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Genetics

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Published: September 23rd, 2021

DOI:

10.3791/62304

1College of Forestry, Beijing Forestry University, 2Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University
* These authors contributed equally

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.

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.

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 expres....

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NOTE: Pigeon pea is a diploid legume crop that belongs to the family Fabaceae. The pigeon pea seeds used in this experiment are from the Northeast Forestry University of China and are coded 87119. The primary steps of this protocol are illustrated in Figure 1A. The seedling incubation was performed in a high humidity environment at 25 °C under fluorescent lights at 50 µmol photons per m-2s-1 in a 16 h photoperiod. A. rhizogenes strains K599 (.......

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A. rhizogenes -mediated hairy root transformation on pigeon pea
This study described the step-by-step protocols for the genetic transformation of hairy roots mediated by A. rhizogenes, which has significance in the field of plant molecules. It took about 5 weeks to get hairy roots from the roots of pigeon pea infected by A. rhizogenes. Figure 1A showed an overview of the entire transformation process, from th.......

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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.......

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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.

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Name Company Catalog Number Comments
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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