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作物学报 ›› 2018, Vol. 44 ›› Issue (02): 227-235.doi: 10.3724/SP.J.1006.2018.00227

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基于棉花U6启动子的海岛棉CRISPR/Cas9基因组编辑体系的建立

李继洋, 雷建峰, 代培红, 姚瑞, 曲延英, 陈全家, 李月, 刘晓东*()   

  1. 新疆农业大学农学院 / 新疆农业大学农业生物技术重点实验室, 新疆乌鲁木齐 830052;
  • 收稿日期:2017-05-21 接受日期:2017-11-21 出版日期:2018-02-12 网络出版日期:2017-12-11
  • 通讯作者: 刘晓东
  • 作者简介:

    qinlixia11@163.com, Tel: 0359-2161515

  • 基金资助:
    本研究由国家自然科学基金项目(31660433)和新疆维吾尔自治区研究生科研创新项目(XJGRI2016055)资助

Establishment of CRISPR/Cas9 Genome Editing System Based on GbU6 Promoters in Cotton (Gossypium barbadense L.)

Ji-Yang LI, Jian-Feng LEI, Pei-Hong DAI, Rui YAO, Yan-Ying QU, Quan-Jia CHEN, Yue LI, Xiao-Dong LIU*()   

  1. College of Agronomy, Xinjiang Agricultural University / Key Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China;
  • Received:2017-05-21 Accepted:2017-11-21 Published:2018-02-12 Published online:2017-12-11
  • Contact: Xiao-Dong LIU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31660433) and the Innovation Research Program for Graduate Students in Xinjiang (XJGRI2016055).

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摘要：

CRISPR/Cas9基因组编辑技术是基因功能研究的一种强有力的工具, 目前已在许多生物体中成功实现内源靶向基因的突变。利用已克隆的海岛棉新海16的2个U6启动子, 分别构建带有新海16内源基因(GbGGB和GbERA1)靶位点DNA片段的CRISPR/Cas9基因编辑载体。以新海16的胚性愈伤组织为供试材料, 制备海岛棉的原生质体。通过PCR方法大量富集构建好的CRISPR/Cas9基因编辑载体的核心片段(包括GbU6::sgRNA和CAMV35S::Cas9两部分), 并利用PEG法转化海岛棉的原生质体。对原生质体基因组DNA进行酶切后PCR, 成功检测到内源靶基因的突变现象。对PCR产物进行克隆测序, 结果显示序列突变的类型主要以碱基替换为主, 少数为碱基缺失。结果表明基于海岛棉U6启动子的CRISPR/Cas9基因编辑系统能在海岛棉中实现靶向基因编辑的功能, 为棉花功能基因组学研究提供了重要的技术基础。

关键词: 棉花, 原生质体, CRISPR/Cas9, 基因组编辑

Abstract:

CRISPR/Cas9 genome editing is a powerful tool for genes functional analyses, and the mutation of endogenous genes has been successfully implemented in many organisms using the tool. Two cloned U6 promoter from sea island cotton Xinhai 16 were used to construct CRISPR/Cas9 gene editing vectors with target (GbGGB and GBERA1) DNA fragments from Xinhai 16 respectively. Through PEG method, the core fragments (including GbU6::sgRNA and CAMV35S::Cas9) of the CRISPR/Cas9 gene editing vectors enriched by PCR method were transformed into the cotton protoplast prepared from the embryo callus of Xinhai 16. The mutation of endogenous target genes was successfully detected by a restriction enzyme PCR (RE-PCR) assay of protoplast genome. The cloning and sequencing of the PCR product, showed that the two Cas9-GbU6-sgRNA vectors could both induce targeted mutagenesis. Sequence analysis revealed that most of the mutations were nucleotide substitutions and the few were nucleotide deletion. The results indicate that the CRISPR/Cas9 gene editing vector system based on GbU6 promoter can realize targeted mutagenesis in sea island cotton, which provides an important technical basis for cotton functional genomics research.

Key words: cotton, protoplast, CRISPR/Cas9, genome editing

图1

海岛棉基因组编辑载体结构示意图 A: 含Cas9I编辑载体; B: 含Cas9II编辑载体。"

表1

本研究使用的引物信息"

引物名称
Primer name		 序列
Sequence (5'-3')		 引物名称
Primer name		 序列
Sequence (5'-3')
GbU6-4PERA1-sg1F GATTGTTCGCAGAATGCATGACGG GbU6-5PGGB-sg2F AAGTGCTCTGTCGAAGTACTGAAG
GbU6-5PERA1-sg1F AAGTGTTCGCAGAATGCATGACGG GbU6GGB-sg2R AAACCTTCAGTACTTCGACAGAGC
GbU6ERA1-sg1R AAACCCGTCATGCATTCTGCGAAC Test GGB-sgF AAGTGGAAAGAGAATGGCGAC
GbU6-4PERA1-sg2F GATTGTCTTTCGCAGAATGCATGA Test GGB-sgR AGCTAATTCGCTATTGCAATCAATC
GbU6-5PERA1-sg2F AAGTGTCTTTCGCAGAATGCATGA Test ERA1-sgF GTATCCTCTTACCGCCTTTC
GbU6ERA1-sg2R AAACTCATGCATTCTGCGAAAGAC Test ERA1-sgF CATACTTACTGAGATGGCTGT
GbU6-4PGGB-sg1F GATTGTGTCGAAGTACTGAAGCGG FJ-1F GTAAAACGACGGCCAG
GbU6-5PGGB-sg1F AAGTGTGTCGAAGTACTGAAGCGG FJ-1R CAGGAAACAGCTATGAC
GbU6GGB-sg1R AAACCCGCTTCAGTACTTCGACAC FJ-2F TAAACTGAAGGCGGGAAACG
GbU6-4PGGB-sg2F GATTGCTCTGTCGAAGTACTGAAG FJ-2F CGGTTCTGTCAGTTCCAAACG

图2

基因编辑载体的酶切鉴定 M1, M2: 2K plus DNA marker. A: M-1 to M-4 were GbU6-5P::GGB-sgRNA1-Cas9IIGbU6-5P::GGB-sgRNA2-Cas9II, GbU6-5P::ERA1-sgRNA1-Cas9II, and GbU6-5P:: ERA1-sgRNA2-Cas9II, respectively. B: 35-1 to 35-4 were GbU6-5P::GGB-sgRNA1-Cas9I, GbU6-5P::GGB-SgRNA2-Cas9I, GbU6-5P::ERA1-sgRNA1-Cas9I, and GbU6-5P:: ERA1-sgRNA2-Cas9I."

图3

棉花原生质体转化前后形态图 A: 棉花原生质体酶解过夜镜检结果(×40); B: 转化GbU6-5-GGB-sgRNA1-Cas9I载体核心片段的棉花原生质体过夜孵育后镜检结果(×40)。Bar=100 μm。"

图4

酶切/PCR检测CRISPR/Cas9基因组编辑效应原理示意图红色区域为被编辑突变的酶切位点。"

图5

酶切/PCR检测CRISPR/Cas9基因组编辑效应 M: 2000 plus DNA marker。A: 1、2分别为转化GbU6-5P::ERA1-sgRNA1-Cas9I基因组酶切前、后PCR扩增结果, 3、4分别为转化GbU6-5P::GGB-sgRNA1-Cas9I基因组酶切前、后PCR扩增结果。B: 1、2分别为转化GbU6-5P::ERA1-sgRNA2-Cas9I基因组酶切前、后PCR扩增结果, 3、4分别为转化GbU6-5P::GGB-sgRNA2-Cas9I基因组酶切前、后PCR扩增结果, CK1、CK2为转化阴性对照载体基因组酶切前后PCR扩增结果。"

图6

GbGGB-sgRNA2靶位点编辑效应(碱基置换型)测序检测结果"

图7

GbGGB-sgRNA2靶位点编辑效应(碱基置换型)测序峰图红色方框指示碱基突变的区域; 黑色方框指示PAM位点区域。"

图8

GbGGB-sgRNA2靶位点编辑效应（碱基缺失型）测序峰图红色方框指示突变序列（B）碱基相比对照（A）测序峰图发生碱基缺失区域;C图显示碱基缺失区域。"

图9

GbGGB-sgRNA2靶位点区域(guide RNA)及其两侧位点的突变效率分析 L30、L60、L90分别表示guide DNA靶序列左侧30、60、90 bp区域; R30、R60、R90分别表示guide DNA靶序列右侧30、60、90 bp区域。"

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