CRISPR/Cas9技术在天目地黄RcPDS1基因编辑中的应用  

CRISPR/Cas9 Technology for RcPDS1 Gene Editing in Rehmannia chingii

作　　者：左鑫 李铭铭 李欣容 苗春妍 李炎枋 杨旭 张重义[2] 王丰青[1] ZUO Xin;LI Mingming;LI Xinrong;MIAO Chunyan;LI Yanfang;YANG Xu;ZHANG Zhongyi;WANG Fengqing(College of Agronomy,Henan Agricultural University,Zhengzhou 450046,China;College of Agriculture,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[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

出　　处：《园艺学报》2022年第7期1532-1544,共13页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(81872950,81473299)。

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(Rc PDS1),利用PCR方法扩增其c DNA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Rc PDS1的全长c DNA序列,其具有1个长度为1743 bp的开放阅读框,编码580个氨基酸残基,基因组DNA序列长度8041 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,Rc PDS1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Rc PDS1基因的靶向敲除。To study the application of CRISPR/Cas9 technology in Rehmannia chingii,the phytoene desaturase gene(Rc PDS1)of R.chingii was cloned,and its c DNA 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 c DNA sequence of Rc PDS1 was obtained.The c DNA sequence of Rc PDS1 with a length of 1743 bp contains a single open reading frame,which encoding 580 amino acid residues.The length of genomic DNA sequence of Rc PDS1 gene is 8041 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 Rc PDS1 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.

关 键 词：天目地黄 CRISPR/Cas9 Rc PDS1 基因编辑 

分 类 号：Q78[生物学—分子生物学] R282[医药卫生—中药学]