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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (4): 978-987.doi: 10.3724/SP.J.1006.2023.24071

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花中不同植物病毒介导的VIGE体系的研究

雷建峰1(), 李月2, 代培红2, 赵燚2, 尤扬子2, 贾建国1, 赵帅1, 曲延英1,*(), 刘晓东2,*()   

  1. 1新疆农业大学农学院/教育部棉花工程研究中心, 新疆乌鲁木齐 830052
    2新疆农业大学生命科学学院, 新疆乌鲁木齐 830052
  • 收稿日期:2022-03-30 接受日期:2022-07-21 出版日期:2023-04-12 网络出版日期:2022-08-17
  • 通讯作者: *刘晓东, E-mail: xiaodongliu75@aliyun.com;曲延英, E-mail: xjyyq5322@126.com
  • 作者简介:E-mail: kyleijianfeng@163.com
  • 基金资助:
    新疆维吾尔自治区自然科学基金项目(2022D01B23);自治区高校基本科研业务费科研项目(XJEDU2022J007)

Study on VIGE system mediated by different plant viruses in cotton

LEI Jian-Feng1(), LI Yue2, DAI Pei-Hong2, ZHAO Yi2, YOU Yang-Zi2, JIA Jian-Guo1, ZHAO Shuai1, QU Yan-Ying1,*(), LIU Xiao-Dong2,*()   

  1. 1College of Agronomy, Xinjiang Agricultural University/Research Center of Cotton Engineering, Ministry of Education, Urumqi 830052, Xinjiang, China
    2College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
  • Received:2022-03-30 Accepted:2022-07-21 Published:2023-04-12 Published online:2022-08-17
  • Contact: *E-mail: xiaodongliu75@aliyun.com;E-mail: xjyyq5322@126.com
  • Supported by:
    Natural Science Foundation of Xinjiang Uygur Autonomous Region(2022D01B23);Basic Research Funds for Universities in the Autonomous Region(XJEDU2022J007)

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摘要：

植物病毒介导sgRNA的传递与表达相比传统转化完整的编辑载体进行基因编辑具有巨大优势, 因为sgRNA的表达可以伴随着病毒的复制和移动而快速扩增、累积, 从而产生高效的基因编辑效率。为在棉花中开发应用更多植物病毒介导的基因编辑系统(virus-induced genome editing, VIGE)。本研究以超表达Cas9 (Cas9-OE)棉花作为VIGE受体, 在棉花中分别建立了棉花叶皱缩病毒(cotton leaf crumple virus, CLCrV)和烟草脆裂病毒(tobacco rattle virus, TRV)介导的VIGE系统。首先CLCrV和TRV介导的VIGE均可以靶向敲除棉花GhBsrk1和GhMAPKKK2基因A亚组和D亚组基因组序列, 验证了这2个系统的可行性和有效性。进一步量化这2个系统对GhBsrk1和GhMAPKKK2基因的编辑效率结果发现, CLCrV和TRV介导的VIGE均能够产生高效的基因编辑效率, 并且2种系统基因编辑效率不存在显著差异。本研究还探究了使用棉花内源U6启动子和拟南芥U6启动子驱动sgRNA对VIGE基因编辑效率的影响。结果显示, 这2个启动子介导的VIGE均能够产生高效的基因编辑效率, 并且基于这2种启动子介导的VIGE系统基因编辑效率不存在显著差异。以上结果预示着可以开发更多的植物病毒载体应用于棉花基因编辑的研究, 进而获得高效的sgRNA筛选体系。

关键词: 棉花, CLCrV, TRV, VIGE, 基因编辑

Abstract:

Plant virus-mediated sgRNA delivery and expression has tremendous advantage over traditional transformation of intact editing vectors for gene editing, because sgRNA expression can be rapidly amplified and be accumulated along with virus replication and movement, resulting in efficient gene editing efficiency. In this study, to develop and apply more plant virus-mediated gene editing (VIGE) systems in cotton, the overexpressing Cas9 (Cas9-OE) cotton was used as VIGE receptor and the cotton leaf crumple virus (CLCrV) and tobacco rattle virus (TRV) mediated VIGE systems were established in cotton. First of all, both CLCrV and TRV-mediated VIGE could target and knock out the GhBsrk1 and GhMAPKKK2 in subgroup A and subgroup D genomic sequences, which verified the feasibility and effectiveness of these two systems. Further quantificaiton of the editing efficiency of GhBsrk1 and GhMAPKKK2 genes by these two systems showed that both CLCrV and TRV-mediated VIGE could produce high-efficiency gene editing efficiency, and there was no significant difference in gene editing efficiency between the two systems. This study also explored the effect of sgRNAs driven by cotton endogenous U6 promoter and Arabidopsis U6 promoter on VIGE gene editing efficiency. The results showed that both promoter-mediated VIGE were able to produce high-efficiency gene editing efficiency, and there was no significant difference in gene editing efficiency based on these two promoter-mediated VIGE systems. The above results indicate that more plant virus vectors can be developed for the application of cotton gene editing research, thus obtaining an efficient sgRNA screening system.

Key words: cotton, CLCrV, TRV, VIGE, gene editing

表1

本研究中使用的引物序列"

引物 Primer name 序列 Sequence (5'-3') 目的 Destination
GhBsrk1-sgRNA1F: GATTGTTGCACTTAGCTCCATGGC 构建AtU6-26::GhBsrk1-sgRNA和GhU6-5P::GhBsrk1-sgRNA: 5'-GTTGCACTTAGCTCCATGGC-3'
Construction of AtU6-26::GhBsrk1-sgRNA and GhU6-5P::GhBsrk1-sgRNA: 5'-GTTGCACTTAGCTCCATGGC-3'
GhBsrk1-sgRNA2F: AAGTGTTGCACTTAGCTCCATGGC
GhBsrk1-sgRNA1R: AAACGCCATGGAGCTAAGTGCAAC
GhMAPKKK2-sgRNA1F: GATTGAGGGTTCCCAGCTGACATA 构建AtU6-26::GhMAPKKK2-sgRNA和GhU6-5P::GhMAPKKK2-sgRNA: 5'-GAGGGTTCCCAGCTGACATA-3'
Construction of AtU6-26::GhMAPKKK2-sgRNA and GhU6-5P::GhMAPKKK2-sgRNA: 5'-GAGGGTTCCCAGCTGACATA-3'
GhMAPKKK2-sgRNA2F: AAGTGAGGGTTCCCAGCTGACATA
GhMAPKKK2-sgRNA1R: AAACTATGTCAGCTGGGAACCCTC
M-GhBsrk1F: GTTTAGAATACAATGCAGAAACTTTC PCR扩增涵盖GhBsrk1基因A亚组和D亚组靶位点区域
PCR amplification contains the target site regions of GhBsrk1 gene subgroup A and subgroup D
M-GhBsrk1R: GACAAGTTTAAGCACACACTTC
M-MAPPPK2F: CCATGTCGTAGCTTATAAAGG PCR扩增涵盖GhMAPKKK2基因A亚组和D亚组靶位点区域
PCR amplification contains the target site regions of GhMAPKKK2 gene subgroup A and subgroup D
M-MAPPPK2R: CGATTCATTCACGAACTCATG
GhUBQ7F: GAAGGCATTCCACCTGACCAAC PCR扩增GhUBQ7基因部分片段
PCR amplification of partial fragments of GhUBQ7 gene
GhUBQ7R: CTTGACCTTCTTCTTCTTGTGCTTG
Cas9F: GTCATTACGGACGAGTACAAG qRT-PCR分析Cas9 mRNA表达量
The mRNA relative expression level of Cas9 by qRT-PCR
Cas9R: AGGTAGCAGATCCGATTCTTT

图1

CLCrV-sgRNA和TRV-sgRNA载体构建"

图2

qRT-PCR分析Cas9表达量 在同一指标中不同小写字母表示不同处理在0.05概率水平差异显著。"

图3

不同CLCrV-sgRNA和TRV-sgRNA表达载体的酶切鉴定 M: 2K Plus II DNA 标准分子量; 1: CLCrV-AtU6-26::GhBsrk1- sgRNA; 2: CLCrV-AtU6-26::GhMAPKKK2-sgRNA; 3: CLCrV- GhU6-5P::GhBsrk1-sgRNA; 4: CLCrV-GhU6-5P::GhMAPKKK2- sgRNA; 5: TRV-AtU6-26::GhBsrk1-sgRNA; 6: TRV-AtU6-26:: GhMAPKKK2-sgRNA; 7: TRV-GhU6-5P::GhBsrk1-sgRNA; 8: TRV- GhU6-5P::GhMAPKKK2-sgRNA。"

图4

CLCrV和TRV介导的VIGE靶向敲除棉花GhBsrk1和GhMAPKKK2基因 M: 2K Plus II DNA标准分子量。A和B: GhBsrk1-sgRNA靶向突变检测。A: WT为对照, 1和2分别为接种CLCrV-AtU6-26:: GhBsrk1-sgRNA和TRV-AtU6-26::GhBsrk1-sgRNA植株编号, 凝胶电泳图显示GhBsrk1基因经Nco I酶切消化的产物和未消化的PCR产物。B: 未消化的PCR产物缺乏Nco I位点(由于存在突变), 随后进行纯化、克隆和测序分析。C: GhBsrk1突变测序峰图; D和E: GhMAPKKK2-sgRNA靶向突变检测。D: WT为对照, 1和2分别为接种CLCrV-AtU6-26::GhMAPKKK2-sgRNA和TRV-AtU6-26::GhMAPKKK2-sgRNA植株编号, 凝胶电泳图显示GhMAPKKK2基因经Nde I酶切消化的产物和未消化的PCR产物。E: 未消化的PCR产物缺乏Nde I位点(由于存在突变), 随后进行纯化、克隆和测序分析。F: GhMAPKKK2突变测序峰图。PAM位点为绿色, 蓝色下画线表示靶序列上的酶切位点。‘M’表示突变序列, ‘+’碱基插入用红色大写字母表示, ‘-’碱基缺失用红色线段表示。"

图5

CLCrV和TRV介导的VIGE基因编辑效率比较 M: 2K Plus II DNA标准分子量。A: WT为对照, 1~12为接种CLCrV-AtU6-26::GhMAPKKK2-sgRNA单株编号, 13~25为接种TRV-AtU6-26::GhMAPKKK2-sgRNA单株编号。B: WT为对照, 1~8为接种CLCrV-AtU6-26::GhBsrk1-sgRNA单株编号, 9~15为接种TRV-AtU6-26::GhBsrk1-sgRNA单株编号。"

图6

不同U6启动子介导的VIGE基因编辑效率比较 M: 2K Plus II DNA标准分子量。A: WT为对照, 1~12为接种CLCrV-AtU6-26::GhMAPKKK2-sgRNA单株编号。B: WT为对照, 1~11为接种CLCrV-GhU6-5P::GhMAPKKK2-sgRNA单株编号。"

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