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

预防：（1）预防最有效的办法是接种猪瘟疫苗。在该病的常发地区或受威胁地区,按疫苗标注说明，每头份用灭菌生理盐水1 ml稀释后皮下或肌肉注射一头份，种母猪于配种前或配种后免疫一次；仔猪于20～25日龄首免，50～60日龄二免。在非疫区，对种母猪于配种前或配种后免疫一次;种公猪于春秋两季各免疫一次;仔猪断奶后免疫一次。另外，可以对仔猪进行超前免疫。（2）开展免疫监测。（3）坚持自繁自养，全进全出的饲养管理制度。（4）及时淘汰隐性感染者带毒种猪。（5）做好场舍的消毒杀虫和卫生工作，减少猪瘟病毒的侵入。

Materials and methods

Experimental materials and reagents

Universal Genomic DNA Extraction Kits(Tiangen,China)were used to extract genomic DNA from leaves of T.hispida.Gene-speci fi c primers were designed according to the upstream promoter region of ThCAP in 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 predicted cisacting 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 of cis-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-ThCAP gene 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 into A.tumefaciens strain LBA4404,and then inserted into Arabidopsis 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.

Bioinformatics analyzing for promoter sequence of ThCAP gene

Cloning of 5′upstream sequence of the ThCAP gene

Construction of expression vectors and generation of transgenic plants

Escherichia coli strain DH5α and Agrobacterium tumefaciens strain 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 of ThCAP.ExTaq DNA polymerase,exonuclease III and T4 DNA 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.

The constructed serial deletion recombinant plasmids of the pCAMBIA 3301-ThCAP gene promoter was introduced into A.tumefaciens strain EHA105(Fig.4,P4 data not shown).For introducing the constructed vector of ThCAP gene promoter with the 5′deletion fragments driving the GUS reporter gene into A.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 transgenic A.thaliana,compared with nontransgenic plant(WT),thepromoter fragment of the ThCAP gene 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).

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

By analyzing the regulatory elements controlling the 1538-bp promoter sequence of the ThCAP gene,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 fundamental cis-acting with predicted functions.In addition,a variety of cis-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 of ThCAP gene was regulated by various external environmental signals(low temperature,drought,hormone and light stress).

GUS staining analysis

To investigate the regulatory mechanism of ThCAP gene expression,the sequences of PThCAP was 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 of ThCAP gene,indicating that the repetition sequence was the sequence of PThCAP(Fig.3).

Results

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

The full-length promoter sequence of the ThCAP gene and serial deletion fragments of the T3generation of transgenic were gained through the screening.GUS staining was performed on roots,stems and leaves of A.thaliana to detect promoter activity of the ThCAP gene.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.

在科技迅猛发展的今天，低碳经济已经深入到人们的生活，并且给人们的生活方式带来天翻地覆的变化。各个国家在发展低碳经济方面不甘落后，已经做出了相关的政策调整和出台有关的法律制度。相关企业在积极寻求自身长久发展利益的同时，应该不断探索低碳营销的方法。而作为一个普通的消费者，更加需要建立低碳消费的观念，促进整个社会朝着可持续发展的方向前进。在企业、消费者、国家和非营利组织的共同努力下，低碳营销模式就能取代之前的营销模式，逐渐成为21世纪营销模式的中流砥柱。

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

Total RNA of A.thaliana were 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).

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

桩顶和基础之间应设置褥垫层[12-14]，通常褥垫层厚度宜为桩径的40%～60%[15]。褥垫一般选用最大粒径不大于30 mm的中砂、粗砂、级配砂石和碎石等材料，采用静力压实法，夯填度不应大于0.90。当其他条件一定时，从图5可以看出桩土应力比随褥垫层厚度的增大而减小。当褥垫层厚度大于300 mm时，桩土应力比趋于稳定，充分发挥了桩间土的承载力。由于桩间土表面的荷载作用，会产生水平向和竖向附加应力在桩侧土单元体，水平向附加应力增大了侧阻力作用在桩表面上；竖向附加应力则大大提高了单桩承载力，褥垫层的作用得到充分地发挥。

“由文化和旅游部、国家广播电视总局、国家新闻出版署、北京市人民政府共同主办的“第十三届中国北京国际文化创意产业博览会”于2018年10月25日召开，主题为：引领文化产业高质量发展，助推全国文化中心建设。中国动漫集团有限公司党委书记、董事长庹祖海于25日出席主题报告会并演讲。他呼吁加强动漫和旅游的融合，并指出，动漫和旅游的融合实现了消费产品与消费体验的融合、消费效率与消费深度的融合。

Table 1 Predicted promoter elements of ThCAP gene

Element name Nos.Sequence Biological function ATGCAAAT motif 1 ATACAAAT Cis-acting regulatory element associated to the TGAGTCA motif TATA box 18 TATAAAT,TATATAA,TTATTT,TTATTT,TTATTT Core promoter element around-30 of transcription start CAAT box 30 CAAT/CCAAT/CAATT/CAAAT Common cis-acting element in promoter and enhancer regions CANNTG 1 CANNTG Cold and ABA responsive element MRE 1 AACCTAA MYB binding site involved in light responsiveness MYBCORE 4 CNGTTR Involved in regulation of genes that are responsive to water stress in Arabidopsis MYB2CONSENSUSAT 1 YAACKG MYB recognition site found in promoters of the dehydration responsive gene rd22 and many other genes in Arabidopsis MYCCONSENSUSAT 4 CANNTG MYC recognition site found in the promoters of the dehydration responsive gene ASF1MOTIFCAMV 1 TGACG Abiotic and biotic stress differentially stimulate ‘as-1 element’activity GT1GMSCAM4 7 GAAAAA Plays a role in pathogen and salt induced SCaM-4 gene expression W BOX 1 TTGAC Recognized speci fi cally by salicylic acid(SA)-induced WRKY DNA binding proteins TGACG motif 3 TGACG Cis-acting regulatory element involved in the MeJA responsiveness Circadian 2 CAANNNNATC Cis-acting regulatory element involved in circadian control ABRE 1 ACGT,TACGTG,CACGTG Cis-acting element involved in ABA responsiveness Dof 13 AAAG Dof transcript factor recognition site GARE 1 TAACAAA GA response elements SRE 1 TTATCC Sugar repress elements DRE 1 CCGAC Low temperature responsive element

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 PThCAP subjected 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 of A.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 the ThCAP gene promoter.Furthermore,the MYB recognition site(as shown in Table 1)was found in the promoters of ThCAP by analyzing the cis-acting regulatory elements.Therefore, –1190 to –900 bp of the ThCAP gene 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 the TaDREB3 gene 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,the OsWRKY71 and TdCor39-TaDREB3 promoter 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 from T.hispida to 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-acting elements 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 different cis-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 the ThCAP gene 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 in A.thaliana.To determine the main regulatory functional areas of the promoter,a deletion analysis of the promoter fragments of ThCAP gene was performed.GUS staining showed that the P2 fragment of A.thaliana has 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 PThCAP included elements that inhibited the PThCAP expression in A.thaliana.Therefore,the region-1190 to-900 bp might play a vital role in regulating cold acclimation according to our results.

激电中梯测量采用短导线中间梯度装置，测量仪器为DJF10-2型大功率智能发送机和DJS-9数字直流激电接收机，以及氯化铅固体不极化电极等，采用U形布线原则，每次布极，观测段选在AB的中部，范围不大于2/3AB距。1∶5000激电中梯剖面点距10m，供电极距AB=1000m，MN=20m，供电周期32s，断电延时200ms。1∶2000激电中梯剖面点距5m，供电极距AB=800m，MN=40m，供电周期16s。

Conclusion

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

References

Basehoar AD,Zanton SJ,Pugh BF(2004)Identi fi cation and distinct regulation of yeast TATA box-containing genes.Cell 116:699–709

Company N,Nadal A,Ruiz C,Pla M(2014)Production of phytotoxic cationic α-helical antimicrobial peptides in plant cells using inducible promoters.PLoS One 9:e109990

Doyle JJ,Doyle JL(1990)Isolation of plant DNA from fresh tissue.Focus 12:13–15

Fang ZW,Xu XY,Gao JF,Wang PK,Liu ZX,Feng BL(2015)Characterization of FeDREB1 promoter involved in cold-and drought-inducible expression from common buckwheat(Fagopyrum esculentum).Genet Mol Res 14:7990–8000

Fei J,Wang YS,Jiang ZY,Cheng H,Zhang JD(2015)Identi fi cation of cold tolerance genes from leaves of mangrove plant Kandelia obovata by suppression subtractive hybridization.Ecotoxicology 24:1686–1696

Guo XH,Jiang J,Lin SJ,Wang BC,Wang YC,Liu GF,Yang CP(2009)A ThCAP gene from Tamarix hispida confers cold tolerance in transgenic Populus(P.davidiana×P.bolleana).Biotechnol Lett 31:1079–1087

Jefferson RA (1989)The GUS reporter gene system.Nature 342:837–838

Kovalchuk N,Jia W,Eini O,Morran S,Pyvovarenko T,Fletcher S,Bazanova N,Harris J,Beck-Oldach K,Shavrukov Y,Langridge P,Lopato S(2013)Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters.Plant Biotechnol J 11:659–670

Li H,Wang Y,Jiang J,Liu G,Gao C,Yang C(2009)Identi fi cation of genes responsive to salt stress on Tamarix hispida roots.Gene 433:65–71

Mayer BF,Ali-Benali MA,Demone J,Bertrand A,Charron JB(2015)Cold acclimation induces distinctive changes in the chromatin state and transcript levels of COR genes in Cannabis sativa varieties with contrasting cold acclimation capacities.Physiol Plant 155:281–295

Nataliya KC,Wei J,Omid E,Sarah M,Tatiana P,Stephen F,Natalia B,John H,Kontanze BO,Yuri S,Peter L,Sergiy L(2013)Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters.Plant Biotechnol J 11:659–670

Shinozaki K,Yamaguchi-Shinozaki K(1997)Gene expression and signal transduction in water-stress response.Plant Physiol 115:327–334

Trivedi DK,Gill SS,Tuteja N(2016)Abscisic acid(ABA):biosynthesis,regulation,and role in abiotic stress tolerance.In:Tuteja N,Gill SS(eds)Abiotic stress response in plants,Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim.doi:10.1002/9783527694570.ch15

Yamaguchi-Shinozaki K,Shinozaki K(2005)Organization of cisacting regulatory elements in osmotic-and cold-stress-responsive promoters.Trends Plant Sci 10:88–94

Yu X,Liu Y,Wang S,Tao Y,Wang Z,Shu Y,Ma H(2016)CarNAC4,a NAC-type chickpea transcription factor conferring enhanced drought and salt stress tolerances in Arabidopsis.Plant Cell Rep 35:613–627

Zhang JY,Huang SN,Wang G,Xuan JP,Guo ZR(2016)Overexpression of Actinidia deliciosa pyruvate decarboxylase1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana.Plant Physiol Biochem 106:244–252

Zong JM,Li XW,Zhou YH,Wang FW,Wang N,Dong YY,Yuan YX,Chen H,Liu XM,Yao N,Li HY(2016)The AaDREB1 transcription factor from the cold-tolerant plant adonis amurensisenhances abiotic stress tolerance in transgenic plant.Int J Mol Sci.doi:10.3390/ijms17040611