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

Ling Kui; Haitao Chen; Weixiong Zhang; Simei He; Zijun Xiong; Yesheng Zhang; Liang Yan; Chaofang Zhong; Fengmei He; Junwen Chen; Peng Zeng; Guanghui Zhang; Shengchao Yang; Yang Dong; Wen Wang; Jing Cai

doi:10.3389/fpls.2016.02036

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

Front Plant Sci. 2017 Jan 12:7:2036. doi: 10.3389/fpls.2016.02036. eCollection 2016.

Authors

Ling Kui¹, Haitao Chen², Weixiong Zhang³, Simei He⁴, Zijun Xiong⁵, Yesheng Zhang², Liang Yan⁶, Chaofang Zhong⁷, Fengmei He⁸, Junwen Chen⁴, Peng Zeng³, Guanghui Zhang⁴, Shengchao Yang⁴, Yang Dong⁹, Wen Wang¹, Jing Cai¹⁰

Affiliations

¹ State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences Kunming, China.
² State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of SciencesKunming, China; Nowbio Biotech Inc., KunmingChina.
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 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of SciencesKunming, China; China National GeneBank, BGI-ShenzhenShenzhen, 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 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.
¹⁰ 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.

Abstract

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.

Keywords: CRISPR/Cas9 gene editing; Dendrobium officinale; gene promoters; lignocellulose biosynthesis; overexpression; transgene; vector construction.