Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale

	Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale
	Kui L 1; Chen HT 1,2; Zhang YS 1,2; Yan L[6]6; Zhong CF 7; He FM 8; Chen JW 4; Zeng P 3; Zhang WX 3; Zhang GH 4
刊名	Frontiers in Plant Science
	2017
卷号	7 期号:X 页码:e2036
关键词	CRISPR/Cas9 gene editing Dendrobium officinale gene promoters lignocellulose biosynthesis overexpression transgene vector construction
通讯作者	jingcai@umac.mo ; wwang@mail.kiz.ac.cn
合作状况	其它
英文摘要	Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses.Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D. officinale. These efficient research tools will not only help create novel D. officinale varieties, but will also facilitate the molecular genetic investigation of orchid biology.
收录类别	SCI
资助信息	This work was supported in part by grants from the pilot project for establishing new socialized service system by agricultural science and education combination in Yunnan Province (Medical Plant Unit) (2014NG003), start-up research grant from University of Macau to JC (SRG2015-00062-ICMS- QRCM). Our work was also partially supported by Yunling Industrial Technology Leading Talent.
语种	英语
内容类型	期刊论文
源URL	[http://159.226.149.26:8080/handle/152453/10832]
专题	昆明动物研究所_基因起源组昆明动物研究所_遗传资源与进化国家重点实验室
作者单位	1.State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China 2.Nowbio Biotech Inc., Kunming, China 3.State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China 4.National and Local Joint Engineering Research Center on Gemplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, China 5.China National GeneBank, BGI-Shenzhen, Shenzhen, China 6.Pu’er Institute of Pu-er Tea, Pu’er, China 7.Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China 8.College of Horticulture and Landscape, Yunnan Agricultural University, Kunming, China 9.Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China 10.Province Key Laboratory, Biological Big Data College, Yunnan Agricultural University, Kunming, China
推荐引用方式 GB/T 7714	Kui L,Chen HT,Zhang YS,et al. Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale[J]. Frontiers in Plant Science,2017,7(X):e2036.
APA	Kui L.,Chen HT.,Zhang YS.,Yan L[6].,Zhong CF.,...&Xiong ZJ.(2017).Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale.Frontiers in Plant Science,7(X),e2036.
MLA	Kui L,et al."Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale".Frontiers in Plant Science 7.X(2017):e2036.