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Bulletin of Botanical Research ›› 2021, Vol. 41 ›› Issue (5): 816-823.doi: 10.7525/j.issn.1673-5102.2021.05.020

• Research report • Previous Articles    

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)

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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