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植物研究 ›› 2021, Vol. 41 ›› Issue (5): 816-823.doi: 10.7525/j.issn.1673-5102.2021.05.020

• 研究报告 • 上一篇    

一种带有可视化筛选标记的桉树基因编辑载体构建及验证研究

王泽琛1,2, 刘亚梅1,2, 欧阳乐军1,2(), 李莉梅2, 梁楚炎2, 潘璟茵2   

  1. 1.喀什大学生命与地理科学学院,叶尔羌绿洲生态与生物资源研究高校重点实验室,喀什 844000
    2.广东石油化工学院生物与食品工程学院,茂名 525000
  • 收稿日期:2021-03-04 出版日期:2021-09-20 发布日期:2021-07-05
  • 通讯作者: 欧阳乐军 E-mail:ouyanglejun@gdupt.edu.cn
  • 作者简介:王泽琛(1994—),男,硕士研究生,主要从事植物基因工程方面的研究。
  • 基金资助:
    国家自然科学基金项目(32071780);广东省自然科学基金项目(2019A1515010709);广东科技计划大专项(mmkj2020035);广东省教育厅重点项目(2018KZDXM047)

Construction and Verification of an Eucalyptus Gene Editing Vector with Visual Selection Markers

Ze-Chen WANG1,2, Ya-Mei LIU1,2, Le-Jun OUYANG1,2(), Li-Mei LI2, Chu-Yan LIANG2, Jing-Yin PAN2   

  1. 1.College of Life and Geographic Sciences,the Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at Colleges & Universities under the Department of Education of Xinjiang Uygur Autonomous Region,Kashi University,Kashi 844000
    2.College of Biological and Food Engineering,Guangdong University of Petrochemical Technology,Maoming 525000
  • Received:2021-03-04 Online:2021-09-20 Published:2021-07-05
  • Contact: Le-Jun OUYANG E-mail:ouyanglejun@gdupt.edu.cn
  • About author:WANG Ze-Chen(1994—),male,master,master degree candidate, research direction:plant genetic engineering.
  • Supported by:
    National Natural Science Foundation of China(32071780);Guangdong Natural Science Foundation of China(2019A1515010709);Guangdong Science and Technology Program(mmkj2020035);Guangdong Provincial Department of Education Key Project(2018KZDXM047)

摘要:

桉树作为世界三大速生树种之一,在经济、生态、药用等方面有着较高的价值。由于桉树遗传杂合性高,许多主要的经济性状由多基因共同调控,常规基因编辑手段无法满足对桉树目标基因编辑与转化后高效筛选的要求。通过mCherry荧光蛋白作为筛选标记可极大地减少转化后的鉴定工作量。本研究以尾巨桉为材料,构建含有35S启动子启动mCherry标记基因的CRISPR/Cas9载体,对桉树基因组进行高效的可视化编辑。利用mCherry荧光蛋白作为筛选标记,筛选阳性转化后代,并提取含荧光标记的不定芽基因组进行PCR鉴定分析。结果表明,成功构建了编辑载体PHEE401-35S-mCherry,转化尾巨桉愈伤后,在580 nm的光源下有明显的红色荧光,且经PCR鉴定可扩增得到与35S-mCherry条带大小一致的目的片段。本研究为开展桉树基因编辑提供了一种可视化筛选技术方法。

关键词: 桉树, 基因编辑, mCherry, CRISPR/Cas9

Abstract:

As one of the three fast-growing tree species in the world, Eucalyptus has high value in economic, ecological, medicinal and other aspects. Due to the high genetic heterozygosity of Eucalyptus, many major economic traits might be jointly regulated by multiple genes, and conventional gene editing methods could not meet the requirements for efficient screening of Eucalyptus target gene editing and transformation. Using mCherry fluorescent protein as a selection marker might reduce the identification workload after transformation. In this study, a CRISPR/Cas9 vector containing the 35S promoter to promote the mCherry marker gene was constructed using Eucalyptus urophylla as the material to perform efficient visual editing of the Eucalyptus gene. The mCherry fluorescent protein was used as the selection marker to screen positively transformed progenies, and the adventitious bud genome containing fluorescent markers were extracted for PCR identification and analysis. The results showed that the editing vector PHEE401-35S-mCherry was successfully constructed. After the callus of Eucalyptus urophylla transformed, there was obvious red fluorescence under the light of 580 nm, and the target fragment with the same size as 35S-mCherry band was amplified by PCR identification. The study provided a visual screening method for the Eucalyptus gene editing.

Key words: Eucalyptus, gene editing, mCherry, CRISPR/Cas9

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