利用CRISPR/Cas9技术研究玉米ZmFKF1在开花过程中的作用

doi:10.3864/j.issn.0578-1752.2021.04.003

中国农业科学 ›› 2021, Vol. 54 ›› Issue (4): 696-707.doi: 10.3864/j.issn.0578-1752.2021.04.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

利用CRISPR/Cas9技术研究玉米ZmFKF1在开花过程中的作用

杨敏¹(),胥华伟²,王翠玲²,杨护¹,魏岳荣¹()

¹广东省农业科学院果树研究所/农业部南亚热带果树生物学与遗传资源利用重点实验室/广东省热带亚热带果树研究重点实验室,广州510640
²河南科技大学农学院,河南洛阳471000

收稿日期:2020-07-25 接受日期:2020-09-23 出版日期:2021-02-16 发布日期:2021-02-16
通讯作者: 魏岳荣
作者简介:杨敏,E-mail： minyang_0123@126.com。
基金资助:
国家自然科学基金青年基金(31700252);河南省高等学校重点科研项目(17A180002);河南省重点研发与推广专项科技攻关(202102110010)

Using CRISPR/Cas9-mediated Targeted Mutagenesis of ZmFKF1 Delayed Flowering Time in Maize

YANG Min¹(),XU HuaWei²,WANG CuiLing²,YANG Hu¹,WEI YueRong¹()

¹Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences/ Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization (MOA)/Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research, Guangzhou 510640
²College of Agriculture, Henan University of Science and Technology, Luoyang 471000, Henan

Received:2020-07-25 Accepted:2020-09-23 Online:2021-02-16 Published:2021-02-16
Contact: YueRong WEI

摘要/Abstract

摘要：

【目的】FKF1是多种植物开花途径中发挥重要作用的关键基因。为研究玉米FKF1功能,利用CRISPR/Cas9基因编辑技术将ZmFKF1进行定点编辑,获得ZmFKF1编辑突变体。同时以此为材料,通过表型分析及关键开花基因的表达量变化分析,明确ZmFKF1在玉米开花途径中的作用,为玉米分子育种及遗传改良提供理论依据。【方法】以玉米B104为材料,克隆ZmFKF1,通过序列比对明确ZmFKF1的基因结构。以ZmFKF1为靶标基因,根据CRISPR/Cas9技术原理设计靶点,将设计的靶点序列在玉米参考基因组中进行比对分析,排除非特异性靶位点,最终筛选出在ZmFKF1第1外显子上的靶位点ZmFKF1-T1,构建CRISPR/Cas9基因编辑表达载体。利用农杆菌介导法,转化玉米B104幼胚,通过抗性筛选获得抗性愈伤组织,之后诱导出芽和生根,获得T₀代ZmFKF1编辑阳性植株,并利用cas9特异引物进行验证。利用靶位点扩增测序法,明确T₁代ZmFKF1编辑植株在ZmFKF1预期靶标位点是否发生突变及突变的类型,筛选获得ZmFKF1定点突变纯合株系。获得这些材料后,以野生型B104为对照,统计和分析开花表型。同时,利用实时荧光定量PCR技术检测上述材料中与玉米开花途径相关的关键基因的表达量变化,对表型进行进一步的验证。【结果】在玉米FKF1的第1外显子上设计靶点构建基因编辑表达载体,通过遗传转化获得的转基因株系实现了对ZmFKF1的定点突变,共获得T₀代ZmFKF1编辑阳性株系18株,在预期靶标位点上发生突变的有6株,2种不同的突变类型：单碱基插入和多碱基缺失。通过开花时间统计和分析,与野生型B104相较,3个T₂代ZmFKF1编辑纯合突变体的开花时间延迟,显著（P＜0.05）晚于B104。进一步对这些材料中的开花关键基因ZmGI、conz1和ZmZCN8进行表达量检测,发现突变体中这些基因的表达明显（P＜0.05）低于野生型B104,与晚花表型相符。【结论】可利用CRISPR-Cas9技术对ZmFKF1进行定点编辑获得基因编辑突变体,且突变体开花时间明显延迟。

关键词: CRISPR/Cas9, 基因编辑, 玉米, ZmFKF1, 开花时间

Abstract:

【Objective】FKF1 is a key gene that plays an important role in many plant flowering pathways. In order to study the function of FKF1 in maize, ZmFKF1 editing mutants were obtained by using CRISPR/cas9 technology. In this study, we used these mutants as materials to clarify the role of ZmFKF1 in maize flowering pathway through phenotypic analysis and the expression analysis of key flowering genes. The result provide a theoretical basis for molecular breeding and genetic improvement of maize.【Method】 ZmFKF1 gene was cloned from B104, and its structure was determined by sequence alignment. The targeted sequences of ZmFKF1 were designed according to the principle of CRISPR/Cas9 technology, then these targeted sequences were compared and analyzed in maize reference genome, and the non-specific target site was excluded. Finally, ZmFKF1-T1 on exon 1 of ZmFKF1 was selected to construct CRISPR/cas9 gene editing expression vector. At the same time, B104 immature embryos were selected as explants to transform by Agrobacterium-mediated genetic transformation, the resistant calli were obtained through resistance screening, and then buds and roots were induced. The T₀ generation ZmFKF1 gene editing positive plants were obtained and verified by using the specific primers of cas9. The target site amplification and sequencing analysis was used to determine whether the T₁ transgenic lines had mutation at the expected target site of ZmFKF1 and the type of mutation, and screened the homozygous lines of ZmFKF1 site mutation. After obtaining the above materials, the flowering phenotypes of these materials were statistically analyzed with wild type as control. At the same time, qRT-PCR was used to detect the expression of key genes that related to flowering pathway in the above materials and further verify the phenotype. 【Result】The target sequence was designed on exon1 of ZmFKF1 to construct gene editing expression vector. The transgenic lines were obtained by genetic transformation realized site directed mutagenesis of ZmFKF1. A total of 18 T₀ generation ZmFKF1 gene-edited lines were obtained, and 6 of them were generated mutation on the exon1 with two different mutations types, including single base insertion and multiple bases deletion. The phenotype analysis shown that the flowering time of three T₂ generation ZmFKF1 homozygous lines was delayed compared with wild type B104. Furthermore, the expression levels of three related flowering pathway genes (ZmGI, conz1 and ZmZCN8) in the mutants were significantly lower than those in the wild type B104 (P＜0.05), which was consistent with the late flowering phenotype of the mutants. 【Conclusion】CRISPR/Cas9 technology can be used to edit ZmFKF1 to obtain gene editing mutants, and the flowering time of these mutants is significantly delayed compared to wild type.

Key words: CRISPR/Cas9, gene editing, maize, ZmFKF1, flowering time

杨敏,胥华伟,王翠玲,杨护,魏岳荣. 利用CRISPR/Cas9技术研究玉米ZmFKF1在开花过程中的作用[J]. 中国农业科学, 2021, 54(4): 696-707.

YANG Min,XU HuaWei,WANG CuiLing,YANG Hu,WEI YueRong. Using CRISPR/Cas9-mediated Targeted Mutagenesis of ZmFKF1 Delayed Flowering Time in Maize[J]. Scientia Agricultura Sinica, 2021, 54(4): 696-707.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Usage
ZmFKF1-F	CACCATGTTTGACGACGCGGGGGC	ZmFKF1克隆 Cloning of ZmFKF1 gene
ZmFKF1-R	GACTTTGCCATGCGAATCTACAATGC	ZmFKF1克隆 Cloning of ZmFKF1 gene
FKF1-U6b-F	GTTGAGGTGGACGCGGAGCCGGGA	载体构建及鉴定 Vector construction and verification
FKF1-U6b-R	AAACTCCCGGCTCCGCGTCCACCT
U-F	CTCCGTTTTACCTGTGGAATCG
gR-R	CGGAGGAAAATTCCATCCAC
Pps-R	TTCAGAggtctcTaccgACTAGTCACGCGTATGGAATCGGCAGCAAA
Pgs-L	AGCGTGggtctcGctcgACGCGTATCCATCCACTCCAAGC
SP-L1	GCGGTGTCATCTATGTTACTAG
SP-R	TGCAATAACTTCGTATAGGCT
Cas9-F	CTGACGCTAACCTCGACAAG	鉴定Cas9 PCR detection for Cas9
Cas9-R	CCGATCTAGTAACATAGATGACACC	鉴定Cas9 PCR detection for Cas9
ZmCri-F	CCCTACCTGGCAGCAGAATC	靶点检测及测序引物 Target detection and sequencing primer
ZmCri-R	AGCAGCCCAAATCTGTCAGTC	靶点检测及测序引物 Target detection and sequencing primer
ZmUbi-F	GTCTCCGTGGTGGTCAGTAAGT	qRT-PCR检测引物 Primers used for qRT-PCR assay
ZmUbi-R	GACACGAACAGCAGATACTTTGAC
ZmGI-F	ACTTTCCCAACCAACGC
ZmGI-R	GGGCAGCTACAACAATTTCA
conz1-F	ATCGTCAATGGAGGTGGGTAT
conz1-R	GACCCGACGAGAACAGGC
ZmZCN8-F	ACTTGCACTGGATGGTGACAGATA
ZmZCN8-R	CGGCCTCTCATAAAATATTAGCTCTT

株系 Line	序列 Sequence (5′-3′)	突变类型 Mutation type	是否纯合 Homozygous or not
B104	AGGTGGACGCGGAGCCGGGATGG	---	---
C1-6	AGGTGGACGCGGAGCTATGG	+1（T）,-4（CGGG）	否 No
C2-12	AGGTGGACGCGGAGCCGAGGATGG	+1（A）	是 Yes
C3-3	AGGTGGACGCGGAGCCG	-9（GGATGGCCG）	否 No
C5-7	AGGTGGACGCGGAGCCGGGATGG	-20（AGGTGGACGCGGAGCCGGGA）	否 No
C6-2	AGGTGGACGCGGAGCCATGG	-3（GGG）	是 Yes
C7-1	AGGTGGACGCGGAGCCG	-6（GGGATG）	是 Yes

基因型 Genotype	抽雄时间 Male phase	散粉时间 Pollen phase	吐丝时间 Female phase
B104	62.70±1.37	67.87±1.04	70.53±1.25
C2-12	63.43±1.04*	69.13±1.36*	72.57±1.65*
C6-2	64.07±0.98*	69.37±1.56*	71.90±1.22*
C7-1	63.96±1.30*	69.04±1.81*	72.33±1.12*

利用CRISPR/Cas9技术研究玉米ZmFKF1在开花过程中的作用

Using CRISPR/Cas9-mediated Targeted Mutagenesis of ZmFKF1 Delayed Flowering Time in Maize

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