富含多糖的转基因石斛基因组DNA提取方法_英文_

分子植物育种，2009年，第7卷，第1期，第209－214页Molecular Plant Breeding,2009,Vol.7,No.1,209－214新思路、新技术、新方法Novel Thinking &TechnologyStudy on DNA Isolation from Polysaccharides-rich Transgenic DendrobiumZhang MiaobinPan Lijing *Fan GanqunChen JiminCheng Ping *Zhuhai Agricultural Research Centre,Zhuhai,519070*Correspongding authors,panlijing@,nkpcheng@Abstract Isolation of high-quality genomic DNA of transgenic Dendrobium plantlets for rapid screen or genetic analysis by PCR amplification and Southern blot is difficult,for the transgenic plantlets grow very slowly and contain high-level polysaccharides.In order to overcome such problems as low yield,degradation,poor PCR amplification and poor restriction digestion,three methods developed from hexadecyltrimethylammonium bromide (CTAB)or sodium dodecyl sulfate (SDS)method respectively were optimized and compared in this paper.Of the three modified methods used,method Ⅱproduced pure and high-quantity genomic DNA from small amount samples of transgenic Dendrobium or other plantlets in vitro tissue culture.DNA isolated by method Ⅱwas proved amenable to PCR amplification,restriction digestion and Southern blot analysis of transgenic Dendrobium plantlets.Keywords Dendrobium ,DNA isolation,Polysaccharides,PCR,Southern blot富含多糖的转基因石斛基因组DNA 提取方法张妙彬潘丽晶*范干群陈继敏程萍*珠海市农业科学研究中心,珠海,519070*通讯作者,panlijing@,nkpcheng@,摘要在石斛兰转基因研究中，需通过PCR 和Southern 杂交等手段检测外源基因是否转入并整合到转化植株基因组。

由于转化石斛植株生长缓慢，且含有多糖，因此从转化植株中提取高质量的基因组DNA 以尽快对转基因苗进行分子检测存在较大困难。

本研究旨在通过改良现有的DNA 提取法(CTAB 法或SDS 法)，克服转化石斛植株基因组DNA 提取中碰到的产量低，纯度不高而导致难以进行PCR 或酶切等问题。

在本研究所用的3种改良法中，方法Ⅱ能从少量的转化石斛苗中提取出高产量和高纯度的基因组DNA 。

研究结果表明方法Ⅱ提取的基因组DNA 完全适用于转基因石斛的外源基因PCR 扩增，限制性酶切和Southern 杂交分析。

关键词石斛兰,DNA 提取,多糖,PCR,Southern 杂交Program fund:This study was supported by the Science and Technology Plan Project in Guangdong Province of China (2006B20130004)As important ornamental plants,Dendrobium species are commercially grown globally as cut flowers and pot plants.The demand for their cut flowers has in-creased rapidly over the years,which requires improving the traits of Dendrobium such as flower color,fragrance,florescence.Transgenic engineering of orchids offers an effective way to develop improved cultivars.In or-der to rapid screen of transformed plantlets or verify transgenic integration in their genome,PCR and South-ern blot analysis was usually used.PCR,restriction digestion and Southern hybridiza-tion require high-quality intact DNA free of contamina-tion.Isolation of good-quality genomic DNA is a critical step in these processes.However,Dendrobium usually contains high levels of polysaccharides and other sec-ondary metabolites,which causes DNA unusable for downstream work in molecular biology research (Pirttil 覿et al.,2001).The most effective way to remove polysac-charide inhibition is to dilute the DNA extracts,but ex-cessive dilution of a DNA solution makes it unusable for Southern analysis (Sharma et al.,2002),which re-quires good-quality intact DNA free of polysaccharides,分子植物育种Molecular Plant Breeding proteins,and other secondary metabolites because nu-cleic acids form tight complexes with polysaccharides, creating a gelatinous pellet that contains embedded DNA.In addition,most of Dendrobium species especially the transgenic plantlets grow very slowly whether in open fields or in vitro tissue culture.Up to the present, the time that reports described DNA isolation from transgenic Dendrobium orchids tissues was8~21 months after the transgenic tissues were transformed (Men et al.,2003;Yu et al.,2001),which leads to an extensive delay to provide sufficient tissues for analysis of plantlet genetic traits.In our study on Agrobacterium-mediated transformation of Dendrobium(Zhang et al., 2008),we also descovered that the single transgenic plantlet growing for7~9months was still too small to provide sufficient amounts for high-quality DNA isolat-ed by common protocol(data not shown).So,it is the very necessary to excogitate an efficient procedure of DNA isolation from small amount samples of trans-genic Dendrobium.Although a number of methods for DNA isolation from orchid or other plant species rich in polysaccha-rides and other sticky compounds have been developed (Chen et al.,2006;Peng et al.,2003;Zeng et al.,2002; Wang et al.,2006;Zhan and Zeng,2005),these meth-ods have failed repeatedly to isolate pure,high-quantity DNA from small amount samples of Dendrobium(data not shown).Few reports detailedly specialized in DNA isolation for rapid screen or genetic analysis of trans-genic Dendrobium.In order to develop an efficient procedure of DNA isolation from small amount samples for rapid screen or genetic analysis of polysaccharides-rich transgenic Dendrobium,three DNA extraction methods with modi-fication of several important factors were evaluated based on DNA purity,yield,PCR and Southern blot.Of the three methods,a hexadecyltrimethylammonium bro-mide(CTAB)method(method II)was the most efficient and also applied successfully for PCR-based amplifica-tion,restriction digestion and Southern blot analysis.1Materials and Methods1.1Plant materialSamples were leaves cut from independent lines of transgenic Dendrobium(D.Ekapol“PANDA No.1”) growing on selection medium containing hygromycin and were put into liquid nitrogen immediately.1.2DNA isolation and purificationThree DNA extraction methods namely method I, methodⅡand methodⅢrespectively were optimized from common CTAB method or SDS method.The dif-ference between the methods was specially noted in the corresponding step.(1)Grinding step:Harvest leaves of Dendrobium plantlets in vitro tissue culture;Grind100~150mg of the fresh leaf tissue in liquid nitrogen with a mortar and pestle.(2)Washing step(Only methodⅠneed this step, methodⅡand methodⅢskip to extraction step.): Transfer the ground powder to clean autoclaved2mL centrifuge tubes and add1.5mL of washing buffer (100mmol/L Tris-HCl(pH8.0),5mmol/L ethylene-diaminetetraacetic acid(EDTA pH8.0),0.35mol/L glucose,1%(w/v)polyvinylpyrrolidone(PVP),2% (v/v)-mercaptoethanol);Vortex the samples thoroughly and incubate on ice for30min;Centrifuge at7000×g for10min.Discard supernatants.(3)Extraction step:Add1mL of DNA Extraction buffer1(100mmol/L Tris-HCl(pH8.0),1.5mol/L NaCl, 50mmol/L EDTA(pH8.0),3%(w/v)CTAB)(methodⅠand methodⅡ);Or add1mL of DNA Extraction buffer2(50mol/L Tris-HCl(pH8.0),0.5mol/L NaCl, 50mmol/L EDTA(pH8.0)]and30μL of20%(w/v) SDS(methodⅢ);Incubate in a water bath at65℃for 40min and vortex3-4times during the incubation;Ex-tract with0.8volume(vol)of chloroform-isoamylalcohol (24:1).Gently shake the tubes by hand until the tissue, buffer,and chloroform are homogenized(method I and methodⅡ);Or add1/5vol of5mol/L potassium acetate (pH4.8)and incubate on ice for30min(method III); Spin in centrifuge at10000×g for10min at room tem-perature.(4)Purification step:Transfer supernatant to a new 2mL tube;Add1/10vol of10%(w/v)CTAB solution. Incubate in a water bath at65℃for another15min (Only MethodⅡneeds this step);Add1vol of chloro-form-isoamylalcohol(24:1),centrifuge,and transfer as210above.Repeat this step once more.(5)Precipitation step:To the supernatant,add1 vol of isopropanol and1/103mol/L sodium acetate (pH5.2).Mix by gentle inversion for10min;Centrifuge at10000×g for10min at room temperature and wash the pellet3times with1mL of70%ethanol each time; Air-dry the pellet for10min at room temperature. Re-suspend the pellet in100μL of TE buffer and add 2μL of10mg/mL DNase-free RNase A.Incubate at 37℃for30min.(6)Re-precipitation step:Add300μL of TE(pH8.0) buffer to the DNA solution and incubate at4℃for30min; Centrifuge at10000×g for10min.Transfer4/5super-natant to a new2mL tube;Add2vol of ethanol.Mix bygentle inversion for10min;Centrifuge at10000×g for 15min at room temperature and wash the pellet3times with1mL of70%ethanol each time;Re-suspend the pellet in40μL of ddH2O buffer;Check the quality and concentration of DNA with electrophoresis and spec-trometry.1.3Assessment of DNA purity and yieldDNA purity was measured by absorbance(A)at 260nm and280nm(background absorbance at320nm) using a Beckman Spectrophotometer(Model No.DU640) andbyagarosegelelectrophoresis.DNAconcentrationwas mainly estimated by agarose gel electrophoresis of sam-plesand comparison with known DNA concentrations.1.4Assessment of PCR am plification,restriction enzymes digestion and Southern blot analysisPCR was performed using specific primers for the hygromycin phosphotransferase(hpt)gene which was transferred into Dendrobium.The amplification was per-formed on a DNA Thermal Cycler(MJ research,US-A)in a total volume of50μL containing~100ng tem-plates DNA isolated by methodⅡ,5μL10×PCR Buffer containing MgCl2(Takara company),1μL200μmol/L dNTPs,5μL2.5μmol/L primers and1U Taq Poly-merase(TakaRa Company).Samples were pre-denatured at94℃for5min and then subjected to30cycles of amplification.Termocycling conditions were as fol-lows:denaturation at94℃for1min,annealing at60℃for1min and extension at72℃for1min.The final cycle was followed by an extra extension step at72℃for10min.For the htp gene,the primer sequences were (F)5'－GCT GGG GCG TCG GTT TCC ACT ATC CG－3'and(R)5'－CGC ATA ACA GCG CTC ATT GAC TGG AGC－3',and approximate fragment length was375bp.PCR products were separated in1.0% (w/v)agarose gels.The extracted DNA(two DNA samples isolated from methodⅡ)was subjected to Hin dⅢ(Takara Company)digestion.The reaction was conducted at 37℃overnight in50μLvolume containing30μL (10~15μg)templates DNA,2μL Hin dⅢ,5μL its10×buffer,13μL ddH2O.For Southern analysis,50μL restriction digests were separated in0.8%(w/v)agarose gel at2v/cm for about8hours and were transferred onto a positively charged nylon membrane(Roche Company).Hybridiza-tion was made by the DIG System User's Guide for Fil-ter Hybridization(Roche Company).DIG-Labeled DNA probes of a675bp Hin dⅢfragment containing the hpt sequence isolated from pCAMBIA1301plas-mid,using a random-primed DIG DNA labelling kit (Roche Company).Prehybridisation and hybridisation were performed according the manufacturer's sugges-tions.Washing conditions were twice for5min in room temperature with2×SSC,0.1%(w/v)SDS solution and twice for15min in42℃with0.5×SSC,0.1%(w/v) SDS solution.2Results2.1Purity and YieldThe purity of DNA isolated by the three modified methods was tested by electrophoresis and spectropho-tometry.The pellets were all colorless and dissoluble in TE or ddH2O.Spectrophotometric analysis of DNA samples(two repeats from each modified method)at A260/A280ratio ranged from1.7to2.0,indicating little contamination of proteins and macromolecules.Figure1 showed the DNA samples bands were sharp,no RNA contamination and not any sign of degraded DNA in all samples.The yield of DNA produced from the three modi-fied methods was different and that from methodⅡwasStudy on DNA Isolation from Polysaccharides-rich Transgenic Dendrobium富含多糖的转基因石斛基因组DNA提取方法211分子植物育种Molecular Plant Breeding the highest.Method II yielded approximately15μg DNAper150milligram of fresh weight of leaves(Figure1),e nough to approximately conduct100PCRs,or oneSouthern blot analysis.DNA yielded from methodⅢor methodⅠwas1/3or1/4of that from methodⅡ(Figure1),insufficient for one Southern blotanalysis.Figure1Electrophoresis of extracted genomic DNANote:M:8μL(50ng/μL)λ-Hin dⅢdigest DNA marker (Takara);1~2:DNA isolated by MethodⅠ;3~4:DNA isolated by MethodⅡ;5~6:DNA isolated by MethodⅢ;1μl DNA samples were loaded per lane;The samples were separated on a 0.8%(w/v)agarose gel2.2Assessment of PCR amplification,restriction enzymes digestion and Southern blot analysisThe quality of DNA isolated from two indepen-dent lines of transgenic Dendrobium by methodⅡwas further confirmed by molecular detection such as PCR, restriction digestion and Southern blot analysis.Figure 2A showed the amplification with hpt gene primers. The major band is of the expected size of375bp.The purity of the DNA was also confirmed by means of complete Hin dⅢdigestion after incubating the reaction tube at37℃overnight(Figure2B).It was indicated that the DNA was amenable for further downstream ap-plications.In Southern hybridization,one(Figure2C) or two(Figure2C)positive signal for hpt gene trans-ferred into Dendrobium was obtained.3DiscussionIn our early experiments,the yield of DNA isolated by some existing methods was low and the pellets were obviously contaminated by polysaccharides or other secondary metabolites.It was difficult to dissolve such DNA samples in TE and load them into the elec-trophoresis wells.Three methods modified in thispaper Figure2PCR,restriction digestion and Southern blot analysis of genomic DNA isolated from transgenic Dendrobium by MethodⅡNote:(A)PCR analysis;M:DL2000DNA marker(TakaRa);P: positive pCAMBIA1301control;1~2:Independent lines of trans-genic Dendrobium;The specific primers were used to get hpt gene transferred into Dendrobium and the fragment length was 375bp;(B)Hin dⅢdigestion analysis;M:λ-Hin dⅢdigest DNA marker(TakaRa);1~2:Two DNA samples from indepen-dent lines of transgenic Dendrobium digested with Hin dⅢ; About10μg Hin dⅢ-digested DNA was loaded per lane.The samples were separated on a0.8%(w/v)agarose gel at2v/cm for about8h;(C)Southern blot analysis;The50μL Hin dⅢdigests resolved on a0.8%agarose gel were transferred onto a positively charged nylon membrane(Roche)and then subjected to pre-hybridization and hybridization;DNA probe of a675bp hpt gene from pCAMBIA1301plasmid was labelled using a random-primed DIG DNA labelling kit(Roche)and the following proce-dures were done according to the manufacturer's instructions (Roche)were optimized with several factors which were consid-ered to be important for obtaining good-quality and high-quantity DNA.Firstly,the DNA pellets dissolved212in TE was kept in4℃followed by centrifuging to re-move polysaccharides or other contaminants.Secondly, CTAB concentration was increased to3%(w/v)in ex-traction buffer1of method I and method II.High con-centration of CTAB,together with a high salt concen-tration,facilitated removal of polysaccharides,which was useful in DNA isolation from polysaccharides-rich species(Zeng et al.,2002;Wang et al.,2006;Xu et al., 2004).Thirdly,treatment with a washing step before extraction in methodⅠ.This step was also successfully applied in the RNA isolation of fruit trees containing high levels of polysaccharides and polyphenols(Hu et al., 2002;Xu et al.,2004).Fourthly,10%(w/v)CTAB so-lution was added to thoroughly isolate DNA from polysaccharides contamination(Murray and Thompson, 1986)in methodⅡ.Finally,potassium acetate following SDS extraction step and low temperature in methodⅢwas used to remove polysaccharides,which was useful in RNA or DNA extraction(Bahloul and Burkard, 1993;Ren et al.,2006).For methodⅡthe treatments included higher CTAB concentration,the addition of10%CTAB to isolate DNA again contributed to less polysaccharides and high-quantity DNA isolation from small amount samples of transgenic Dendrobium.The addition of potassium acetate following SDS extraction used in methodⅢreduced the DNA yield,which was similar to the description by Ren et al.(2006).Treatment with a washing step before extraction was extensively useful for eliminating polysacchar ide from DNA of plant species(Xu et al.,2004;Sharma et al.,2002;Zeng et al., 2002),which was proved by method I used in this study.But such treatment resulted in a lower DNA yield.In addition,some reports indicated that the addi-tion of high concentration of NaCl to the DNA suspen-sion before isopropanol precipitation could greatly pre-vent polysaccharides from co-precipitating with DNA (Wang et al.,2006).But in our study,the addition of 2.5mol/L(final concentration)NaCl resulted in no or few DNA precipitation(data not shown),which was similar to an existing report(Zhan and Zeng,2005).Compared with other methods,our three modified methods all produced pure DNA.Of the methods used in this paper,the yield of DNA isolated by methodⅡwas the highest.In a word,methodⅡsolved the prob lems of DNA degradation,contamination,and low yield due to binding and/or coprecipitation with polysaccharides and other sticky compounds.When ap-plied to isolate DNA from small amount samples of other Dendrobium species in vitro tissue culture, method II also yielded good-quality DNA.It is indicat-ed that methodⅡis the most suitable for high-quality DNA isolation for rapid screen or genetic analysis of transgenic Dendrobium in vitro tissue culture.ReferencesBahloul M.,and Burkard C.,1993,An improved method for the isolation of total RNA from spruce tissues,Plant Mol.Biol.Rep.,11(3):212-215Chen H.Y.,Sun Z.D.,Mao Y.J.,Wang X.W.,and Ge H.,2006, Studies on extraction of genome DNA in Cymbidium go－eringii(Rchb.f.)Rchb.f,Fenzi Zhiwu Yuzhong(Mol.Plant Breeding),4(1):135-142(陈惠云,孙志栋,茅轶俊,王晓武,葛红,2006,春兰基因组DNA提取方法的研究,分子植物育种,4(1):135-142)Hu C.G.,Honda C.,Kita M.,Zhang Z.L.,Tsuda T.,and Moriguchi T.,2002,A simple protocol for RNA isolation from fruit trees containing high levels of polysaccharides and polyphenol compounds,Plant Mol.Biol.Rep.,20(1):69 Men S.,Ming X.,Wang Y.,Liu R.,Wei C.,and Li Y.,2003,Ge-netic transformation of two species of orchid by biolistic bombardment,Plant Cell Rep.,21(6):592-598Murray M.G.,and Thompson W.F.,1986,Rapid isolation of high molecular weight plant DNA,Nucl.Acids Res.,8:4321-4326 Peng R.,Song H.Y.,Li Q.S.,and Wang Y.,2003,Extraction and characterization of total DNA from Dendrobium,Zhong-guo Zhongyao Zazhi(China Journal of Chinese Material Medica),28(12):1129-1131(彭锐,宋洪元,李泉森,王宇, 2003,石斛总DNA的提取及鉴定,中国中药杂志,28(12): 1129-1131)Pirttil覿A.M.,Hirsikorpi M.,K覿m覿r覿inen T.,Jaakola L.,and Ho-htola A.,2001,DNA isolation methods for medicinal and aromatic plants,Plant Mol.Biol.Rep.,19:273Ren Y.,Yang G.S.,and Yin J.M.,2006,A simple method of Dendrobium DNA preparation,Hainan Shifan Xueyuan Xuebao(Journal of Hainan Normal University(Natural Sci-ence)),19(4):359-361(任羽,杨光穗,尹俊梅,2006,一种简便的石斛DNA提取方法,海南师范学院学报(自然科学版),19(4):359-361)Sharma A.D.,Gill P.K.,and Singh P.,2002,DNA isolation from dry and fresh samples of polysaccharide-rich plants,Plant Mol.Biol.Rep.,20:415Study on DNA Isolation from Polysaccharides-rich Transgenic Dendrobium富含多糖的转基因石斛基因组DNA提取方法213分子植物育种Molecular Plant Breeding Wang J.B.,Du Z.J.,Lei X.T.,and Xu B.Y.,2006,Extracting total DNA from guava(Psidium guajava L.)leaves by improved CTAB method,Shengwu Jishu Tongxun(Letters in Biotech-nology),17(5):757-759(王家保,杜中军,雷心涛,徐碧玉, 2006,改良CTAB法提取番石榴叶片总DNA,生物技术通讯,17(5):757-759)Xu Q.,Wen X.P.,and Deng X.X.,2004,A simple protocol for isolating genomic DNA from chestnut rose(Rosa roxburghii Tratt)for RFLP and PCR analyses,Plant Mol.Biol.Rep., 22:301Yu H.,Yang S.H.,and Goh C.J.,2001,Agrobacterium-mediated transformation of a Dendrobium orchid with the class1knox gene DOH1,Plant Cell Rep.,20:301-305Zeng J.,Zou Y.P.,Bai J.Y.,and Zheng H.S.,2002,Preparation of total DNA from“recalcitrant plant taxa”,Zhiwu Xuebao(Acta Botanica Sinica),44(6):694-697(曾杰,邹喻苹,白嘉雨,郑海水,2002,顽拗植物类群的总DNA制备,植物学报,44(6):694-697)Zhang M.B.,Liang Q.Z.,Xiao H.,Cen P.,Fan G.Q.,and Pan L.J., 2008,Study on Agrobacterium-mediated transformation of Dendrobium,Yuanyi Xuebao(Acta Horticulturae Sinica), 35:565-570(张妙彬,梁擎中,肖浩,岑鹏,范干群,潘丽晶, 2008,农杆菌介导石斛兰遗传转化的研究,园艺学报,35(4):565-570)Zhan Y.G.,and Zeng F.S.,2005,A method for DNA extraction from mature birch leaves rich in polysaccharide,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry Univer-sity),33(3):24-25(詹亚光,曾凡锁,2005,富含多糖的白桦成熟叶片DNA的提取方法,东北林业大学学报,33(3): 24-25)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!多萜醇的生物合成及其对拟南芥中未折叠蛋白应答和非生物胁迫抗性的影响多萜醇是一类长链非饱和的类异戊二烯聚合物，广泛存在于所有的真核细胞中。