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CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Published: September 23rd, 2021



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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NameCompanyCatalog NumberComments
0.1 mL qPCR 8-strip tube (with optical caps)KIRGEN, Shanghai, ChinaKJ2541
ABASolarbio Life Science, Beijing, ChinaA8060
Agar powderSolarbio Life Science, Beijing, ChinaA8190
CentrifugeOsterode am Harz, Germanyd37520
CFX Connect TW Optics ModuleBio-rad, US1855200
constant temperature incubatorShanghai Boxun Industry & Commerce Co., Ltd, Shanghai,ChinaBPX-82
Diposable Petri dishCorning, US
Dry BathGingko Bioscience Company/Coyote bioscience, ChinaH2H3-100C
Eastep Total RNA Extraction Kit50Promega, Beijing, ChinaLS1030
Electronic balanceTianjin, ChinaTD50020
Filter papeHangzhou wohua Filter Paper Co., Ltd, China
FiveEasy PlusMettler Toledo, Shanghai, China30254105
Flowerpot 9*9China
JASolarbio Life Science, Beijing, ChinaJ8070
KanSolarbio Life Science, Beijing, ChinaK8020
MagicSYBR MixtureCWBIO, Beijing, ChinaCW3008M
Mini MicrocentrifugeScilogex, Beijing, ChinaS1010E
NaClSolarbio Life Science, Beijing, ChinaS8210
NanPhotometer N50 TouchIMPLEN GMBH, GermanyT51082
Purelab untra
RifampicinSolarbio Life Science, Beijing, ChinaR8010
Seedling box 30*200China
Thermal Cycler PCRBio-rad, UST100
Thermostatic oscillatorBeijing donglian Har lnstrument Manufacture Co.,Ltd,ChinaDLHE-Q200
Tomy AutoclaveTomy, JapanSX-500
TryptoneSolarbio Life Science, Beijing, ChinaLP0042
UEIris II RT-PCR System for First-Strand cDNA Synthesis( with dsDNase)US Everbright INC, Jiangsu, ChinaR2028
Yeast Extract powderSolarbio Life Science, Beijing, ChinaLP0021

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