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

doi:10.7525/j.issn.1673-5102.2021.05.020

Abstract

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

CLC Number:

Q782

Ze-Chen WANG, Ya-Mei LIU, Le-Jun OUYANG, Li-Mei LI, Chu-Yan LIANG, Jing-Yin PAN. Construction and Verification of an Eucalyptus Gene Editing Vector with Visual Selection Markers[J]. Bulletin of Botanical Research, 2021, 41(5): 816-823.

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