基于CRISPR/Cas9技术创建甜菜BvCENH3基因突变体的研究

doi:10.12190/j.issn.2096-1197.2023.02.01

北方农业学报 ›› 2023, Vol. 51 ›› Issue (2): 1-11.doi: 10.12190/j.issn.2096-1197.2023.02.01

• 分子生物学·种质资源 • 下一篇

基于CRISPR/Cas9技术创建甜菜BvCENH3基因突变体的研究

韩平安^1,2, 唐宽刚^1,2, 常悦^1,2, 孙瑞芬^1,2, 王良^1,2, 张自强^1,2, 付增娟^1,2, 赵尚敏^1,2, 吴新荣^1,2, 李晓东^1,2

1.内蒙古自治区农牧业科学院,内蒙古呼和浩特 010031;
2.内蒙古自治区甜菜品种遗传改良与种质创制重点实验室,内蒙古呼和浩特 010031

收稿日期:2023-01-31 出版日期:2023-04-20 发布日期:2023-07-05
通讯作者: 吴新荣（1967—）,男,研究员,博士,主要从事甜菜分子育种的研究工作。李晓东（1977—）,男,研究员,博士,主要从事甜菜分子育种的研究工作。
作者简介:韩平安（1987—）,女,副研究员,博士,主要从事甜菜分子育种的研究工作。
基金资助:
内蒙古自治区 “揭榜挂帅”项目（2022JBGS0029）; 国家糖料产业技术体系项目（CARS-170104）; 国家自然科学基金项目（31860407）; 内蒙古自治区“草原英才”创新人才团队项目（甜菜生物技术产业创新团队）; 内蒙古农牧业创新基金项目（2022CXJJN09）

Research on the construction of sugar beet BvCENH3 gene mutants based on CRISPR/Cas9 technology

HAN Ping′an^1,2, TANG Kuan′gang^1,2, CHANG Yue^1,2, SUN Ruifen^1,2, WANG Liang^1,2, ZHANG Ziqiang^1,2, FU Zengjuan^1,2, ZHAO Shangmin^1,2, WU Xinrong^1,2, LI Xiaodong^1,2

1. Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China;
2. Inner Mongolia Key Laboratory of Sugar Beet Genetics and Germplasm Enhancement,Hohhot 010031,China

Received:2023-01-31 Online:2023-04-20 Published:2023-07-05

摘要/Abstract

摘要： 【目的】通过CRISPR/Cas9技术对甜菜BvCENH3基因进行编辑,建立甜菜高效基因组编辑技术体系。【方法】以BvCENH3为编辑目标,设计双靶标构建基因编辑载体,通过农杆菌侵染甜菜叶柄获得转基因植株,利用二代测序技术检测转基因植株突变情况,通过微滴数字PCR技术筛选低拷贝编辑植株。【结果】获得82株甜菜转基因植株,其中40株被成功编辑,编辑效率为48.78%,靶标1效率优于靶标2。突变类型有单碱基替换（T→G、A→C）、碱基缺失（TC、TCTC缺失）等5种。筛选出23株低拷贝编辑植株,BvCENH3插入拷贝数为1.1~1.9。【结论】成功对甜菜BvCENH3进行定点编辑,获得40株BvCENH3基因突变体。初步创建了甜菜基因组编辑技术体系,为甜菜单倍育种奠定了理论与技术基础。

关键词: 甜菜, BvCENH3, CRISPR/Cas9, 基因组编辑, 基因突变体

Abstract: 【Objective】The CRISPR/Cas9 technology was used to edit the BvCENH3 gene in sugar beet aiming to establish an efficient genome editing system.【Methods】Taking sugar beet BvCENH3 gene as the editing target,double candidate targets were selected to construct gene editing vector. Transgenic sugar beet plants were produced through agrobacterium-mediated transformation. Next generation sequencing technology was utilized to identify the mutation types and droplet digital PCR was employed to screen low-copy mutant plants.【Results】82 transgenic sugar beet plants were obtained,40 of which were successfully edited,with an editing efficiency of 48.78%. Target 1 was more efficient than target 2. There were five mutation types,including single base substitution（T→G、A→C） and base deletion（TC、TCTC deletion）. 23 low-copy edited plants were selected,with BvCENH3 insertion copy numbers ranging from 1.1 to 1.9.【Conclusion】Successfully edited BvCENH3 in sugar beet with 40 BvCENH3 gene mutants obtained. Preliminary established sugar beet genome editing system,laying theoretical and technical foundations for sugar beet haploid breeding.

Key words: Sugar beet, BvCENH3, CRISPR/Cas9, Genome editing, Mutants

中图分类号:

S566.335.3

韩平安, 唐宽刚, 常悦, 孙瑞芬, 王良, 张自强, 付增娟, 赵尚敏, 吴新荣, 李晓东. 基于CRISPR/Cas9技术创建甜菜BvCENH3基因突变体的研究[J]. 北方农业学报, 2023, 51(2): 1-11.

HAN Ping′an, TANG Kuan′gang, CHANG Yue, SUN Ruifen, WANG Liang, ZHANG Ziqiang, FU Zengjuan, ZHAO Shangmin, WU Xinrong, LI Xiaodong. Research on the construction of sugar beet BvCENH3 gene mutants based on CRISPR/Cas9 technology[J]. Journal of Northern Agriculture, 2023, 51(2): 1-11.

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基于CRISPR/Cas9技术创建甜菜BvCENH3基因突变体的研究

Research on the construction of sugar beet BvCENH3 gene mutants based on CRISPR/Cas9 technology

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