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 genes^1,2. 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 plants^3,4. Plant cells can sense various signals that enable plants to show specific changes in gene expres....

Protocol

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 (NCPPB2659) were preserved in the laboratory. They were stored in yeast mannitol medium (YEP) with 15% glycerol at -80 °C. The protocol described ....

Results

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 the .......

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 engineering^18,28. The application of this technology is mainly limited to the function o.......

Materials

List of materials used in this article

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