基于CRISPR-dCas9转录激活系统的毛果杨ANT转录因子功能分析

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

摘要

基于CRISPR-dCas9的基因转录激活表达能够避免基因异位表达带来的表型干扰，同时使基因有效地特异性表达。该研究利用新型CRISPR-Act3.0表达系统，在毛果杨（Populus trichocarpa）中对维管形成层特异表达转录因子ANT（AINTEGUMENTA）进行基因转录激活，创制遗传材料，并对基因的功能进行分析。首先对毛果杨PtrANTs转录因子进行同源分析，选取其中的PtrANT-4进行后续研究，对该基因进行克隆并利用荧光定量PCR分析其在各组织中的表达情况；其次在PtrANT-4启动子上设计3条gRNAs，构建CRISPR-dCas9转录激活表达载体，利用原生质体瞬时转化法检测该载体的表达；最后将该表达载体利用农杆菌介导法转化毛果杨，获得PtrANT-4转录激活遗传材料。结果表明：毛果杨中有4个PtrANTs转录因子，选取的PtrANT-4基因CDS序列全长为2 058 bp，编码685个氨基酸，在毛果杨侧生分生组织维管形成层特异表达。基于CRISPR-Act3.0表达系统成功构建的转录激活载体，在毛果杨木质部原生质体中转化后具有激活PtrANT-4表达的作用。获得的遗传转化植株中PtrANT-4基因仅在茎维管形成层中的表达量显著提高，说明PtrANT-4在茎维管形成层发育过程中可能起重要作用。该研究为PtrANT的功能研究奠定了一定研究基础，同时为维管形成层干细胞发育的机制研究提供了重要的遗传材料。

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.

关键词

CRISPR-dCas9 / 基因激活表达 / PtrANT / 毛果杨

Key words

CRISPR-dCas9 / transcriptional activation / PtrANT / Populus trichocarpa

Author summay

孟令桐（2002—），男，本科生，主要从事林木遗传育种研究。