CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System
Summary
Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.
Abstract
An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.
Introduction
Transformation is a basic tool to evaluate the expression of exogenous genes1,2. Many biological aspects of resource plants are common to all plants; therefore, functional studies of certain genes canbe carried out in model plants (such as Arabidopsis)3. Yet, many genes in plants are unique in their function and expression patterns, requiring studies in their own or closely related species, especially for resource plants3,4. Plant cells can sense various signals that enable plants to show specific changes in gene expression, metabolism, and physiology in response to different environmental stress conditions5,6,7. Flavonoids are crucial players in the signaling process of plants that is responsive to environmental stresses5,8,9. In addition, the flavonoid content in horticultural and medicinal plants is also an important indicator for quality evaluation10. Identification of genes involved in the regulation of flavonoid synthesis in response to external signals is crucial for understanding the mechanism of flavonoid synthesis in plants. Several studies have revealed that the application of exogenous hormones can promote the accumulation of flavonoids6,11. A stable transformation system and gene function validation method are essential to demonstrate the function of genes and to understand secondary metabolism in plants.
Agrobacterium-mediated transformation is widely used in DNA insertion5,8,9. Agrobacterium tumefacient can transfer ring genes into the chromosomes of plant cells, and exogenous phytohormones induce single or a few host cells that can regenerate plants to obtain stable transformants12,13,14. Agrobacterium tumefacient-mediated transformation methods are more applicable to plant species suitable for in vitro manipulation, while most perennial woody plants limit the application of this method because of their regeneration difficulty4,15. A. rhizogenes is also able to modify the genome of host cells16. In the present study, we have developed an efficient and stable A. rhizogenes-mediated transformation procedure. A. rhizogenes contains a second binary plasmid carrying non-natural gene T-DNA in addition to the Ri plasmid. The host plant is infected, and a composite plant can be obtained with transgenic hairy roots emerging from the wild-type shoot16,17. The A. rhizogenes-mediated transformation systems are suitable for application in woody plant research due to their fast, low cost and non-required plant regeneration. More than 160 kindsof plants have successfully induced hairy roots, and most of which are in Solanaceae, Compositae, Cruciferae, Convolvulaceae, Umbelliferae, Leguminosae, Caryophyllaceae, and Polygonaceae18,19. Compared with A. tumefaciens, A. rhizogenes showed higher efficiency in the mediated transformation of pigeon pea17,20.
In this study, pigeon pea was used as an example to introduce the transformation process mediated by A. rhizogenes. From inoculation to rooting, the experiments lasted for 5 weeks. We identified the transformation of the adventitious root through morphology and the GFP reporter gene, and the transformation efficiency was as high as 75%. Also, we treated the composite plant with JA and ABA, as well as detected transcripts and secondary metabolites by quantitative real-PCR and HPLC (high performance liquid chromatography). It is confirmed that the expression level of CcCIPK14 responds not only to JA and ABA but also affects the biosynthesis of flavonoids. This system is adequate for studying function genes associated with secondary metabolism. It also provides a new approach to studying non-model plants in lack of a sufficient stable transformation system17,21,22.
Protocol
Representative Results
Discussion
The rapid characterization of gene function is the common goal in the study of most species, and it is particularly important for the development of resource plants. The A. rhizogenes-mediated transformation has been widely used in the hairy root culture. The hairy root culture (HRC), as a unique source of metabolite production, plays a pivotal role in metabolic engineering18,28. The application of this technology is mainly limited to the function o…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors gratefully acknowledge the financial support by National Natural Science Foundation of China (31800509, 31922058), Outstanding Young Talent Fund in Beijing Forestry University" (2019JQ03009), the Fundamental Research Funds for the Central Universities (2021ZY16), Beijing Municipal Natural Science Foundation (6212023), and National Key R&D Program of China (2018YFD1000602,2019YFD1000605-1) and Beijing Advanced Innovation Center for Tree Breeding by Molecular Design. I wish to thank Zhengyang Hou for his guidance in writing the article and to Professor Meng Dong for his guidance on the article idea.
Materials
| 0.1 mL qPCR 8-strip tube (with optical caps) | KIRGEN, Shanghai, China | KJ2541 | |
| ABA | Solarbio Life Science, Beijing, China | A8060 | |
| Agar powder | Solarbio Life Science, Beijing, China | A8190 | |
| Centrifuge | Osterode am Harz, Germany | d37520 | |
| CFX Connect TW Optics Module | Bio-rad, US | 1855200 | |
| constant temperature incubator | Shanghai Boxun Industry & Commerce Co., Ltd, Shanghai,China | BPX-82 | |
| Diposable Petri dish | Corning, US | ||
| Dry Bath | Gingko Bioscience Company/Coyote bioscience, China | H2H3-100C | |
| Eastep Total RNA Extraction Kit50 | Promega, Beijing, China | LS1030 | |
| Electronic balance | Tianjin, China | TD50020 | |
| Filter pape | Hangzhou wohua Filter Paper Co., Ltd, China | ||
| FiveEasy Plus | Mettler Toledo, Shanghai, China | 30254105 | |
| Flowerpot 9*9 | China | ||
| JA | Solarbio Life Science, Beijing, China | J8070 | |
| Kan | Solarbio Life Science, Beijing, China | K8020 | |
| MagicSYBR Mixture | CWBIO, Beijing, China | CW3008M | |
| Mini Microcentrifuge | Scilogex, Beijing, China | S1010E | |
| NaCl | Solarbio Life Science, Beijing, China | S8210 | |
| NanPhotometer N50 Touch | IMPLEN GMBH, Germany | T51082 | |
| Purelab untra | |||
| Rifampicin | Solarbio Life Science, Beijing, China | R8010 | |
| Seedling box 30*200 | China | ||
| Thermal Cycler PCR | Bio-rad, US | T100 | |
| Thermostatic oscillator | Beijing donglian Har lnstrument Manufacture Co.,Ltd,China | DLHE-Q200 | |
| Tomy Autoclave | Tomy, Japan | SX-500 | |
| Tryptone | Solarbio Life Science, Beijing, China | LP0042 | |
| UEIris II RT-PCR System for First-Strand cDNA Synthesis( with dsDNase) | US Everbright INC, Jiangsu, China | R2028 | |
| Yeast Extract powder | Solarbio Life Science, Beijing, China | LP0021 |
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