Guangning LU,Rui YANG,Rong JIANG,Shaohui WANG,Fukuan ZHAO

1.College of Plant Science and Technology,Beijing University of Agriculture,Beijing 102206,China;

2.College of Biological Science and Engineering,Beijing University of Agriculture,Beijing 102206,China

Analysis on Expression Patterns of NAC1 Gene in Tomato Induced by Low Temperature

Guangning LU1,Rui YANG1,Rong JIANG1,Shaohui WANG1,Fukuan ZHAO2*

1.College of Plant Science and Technology,Beijing University of Agriculture,Beijing 102206,China;

2.College of Biological Science and Engineering,Beijing University of Agriculture,Beijing 102206,China

[Objective]This study aimed to explore the relationship between NAC transcription factor and resistance to low temperature in tomato.[Method]Tomato seedlings were used as experimental materials to clone the N-terminal conserved domain of NAC1 gene with RT-PCR and investigate the expression patterns of NAC1 gene under 4℃with real-time quantitative PCR(RT-qPCR).[Result]NAC1 gene was expressed in roots,stems and leaves of tomato,but the expression level varied with the change of treatment duration.Moreover,the expression levels of NAC1 varied among different organs of tomato.The expression level of NAC1 in roots was much higher than that in stems and leaves.[Conclusion]This study laid a theoretical foundation for further investigation of the biological function of NAC1 gene in tomato.

Tomatoes;NAC;Low temperature

N AC transcription factors are unique to higher plants,which contain a highly conserved domain at the N-terminus that can bind to DNA and other proteins[1],and a highly variable transcriptionalactivation domain at the C-terminus[2].The conserved domain of NAC is divided into five sub-domains(A,B,C,D,E), among which sub-domains A,C and D are highly conserved,while sub-domains B and E exhibit no significant conservatism[3].Three kinds of transcription factors have been found in Arabidopsis,including NAM,CUC2 and ATAF1/2.In 1997,Aida et al.[4]found NAM,ATAF1/2 and CUC2 genes in Arabidopsis and names them NAC based on the acronyms.NAC transcription factors have also been divided into three subfamilies,including ATAF,OsNAC3 and NAM[5].In 1996, Souer et al.[6]cloned the first NAC gene and found that Petunia embryos harboring mutant nam(no apical meristem)failed to develop a shoot apical meristem and Petunia plantlets harboring nam failed to develop roots and leaves.Kim et al.[7]found that NTL8 from Arabidopsis belonged to membrane-associated transcription factors;under salt stress,the expression levelof NTL8 increased,which inhibited the expression offlowering signal protein gene,thereby delaying flowering time.Hu et al.[8]cloned salt and drought tolerance-related gene SNAC1 from rice and found that drought stress could induce specific expression of SNAC1 gene in stomatal guard cells of rice and promote stomatal closure without affecting the photosynthetic rate,thereby improving drought tolerance in plants;moreover, salt tolerance in rice plants overexpressing SNAC1 gene was significantly enhanced.In this study,low temperature resistance-related NAC1 gene was obtained from tomato to analyze its expression patterns under low temperature conditions,which laid a theoretical foundation for further investigation of the biological function of NAC1 gene.

Materials and Methods

Materials

Experimental tomato variety was provided by College of Plant Science and Technology,Beijing University of Agriculture.Plump seeds were sown in trays;after emergence of 2-3 true leaves,allthe young leaves were collected,mixed,wrapped with silver paper into 1 g/share,frozen with liquid nitrogen and preserved at-80℃in a refrigerator for cloning N-terminalconserved domain of NAC1 gene.

In mid-May 2014,pest-free tomato seeds with uniform size were sown in the culture medium and incubated in the Agricultural Biological Tissue Culture Laboratory of Beijing University of Agriculture.After emergence of 2-3 true leaves,tomato plantlets were incubated at 4℃in an incubator.Respectively at1,3,6,9,12 h post-treatment,samples were collected,frozen in liquid nitrogen and preserved at-80℃for analyzing the expression levelof NAC1 gene.

Reagents

TransScript First-Strand CDNA Synthesis,EasyPure PCR Purification Kit,pEASY-T1 cloning kit,competent cells,ampicillin,IPTG and X-Gal were purchased from Beijing TransGen Biotech Co.,Ltd.;nucleic acid dyes, peptone and yeast powder were purchased from Beijing chemical reagent Company;La Taq DNA polymerase was purchased from TaKaRa Biotechnology(Dalian)Co.,Ltd.

Total RNA extraction and cDNA synthesis

Total RNA was extracted from experimentalsamples using TrizoltotalRNA Extraction Kit.The integrity of RNA was detected by 1%agarose gel electrophoresis and the concentration was detected with NANODROP 2000 (Thermo Scientific).The amount of RNA for cDNA synthesis was calculated according to RNA concentration. The cDNA synthesis reaction volume was 20μl,containing 5μlof totalRNA, 1μlof Oligo(dT)18(0.5μg/μl),10μlof 2＊TS Reaction Mix,1μlof TransScript RT/RIEnzyme Mix,and 3μlof RNasefree water.The cDNA synthesis reaction was started with incubation at 42℃for 30 min,followed by heating at85℃for 5 min to inactivate Trans Script RT. Primer design

The cDNA sequence of tomato NAC1 was obtained on NCBI(http:// www.ncbi.nlm.nih.gov/)as a reference sequence for designing primers to clone the intermediate conserved domain oftomato NAC1 gene.Upstream primer N1:5’-CAYCCDACKGAYGAAGA-3’;downstream primer N2:5’-TTCTTSTTGTADATTCGACA-3’.Primers used in this study were synthesized by Sangon Biotech(Shanghai) Co.,Ltd.

RT-PCRamplification

cDNA obtained by reverse transcription was used as a template for RT-PCRusing specific primers N1 and N2.The total RT-PCR volume was 20μl,containing 1.5μlof cDNA,0.5μl of LA Taq(5 U/μl),2.5μl of 10×LA PCR BufferⅡ(Mg2+Plus),1.5μlof 10 mmol/L dNTP Mixture,1μl of each of primers,and 12μl of ddH2O.The RTPCRamplification was started with initialdenaturation at94℃for 4 min,followed by 35 cycles of denaturation at 94℃for 30 s,annealing at 53.5℃for 40 s,and extension at 72℃for 60 s; the amplification was completed by holding the reaction mixture at 72℃for 10 min to allow complete extension ofPCR products.

Cloning and sequencing of NAC1 gene

PCR products were detected by 1%agarose gel electrophoresis and purified using PCR purification kit.The purified PCR products were ligated with Transl-T1 vector at room temperature for 5 min.Subsequently,the constructed recombinant plasmid was transformed into Transl-T1 competent cells for blue-white screening and colony PCR in accordance with RTPCR method.Finally,PCR products were detected by 1%agarose gel electrophoresis;positive bacterial liquid was sentto Beijing Genomics Institute(BGI)forsequencing.

Real-time fluorescent quantitative PCR

With SYBRGreen fluorescentdye method,the expression levels of NAC1 gene under different treatments were detected using CFX96(Bio-Rad) real-time fluorescence quantitative PCR instrument.Upstream primer NAC1-S:ACGATCTCAGGCTTTACATCACC;downstream primer NAC1-X: AAACAACAGCCTAAGACTCGAT.Tomato reference gene primer ACT(a): GGAATGGGACAGAAGGAT;ACT(b): CAGTCAGGAGAACAGGGT.In accordance with the introductions of Real Master Mix(SYBR Green)Kit(Takara),cDNA obtained by reverse transcription was diluted(1,1∶10,1∶100, 1∶1 000,1∶100 000)for real-time fluorescence quantitative PCR,to drawn the relative standard curve.Three replications were set for standard sample cDNA and experimentalsample.The RT-qPCR volume was 20μl, containing 10μlof Real Master Mix,2 μl of cDNA,0.5μl of NAC1-S(10 μmol/L),0.5μlof NAC1-X(10μmol/L), and 7μlofddH2O.The RT-qPCR amplification was started with initial denaturation at 94℃for 2 min,followed by 40 cycles of denaturation at 94℃for 20 s,annealing at 53℃for 25 s, and extension at 72℃for 20 s;the fluorescence was collected atthe third step in each cycle.PCR reaction volume of reference gene was 20μl, containing 10μl of Real Master Mix, 2μl of cDNA,0.5μl of Smβ-S(10 μmol/L),0.5μlof Smβ-A(10μmol/L), and 7μlof ddH2O.The specific procedures ofreference gene were consistentwith PCR amplification ofNAC1.

Results and Analysis

Extraction of total RNA from tomato and synthesis of N-terminal conserved domain cDNAof NAC1 gene

Total RNA of tomato was detected by 1%agarose gel electrophoresis and results showed that three bright bands were observed(Fig.1):the top band was 28S rRNA,the middle band was 18S rRNA,and the bottom was 5S rRNA.Electrophoresis patterns indicated thatthe extracted RNA sample was intactwith no degradation.

Using first-strand cDNA of tomato as a template,PCR amplification was performed with specific primers N1 and N2.According to the results of 1%agarose gel electrophoresis (Fig.2),a distinct bright band was observed at approximately 500 bp, which exhibited a similar length to the target DNA fragment.

After recovery,ligation,transformation,blue-white screening and sequencing of the obtained band,PCR amplification results showed that aband was observed between 250-500 bp(Fig.3),which was basically identified as the target DNA fragment.

Analysis of the conserved domain sequence of tomatoNAC1 gene

Homology analysis of the sequencing results was performed by BLAST on NCBI.Results showed that the target DNA sequence shared above 95%homology with NAC from Arabidopsis and other plants.Multiple alignment of the target DNA sequence with NAC conserved amino acid sequences from other plants using DNAMANsoftware showed a high degree ofhomology(Fig.4).

Analysis of the expression patterns of NAC1 under low temperature treatments

Under 4℃cold stress conditions, NAC1 was expressed in roots,stems and leaves of tomato(Fig.5).To be specific,the expression levels of NAC1 in stems and leaves of tomato both reached the peak at 6 h posttreatment and then declined to a lower level;the expression level of NAC1 in tomato roots increased rapidly at 1 h post-treatment,declined slightly at 3 h post-treatment,rose dramatically at 6 h post-treatment,and reached the peak at 12 h post-treatment,indicating that low temperature treatments exhibited certain induction effects on the expression of NAC1 in various organs of tomato,such as roots,stems and leaves.

Experimental results showed that NAC1 gene could be expressed in roots,stems and leaves of tomato, which suggested that the protein encoded by NAC1 might be a transcription factor ubiquitous in various organs of tomato.The expression levels of NAC1 varied with the change of treatment duration,which indicated that NAC1 might be an expression-inducing transcription factor.Moreover, the expression levels of NAC1 varied among different organs.Specifically, the expression level of NAC1 in roots was much higher than that in stems and leaves,suggesting thatNAC1 was expressed primarily in roots;the expression level of NAC1 in stems was relatively stable,which inferred that transcription factor NAC1 might playdifferentroles in the growth and developmentprocess ofvarious organs.

Discussions

Responses of the growth and development of plants to adverse stress constitutes a complex regulatory network,in which the regulation of transcription factors plays an important role.Studies have shown that NAC transcription factors have played a criticalrole in plant defense[9].Sun et al.[10]analyzed the expression levels of rice OsNAC family genes under high salt, drought,low temperature stresses and found that among 45 salt stress-responsive OsNAC genes,the expression of 33 genes was upregulated and that of 12 genes was downregulated; among 44 low temperature stress-responsive OsNAC genes,the expression of 31 genes was upregulated and that of 13 genes was downregulated. In this study,tomato seedlings were treated under low temperature stress at 4℃.Experimental results showed that NAC1 was expressed in various organs(roots,stems,leaves),especially in roots,which indicates that NAC1 is an expression-inducing transcription factor ubiquitous in various organs of tomato,whose expression can be upregulated by low temperature stress.Furthermore,NAC1 may be involved in the regulation process of tolerance to low temperature in tomato.It is preliminarily predicted that overexpressing NAC1 may improve the tolerance oftomato to low temperature stress.The results laid a theoretical foundation for further breeding of cold-toleranttomato cultivars.

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Responsible editor:Xiaohui FAN

Responsible proofreader:Xiaoyan WU

低溫誘導(dǎo)的番茄NAC1基因表達(dá)特征分析

魯廣寧1，楊 瑞1，姜 榮1，王紹輝1，趙福寬2*
（1.北京農(nóng)學(xué)院植物科學(xué)技術(shù)學(xué)院，北京102206；2.北京農(nóng)學(xué)院生物科學(xué)㈦工程學(xué)院，北京102206）

[目的]該研究旨在探索番茄NAC轉(zhuǎn)錄因子㈦抗低溫的關(guān)系。[方法]以番茄幼苗為試材，采⒚RT-PCR方法克隆NAC1基因N端保守Ⅱ，并利⒚RT-qPCR技術(shù)檢測(cè)該基因在4℃低溫l處理下的表達(dá)情況。[結(jié)果]在番茄根、莖、葉中NAC1基因均能表達(dá)，表達(dá)量隨低溫處理時(shí)間的變化而發(fā)生相應(yīng)的變化。在番茄的不同器官中NAC1表達(dá)水平也有所不同，根中的表達(dá)水平整體高于莖和葉片中。[結(jié)論]該研究為進(jìn)一步研究番茄NAC1基因的生物學(xué)功能奠定理論基礎(chǔ)。

番茄；NAC；低溫

現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系北京市果類(lèi)蔬菜創(chuàng)新團(tuán)隊(duì)（GCTDZJ2014033055）；國(guó)家自然科學(xué)基金（31171952）；北京市長(zhǎng)城學(xué)者培養(yǎng)計(jì)劃（CIT&TCD20130323）。

魯廣寧（1991-），女，北京人，碩士研究生，研究方向：蔬菜學(xué)。*通訊作者，博士，教授，從事蔬菜育種及生物技術(shù)方面研究，E-mail：zhaofukuan@sina.com。

2014-11-13

Supported by Beijing Fruit Vegetable Innovation Team of Modern Agriculture Industry Technology System(GCTDZJ2014033055);National Natural Science Foundation of China(31171952);Great Wall Scholar Training Program of Beijing Municipality (CIT&TCD20130323).

*Corresponding author.E-mail:zhaofukuan@sina.com

Received:November 13,2014 Accepted:December 30,2014

修回日期2014-12-30