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园艺学报 ›› 2019, Vol. 46 ›› Issue (4): 623-634.doi: 10.16420/j.issn.0513-353x.2018-0626

• 研究论文 • 上一篇    下一篇

利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究

郭 晔,万东艳,柴壮壮,王跃进,文颖强*   

  1. 西北农林科技大学园艺学院,农业部西北地区园艺作物生物学与种质创新重点实验室,旱区作物逆境生物学国家重点实验室,陕西杨凌 712100
  • 出版日期:2019-04-25 发布日期:2019-04-25
  • 基金资助:
    国家自然科学基金面上项目(31772264);杨凌示范区农业科技示范推广项目(2017-TS-24)

Knock-out Analysis of VviPDS1 Gene Using CRISPR/Cas9 in Grapevine

GUO Ye,WAN Dongyan,CHAI Zhuangzhuang,WANG Yuejin,and WEN Yingqiang*   

  1. College of Horticulture,Northwest A & F University,Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China,Ministry of Agriculture,State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling,Shaanxi 712100,China
  • Online:2019-04-25 Published:2019-04-25

摘要: 选择葡萄八氢番茄红素脱氢酶(Phytoene desaturase)基因VviPDS1为靶标,利用CRISPR/Cas9系统构建基因敲除载体,瞬时转化葡萄叶片原生质体,检测到不同类型的突变。通过农杆菌介导转化‘无核白’葡萄胚性愈伤组织,筛选获得卡那霉素抗性植株71株。经PCR鉴定,其中53株为阳性植株,阳性率为74.64%。测序结果表明,共有20株在靶点发生不同类型的突变,编辑效率为37.74%;其中9株产生了双等位基因突变。对其进行氨基酸序列预测,在第202位氨基酸之后发生了不同程度的变异。利用CRISPR/Cas9系统敲除VviPDS1获得的突变体植株呈现整体矮化,其叶片出现不同程度白化。表明CRISPR/Cas9系统可以通过细胞中的瞬时或稳定表达进行基因编辑,可以实现在葡萄编辑植株中产生纯合敲除。

关键词: 葡萄, CRISPR/Cas9, PDS1, 基因编辑

Abstract: VviPDS1,phytoene desaturase in grapevine,was selected as the target gene. A gene knockout vector,using the CRISPR/Cas9 system,was constructed. Different types of mutations were detected in grape protoplasts by transient transformation of protoplasts from grape leaves. In addition,the vector was constructed and transformed to Vitis vinifera L.‘Thompson Seedless’through Agrobacterium- mediated transformation. Seventy-one kanamycin resistant lines were obtained through resistance screening. Fifty-three transgenic lines,74.64%,were confirmed by T-DNA specific PCR. Sequencing revealed that twenty transgenic lines had different types of mutations at the target site and the editing efficiency was 37.74%. Among them,9 lines with biallelic mutations were shown the albino phenotype. The prediction of amino acids of biallelic mutations showed them have different degrees of mutation behind the 202th amino acids. These biallelic mutations were shown dwarf and different albino phenotypes. These indicated that the CRISPR/Cas9 system allows genome editing in the transient of grape cells or stable expression,which can produce homozygous knockout in transgenic plants of grape.

Key words: grapevine, CRISPR/Cas9, PDS1, gene editing

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