墨兰B类PI基因多靶点CRISPR/Cas9载体构建  

Construction of Multi-Target CRISPR/Cas9 Vector for PI Gene in Cymbidium sinense

作　　者：吴林鲜 兰晓天 朱永平[1] 胡淞 田英男 冯磊 刘修远 万希文 何静 和凤美[1] Wu Linxian ,Lan Xiaotian ,Zhu Yongping, Hu Song, Tian Yingnan, Feng Lei, Liu Xiuyuan, Wan Xiwen ,He Jing ,He Fengmei(Yunnan Agricultural University, Kunming, 65020)

机构地区：[1]云南农业大学,昆明650201

出　　处：《分子植物育种》2018年第21期7027-7031,共5页Molecular Plant Breeding

基　　金：国家自然科学基金项目(30860178)资助

摘　　要：MADS基因家族对植物成花具有重要作用,在兰花中,目前还没有利用CRISPR/Cas9进行基因编辑的相关报道。墨兰(Cymbidium sinense) B类PISTILLATA (PI)基因可能与墨兰舌瓣的形成有关,为了进一步验证其PI基因的功能,本研究利用在线软件CRISPR-P设计3个靶点,并采用Overlapping-PCR技术和Golden Gate cloning策略构建了墨兰PI基因CRISPR/Cas9的多重sg RNA载体系统,经PCR、酶切和测序验证,植物编辑载体已构建成功。为在墨兰中实现该基因的定向突变提供了理论依据,同时也为在兰花基因组中应用CRISPR/Cas9基因编辑技术进行基因的定点突变修饰提供参考。The MADS gene family plays an important role in plant flower formation, but there is no report on the use of CRISPR/Cas9 for MADS gene editing in Orchidaceae. Cymbidium sinense B-type PISTILLA TA （PI） gene may be related to the formation of tongue flaps. In order to further investigate the function ofPI gene, three targets of PI gene locus were designed by using online software CRISPR-P, and the multiplex sgRNA vector system of CRISPR/Cas9 vector for PI gene was constructed by Overlapping-PCR technique and Golden Gate cloning strategy. The plant editing vector had been constructed successfully by PCR, enzyme digestion and sequencing. This study would lay a foundation for directional mutation of Cymbidium sinense PI gene, and provide a reference for the application of CRISPR/Cas9 gene editing to the site-directed mutation modification of genes in Orchidaceae genome.

关 键 词：CRISPR/Cas9 墨兰 MADS-BOX PI 基因功能 

分 类 号：S682.31[农业科学—观赏园艺]