO2-KO与野生型相比，其对相同浓度致病菌的抵抗力明显增强；副溶血弧菌刺激48h组的野生型脊尾白虾死亡率为50%，EcBCO2基因敲除组死亡率为20%左右；嗜水气单胞菌刺激组的野生型脊尾白虾死亡率为65%，EcBCO2基因敲除组死亡率为25%左右；阴性对照PBS组的EcBCO2基因敲除组与野生型组脊尾白虾累积死亡率均在10%左右。（3）纯合型突变体EcNinaB-X1-KO与野生群体相比，其对相同致病菌的抵抗力也明显增强，副溶血弧菌刺激48h组的野生型脊尾白虾死亡率为50%，EcNinaB-X1基因敲除组死亡率为20%左右；嗜水气单胞菌刺激组的野生型脊尾白虾死亡率为65%，EcNinaB-X1基因敲除组死亡率为25%左右；阴性对照PBS组的EcNinaB-X1基因敲除组与野生型组脊尾白虾累积死亡率均在10%左右。（4）EcNinaB-X1基因敲除与EcBCO2基因敲除后的突变体，在受到副溶血弧菌与嗜水气单胞菌的刺激后，其死亡率趋势基本一致，提示2个基因之间可能存在着补偿机制，团队开展了EcNinaB-X1与EcBCO2纯合突变体的杂交实验，获得了EcNinaB-X1与EcBCO2双位点突变的突变体，目前相关研究正在进行中。（5）利用RNA-seq技术，初步解析了EcNinaB-X1-KO与EcBCO2-KO颜色改变与抗病力增强的分子机制。综上，研究结果表明，类胡萝卜素氧化酶在脊尾白虾遗传育种中具有潜在的应用价值。通过本项目实施，为后续开展其他甲壳动物类胡萝卜素代谢的生理功能研究及培育体色与肌肉颜色美观、抗逆性强的新品系提供参考。