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.
国家科技期刊平台
NSTL
万方数据
维普
