Xiaohong Guo?Yandong Lv?Hongyu Li?Nan Fu?Guiping Zheng?Lihua Liu?Yuhua Li

Introduction

Low temperature is a critical environmental factor that limits plant growth and development and distribution ranges(Zong et al.2016).To adapt to the seasonal variations in temperature,most temperate plants have evolved mechanisms for cold acclimation by which they can increase their cold resistance to survive subsequent freezing temperatures(Shinozaki and Yamaguchi-Shinozaki 1997).Cold acclimation improves the transcription of several cold-regulated(COR)genes that encode proteins and enzymes that mediate lipid and sugar metabolism to protect plant cells from freezing injury(Mayer et al.2015).

Although conventional cross-breeding has generated someanti-coldcropvarieties,manyproblemsstillneedtobe solved,especially the lack of drought-resistant resources,high experimental costs and long time needed for selection.Advanced techniques using genetic engineering could help broaden the genetic base of plants by identifying new sources of resistant genes(Zhang et al.2016).Although increasing the expression of stress-defense genes can enhance cold tolerance of the plant,the overexpression of exogenous genes,leading to excessive energy loss,thereby reducing plant growth.Using inducible promoters to control the expression of target gene is an effective way to avoid potential negative effects caused by constitutive promoters(Company et al.2014).A few studies on crop biotechnology have obtained strong and reliable expression with inducible promoters(Fang et al.2015).Therefore,the study of a coldinduced promoter in plants not only contributes to understanding the regulatory mechanism of gene expression,but also facilitates the expression of exogenous genes in genetic engineering.

Tamarix hispidais widely distributed in Central Asia and Chinabecauseitishighlytoleranttoabioticstressesincluding cold,drought,salinity and other abiotic stresses(Li et al.2009).In our previous study,ThCAPtranscription levels increasednotonlyinleavesbutalsoinrootsofT.hispidaafter exposureto4°C.Wealsodiscoveredthatthisgeneenhanced cold tolerance of transgenicPopulus(P.davidiana×P.bolleana)(Guo et al.2009).The present study was aimed at cloningandanalyzingthepromoterregionoftheThCAPgene to understand the regulatory mechanism ofThCAPgene and to develop transgenic plants that tolerate cold stress.

Materials and methods

Experimental materials and reagents

Escherichia colistrain DH5α andAgrobacterium tumefaciensstrain EHA105 were provided by the Laboratory of Rice Biology and Molecular Breeding of Heilongjiang Bayi Agricultural University.Plasmid pCAMBIA3301 was obtained from the Institute of Crop Sciences,Chinese Academy of Agricultural Sciences.A Genome Walking Kit(TaKaRa,Japan)was used to isolate the promoter region ofThCAP.ExTaqDNA polymerase,exonuclease III and T4DNA polymerase were obtained from the New England Biolabs(Beijing).DNA was extracted using a DNA extraction kit(Tiangen Biotech Co.,Ltd.,Beijing,China);endonuclease,5-Bromo-4-chloro-3-indolylβ-D-galactopyranoside(X-gal),Isopropyl β-D-1-thiogalactopyranoside(IPTG),DNA Marker DL2000,plasmid extraction kits and gel extraction kits were purchased from Dalian TaKaRa and OMEGA Bio-Tek,respectively.

Bioinformatics analyzing for promoter sequence of ThCAP gene

Putativecis-acting elements in the promoter region were analyzed using the Plant CARE database(http://bioinfor matics.psb.ugent.be/webtools/plantcare/html/).

Construction of expression vectors and generation of transgenic plants

Universal Genomic DNA Extraction Kits(Tiangen,China)were used to extract genomic DNA from leaves ofT.hispida.Gene-speci fi c primers were designed according to the upstream promoter region ofThCAPin the 5′untranslated region (UTR). (SP1: 5′-GGGCGCGA GGTGGGAAGGT-3′;SP2:5′-TTCACGCGCTCTAGCT CGGATAAG-3′;SP3:5′-CGGAGGAGGAGGCCAGAG CAG-3′).The reaction volume and genome-walking conditions were set according to the manufacturer’s instructions.The tertiary PCR product was separated and puri fi ed using DNA puri fi cation kits(DP241),and then cloned into the pMD18-T vector(TaKaRa,China).A new fragment was isolated from each walking when the sequence of the fragment was 100%similar to the known sequence in the overlapping region.Based on prioritizing the predictedcisacting elements,a serial deletion analysis was carried out and 5′deletion fragments of PThCAP(P1,P2,P3,P4 and P5 means that the length of promoter was-1538,-1190 and-900,-718 and-375 bp,respectively),which were cloned in accordance with sites ofcis-acting elements and submitted to the GenBank(NCBI)database(accession number KY113121).The results were veri fi ed by relative gene expression using qRT-PCR(Supplementary Fig.S1).Plant expression vectors were constructed as shown in Fig.1.Cauli fl ower mosaic virus 35S(CaMV35S)promoter on pCAMBIA3301 vectors were replaced with the fi ve cloned serial deletion fragments of PThCAP.The pMD 18-T-ThCAPgene promoter’s serial deletion recombinant plasmidsandpCAMBIA3301vectorplasmidswere identi fi ed through sequencing as correct positive plasmids and kept at 4°C.Together with pCAMBIA3301(CaMV 35S::GUS),recombinant plasmids were introduced intoA.tumefaciensstrain LBA4404,and then inserted intoArabidopsis thaliana.Transformants were selected by spraying with Basta.Subsequent selection was achieved on 1/2 MS medium(pH 5.7,1%sucrose and 0.8%agar).Eight to forty individual lines for each construct were analyzed by PCR.Independent transgenic plants for each vector were used to reduce the variation generated by the insertion of foreign genes.

Validation of relative gene expression using quantitative real-time-PCR

Total RNA ofA.thalianawere obtained using the CTAB method(Doyle and Doyle 1990).Three independent biological replicates of each sample and three technical replicates of each biological replicate were used in the RTPCR.PCR ampli fi cations were performed in 20 μL total volume reactions containing 3 μL templates,2× SYBR Mix 10,0.15 μL Taq polymerase(5 μ/μL)and 1 μL of each primer.The reaction conditions were 2 min at 94°C,followed by 45 cycles of 72 °C for 30 s and 75 °C for 1 s.To determine the relative fold-change for each sample in each experiment,the Ct was calculated according the manufacturer’s recommendations(TaKaRa,Japan).

GUS staining analysis

The full-length promoter sequence of theThCAPgene and serial deletion fragments of the T3generation of transgenic were gained through the screening.GUS staining was performed on roots,stems and leaves ofA.thalianato detect promoter activity of theThCAPgene.The Jefferson method was used as previously described(Jefferson 1989),and the GUS activity test solution included 0.1 mol/L K4-Fe(CN)6,0.1 mol/L K3Fe(CN)6,50 mmol/L sodium phosphate buffer(pH 7.0),10 mmol/L Na2EDTA,0.001%(v/v)TritonX-100,0.5 mg/ml X-Gluc and 20%methyl alcohol.Samples were placed in 1.5 mL centrifuge tubes,and then the GUS staining solution was added to cover the materials fully.Aluminum foil was used to wrap the tube to prevent light exposure.The centrifuge tube was placed into an incubator at 37°C for 24 h,then 70%ethyl alcohol was added before incubation at 37 °C for another 5–6 h,when 90%ethyl alcohol 1 mL was added.The samples were then incubated at 37°C for 10 h until no chlorophyll was found in the samples.The samples were immersed in 70%ethyl alcohol and stored at room temperature.

Results

Cloning of 5′upstream sequence of the ThCAP gene

To investigate the regulatory mechanism ofThCAPgene expression,the sequences of PThCAPwas ampli fi ed using a genome-walking approach.The homogeneous bands gained from the third-round PCR reaction product had lower molecular weight than in the second nested PCR(Fig.2).The sequencing results showed a repetition sequence of 615 bp between the gained sequence(2153 bp)and sequence ofThCAPgene,indicating that the repetition sequence was the sequence of PThCAP(Fig.3).

Sequence analysis of the regulatory elements of the ThCAP gene promoter

Fig.1 Diagram of plant expression vector construction of deletion fragments of ThCAP gene promoter

Fig.2 Cloning of ThCAP promoter by the TAIL-PCR method.1 Electrophoresis of second-round PCR product;2 electrophoresis of third-round PCR product;3 DL2000 bp Marker(TaKaRa,Japan)

Fig.3 Gene promoter sequence of ThCAP

By analyzing the regulatory elements controlling the 1538-bp promoter sequence of theThCAPgene,we found that the sequence contained 18 TATA boxes(a TATA box was located in position-20 to approximately-30)of the promoter,which facilitates the transcription initiation complex to start transcription from the correct transcriptional start site)and 30 CAAT boxes(concerning transcriptional initiation frequency).The gained sequence included different fundamentalcis-acting with predicted functions.In addition,a variety ofcis-acting elements were associated with abiotic stress were identi fi ed in the promoter region(Table 1).For example,a cold-induced element(ACCGAC)and MYC recognition site were related to drought,abscisic acid and freezing injury.MBS element of MYB binding site was involved in drought-induced stress.A G-box element was involved in abscisic acid stress,and binding sites of transcription factors participated in light reply or adverse situation.Therefore,the expression of promoter ofThCAPgene was regulated by various external environmental signals(low temperature,drought,hormone and light stress).

Generation of plants with 5′deletion in ThCAP gene promoter expression vector

The constructed serial deletion recombinant plasmids of the pCAMBIA 3301-ThCAPgene promoter was introduced intoA.tumefaciensstrain EHA105(Fig.4,P4 data not shown).For introducing the constructed vector ofThCAPgene promoter with the 5′deletion fragments driving the GUS reporter gene intoA.tumefaciens,several transgenic pure lines(three Pl::GUS,two P2::GUS, fi ve P3::GUS,four P4::GUS and three P5::GUS)were obtained in this study.After a week of GUS staining of transgenicA.thaliana,compared with nontransgenic plant(WT),thepromoter fragment of theThCAPgene had GUS expression activity in leaves and roots of transgenic plants(lines P1–P4),indicating that lines P1–P4 had promoter activity,verifying the biological function of the promoter in the transgenic plants(Fig.5).However,there was no signi ficant difference in GUS activity among transgenic plants harboring the deletion constructs of PThCAP(Supplementary Fig.S2).

Table 1 Predicted promoter elements of ThCAP gene

Fig.4 Veri fi cation of the recombinant plasmids with speci fi c primer of PThCAP promoter sequence by PCR.Lanes 2–5 showed the PCR products of successive deletions of the PThCAP(1538,1190,900 and 375 bp,respectively).Lanes 1 showed the DL2000 DNA marker.1 DL2000 bp Marker;2 pCAMBIA-ThCAP P1 PCR(1538 bp);3 P2 PCR(1190 bp);4 P3 PCR(900 bp);5 P5 PCR(375 bp)

Fig.5 TransgenicArabidopsisGUS staining.1 Nontransgenic Arabidopsis thaliana(WT);2–5 5 weeks of the T3generation of P1–P4 transgenic Arabidopsis thaliana

Identi fi cation of the critical regulatory area in the ThCAP gene promoter in response to cold stress

To identify the regulatory area in PThCAPsubjected to cold stress,transgenic lines and nontransgenic lines were treated with low temperature stress at 4°C for 2 or 4 h.Expression of the GUS gene in transgenic plants was used to determine the critical cold-responsive regulatory area in PThCAP.As shown in Fig.6,only the P2 fragment had strong GUS expression activity in leaves and roots ofA.thaliana,while the P1,P3 and P4 fragments lacked any signi fi cant GUS expression.It is clear that these regions(-1538 to-1190 bp and-900 to 0 bp)inhibited the expression of theThCAPgene promoter.Furthermore,the MYB recognition site(as shown in Table 1)was found in the promoters ofThCAPby analyzing thecis-acting regulatory elements.Therefore, –1190 to –900 bp of theThCAPgene promoter might play a vital role in regulating cold acclimation associated with changes in gene expression.

Fig.6 P2 fragments can be induced to be expressed at low temperature.1–4:T3generation of P1–P4 transgenic Arabidopsis thaliana at 5 weeks

Discussion

Plant genetic engineering technology is used not only for overcoming interspeci fi c incompatibility during conventional breeding but also greatly broadens the sources of candidate genes for abiotic stress studies.Cold-related genes encoding regulatory proteins might contribute to improving cold tolerance of plants(Fei et al.2015).Li et al.reported that expression of wheat driven by the RD29 promoter enhanced water-stress tolerance without impacting growth and development of tomato.Constitutive overexpression of theTaDREB3gene in barley can improve frost tolerance of transgenic plants at the vegetative stage of plant development,but leads to stunted phenotypes compared with the control(Kovalchuk et al.2013).Similarly,theOsWRKY71andTdCor39-TaDREB3promoter enhanced cold tolerance in barley(Nataliya et al.2013).In this study,we used plant genetic engineering techniques to study of a cold-induced promoter fromT.hispidato better understand the regulatory mechanism of a cold-responsive gene.The TATA box and CAAT box play vital roles in assembling the transcription machinery at promoters that previously have been shown to be induced by cold stress(Basehoar et al.2004).Cis-actingelements are molecular switches having stress-responsive promoters that function for plant adaptation to environmental stresses(Yamaguchi-Shinozaki and Shinozaki 2005).The stress-induced promoter region contained differentcis-elements including abscisic-acid-responsive element(ABRE)(PyACGTGGC),MYB recognition site(MYBRS-C/TAACNA/G),MYC recognitionsite(MYCRS-CANNTG),andDRE (A/GCCGAC)(Trivedi et al.2016).Among these elements,the MYC recognition site and the MYB site function are involved in ABA-independent gene expression under lowtemperature conditions(Yu et al.2016).In our study,we ampli fi ed a 1538-bp fragment upstream of theThCAPgene through PCR ampli fi cation.We found several general promoter elements in multiple plant promoters,e.g.,TATA box,CAAT box and cold-induced element(ACCGAC),MYC recognition site,MBS element of MYB binding site and binding sites of transcription factors,indicating that the promoter sequence had the general biological function related to abiotic stress resistance inA.thaliana.To determine the main regulatory functional areas of the promoter,a deletion analysis of the promoter fragments ofThCAPgene was performed.GUS staining showed that the P2 fragment ofA.thalianahas strong GUS expression activity in leaves and roots of transgenic lines.We confi rmed that the regions-1538 to-1190 bp and-900 to 0 bp of PThCAPincluded elements that inhibited the PThCAPexpression inA.thaliana.Therefore,the region-1190 to-900 bp might play a vital role in regulating cold acclimation according to our results.

Conclusion

PThCAP,a novel cold-inducible promoter fromT.hispidawas cloned in this study.This study provides evidence for the regulatory mechanism of theThCAPgene involved in plant response to cold stress and also provides a candidate gene for genetically improving plants.

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