CRISPR/Cas9技术在天目地黄RcPDS1基因编辑中的应用

doi:10.16420/j.issn.0513-353x.2021-0475

摘要/Abstract

摘要：

为了研究CRISPR/Cas9技术在天目地黄基因编辑中的应用,克隆了天目地黄（Rehmannia chingii）的八氢番茄红素脱氢酶基因（RcPDS1）,利用PCR方法扩增其cDNA序列和基因组DNA序列。通过构建单靶点CRISPR/Cas9载体,利用根癌农杆菌介导的遗传转化方法侵染天目地黄无菌苗叶盘,通过TA克隆测序法分析基因编辑的类型。结果显示,克隆获得了1个天目地黄RcPDS1的全长cDNA序列,其具有1个长度为1 743 bp的开放阅读框,编码580个氨基酸残基,基因组DNA序列长度8 041 bp,包含14个内含子和15个外显子。通过遗传转化共获得57个转基因再生株系,其中具明显白化表型的株系有20个（35.09%）。TA克隆测序结果显示,RcPDS1靶位点突变类型主要包括碱基缺失、替换和插入。利用CRISPR/Cas9基因编辑技术在天目地黄中成功实现了对RcPDS1基因的靶向敲除。

关键词: 天目地黄, CRISPR/Cas9, RcPDS1, 基因编辑

Abstract:

To study the application of CRISPR/Cas9 technology in Rehmannia chingii,the phytoene desaturase gene（RcPDS1）of R. chingii was cloned,and its cDNA sequence and genomic DNA sequence were amplified by polymerase chain reaction（PCR）. The constructed single-target CRISPR/Cas9 plasmid was transformed into R. chingii genome using Agrobacterium-mediated genetic transformation method,and the types of gene editing were analyzed by TA clone sequencing. The results showed that the full-length cDNA sequence of RcPDS1 was obtained. The cDNA sequence of RcPDS1 with a length of 1 743 bp contains a single open reading frame,which encoding 580 amino acid residues. The length of genomic DNA sequence of RcPDS1 gene is 8 041 bp,which contains 14 introns and 15 exons. A total of 57 transgenic lines were obtained through A. tumefaciens-mediated genetic transformation,of which 20 lines（35.09%）had the obvious albino phenotype. The results of sequence analysis showed that the target sites mutation modes of RcPDS1 were mainly deletions,substitutions and insertions. It showed that the CRISPR/Cas9 system is a powerful tool to achieve targeted knockout of target genes in R. chingii genome.

Key words: Rehmannia chingii, CRISPR/Cas9, RcPDS1, gene editing

中图分类号:

Q78
R 282

左鑫, 李铭铭, 李欣容, 苗春妍, 李炎枋, 杨旭, 张重义, 王丰青. CRISPR/Cas9技术在天目地黄RcPDS1基因编辑中的应用[J]. 园艺学报, 2022, 49(7): 1532-1544.

ZUO Xin, LI Mingming, LI Xinrong, MIAO Chunyan, LI Yanfang, YANG Xu, ZHANG Zhongyi, WANG Fengqing. CRISPR/Cas9 Technology for RcPDS1 Gene Editing in Rehmannia chingii[J]. Acta Horticulturae Sinica, 2022, 49(7): 1532-1544.

图/表 10

表1 引物名称及序列

Table 1 Primer names and sequences

用途 Aim	产物长度/bp Product size	引物名称 Primer name	引物序列（5′-3′） Primer sequence
RcPDS1扩增 Amplification of RcPDS1	6 781（DNA） 1 882（cDNA）	RcPDS1_F	CAGTTGCCTGAGCTGTTGAA
RcPDS1扩增 Amplification of RcPDS1	6 781（DNA） 1 882（cDNA）	RcPDS1_R	GCTTCCCTATCTTCTGTCTTCC
RcPDS1 qRT-PCR	103	RcPDS1_qF	TATCATTTGCAGTTAGTG
RcPDS1 qRT-PCR	103	RcPDS1_qR	ACAACTTTCAAAGGGATTGC
靶序列的合成 Construction of target site	19	sgRcPDS1_F	AAGCAAGGGATGTGCTGGG
靶序列的合成 Construction of target site	19	sgRcPDS1_R	CCCAGCACATCCCTTGCTT
转基因鉴定 Identification of the transformation	474	Cas9_F	TCAACGGCATTCGGGACAAG
	474	Cas9_R	CCACATACATATCGCGGCCA
	281	pKSE401_F	TGTCCCAGGATTAGAATGATTAGGC
	281	sgRcPDS1_R	CCCAGCACATCCCTTGCTT
靶位点序列扩增 Amplification of target region	1 387	RcPDS1_TF	CTTCTCCTCGTCCAAACAAG
靶位点序列扩增 Amplification of target region	1 387	RcPDS1_TR	AGCTCTCCAAACAGGTTCTG
Gap序列扩增 Amplification of the gap sequence	828	PDSgap1_F	CAGCGAAAACAAGCTGAATCTG
	828	PDSgap1_R	GATTTCCTCCAAACTAACCGTG
		PDSgap2_F	TCGGACATGTTTCTGCTATCAA
	2 091	PDSgap2_R	TCCTTCCATTGCAACCGATC
	1 012	PDSgap3_F	ACAAACCAGGAGAGTTCAGC
	1 012	PDSgap3_R	GCTTCAACATAAGACTGACCG
	278	PDSgap4_F	CCTGGTACCGAACCTTGTC
	278	RcPDS1_R	GCTTCCCTATCTTCTGTCTTCC

图1 RcPDS1基因cDNA（A）和DNA（B）的PCR扩增产物凝胶电泳图

Fig. 1 Gel electrophoresis diagram of PCR amplified products of cDNA（A）and DNA（B）of RcPDS1 gene

图2 RcPDS1与AtPDS3的序列联配

Fig. 2 Alignment of RcPDS1 and AtPDS3

图3 RcPDS1的系统发育树

Fig. 3 Phylogenetic tree of RcPDS1

图4 天目地黄RcPDS1在不同组织中的表达量

Fig. 4 The expression level of RcPDS1 gene in different tissues of Rehmannia chingii

图5 CRISPR/Cas9靶位点选择与重组质粒的构建A:RcPDS1基因及靶点设计;B:PCR鉴定结果;C:重组质粒结构。

Fig. 5 CRISPR/Cas9 target site selection and recombinant plasmid constructionA:Schematic diagram showing RcPDS1 gene structure;B:Result of transformation detection by PCR;C:Schematic of the CRISPR/Cas9 binary vector pKSE401.

图6 天目地黄遗传转化与白化表型突变体A:抗性筛选;B:抗性白化愈伤组织;C:抗性白化芽再生;D:白化芽与绿芽共生;E:白化苗在光照下生长;F:褐化的白化苗;G:完全白化的分化芽;H:白绿嵌合苗。

Fig. 6 Genetic transformation of Rehmannia chingii and albino phenotype mutantsA:Embryogenic calluses were cultured on the medium with kanamycine;B:Positive albino callus;C:Positive albino shoot;D:Positive albino shoot and green shoot regenerated from the same callus;E:Positive albino shoot cultured under light;F:Browning albino shoots;G:Fully albino shoot;H:Chimeric albino shoot.

图7 转基因株系的阳性鉴定

Fig. 7 Positive identification of transgenic plants

图8 天目地黄野生型（WT）和RcPDS1敲除植株表型

Fig. 8 The phenotype of wild type and RcPDS1 knockout mutants plants of Rehmannia chingii

图9 天目地黄转基因植株RcPDS1靶点突变绿色字母为插入碱基,粉红色字母为替换碱基,短横线为缺失碱基,右边数字为变异碱基 × 克隆数。

Fig. 9 RcPDS1 target mutations in transgenic plants of Rehmannia chingiiGreen letters are insertions,pink letters are substitutions,dashes are deletions,and the number at the right-hand side represent types of base mutation.

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