TANG Jian-min, QI Li-wang, HE Xiao-ting, JIANG Yu-xin, LI Zhe-xin*

1. College of Landscape Architecture and Life Science, Institute of Special Plants, Chongqing Special Plant Industry Collaborative Innovation Center, Chongqing Key Laboratory of Economic Plant Biotechnology, Chongqing University of Arts and Sciences,

Yongchuan 402160, PRC;2. State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091,PRC

Abstract Ginger (Zingiber officinale Roscoe) has high economic value as medicinal and food resources. 6-gingerol is the core medicinal constituents of ginger. In the present study, a local ginger cultivar of Chongqing was taken as the research material.The ZoWRKY1 gene was cloned to determine its expression level in different ginger developmental phases and to analyze its correlation with 6-gingerol content. The expression level of ZoWRKY1 under different concentrations of NaCl stress was tested, and so was the correlation between ZoWRKY1’s expression level and the contents of 6-gingerol synthase genes, i.e. ZoPAL, ZoC4H and Zo4CL. The results showed that the cDNA of the cloned ZoWRKY1 gene is 1 026 bp in total length, and ZoWRKY1 belongs to the second type member of the WRKY family; the expression level of ZoWRKY1 rose sharply in the second developmental phase of the ginger which was about one month after sowing, and there was a significant correlation between the expression level of ZoWRKY1 and the increase of 6-gingerol content; the expressions of ZoWRKY1 and 6-gingerol synthase genes ZoPAL, ZoC4H and Zo4CL had sharp rises under 25 g/L NaCl stress, and the expression level of ZoWRKY1 was closely related to that of ZoC4H or Zo4CL. Therefore, it was speculated that there was a regulatory correlation between ZoWRKY1 and ZoC4H or Zo4CL that can further affect the biosynthesis of 6-gingerol.

Key words Ginger; WRKY transcription factor; 6-gingerol synthesis; Correlation

1. Introduction

Ginger (

Zingiber officinale

Roscoe), a perennial herbaceous monocot in family Zingiberaceae and genus

Zingiber

, has high medicinal and culinary value. Gingerol is the pungent substance and core component of gingerand gingerols are the main components of gingerol, especially 6-gingerols, accounting for over 75% of the total gingerol. Substances containing phenylpropanoids are direct or indirect products of phenylpropane metabolism.According to the existing findings, enzymes like phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (

C4H

) and 4-coumarate: CoA ligase(

4CL

) play an important role in the biosynthesis of phenylpropanoids-containing substances (including 6-gingerol). A tremendous amount of experimental data has proved that these enzyme genes have control over the biosynthesis of phenylpropane metabolites. The expression quantity of PAL gene is positively correlated with the content of flavonoids in both pistachio and ginkgo. Lowering of chrysanthemum’s

C4H

gene expression level by stress could also reduce the mass of flavonoids. The lignin eucalypt would decrease by 18.9% with the reduction of

C4H

expression. Strong positive correlation was also observed between the expression level of

4CL

gene and the accumulation of flavonoids in Erigeron breviscapus and Boesenbergia. The suppression of PAL,

C4H

, and

4CL

expression in tobacco or Arabidopsis can also reduce the content of lignin,leading to the changes in lignin composition. When C4H antisense gene is transferred into alfalfa, the contents of lignin in gene silencing plants are lower than those in wild plants.The expression of inducible gene in plants is subject to transcriptional level, while the transcriptional levels are dominated by various transcription factors. WRKY transcription factor plays a critical role in regulating the biosynthesis of different phenylpropanoids (

e

.

g

. lignin and flavonoids). The

VvWRKY2

gene of wine grape could regulate the formation and accumulation of lignin by activating the

VvC4H

expression, and this function has been proved in tobacco.The contents of lignin in wild populus tomentosa are almost twice as much as those in transgenic populus tomentosa with

PtrWRKY25

over-expression, which means

PtrWRKY25

is likely to work mainly during the biosynthesis route of lignin. The regulation of lignin biosynthesis by WRKY

transcription factors in other plants such as alfalfa and spruce is also reported.

AtWRKY23

could stimulate the formation of flavonoids in arabidopsis by increasing the expression of related enzyme genes, and this kind of regulation is closely connected with auxin signal pathways. The

VvWRKY26

transcription factor extracted from grapes is fundamental to the formation of anthocyanin. The over-expressed

MdWRKY11

of alfalfa could promote the expression of

F3H

,

FLS

,

DFR

,

ANS

and

UFGT

, thus leading to the rise of flavonoids and anthocyanin accumulation.Plants acquire a series of defense response mechanisms during their interactions with the environment, allowing them to produce and accumulate secondary metabolites to resist various biotic or non-biotic adverse stresses through the expression of internal resistance genes. 23

WRKY genes (

ZoWRKY

) were obtained and numbered from 1 to 23 according to the RNA-seq of local ginger’s fleshy root, underground rhizome, aerial stem and leaf.

ZoWRKY1

to

ZoWRKY5

are the ones with high levels of gene expression in underground rhizomes. The expression level of

ZoWRKY1

changes significantly under NaCl stress induction. This paper,focusing on the cloning and bioinformatics analysis of

ZoWRKY1

, aims for the expression of

ZoWRKY1

under different levels of NaCl stress and in various developmental phases of local ginger. Besides, the contents of 6-gingerol and the key biosynthesisrelated enzyme genes were analyzed to illustrate the possible correlations between

ZoWRKY1

and 6-gingerol biosynthesis. The findings could lay the foundation for further researches on the regulation of 6-gingerol biosynthesis by

ZoWRKY1

in ginger and provide theoretical basis for the realization of artificial regulation on chemical synthesis and accumulation in plants.

2. Materials and Methods

2.1. Materials and sampling

Local Sichuan-Chongqing ginger variety“Yujiang 1” was selected as the experimental material.The female ginger was grown in April (pregermination for 1 month) and harvested in October. Different samples were gathered according to the research contents. (1) Sampling of ginger in different developmental phases: sample the rhizome of the first branch tender ginger on the 15of each month;the six phases from April 15to September 15were numbered from Stage 1 to 6. (2) Sampling of ginger under NaCl stress: one month after the sowing of the female ginger, spray the leaves of ginger plants with NaCl solution at different concentrations (0, 25, 50,75, 100, and 125 g/L) every 2 d; harvest the rhizome on the 10day of the spray treatment. Quick freeze the above samples by liquid nitrogen and store them in refrigerator at -80℃.

2.2. Total RNA extraction and cDNA synthesis

The total RNA was extracted by RNAiso Plus Total RNA kit (Takara, Dalian). The results of agarose gel electrophoresis showed clear and undegraded 28S and 18S RNAs of different samples. The inverse transcription and synthesis of cDNA were done by TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit(TransGen, Beijing).

2.3. Gene cloning and sequencing

The specific primer of

ZoWRKY1

sequence was designed according to the transcriptome sequencing analysis (Table 1); the mixed cDNA of rhizomes sampled at different developmental phases was taken as the PCR template; the reaction system (20 μL): 10×Buffer (10 μL), 1.5 mmol/L Mg(1.5 μL), 0.1 mmol/L dNTPs (4 μL), 0.4 μmol/ L F primer and R primer (0.5 μL for each), 0.5 U rTaq DNA polymerase (0.2 μL),200 ng/μL cDNA template (1 μL), and ddHO (2.3 μL). The process of PCR reaction: 95℃ 5 min; 95℃10 s, 56℃ 30 s, 72 ℃ 1 min, 35 cycles; 72℃ 7 min.The PCR products were analyzed by 1.0% agarose gel electrophoresis. A single band was obtained and connected to the cloning vector after gel extraction.The cloning kit was the pEASY-Blunt Simple Cloning Kit; transform the Top 10 Escherichia coli; screen the positive clones; carry out PCR test and sequencing verification for the bacteria solution. Analyze the sequence of

ZoWRKY1

by DNAman and forecast its protein structure by the online tool at http://smart.embl-heidelberg.de/http://smart.embl-heidelberg.de/.

2.4. qRT-PCR analysis

The tenfold-diluted cDNA solutions of different samples (namely, samples collected at different developmental phases of ginger or those treated by NaCl stress) were prepared respectively; qRT-PCR test was prepared and the results were analyzed by TB GreenTM Premix Ex TaqTM (Tli RNaseH Plus) kit(Takara, Dalian) with the qTOWER2.0 real-time PCR system (Analytik Jena, Germany). The sequences of the primers were listed in Table 1. The reaction system (20 μL): cDNA (2 μL), TB Green Ⅱ (10 μL),primers (0.8 μL for each), ddH2O (6.4 μL). Reaction conditions: 95℃ 30 s; 95℃ 5 s, 55℃ 15 s, 72℃ 10 s,40 cycles.

Table 1 Related primer sequence

2.5. Correlation analysis

During the early stage of research, the contents of 6-gingerol in ginger rhizomes of different developmental phases were measured by LC-MS method. Calculate the increment of 6-gingerol in each phase by subtracting the total 6-gingerol content of the prior phase from the total 6-gingerol content of all the phases. Perform correlation analysis by IBM SPSS Statistics18.0.

3. Results and Analysis

3.1. Cloning and sequencing of ginger ZoWRKY1 gene

BLASTx comparison and ORF analysis were performed in NCBI based on the cDNA sequence of

ZoWRKY1

in the transcriptome sequencing library.The results revealed that this sequence had integral ORF and therefore was reckoned a full-length gene.Specific primers were designed for this gene; the mixed cDNA of rhizome samples was taken from different developmental phases as the PCR template.The size of PCR product was proved to be in line with expectation (Fig. 1A). The PCR product was connected to the pMD19-T carrier for sequencing.The total length of the obtained cDNA sequence was 1 026 bp. The

ZoWRKY1

sequnce was analyzed with software DNAman. It was found that the protein was composed of 341 amino acids. The cDNA sequence of

ZoWRKY1

was submitted to NCBI Genbank with the accession number of MT414710. This gene was 92% homologous with the

MdWRKY21

gene(XM_018829735) of Musa acuminate (Zingiberales,musaceae) by NCBI comparison. According to the sequencing of amino acids,

ZoWRKY1

had 1 WRKY conserved domain, 2 LCRs (low complexity region), and CH-type zinc fingers (Fig. 1B and 1C).Therefore,

ZoWRKY1

belonged to the member of the second kind in WRKY family.

Fig. 1 Cloning and bioinformatics analysis of ZoWRKY1

3.2. ZoWRKY1 expression at ginger’s different developmental phases

The expression levels of

ZoWRKY1

at ginger’s different development phases were measured by qRTPCR (Fig. 2). The changes of

ZoWRKY1

expression were apparent: a sharp rise occurred at the early developmental phase, namely, 1～2 month(s) after pregermination and sowing (Stage 2～3), which increased by five times in comparison with the expression level at the sowing phase; whereas large reductions were observed during the following months and the expression levels were similar to that of Stage 1.

Fig. 2 ZoWRKY1 expression at different developmental phase of ginger

3.3. Correlation analysis of ZoWRKY1 expression level and 6-gingerol content

According to the results, the contents of 6-gingerol in ginger rhizomes from Stage 1 to 6 were 182.37, 498.47, 528.56, 486.70, 478.14, and 489.34 μg/g. The findings of correlation analysis by IBM SPSS Statistics18.0 suggested a positive correlation between the expression level of

ZoWRKY1

and the increase of 6-gingerol contents, i.e. a correlation coefficient of 0.831 (

P

=0.040<0.05).

3.4. Expressions of ZoWRKY1 and 6-gingerol biosynthetic pathway-related enzyme genes under NaCl stress

The cDNA of the NaCl-treated ginger rhizomes were used as template to carry out qRT-PCR analysis for

ZoWRKY1

and the three enzyme genes (

i

.

e

.

ZoPAL

,

ZoC4H

and

Zo4CL

) related to 6-gingerol biosynthesis.The treatment of 0 g/L NaCl was used as the control group. As shown in Fig. 3, the expression level of

ZoWRKY1

rose sharply at the NaCl concentration of 25 and 50 g/L, increasing by 2.73 and 1.58 times respectively, and then, the figure reduced to a level similar to that of the control. For the three enzyme genes (

i

.

e

.

ZoPAL

,

ZoC4H

and

Zo4CL

) related to 6-gingerol biosynthesis, high expression levels were observed under low NaCl stress (25 g/L), and the difference was extremely significant compared with the expression level of the control.IBM SPSS Statistics18.0 was applied to analyze the correlations between the expression level of

ZoWRKY1

and those of the three enzyme genes (

i.e

.

ZoPAL

,

ZoC4H

and

Zo4CL

) related to 6-gingerol biosynthesis. The results showed that the expression levels of

ZoC4H

and

Zo4CL

had a correlation coefficient of 0.842 (

P

=0.036<0.05) and 0.965 (

P

=0.002<0.01) to the expression level of

ZoWRKY1

, both representing significant correlations.In contrast, the correlation coefficient between the expression level of

ZoPAL

and

ZoWRKY1

was 0.794(

P

=0.059>0.05), indicating no significant correlation.From these findings we can infer that there are positive correlations between the expression level of

ZoWRKY1

and those of

ZoC4H

and

Zo4CL

.

Fig. 3 Expressions of ZoWRKY1 and 6-gingerol biosynthetic pathway-related enzyme genes under NaCl stress

4. Conclusion and Discussion

Transcription factor gene technology is an effective method for genetic improvements of plants’ secondary metabolism. Transcription factors,compared with synthetase genes, can better regulate secondary metabolism and offer superior potential for genetic improvements. Many transcription factor families have been proved to be involved in the regulation of plants’secondary metabolites in recent years, among which WRKY transcription factor is a very hot topic.

In this paper, a WRKY transcription factor

ZoWRKY1

is cloned to verify its regulatory effects on the biosynthesis of ginger’s 6-gingerol. The results of qRT-PCR and correlation analysis reveal that the expression level of

ZoWRKY1

, as shown in Fig. 2,rises sharply at Stage 2 (a month after the sowing of ginger) and presents significant correlations with the increase of 6-gingerol content. This means the biosynthesis of 6-gingerol has staged specificity:6-gingerol is produced in abundance one month after the sowing of ginger, and the production goes down to very low levels and is accompanied by gradual accumulation. There is a saying in China: the older the ginger is, the spicier it tastes. According to this saying,if gingerol is the major source of ginger’s strong hot taste, the content of gingerol should have a tendency to increase with the growth and development of ginger rhizomes, and therefore theoretically the rhizomes should not be very spicy in early developmental stages because of low gingerol contents. Nevertheless, the findings suggest that large amounts of 6-gingerol are formed in the early developmental stage, which means 6-gingerol is not the only factor deciding the hot taste of ginger despite its key role in ginger’s medicinal value. Other gingerol substances could be the major source of ginger’s spicy taste.NaCl stress is the most common form of stress among crops. The expression levels of

ZoWRKY1

are very high in ginger rhizomes under 25 g/L NaCl stress,indicating the involvement of

ZoWRKY1

in ginger’s low-salt resistance stress response. This finding is in accord with earlier research of WRKY

transcription factor and plant’s tolerance to stress. According to the significant correlations between the expression level of

ZoWRKY1

and the increase of 6-gingerol content, the expression level of

ZoWRKY1

would increase with the biosynthesis of 6-gingerol under low NaCl stress. This surmise is further confirmed by the fact that the treatment of 25 or 50 g/L NaCl(as shown in Fig. 3) could induce sharp rises in the expression levels of 6-gingerol synthetase genes (

i

.

e

.

ZoPAL

,

ZoC4H

and

Zo4CL

). The correlation analysis have also proved significant correlations between the expression level of

ZoWRKY1

and those of

ZoC4H

and

Zo4CL

, from which we can infer that

ZoWRKY1

is capable of regulating

ZoC4H

and

Zo4CL

as well as further affecting the biosynthesis of 6-gingerol,whereas this speculation needs further evidence.The WRKY transcription factor

ZoWRKY1

of ginger is cloned and found to have sharp rises in its expression level under low NaCl stress. Significant correlations between

ZoWRKY1

and the synthetase genes of 6-gingerol (

i

.

e

.

ZoC4H

and

Zo4CL

) are verified, indicating possible regulatory effects of

ZoWRKY1

on the biosynthesis of 6-gingerol.