Functional Analysis of ANT Transcription Factor of Populus trichocarpa Based on CRISPR-dCas9 Transcription Activation System

doi:10.7525/j.issn.1673-5102.2024.03.012

Abstract

Abstract:

The expression of gene transcription activation based on CRISPR-dCas9 might avoid phenotypic interference caused by gene ectopic expression， and made genes expressed efficiently and specifically. In this study， a novel CRISPR-Act3.0 expression system based on CRISPR-dCas9 was used to perform transcriptional activation of the vascular cambium-specific transcription factor ANT（AINTEGUMENTA） in Populus trichocarpa to create genetic materials and function analysis. First， homology analysis was conducted on the PtrANTs transcription factors of P. trichocarpa， and PtrANT-4 was selected for subsequent research. PtrANT-4 gene was cloned and its expression in various tissues was analyzed using fluorescence quantitative PCR. Secondly， three gRNAs were designed on the gene promoter of PtrANT-4， and the transcriptional activation expression vector CRISPR-dCas9/ANTprogRNAs was constructed. The expression of the vector was detected by transient protoplast transformation method. Finally， the expression vector was transformed into P. trichocarpa using Agrobacterium-mediated method， and transcription-activated genetic plants of PtrANT-4 were obtained. The results showed that there were four PtrANTs transcription factors in P. trichocarpa. PtrANT-4 was specifically expressed in vascular cambium of lateral meristem of P. trichocarpa. The transcription activation vector successfully constructed based on the CRISPR-Act3.0 expression system has the transcriptional activation effect of PtrANT-4 after transformation in xylem protoplasts of P. trichocarpa. The expression level of the PtrANT-4 gene in the genetically transformed plants was significantly increased only in the vascular cambium， suggesting that PtrANT-4 might play an important role in the development of stem vascular cambium This study lays a foundation for the functional study of PtrANT， and provides important genetic materials for the study of the mechanism of vascular cambial stem cell development.

Key words: CRISPR-dCas9, transcriptional activation, PtrANT, Populus trichocarpa

CLC Number:

S791.11

Lingtong MENG, Liwei SU, Xiangxin LI, Tiansheng XIONG, Panpeng CHANG, Mengzhuo LIU, Chenguang ZHOU. Functional Analysis of ANT Transcription Factor of Populus trichocarpa Based on CRISPR-dCas9 Transcription Activation System[J]. Bulletin of Botanical Research, 2024, 44(3): 431-440.

Figures/Tables 7

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引物名称 Primer name	引物序列（5′→3′） Primer sequences（5′→3′）
PtrANT-F	CACCGTGGCTAGACTTGGTCCTGCG
PtrANT-R	GCTTCCCTCCACCAATAAGGTCA
M13-F	GTAAAACGACGGCCAGT
M13-R	GTCATAGCTGTTTCCTG
D-ANT-4-F	GTGCTGGTCACCATCACCAA
D-ANT-4-R	GGCGTTGATGATCAGGGTGA
RT-18s-F	CGAAGACGATCAGATACCGTCCTA
RT-18s-R	TTTCTCATAAGGTGCTGGCGGAGGT
RT-PtrActin-F	TGTTGCCCTTGACTATGAGCAGGA
RT-PtrActin-R	ACGGAATCTCTCAGCTCCAATGGT
gRNA1-F	GGTCAAGGGTTCGTTTGTCACACA
gRNA1-R	AAACTGTGTGACAAACGAACCCTT
gRNA2-F	GGTCAAACACACAAATATTTCTGG
gRNA2-R	AAACCCAGAAATATTTGTGTGTTT
gRNA3-F	GGTCAGAGACTTCGTTATGTCTGC
gRNA3-R	AAACGCAGACATAACGAAGTCTCT
TC14-F2	CAAGCCTGATTGGGAGAAAA
zCas9-F12	GCTGACAAGAAGTACTCGAT
Hyg-F	TCCACTATCGGCGAGTACTTC
Hyg-R	CAGGACATATCCACGCCCTC

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