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植物研究 ›› 2024, Vol. 44 ›› Issue (3): 431-440.doi: 10.7525/j.issn.1673-5102.2024.03.012

• 分子生物学 • 上一篇    下一篇

基于CRISPR-dCas9转录激活系统的毛果杨ANT转录因子功能分析

孟令桐1, 苏丽伟1, 李祥欣2, 熊天圣1, 常攀鹏1, 刘孟卓1, 周晨光1()   

  1. 1.林木遗传育种全国重点实验室(东北林业大学),哈尔滨 150040
    2.上海科技大学生命科学与技术学院,上海 201210
  • 收稿日期:2023-11-28 出版日期:2024-05-20 发布日期:2024-05-14
  • 通讯作者: 周晨光 E-mail:zhouchenguang@nefu.edu.cn
  • 作者简介:孟令桐(2002—),男,本科生,主要从事林木遗传育种研究。
  • 基金资助:
    国家级大学生创新训练项目(202210225168);国家自然科学基金项目(32001332)

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

Lingtong MENG1, Liwei SU1, Xiangxin LI2, Tiansheng XIONG1, Panpeng CHANG1, Mengzhuo LIU1, Chenguang ZHOU1()   

  1. 1.State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University,Harbin 150040
    2.School of Life Science and Technology,Shanghai Tech University,Shanghai 201210
  • Received:2023-11-28 Online:2024-05-20 Published:2024-05-14
  • Contact: Chenguang ZHOU E-mail:zhouchenguang@nefu.edu.cn

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。

关键词: CRISPR-dCas9, 基因激活表达, PtrANT, 毛果杨

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

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