利用CRISPR_Cas9技术创建拟南芥Argonaute2基因缺失突变体

doi:10.3969/j.issn.1004-1524.2021.11.01

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (11): 2001-2008.DOI: 10.3969/j.issn.1004-1524.2021.11.01

利用CRISPR_Cas9技术创建拟南芥Argonaute2基因缺失突变体

李红英(), 高延武, 于茹恩, 王政博, 李雪萍, 刘龙昌^*()

河南科技大学园艺与植物保护学院,河南洛阳 471023

收稿日期:2020-12-11 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 刘龙昌
作者简介:*刘龙昌,E-mail: snowliu@126.com
李红英(1982—),男,河南南阳人,博士,主要从事农林植物发育相关基因调控机理研究。E-mail: lihy@haust.edu.cn
基金资助:
国家自然科学基金(31400568);河南科技大学2019年研究生创新基金

Argonaute2 mutants in Arabidopsis created by CRISPR_Cas9 technology

LI Hongying(), GAO Yanwu, YU Ru’en, WANG Zhengbo, LI Xueping, LIU Longchang^*()

College of Horticulture and Plant Protection, Henan University of Science & Technology, Luoyang 471023, China

Received:2020-12-11 Online:2021-11-25 Published:2021-11-26
Contact: LIU Longchang

摘要/Abstract

摘要：

Argonaute2(AGO2)在植物抗病和发育过程中发挥重要作用。为创制拟南芥ago2核苷酸插入/缺失突变体材料,分析了拟南芥AGO2基因结构,选择其外显子上3个靶点构建了CRISPR_Cas9基因编辑载体,并通过农杆菌介导的花序浸染法转化野生型拟南芥,利用潮霉素对T₀代种子进行筛选,获得62株T₁代抗性苗;然后提取T₁代抗性苗DNA,进行潮霉素特异引物PCR扩增检测,确定获得53棵转基因阳性苗。随机选择10株T₁代阳性苗,扩增包含靶点的基因片段进行测序,结果显示,在第1个靶点附近6株苗产生了编辑,第2靶点附近10株苗全部成功编辑,第3个靶点未发生编辑。编辑位点附近产生了多种编辑形式,以PAM前删除或者增加1个碱基的形式出现频率最高,也有删除大于10碱基的编辑形式,最长可删除106个碱基。这些突变株系的获得为深入研究拟南芥AGO2的功能提供了丰富的遗传材料。

关键词: Argonaute2, 拟南芥, 突变体, 花序浸染法, 基因编辑, CRISPR_Cas9

Abstract:

Argonaute2 (AGO2) plays an important role in plant resistance and development. In order to create Arabidopsis ago2 nucleotides insertion/deletion mutant materials, structure of AtAGO2 gene was analysed firstly, and then the CRISPR_Cas9 based gene editing expression vector which contains three target sites of AtAGO2 was constructed, and transferred into Arabidopsis by Agrobacterium mediated floral-dip. 62 anti-hygromycin resistant seedlings were obtained by screening the seeds of T₀ generation plants on the medium containing hygromycin antibiotic. And 53 definitively transgenic positive seedlings were further confirmed by PCR amplification using hygromycin B gene specific primers. The fragments of AtAGO2 containing the target sites of 10 random selected transgenic progenies were sequenced, and the results showed that six transgenic lines were successfully edited around the T₁ site, all the 10 lines were edited around the T₂ site, and the third target site was not edited. Varied forms of mutation happened in the nearby area of the target sites, and the most frequent ones were deleting or adding one base before PAM. There were also more than 10 bases deletion forms, and the longest one was 106 bases deletion. This study provided abundant useful genetic materials for further study of the function of AGO2 in Arabidopsis.

Key words: Argonaute2, Arabidopsis, mutant, floral dip, gene editing, CRISPR_Cas9

中图分类号:

S184

李红英, 高延武, 于茹恩, 王政博, 李雪萍, 刘龙昌. 利用CRISPR_Cas9技术创建拟南芥Argonaute2基因缺失突变体[J]. 浙江农业学报, 2021, 33(11): 2001-2008.

LI Hongying, GAO Yanwu, YU Ru’en, WANG Zhengbo, LI Xueping, LIU Longchang. Argonaute2 mutants in Arabidopsis created by CRISPR_Cas9 technology[J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2001-2008.

图/表 7

表1 引物序列

Table 1 Sequence of primers

引物名称 Primer ID	引物序列(5'→3') Nucleotide sequence (5'→3')	引物名称 Primer ID	引物序列(5'→3') Nucleotide sequence (5'→3')
AtAGO2-T1F	GTCAATGGAGAGAGGTGGTTATCG	gRNA-R	CGGAGGAAAATTCCATCCAC
AtAGO2-T1R	AAACCGATAACCACCTCTCTCCAT	Uctcg-B1'	TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG
AtAGO2-T2F	GTCATCCGTCCACCAGCACCACCG	gRctga-B2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
AtAGO2-T2R	AAACCGGTGGTGCTGGTGGACGGA	Uctga-B2'	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
AtAGO2-T3F	ATTGCCACAACTCCGCCTCTATC	gRaaga-B3	AGCGTGGGTCTCGTCTTGGTCCATCCACTCCAAGCTC
AtAGO2-T3R	AAACGATAGAGGCGGAGTTGTGG	Uaaga-B3'	TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG
C9AtAGO2-seq-F	GCTTTGTTTCACTCAGTGTTTCC	gRcggt-BL	AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC
C9-AtAGO2-seq-R	AACCTTCTTGGTAGGAATTTCTC	Hyg-F	ATCCTGCAAGCTCCGGATGCCTC
U-F	CTCCGTTTTACCTGTGGAATCG	Hyg-R	CGTCTCCGACCTGATGCAGCTCT

图1 AtAGO2基因结构与敲除靶点位置信息

Fig.1 Structure of AtAGO2 and location of target sites

图2 AtAGO2 3个靶点sgRNA表达盒PCR扩增产物琼脂糖凝胶电泳检测 M,DL 2000 marker;T1,靶点1;T2,靶点2;T3,靶点3。

Fig.2 Gel electrophoresis detection of PCR amplification products of three target sgRNA expression cassettes M, DL 2000 marker; T1, Target site 1; T2, Target site 2; T3, Target site 3.

图3 pCC9AtAGO2质粒靶位点序列与载体结构示意图 a,pCC9AtAGO2质粒靶位点测序;b,pCC9AtAGO2载体结构示意图。

Fig.3 Sequence of target sites and structure of pCC9AtAGO2 vector a, Sequencing of target sites of pCC9AtAGO2; b, Structure of pCC9AtAGO2.

图4 部分抗性苗潮霉素基因特异片段PCR扩增检测 M,DL 2000 DNA marker;1~19,转基因拟南芥潮霉素抗性阳性苗;wt,野生型。

Fig.4 PCR amplification detection of hygromycin B gene in T0 transgenic plants with hygromycin B tolerance M, DL 2000 DNA marker; 1-19, Positive seedlings of transgenic lines with hygromycin B tolerance; wt, Wild type.

图5 部分转基因T1代植株AGO2基因编辑靶点所在DNA片段PCR扩增结果 M,DL 2000 DNA marker;#2~#54,转基因T1代植株。

Fig.5 PCR amplification of AGO2 gene fragments containing editing target sites of selected transgenic lines M,DL 2000 DNA marker; #2-#54, T1 transgenic plants.

图6 部分转基因T1代植株AGO2靶点编辑形式 a,靶点1编辑形式;b,靶点2编辑形式。

Fig.6 Editing forms of AGO2 target site of selected T1 transgenic progenies a, Editing forms of target T1; b, Editing forms of target T2.

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利用CRISPR_Cas9技术创建拟南芥Argonaute2基因缺失突变体

Argonaute2 mutants in Arabidopsis created by CRISPR_Cas9 technology

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