Li Biao

Liang Yilong

Shao Hongbo

Tang Kun

Wang Bochu

CR Biologies

C. R. Biologies 331 (2008) 329–333http://france.elsevier.com/direct/CRASS3/Molecular biology and genetics / Biologie et génétique moléculairesA study on differentially expressed gene screening ofChrysanthemum plants under sound stressShao Hongbo a,b,c,∗,1, Li Biao d,e,∗,1, Wang Bochu d,e, Tang Kun d,e, Liang Yilong da State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation,Chinese Academy of Sciences, Northwest A&F University, Yangling 712100, Chinab Binzhou University, Binzhou 256603, Chinac Institute of Life Sciences, Qingdao University of Science & Technology, Qingdao 266042, Chinad College of Bioinformatics, Chongqing University of Posts & Telecommunications, Chongqing 400065, Chinae Key Laboratory for Biomechanics & Tissue Engineering under the State Ministry of Education, College of Bioengineering,Chongqing University, Chongqing 400044, ChinaReceived 13 January 2008; accepted after revision 21 February 2008Available online 21 March 2008Presented by Philippe MoratAbstractEnvironmental stress can induce differential expression of genes of flower plants. It had been found that sound stimulation hadan obvious effect on the growth and development of flower plants, but it is not reported on the differentially expressed genesand their expressing characteristics under sound stimulation. This is one of the few reports in terms of using the DDRT-PCRtechnique for screening the differentially expressed cDNA fragments responding to sound-wave stress on Chrysanthemum. Sixdifferentially expressed cDNA fragments were obtained. Molecular weight of fragments was from 200 to 600 bp, respectively.Among differential fragments acquired, three of them (SA3, SG7–1, and CA2) were found to be positive fragments by northerndot hybridization, whose molecular weight are 270, 580 and 370 bp, respectively. SA3 was differentially expressed and SG7–1was preferably expressed, while CA2 was restrained by the sound wave. These results indicated that expression of some genes wasturned on, meanwhile the stress restrained some genes from expression under the mode of sound-stress stimulation. To cite thisarticle: S. Hongbo et al., C. R. Biologies 331 (2008).© 2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.Keywords: DDRT-PCR (Differentially Display Reverse Transcription-Polymerase Chain Reaction); Northern dot hybridization; Sound stress;Physiological response; Chrysanthemum* Corresponding author.E-mail addresses: shaohongbochu@126.com (S. Hongbo),libiao@126.com (L. Biao).1 Li Biao and Shao Hongbo are the co-first authors of this paperbecause of equal contribution.1. IntroductionBecause of immovability, the plants are inevitably af-fected by various environmental factors in the processof evolution [1–18]. Plants are remarkably sensitive toenvironmental conditions and have evolved the abilityto undergo adaptive developmental and physiologicalchanges in response to environmental stimuli [19–26].1631-0691/$ – see front matter © 2008 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.doi:10.1016/j.crvi.2008.02.007330 S. Hongbo et al. / C. R. Biologies 331 (2008) 329–333The sound wave, as a form of alternative mechanicstress, has obvious effects on the growth and develop-ment of plants [1,2]. It was reported that some soundwave could accelerate the growth of plants and changethe transition temperature of cell walls that were tightlyrelated to the division of plant cells [3]. In the study ofthe relation of sound wave and deformability of plas-malemma [4], it was found that the deformability wasclose to the frequency of the sound, and with the in-crease of frequency, the deformability decreased. Fur-thermore, the study of the effects of sound stress onnucleic acid and soluble proteins shows that the contentof DNA did not change evidently, that of RNA and solu-ble proteins increasing with varying degrees. The genesin response to sound are started up under sound stim-ulation [5]. However, the started-up genes sensitive tosound stimulation are not clear [1,22–26].In the present work, we used DDRT-PCR to isolatethe differentially expressed gene fragments respondingto sound-wave stress on Chrysanthemum. The relationof plant growth and development and sound-wave stresswas also discussed, which would provide a solid basisfor investigating molecular machinery of flower plantadaptation to environmental stresses [20–25].2. Materials and methods2.1. Chrysanthemum callus cultureStems from Chrysanthemum seedlings were inocu-lated in conical flasks with 20 ml of MS solid mediumeach and cultured in an illumination incubator at 26 ◦C[6].2.2. Sound-stress stimulation and plant materialThe sound generator in our laboratory achieved al-ternating stress. Inoculated stems were stimulated bysound wave with certain intensity (100 dB) and fre-quency (1000 Hz) for nine days, respectively, and eachday for 60 min. The control group was placed in thesame environment as stressed groups. The strong soundfield apparatus refers to [6]. Fresh leaves of stressedgroups and control groups were frozen in liquid nitro-gen before the extraction of total RNA, respectively.2.3. Total RNA extractionTotal RNA was extracted from leaves, as describedby Burgermeister et al. [1]. 0.5 g of leaves from sev-eral plant materials were pooled for one isolation af-ter purification by phenol:chloroform:isoamyl alcohol(50:49:1, v/v) and ethanol precipitation. RNA was re-suspended in a total volume of 100 ul of double-distilledwater. Agarose gel electrophoresis was used to test theintegrity and a UV spectrophotometer was used to de-tect the purity of the RNA extracted.2.4. mRNA differential displaymRNA differential display was carried out as de-scribed in the handbook of GenHunter Corporation(1994). Anchored primers (HT11M: 5′-AAGCTTTTT-TTTTTTA/G/C-3′) were synthesized by Sangon. In a0.5-ml microcentrifugation tube were added 2 ug of to-tal RNA, 4.96 ul of 20 uM anchored oligo-dt primer; themixture was brought to 11 ul with DEPC-treated water.The tube was heated in a water bath at 70 ◦C for 5 min,and then cooled on ice. 4 ul of 5 M MuLV RT buffer,2 ul of 10 mM 4dNTP mix, 0.5 ul of 20 U RNase in-hibitor, and 1.5 ul of DEPC-treated water were added.The mixture was incubated at 37 ◦C for 5 min. 1 µlMMLV reverse transcriptase (200 U/µl) was added for60 min at 42 ◦C. The reaction was heated in a water bathat 70 ◦C for 10 min, and then cooled on ice. The prod-ucts of the reverse transcriptions were stored at 4 ◦C forimmediate use or at −20 ◦C for later use.PCR amplification of each reverse transcriptionproducts was carried out in combination with one ofeight arbitrary primers H-AP (HAP1, AGCTTGATT-GCC; HAP2, AAGCTTCGACTGT; HAP3, AAGCT-TTGGTCGA; HAP4, AAGCTTCTCAACG; HAP5,AAGCTTAGTAGGC; HAP6, AAGCTTCGACCAT;HAP7, AAGCTTAACGAGG; HAP8, AAGCTTTTAC-CGC) synthesized by Sangon. PCR was carried out asfollowed: 94 ◦C for 30 s; 40 ◦C for 2 min; 72 ◦C for 30 sfor 40 cycles, and finally 72 ◦C for 5 min.The amplified cDNA subpopulations of 30-terminalsof mRNAs as defined by this pair of primers weresize-fractionated by denaturing in polyacrylamide elec-trophoresis using Bio-Rad Mini-PROTEAN Electro-phoresis Cell. 3.5 ul of each PCR sample were incu-bated with 2 ul of loading dye (95% formamide, 10 mMEDTA, pH 8.0, 0.01% bromophenol blue) at 80 ◦C for2 min immediately before loading.2.5. Silver stainingSilver staining of the polyacrylamide gel was per-formed as described by the Bio-Rad Silver Stain Hand-book, with the modification that the gel was fixed using7.5% acetic acid (v/v) immediately after electrophore-sis; the developmental reaction was stored using 7.5%acetic acid (v/v).S. Hongbo et al. / C. R. Biologies 331 (2008) 329–333 331Table 1RNA quantity and quality on UV spectrophotometerTissues Absorbency ratios Total RNA(µg/g FW)A260/A230 A260/A280Sound stimulating samples* 3.98 1.99 201.3 (45)Control samples* 3.85 1.87 198.6 (43)* Results are expressed as the mean of five samples (standard devi-ation).2.6. Re-amplification and northern blot hybridizationThe gel band of differentially expressed cDNA frag-ments was cut down with a knife and added to anEnppendorf tube in which it was boiled for 15 min.The extract served as the template to re-amplify withthe same pair of primes as in PCR.2.0 ug of total RNA from the treated and the controlsamples were denatured at 70 ◦C for 5 min, and spot-ted onto a nylon membrane (Hybond-N+, Amersham,UK). The RNA on the membrane between two sheets offilter paper was fixed in an oven at 80 ◦C for 2 h. Probe-labelling and hybridization conditions were described inthe instruction of the northern and southern direct HRPlabelling and detection kit (Pierce, USA). The exposuretime was 30 min.3. ResultsWe made use of the total RNA extraction method de-scribed; 200 µg of total RNA were routinely obtainedfrom 1 g (FW) of the callus of Chrysanthemum (Ta-ble 1). Results of RNA isolation from the sound stimu-lating samples and the control samples were 201.3 µg/gand 198.6 µg/g as the mean of five samples, respec-tively. The A260/A280 ratio ranged between 1.84 andFig. 1. Total RNA on 1.2% agarose gel electrophoresis.2.01, indicating little or no protein contamination. TheRNA yield of sound-stimulating samples was more thanthat of control samples.After total RNA extraction, 3.5 ul of total RNAswere separated on 1.2% denaturing agarose gels andphotographed under UV light. RNA examined by elec-trophoresis on 1% agarose/TAE gels showed that thelightness of the 28s rRNA band was double-fold thanthat of the 18s rRNA band, indicating that little orno RNA degradation occurred during the extraction(Fig. 1). In Fig. 1, the RNA bands of the treated groupwere coloured brighter than those of the control group.The quality of the total RNA could assure the need forsubsequencing tests.Through separating these PCR products on dena-turing polyacrylamide gel electrophoresis, six differ-entially expressed cDNA fragments were obtained,which were SA3, CA2, SG2–1, SG2–2, SG7–1, SG7–2(Fig. 2). The fragments’ molecular weight was between200 and 600 bp, respectively. After being re-amplified(Fig. 3), the SG2–2 band had not outcome, others hada similar molecular weight on the agarose gel and onthe denaturing polyacrylamide gel. SG2–2 was fakeFig. 2. mRNA differential display visualized by silver staining. SA3, SA2, SG2, SG7 are the treated groups; CA3, CA2, CG2, CG7 are the controlgroups; A3, A2, G2, G7 are respectively the primer pairs of anchored primer and arbitrary primer (HT11A and HAP3, HT11A and HAP2, HT11Gand HAP2, HT11G and HAP7).332 S. Hongbo et al. / C. R. Biologies 331 (2008) 329–333Fig. 3. Agarose gel electrophoresis of re-amplification products.M, molecular size marker.Fig. 4. Northern dot hybridization analysis of cDNA fragments.positive and discarded. Northern dot hybridization wasperformed (Fig. 4).4. Discussions and conclusionsFor the successful isolation of intact RNA from planttissues, it is important to prevent the contaminating sub-stances from binding to nucleic acids. In our exper-iments, many difficulties were encountered from thestage of cell lyses to RNA separation in the super-natant and subsequent reactions. The major problemwas low yield, degradation, and poor RT-PCR ampli-fication. The success of the RNA isolation proceduremay be judged by the quantity, quality and integrityof the RNA recovered [1–7]. The fine quality of RNAisolated as described in this study was confirmed in sev-eral ways. First, the A260/A280 absorbance ratio was1.9–2.0 and the A260/A230 absorbance ratio was 3.1–4.0, indicating that RNA was relatively free of proteinand polysaccharide contamination. Second, 1.2% dena-turing agarose gel electrophoresis showed clear, discreteribosomal RNA with no apparent RNA degradation,suggesting that RNA was also relatively free of RNases.Third, the RNA quality was tested by DDRT-PCR. Re-sults showed that, as expected, about 200 and 600 bpDNA differential display fragments were detected in theDDRT-PCR products when RNA was used as a tem-plate. RNA was also used for northern analysis.In Fig. 1, the results demonstrated that the contentof RNA increased under sound stimulation. The sameresult was obtained by Wang [5]. As shown in Fig. 2,a total of six bands were displayed, ranging in sizefrom 200 to 600 bp. Five differential bands were foundfrom stressed group, when control group had one (Fig. 2indicated the differential bands clearly). To determinetrue differentially display bands among them, we car-ried out northern dot hybridization. Three of them werefound to be positive fragments, SA3, SG7–1 and CA2,whose molecular weights are 260, 590, and 360 bp, re-spectively. SA3 was differentially expressed, and SG7–1 was advantageously expressed, while CA2 was re-strained by the sound wave. The fact indicated that ex-pression of some genes was turned on; meanwhile, thestress restrained some genes from expression under thismode of sound stress. Wang [7] considered that soundstress have double effects on plant growth, namely, notonly can sound stimuli hinder the plant growth, but alsosound at a proper frequency and intensity can promotecell division. Our experimental results hold up the view.The three gene fragments acquired will be cloned, andsequenced, and then their functions will be further ana-lyzed in the next experimentation.In a word, precise elucidation of plant gene regu-latory network system under abiotic stresses is of im-portance to molecularly engineering plant resistance;it is the reason why many excellent scientists world-wide have been engaged in this frontier field, whichresulted in a long-step progress [8–26]. One of the great-est challenges is to screen for differentially expressedgenes under corresponding environmental stimuli andanalyze them, which is the pre-condition for elucidatingthe molecular mechanism of the phenomenon [9–21].Performance and change of physiological functions un-der environmental stresses are phenotypes of potentialgene expression underlying this mechanism for flowerplants [21–26]. We have established a powerful plat-form to further solve this hot topic. There are also manyissues remaining to be solved and needing to make ef-forts. The scope of tested plants needs to be extended;comprehensive study on a combination of environmen-tal stress factors in laboratories and in the field shouldbe given much attention; system development viewpointand computer simulation analysis should also be ap-plied.AcknowledgementsWe thank the National Natural Science Foundationof China (No. 30470431), the Natural Science Foun-dation of Chongqing (CSTC 2005 BB1094), and theDoctoral Fund of Qingdao University of Science andTechnology (0022221).S. Hongbo et al. / C. R. Biologies 331 (2008) 329–333 333References[1] J. Braam, M.L. Sistrunk, D.H. Polisensky, et al., Plant responsesto environmental stress: regulation and functions of the Ara-bidopsis TCH genes, Planta 203 (1997) S35–S41.[2] B.C. Wang, H.C. Zhao, C.R. Duan, et al., Effects of cell wallcalcium on the growth of Chrysanthemum callus under soundstimulation, Biointerfaces 25 (2002) 189–195.[3] K.L. 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A study on differentially expressed gene screening of <i>Chrysanthemum</i> plants under sound stress

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A study on differentially expressed gene screening of <i>Chrysanthemum</i> plants under sound stress

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