生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 210-219.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0173

• 研究报告 • 上一篇    下一篇

CLCrV介导的VIGE体系承载力的研究

赵燚1(), 雷建峰2, 刘敏1, 胡子曜1, 代培红1, 刘超1, 李月1, 刘晓东1()   

  1. 1.新疆农业大学生命科学学院,乌鲁木齐 830052
    2.新疆农业大学农学院 教育部棉花工程研究中心 农业生物技术重点实验室,乌鲁木齐 830052
  • 收稿日期:2022-02-15 出版日期:2022-11-26 发布日期:2022-12-01
  • 作者简介:赵燚,男,硕士研究生,研究方向:作物逆境分子生物学;E-mail:277092776@qq.com
  • 基金资助:
    新疆维吾尔自治区重大科技专项计划项目(2021A02001-3)

Research on the Carrying Capacity of CLCrV-mediated VIGE System

ZHAO Yi1(), LEI Jian-feng2, LIU Min1, HU Zi-yao1, DAI Pei-hong1, LIU Chao1, LI Yue1, LIU Xiao-dong1()   

  1. 1. College of Life Sciences,Xinjiang Agricultural University,Urumqi 830052
    2. College of Agronomy/Research Center of Cotton Engineering,Ministry of Education/Laboratory of Agricultural Biotechnology,Xinjiang Agricultural University,Urumqi 830052
  • Received:2022-02-15 Published:2022-11-26 Online:2022-12-01

摘要:

探索CLCrV介导的VIGE体系的承载力。通过CLCrV介导的sgRNA投送系统将基因编辑载体注射过表达Cas9的棉花,提取棉花基因组DNA,利用PCR/RE的方法筛选有效的sgRNA。然后利用同样的方法来检测CLCrV介导的VIGE体系的承载力。基于棉花GhBsr-k1基因成功构建了6个基因编辑载体,其中2个基因编辑载体实现了对棉花GhBsr-k1基因的靶向编辑;将完整的基因编辑载体元件构建到CLCrV病毒载体上并没有检测到棉花细胞中发生基因编辑。针对棉花GhBsr-k1基因,筛选到两个有效的sgRNA。但承载整个CRISPR/Cas9系统的CLCrV载体难以在棉花叶片中实现有效的基因编辑。

关键词: 棉花叶皱缩病毒, VIGE, 棉花, GhBsr-k1

Abstract:

This work aims to explore the carrying capacity of CLCrV-mediated VIGE system. Through CLCrV-mediated sgRNA delivery system,the gene editing vector was injected into cotton overexpressing Cas9,and the genomic DNA of cotton was extracted. The effective sgRNA was screened by PCR/RE method. Then the same method was used to detect the carrying capacity of the CLCrV-mediated VIGE system. Six gene-editing vectors were successfully constructed based on the cotton GhBsr-k1 gene,and two of them achieved targeted editing of cotton GhBsr-k1 gene. The complete gene editing vector components were constructed on the CLCrV vector and no gene editing was detected in cotton cells. Two effective sgRNAs were screened for the GhBsr-k1 gene in cotton. However,CLCrV vectors carrying the entire CRISPR/Cas9 system are difficult to achieve efficient gene editing in cotton leaves.

Key words: cotton leaf crumple virus, VIGE, cotton, GhBsr-k1