title: Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid,Dendrobium officinale.
abstract: Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses.Dendrobium officinaleis a special orchid species that can grow without seed vernalization. Because the whole-genome sequence ofD. officinaleis 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 inD. officinale. In this study, based on the previously developedAgrobacterium-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 ofD. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and theβ-glucuronidase(GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by theAgrobacterium-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 inD. 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 inD. officinale. These efficient research tools will not only help create novelD. officinalevarieties, but will also facilitate the molecular genetic investigation of orchid biology.
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences Kunming, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Kui
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of SciencesKunming, China; Nowbio Biotech Inc., KunmingChina.
authors.forename: Haitao
authors.initials: H
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau Macau, China.
authors.forename: Weixiong
authors.initials: W
authors.lastname: Zhang
authors.affiliation: National and Local Joint Engineering Research Center on Gemplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University Kunming, China.
authors.forename: Simei
authors.initials: S
authors.lastname: He
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of SciencesKunming, China; China National GeneBank, BGI-ShenzhenShenzhen, China.
authors.forename: Zijun
authors.initials: Z
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of SciencesKunming, China; Nowbio Biotech Inc., KunmingChina.
authors.forename: Yesheng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Pu'er Institute of Pu-er Tea Pu'er, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Yan
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology Wuhan, China.
authors.forename: Chaofang
authors.initials: C
authors.lastname: Zhong
authors.affiliation: College of Horticulture and Landscape, Yunnan Agricultural University Kunming, China.
authors.forename: Fengmei
authors.initials: F
authors.lastname: He
authors.affiliation: National and Local Joint Engineering Research Center on Gemplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University Kunming, China.
authors.forename: Junwen
authors.initials: J
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau Macau, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zeng
authors.affiliation: National and Local Joint Engineering Research Center on Gemplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University Kunming, China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Zhang
authors.affiliation: National and Local Joint Engineering Research Center on Gemplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University Kunming, China.
authors.forename: Shengchao
authors.initials: S
authors.lastname: Yang
authors.affiliation: Nowbio Biotech Inc., KunmingChina; Faculty of Life Science and Technology, Kunming University of Science and TechnologyKunming, China; Province Key Laboratory, Biological Big Data College, Yunnan Agricultural UniversityKunming, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences Kunming, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of MacauMacau, China; Shenzhen Key Laboratory for Orchid Conservation and Utilization, National Orchid Conservation Center of China and Orchid Conservation and Research Center of ShenzhenShenzhen, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Cai
keywords: CRISPR/Cas9 gene editing; Dendrobium officinale; gene promoters; lignocellulose biosynthesis; overexpression; transgene; vector construction