湖北地黄CRISPR/Cas9基因编辑体系的建立

doi:10.11983/CBB22250

植物学报 ›› 2023, Vol. 58 ›› Issue (6): 905-916.DOI: 10.11983/CBB22250

湖北地黄CRISPR/Cas9基因编辑体系的建立

苗春妍¹^,^†, 李铭铭¹^,^†, 左鑫¹, 丁宁¹, 杜家方¹, 李娟¹, 张重义², 王丰青¹^,^*()

¹河南农业大学农学院, 郑州 450046
²福建农林大学农学院, 福州 350002

收稿日期:2022-10-21 接受日期:2023-03-17 出版日期:2023-11-01 发布日期:2023-11-27
通讯作者: * E-mail: fqwang@henau.edu.cn
作者简介:^† 共同第一作者
基金资助:
国家自然科学基金(81473299);河南省高等学校重点科研项目(22A360009)

Establishment of CRISPR/Cas9 Gene Editing System in Rehmannia henryi

Chunyan Miao¹^,^†, Mingming Li¹^,^†, Xin Zuo¹, Ning Ding¹, Jiafang Du¹, Juan Li¹, Zhongyi Zhang², Fengqing Wang¹^,^*()

¹College of Agronomy, Henan Agricultural University, Zhengzhou 450046, China
²College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Received:2022-10-21 Accepted:2023-03-17 Online:2023-11-01 Published:2023-11-27
Contact: * E-mail: fqwang@henau.edu.cn
About author:^† These authors contributed equally to this paper

摘要/Abstract

摘要： 湖北地黄(Rehmannia henryi)是一种具有重要价值的药用植物, 其基因编辑研究还未见报道。为建立湖北地黄基因编辑体系, 克隆了类胡萝卜素合成途径中八氢番茄红素脱氢酶(PDS)的编码基因, 并构建RhPDS1基因的CRISPR/Cas9载体, 以根癌农杆菌介导法转化湖北地黄基因组。结果表明, RhPDS1转录本含1 764 bp的开放阅读框(ORF), 其推测的氨基酸序列具有八氢番茄红素脱氢酶的典型结构域。RhPDS1基因在蕾、花和嫩叶中的相对表达量较高。利用基因编辑获得3个具有白化表型的再生株系, 白化苗分化率为3.7%。测序结果表明, 3个突变体属于2个基因编辑事件, 靶点序列分别为1 bp或/和5 bp碱基缺失, 造成移码突变。白化苗突变体的叶绿素和类胡萝卜素含量极显著低于野生型, RhPDS1基因的表达量也显著下降。综上, 该研究克隆了RhPDS1基因, 并利用CRISPR/Cas9技术实现了对RhPDS1基因的靶向敲除, 为湖北地黄的功能基因组学研究和野生驯化奠定了技术基础。

关键词: 湖北地黄, RhPDS1, CRISPR/Cas9, 基因敲除

Abstract: Rehmannia henryi is an important plant with great medicinal value, but no research of CRISPR/Cas9 has been done on this species. To establish the gene editing system of R. henryi, the gene encoding phytoene desaturase (PDS) in carotenoid biosynthesis was screened, and the CRISPR/Cas9 vector of RhPDS1 was constructed and transformed into R. henryi genome by Agrobacterium-mediated transformation method. The transcript of RhPDS1 with a 1 764 bp open reading frame (ORF) of RhPDS1 was obtained, the deduced amino acid sequence of RhPDS1 has the typical structural domains of phytoene desaturase. RhPDS1 showed higher expression levels in bud, flower and new leaf. Using CRISPR/Cas9 method, three regenerated shoots with albino phenotype were finally obtained, the differentiation rate of albino shoot was 3.7%. Sequencing analysis revealed that the three albino shoots belong to 2 editing events, in which deletion of 1 bp or (and) 5 bp occurred, respectively, which caused frame shift mutations. The contents of chlorophylls and carotenoids were significantly decreased in the albino mutants as compared to wild type, and the expression levels of RhPDS1 were also decreased in the albino mutants. Taken together, the RhPDS1 gene was cloned and knocked out by using CRISPR/Cas9 method, which laid down the foundations for functional genomics studies and de novo domestication of R. henryi.

Key words: Rehmannia henryi, RhPDS1, CRISPR/Cas9, gene knock out

苗春妍, 李铭铭, 左鑫, 丁宁, 杜家方, 李娟, 张重义, 王丰青. 湖北地黄CRISPR/Cas9基因编辑体系的建立. 植物学报, 2023, 58(6): 905-916.

Chunyan Miao, Mingming Li, Xin Zuo, Ning Ding, Jiafang Du, Juan Li, Zhongyi Zhang, Fengqing Wang. Establishment of CRISPR/Cas9 Gene Editing System in Rehmannia henryi. Chinese Bulletin of Botany, 2023, 58(6): 905-916.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22250

https://www.chinbullbotany.com/CN/Y2023/V58/I6/905

图/表 6

表1 引物名称及序列

Table 1 Primer names and sequences

Primer name	Primer sequence (5′-3′)	Aim
RhPDS1_orf_F	CCAAAAGTTGCTCCACCTGT	Amplification of RhPDS1 from cDNA
RhPDS1_orf_R	TGCTGCTTCCCTATCTTCTG	Amplification of RhPDS1 from cDNA
RhPDS1_F	ATATCAAAATGGCCCAATTCGGA	Amplification of RhPDS1 from genomic DNA
RhPDS1_R	TCCACACTCGAAACATATTAGGC	Amplification of RhPDS1 from genomic DNA
RhPDS1q_F	GCTAGCAGATGTGGCAGC	qRT-PCR analysis of RhPDS1
RhPDS1q_R	CAATGACCACTTGCAATGGC	qRT-PCR analysis of RhPDS1
RhActin_F	GTGAGGAGATGCAGAAGCAA	qRT-PCR analysis of RhActin
RhActin_R	CCTCGTGTGTTCACTGACAA	qRT-PCR analysis of RhActin
Rh18S_F	AGCAAGGGCAGTGTTACAAG	qRT-PCR analysis of Rh18S
Rh18S_R	GACCACAAGTGAGGACAAGG	qRT-PCR analysis of Rh18S
sgRhPDS1_F	attgAAGCAAGGGATGTGCTGGG	Construction of target site
sgRhPDS1_R	aaacCCCAGCACATCCCTTGCTT	Construction of target site
Detect1_F	TCAACGGCATTCGGGACAAG	Identification of the transformation
Detect1_R	CCACATACATATCGCGGCCA	Identification of the transformation
Detect2_F	TGTCCCAGGATTAGAATGATTAGGC	Identification of the transformation
Detect2_R	CCCAGCACATCCCTTGCTT	Identification of the transformation
RhPDS1t_F	CGACTATCCGAGACCAGAGC	Amplification of target region
RhPDS1t_R	ATGTGCAACCCAGTCTCGTA	Amplification of target region

图1 RhPDS1基因的PCR扩增产物电泳检测 (A) 以湖北地黄叶片cDNA为模板扩增RhPDS1; (B) 以湖北地黄叶片基因组DNA为模板扩增RhPDS1基因

Figure 1 Electrophoresis detection of PCR amplified products of RhPDS1 gene (A) RhPDS1 was amplified with the cDNA of Rehmannia henryi leaves as a template; (B) RhPDS1 was amplified with the genomic DNA of R. henryi leaves as a template

图2 RhPDS1的序列特征及基因表达特性 (A) RhPDS1与拟南芥、地黄和天目地黄同源蛋白的多序列联配; (B) RhPDS1及其同源蛋白的系统进化树; (C) 湖北地黄RhPDS1基因的组织表达特性(不同小写字母表示不同组织间基因表达差异显著(P<0.05))。

Figure 2 Sequence characteristics and gene expression patterns of RhPDS1 (A) Multiple sequence alignment of RhPDS1 with its homologs from Arabidopsis thaliana, Rehmannia glutinosa and R. chingii; (B) Phylogenetic tree of RhPDS1 and its homologous proteins; (C) Expression profiles of RhPDS1 in different tissues of R. henryi (different lowercase letters represent significant differences among different tissues (P<0.05)).

图3 RhPDS1基因编辑载体构建及湖北地黄遗传转化 (A) RhPDS1基因编辑载体框架图; (B) 抗性愈伤组织筛选(bar=1 cm); (C), (D) 抗性芽分化(bars=1 cm); (E) 白化芽再生(bar=1 cm); (F) 白化苗的PCR鉴定。PAM: 原间隔序列邻近基序; WT: 野生型

Figure 3 Construction of genome editing vector and genetic transformation of Rehmannia henryi (A) Schematic map of CRISPR/Cas9 construct for RhPDS1; (B) Embryogenic calli were screened on the medium with kanamycine (bar=1 cm); (C), (D) Resistant shoots differentiation (bars=1 cm); (E) Albino shoot regeneration (bar=1 cm); (F) Positive identification of albino shoots by PCR. PAM: Protospacer adjacent motif; WT: Wild type

图4 转基因湖北地黄RhPDS1基因靶点突变分析 (A) 3株白化苗的表型(bars=1 cm); (B) 2种白化突变体的靶点基因组碱基序列特征(短横线代表缺失碱基); (C) 2种突变的T克隆测序峰图; (D) 2种突变体的氨基酸序列特征。PAM和WT同表3。

Figure 4 Analysis of target mutations of RhPDS1 gene in transgenic Rehmannia henryi (A) Phenotypes of the three albino shoots (bars=1 cm); (B) cDNA sequence characteristics of target site in the genome of two albino mutants (short horizontal lines represent base deletions); (C) Sanger sequencing results for T clone of two kinds of mutant; (D) Amino acids sequence characteristics of the two albino mutants. PAM and WT are the same as shown in Figure 3.

图5 基因编辑突变体的叶绿素、类胡萝卜素含量及RhPDS1的表达量 (A) 突变体的叶绿素和类胡萝卜素含量; (B) 突变体中RhPDS1的相对表达量。数据为3次生物学重复平均值±标准差(n=3)。 **表示与野生型(WT)相比在P<0.01水平差异显著。

Figure 5 Detection of chlorophylls and carotenoids contents in RhPDS1 mutants and the expression of RhPDS1 (A) Chlorophylls and carotenoids contents in RhPDS1 mutants; (B) The relative expression levels of RhPDS1 in CRISPR/Cas9 edited shoots. Values are means±SD (n=3). ** indicate significant differences compared to wild type (WT) at P<0.01.

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湖北地黄CRISPR/Cas9基因编辑体系的建立

Establishment of CRISPR/Cas9 Gene Editing System in Rehmannia henryi

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