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Functional Analysis of ANT Transcription Factor of Populus trichocarpa Based on CRISPR-dCas9 Transcription Activation System
Lingtong MENG, Liwei SU, Xiangxin LI, Tiansheng XIONG, Panpeng CHANG, Mengzhuo LIU, Chenguang ZHOU
PDF(1563 KB)
PDF(1563 KB)
Functional Analysis of ANT Transcription Factor of Populus trichocarpa Based on CRISPR-dCas9 Transcription Activation System
)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.
CRISPR-dCas9 / transcriptional activation / PtrANT / Populus trichocarpa
S791.11
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