CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建

doi:10.6048/j.issn.1001-4330.2018.05.005

新疆农业科学 ›› 2018, Vol. 55 ›› Issue (5): 821-828.DOI: 10.6048/j.issn.1001-4330.2018.05.005

CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建

杨晶¹,王旭辉^1,2,王东³,李冠¹

1.新疆大学生物工程研究中心,乌鲁木齐 830046;
2.新疆农业科学院生物质能源研究所,乌鲁木齐 830091;
3.新疆喀什大学,新疆喀什 844006

出版日期:2018-05-20 发布日期:2018-07-25
通信作者: 李冠(1949-),男,新疆人,教授,博士生导师,研究方向为植物生理生化与分子生物学,(E-mail)guanli@xju.Edu.cn
作者简介:杨晶(1992-),女,新疆人,硕士研究生,研究方向为生物化学与分子生物学,(E-mail)1605670957@qq.com
基金资助:
国家自然科学基金项目“甜瓜抗白粉病基因挖掘与聚合育种”(31260258);国家自然科学基金项目“甜瓜果实糖积累的遗传学与关键调控因子研究”(31660297);自治区自然科学基金项目“哈密瓜抗白粉病、霜霉病新种质的分子选育”(2016D01C066)

Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9

YANG Jing¹, WANG Xu-hui^1,2, WANG Dong³, LI Guan¹

1.Bioengineering Research Center, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China;
2. Research Institutes of Bioenergy, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China;
3. Kashi University, Kashi Xinjiang 844006, China

Online:2018-05-20 Published:2018-07-25
Correspondence author: LI Guan(1949-), native place, Xinjiang, PhD, Doctoral supervisor, research field:Plant physiology,biochemistry and molecular biology. (E-mail) guanli@xju.Edu.cn
Supported by:
The National Natural Science Foundation of China"Gene digging and polymerization of powdery mildew resistance of melon"(31260258); the National Natural Science Foundation of China "Study on genetics and key regulatory factors of sugar accumulation in melon fruit"(31660297); Natural Science Foundation of Xinjiang Uygur Autonomous Region of China "Molecular selection of new germplasm for powdery mildew resistant and downy mildew of Hami melon"(2016D01C066)

摘要/Abstract

摘要： 【目的】构建甜瓜eIF4E基因的CRISPR-Cas9植物基因编辑系统的gRNA 表达载体。为研究eIF4E基因功能奠定基础。【方法】以新疆主栽甜瓜皇后为材料,在eIF4E基因的第一个外显子区设计gRNA引物,以载体pP1C.4为模板,扩增获得sgRNA克隆框,使用EcoR I、Xba I双酶切pP1C.4载体,利用DNA重组酶构建重组载体pP1C.4-eIF4E。【结果】经菌落PCR检测和测序,gRNA 已经成功连接到植物基因敲除载体pP1C.4。【结论】构建了植物基因敲除载体pP1C.4-eIF4E,pP1C.4-eIF4E能对eIF4E基因进行定向编辑。

关键词: 甜瓜; CRISPR/Cas9; eIF4E ; 载体构建

Abstract: 【Objective】 The objective of this study is to construct gRNA expression vector of eIF4E gene in melon by using CRISPR-Cas9 plant gene editing system.【Method】 The gRNA primer was designed in the first exon region of eIF4E gene from "Queen" of melon in Xinjiang. The sgRNA cloning box was amplified by using the vector pP1C.4 as a template, the pP1C.4 vector was cut by EcoRI and XbaI enzymes, and the recombinant vector pP1C.4-eIF4E was constructed using DNA recombinase.【Result】 The results of colony PCR detection and sequencing showed that gRNA had been successfully linked into plant gene knockout vector pP1C.4.【Conclusion】 The plant gene knockout vector pP1C.4-eIF4E is constructed successfully and eIF4E can be directionally edited by pP1C.4-eIF4E, which is of great significance for further study on the function of eIF4E gene.

Key words: melon; CRISPR / Cas9; eIF4E; vector construction

中图分类号:

S652
S503.52

杨晶, 王旭辉, 王东, 李冠. CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建[J]. 新疆农业科学, 2018, 55(5): 821-828.

YANG Jing, WANG Xu-hui, WANG Dong, LI Guan. Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9[J]. Xinjiang Agricultural Sciences, 2018, 55(5): 821-828.

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CRISPR-Cas9技术敲除甜瓜eIF4E基因表达载体的构建

Construction of Expression Vector for Melon eIF4E Gene Knockout Using CRISPR-Cas9

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