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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2024, Vol. 44 Issue (4): 102-111    DOI: 10.13523/j.cb.2309027
综述     
新型基因编辑技术加速盐藻表达系统的成熟和应用*
黄晓雯1,皇甫雨静1,姬聪浩1,代月优1,李姝璇1,冯书营1,2,3,**()
1 河南中医药大学医学院 郑州 450046
2 河南省中医药特医食品工程研究中心 郑州 450046
3 河南省药食同源中药工程研究中心 郑州 450046
Novel Gene Editing Technology Accelerates Maturation and Application of Dunaliella salina Expression Systems
HUANG Xiaowen1,HUANGFU Yujing1,JI Conghao1,DAI Yueyou1,LI Shuxuan1,FENG Shuying1,2,3,**()
1 Henan University of Chinese Medicine, Medical College, Zhengzhou 450046, China
2 Henan Engineering Research Center of Special Medical Food of Traditional Chinese Medicine, Zhengzhou 450046, China
3 Henan Engineering Research Center of Medicinal and Food Homologous Traditional Chinese Medicine, Zhengzhou 450046, China
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摘要：

盐藻因具备低成本、易培养、便于转基因操作、可调控的转录和翻译等优点,已成为基因工程领域备受关注的新型表达系统。但盐藻表达系统存在低表达和不稳定遗传等技术瓶颈,严重限制了其应用。CRISPR/Cas系统凭借高稳定性、高靶点特异性、可编程性等优点,可作为助推盐藻表达系统改进和应用的重要工具,从而大幅提高盐藻表达系统重组蛋白、脂质积累和胡萝卜素等高附加值产物的产量。由于盐藻兼具原核细胞和真核细胞的双重特点,基因编辑存在较大的难度和限制性,因而必须不断提高基因编辑技术在盐藻中的精确性和编辑效率,以确保目标基因得到准确修饰。探讨盐藻表达系统的基因工程研究现状,包括盐藻转化方法、外源基因的表达和基因编辑的研究现状等,分析CRISPR/Cas编辑技术在盐藻中面临的主要挑战,提出未来的发展方向和增效策略,以期加速盐藻表达系统的成熟和应用。
关键词： 盐藻;  新型表达系统;  基因编辑;  CRISPR/Cas9系统    
Abstract:

Dunaliella salina (D. salina) has become a promising expression system in genetic engineering due to its low cost, easy cultivation, simple transgenic operation, and controllable transcription and translation characteristics. However, there are technical bottlenecks such as low expression and unstable inheritance in the D. salina expression system, which severely limit its application. With the advantages of high stability, the high target specificity and programmability, the CRISPR/Cas system can be used as an important tool to promote the improvement and application of the D. salina expression system, thereby greatly increasing the production of high value-added products such as recombinant proteins, lipid accumulation and carotene. Due to the dual characteristics of prokaryotic and eukaryotic cells, salt algae have greater difficulties and limitations in the gene editing process. Therefore, it is necessary to continuously improve the accuracy and editing efficiency of gene editing technology in D. salina to ensure that the target genes are accurately modified. On this basis, the advantages of genetic engineering of D. salina expression system were comprehensively reviewed, including the transformation methods, exogenous gene expression and the current research status of gene editing. Meanwhile, the main challenges of CRISPR/Cas editing technology in D. salina are highlighted, the future development direction and improvement strategy are proposed, and the application prospect of this technology is envisioned to accelerate the early maturation and popularization of D. salina expression system.
Key words: Dunaliella salina    Novel expression system    Gene editing    CRISPR/Cas9 system
收稿日期: 2023-09-22 出版日期: 2024-04-30
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(U1804112);河南省自然科学基金(232300421164);河南省高等学校重点科研项目计划基础研究专项(23ZX005)
通讯作者: ** 电子信箱:fsy@hactcm.edu.cn   
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黄晓雯
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引用本文:

黄晓雯, 皇甫雨静, 姬聪浩, 代月优, 李姝璇, 冯书营. 新型基因编辑技术加速盐藻表达系统的成熟和应用*[J]. 中国生物工程杂志, 2024, 44(4): 102-111.

HUANG Xiaowen, HUANGFU Yujing, JI Conghao, DAI Yueyou, LI Shuxuan, FENG Shuying. Novel Gene Editing Technology Accelerates Maturation and Application of Dunaliella salina Expression Systems. China Biotechnology, 2024, 44(4): 102-111.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2309027    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I4/102

图1  盐藻表达系统的应用现状及前景
转化方法 转化基因 转化效率 转化系统 优缺点 参考文献
电穿孔 CAT、ble、bar、GUS基因 约2.6% 细胞核、叶绿体 优点:无宿主限制、可重复性、简便、经济
缺点:损伤细胞		 [21-22]
基因枪 Pat、EGFP基因 - 细胞核、叶绿体 优点:简便、快捷、经济
缺点:重复性差、费用高、损伤细胞		 [8,23]
玻璃微珠 Bar、GUS基因 5.9% 细胞核 优点:简便、经济、可控、细胞损伤较小 [24]
农杆菌转化法 β-胡萝卜素羟化酶基因 11.5% 细胞核 优点:简便、经济
缺点:重复性差、易污染、培养环节要求高		 [25]
细胞穿膜肽 GUS、β-葡糖醛酸酶基因 近100% 细胞核 优点:简单、快捷、高效、经济、不损伤细胞 [27-28]
盐梯度 GUS、β-胡萝卜素羟化酶基因 近100% 细胞核 优点:简便、经济、高效、不损伤细胞 [27,29]
LiAc/PEG GUS、SKTI基因 7.2% 细胞核 优点:成熟、简便、经济、可重复性好
缺点:一定的细胞毒性		 [30,32]
表1  盐藻细胞常用转化方法及其特点
图2  核酸酶诱导的双链断裂的修复途径
编辑系统 目的基因 产物或机制 应用 参考文献
传统转基因技术 流感病毒基因 重组禽流感病毒蛋白 疫苗制备 [2]
传统转基因技术 Can-N基因 血管能抑素 疾病治疗 [3]
传统转基因技术 VP28 白斑综合征病毒VP28蛋白 疫苗制备 [4]
CRISPR/Cas9 β-胡萝卜素羟化酶基因 β-胡萝卜素 提高天然成分的产量 [7,27]
传统转基因技术 SKTI基因 大豆kunitz胰蛋白酶抑制剂 疾病治疗 [32]
- 甘油-3-磷酸脱氢酶基因 饥饿诱导脂质代谢增加 生物燃料开发 [41]
传统转基因技术 NR基因 NR基因缺陷盐藻株 环保筛选标记 [42]
传统转基因技术 BKT基因 虾青素 提高天然成分的产量 [43]
传统转基因技术 硝酸盐转运蛋白基因 耐盐白菜植株 盐藻育种 [44]
表2  基因编辑技术在盐藻中的应用前景
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