烟草DNA的提取与SRAP反应体系的建立_梁景霞 的英文翻译

DNA extraction and the establishment of SRAP reaction systemin tobaccoLiang Jingxia1Qi Jianmin1Wu Weiren1Zhou Dongxin2Chen Shunhui3Wang Tao3Tao Aifen11. Fujian Agriculture and Forestry University,Fuzhou 3500022. Longyan Branch of Institute of Fujian Tobacco Agricultural Science, Longyan 3640003. Inst itute of Fujian Tobacco Agricultural Science, Fuzhou 350003AbstractMethod of DNA extraction and factors influencing SRAP analysis were studied using ten tobacco cultivars. A reliable, effective and reproductive PCR reaction system for detecting SRAP was developed. Each 25μL PCR reaction mixture consisted of 40ng of genomic DNA, 200μmol of dNTPs, 2.0mmol of MgCl2, 30ng of primer and 1.5 unit of Taq polymerase. Samples were subjected to thermal profile for amplification in an oven thermocycler: 5 min of denaturing at 94 ℃ , five cycles of three steps:1min of denaturing at 94℃ , 1min of annealing at 33℃ and 1min of elongation at 72℃. In the following 30 cycles the annealing temprerature was increased to 53℃,with a final elongation step of 5min at 72℃. Different molecular marks used in tobacco were also analyzed. It was shown that SRAP technology is a useful molecular marker system for mapping and gene tagging in tobacco.Key words:Tobacco DNA extraction SRAP reaction systemTobacco is one of the model plant was first used in molecular biology and genetic engineering, but its breeding rate has lagged behind other crops, and its genetic mapping has not been reported. Presently, most planted tobacco in China are imported species[1]. The reason it that tobacco breeding has objectives inherent complexity and particularity, and mainly breeding tobacco plants also depends on a number of phenotypic selection and determination off physiological and biochemical indicators. Never applied scientific problems from the research-based genetic level, to make progress in tobacco breeding subject to be restricted. But in other crops, marker-assisted selection has reflected its strengths [2]. Currently, the mainly used molecular marker method to identified Tobacco genetic diversity and phylogenetic relationship is RAPD[3~5], in which analysis procedures simple, low cost, but not has much testing sites. Existing molecular markers showed that tobacco has a relatively narrow genetic base[3]. Therefore,molecular-assisted of breeding tobacco need to find a method to detect molecular markers of similar genetic or more difference loci of gene to accelerate the application of molecular-assisted breeding and popularization rate.Sequence-related amplified poly- morphism(SRAP[6]) is a novel PCR-based marker systems which was developed in Brassica crops in 2001 by Quiros and Dr. Li who in vegetable department, the University of California. This tag specific area of gene ORFs (Open reading frames) were amplified by the unique dual primer design , and the upstream primer is 17bp, exon region-specific amplification and Downstream primer length 18bp, for introns, promoter region-specific amplification. From intron and promoter in different individuals or species to the spacing have many vary region of different length so that make polymorphic. The marker characteristics is simple, efficient, high yield, high co-dominant, good repeatability and easy sequencing, cloning easy target fragment . Presently, it can be used in analysis of genetic diversity of crops[7], Genetic mapping[6] [8],Marks an important trait[6]and Cloning genes [9]. Because SRAP is just developed this year and its detailed reported about its reaction system is less. And primers used in different species have its different reaction conditions, so screening andoptimization of reaction conditions primer is necessary. In our study, we used several cultivars germplasm as our experimental materials, discussing the best program for DNA extracted tobacco and SRAP analysis. In order to provide scientific basis of analyzing of genetic diversity in tobacco and constructing the genetic linkage map.1 Materials and Methods1.1 MaterialsMaterials are 10 common Nicotiana (Nicotiana tabacum L.) cultivars, there are 4cured tobacco , 4 Sun-cured Tobacco, 2 burley tobacco (Table 1).1.2 Extracted the genomic DNAUsing the modified CTAB method and genomic DNA extraction step of the tobacco : Weighed 5g fresh young tobacco leaves which were preserved at 4℃and added 0.2g water-insoluble PVP, then grounded into powder in liquid nitrogen and quickly loaded into a 50mL centrifuge tube. First added 150 ~ 300μL B-mercaptoethanol into the tube , and then added 15mL the extracted DNA[ 2%CTAB, 100mmol Tris-HCl (pH810) , 114mol NaCl, 20mmol EDTA( pH8.0) ] which had preheated in 65℃. Shaking gently and heating it at 65℃ water for 30min, cooled to room temperature. Added the equal volume of chloroform / isoamyl alcohol (24: 1), moderate upside down for 30 to 50 times, and centrifuged in the room temperature and 8000r / min for 10min. Transferred the supernatant into another tube, repeated. Took the supernatant in a centrifuge tube and added equal volume of cold isoamyl alcohol, reversed mildly for several times then stand until the flocculent precipitate built. After washing with 70% ethanol twice, dried it in the Clean Benches. Dissolved the dried DNA into 300μLTE. Detected the quality and size of fragments of the extracted DNA and stored at - 20℃ for sparing.Table 1 test material name and numberNumber Variety name Type Number Variety name Type1 Jade Bi No.1 Cured Tobacco 6 Longyan Sun - cured Tobacco2 ChangBo Huang Cured Tobacco 7 Xianyou Sun - cured Tobacco3 K326 Cured Tobacco 8 Xiaohuaqing Sun - cured Tobacco4 HongHuaDaJinYuan Cured Tobacco 9 Dahuangyan Burley tobacco5 TieBaZi Sun - curedTobacco10 TN90 Burley tobacco1.3 DNA amplificationSRAP reaction system was referenced Li and Quiros et al [6] method, and has been improved. Reaction volume is 25 μL, Tris-HCl (pH8.0) 10mmol, KCl 50mmol, MgCl2210mmol, dNTP200μmol , both Upper and lower primers are 30ng , DNA template 40ng and DNA polymerase 1. 5U. In order to prevent the reaction process of liquid evaporation, finally add 30μL paraffin oil to cover the reaction mixture. Primers we used was from American Operon products, Taq polymerase and dNTP was from Shanghai ShengGong product, PCR reaction was played in PTC - 100 functional PCR. Response procedures as follows: Degenerated in 94℃for 5 min, 5 cycle before the reaction was operated in 94℃ for 1 min, 33 ℃ for 1 min and 72℃for 1 min. Then next 30 cycles the annealing temperature increased to 53℃, and finally 72℃ for 5min. Template DNA, Mg2 +, dNTP, primers and Taq enzyme dosage gradient must strictly follow the test design requirements(Table2). The other parameters also must be strictly to control the standards to ensure the accuracy of the test.1.4 ElectrophoresisPCR products were used Sequi-GenGT electrophoresis and a 6% polyacrylamide gel electrophoresis (containing 7mol/L urea) to separation, and electrophoresis buffer was 1×TBE. When electrophoresis, first pre-electrophoresis in 2000V for 30min. Period, The bubbles in the sample wells must be expel to clean, denatured PCR products in the PCR instrument in 94℃for 2 min before spotting. 1.5h with 2000V voltage electrophoresis and stop until xylene green move to 2/3 of the sheet. Used 10% acetic acid to fixed, washed, silver staining, developing, stop shadow the gel , and the specific steps was referenced the method of Wu GuanYun et al [11] .Table 2 Tobacco SRAP-PCR reactions for each factor gradientDNA Templateng Taq enzymeUPrimerngdNTPμmolMg2 +mmol2 0.5 0 100 0 10 0.5 10 100 0.5 20 1.0 20 200 1.0 40 1.0 30 200 2.0 100 2.0 40 300 4.0 200 2.0 50 300 8.02 Results and analysis2.1 Detection of DNA extraction and amplified productsQuality of the DNA extracted is a key to determinant SRAP-PCR success.Tests showed that the material used that its growing season is very important. DNA of Tobacco in leaf stage extracted by SDS method can accord the test requirements, but not accorded in the late. It was due to that metabolites, proteins, carbohydrates, pigments, and nicotine content of tobacco polyphenols higher in tobacco secondary [12], to bring the hard work in the the work of DNA Isolation and purification. Using SDS extraction method to extract the DNA of tobacco will make the DNA serious browning, or couldn't extract. This phenomenon was particularly prominent in the tobacco, and it may be related to most of the wild plant contains more contaminating proteins and phenols. So it should be completed extract DNA in early seedling leaves of tobacco for spare. This test uses a modified CTAB method to effectively eliminates polysaccharide. Increases the time of add chloroform/isoamyl alcohol to extract in the extraction process to completely remove the proteins, pigments and other impurities. Adding non-water soluble PVP in grinding and adding B-mercaptoethanol when temporary use and other measures to effectively prevent browning. CTAB method has many steps and easy to cause DNA damage after improper operation, so it is necessary to ensure adequate mixing during the operation of the test , but also to prevent severe shock.In PCR product detection , for simplicity, our studies try to use a 2% agarose gel to detect. Compared to a 6% polyacrylamide gel , part of the strip is not detected so that type of the strips are different. Because the PCR products were mostly between 100 ~ 700bp, this study suggests that it is better reflect the real situation PCR amplification by high-resolution polyacrylamide gel electrophoresis. 2.2 Effect of DNA concentration on the PCR amplifiedContent of the template DNA is a factor restricting to the yield and specificity of amplified. Generally speaking, there are a wide range of suitable concentrations , but it will no amplification when too little , and Specific band will hard to appear when too difficult. The trial is a 2~200ng gradient, and the reaction results are shown in section 11A. Dosage of 20~200ng template could amplify the full band, while 2ng amplified bands very weak, while 200ng unspecific. Because the template primer of collision is less while fewer templates , and the chance of collision between the template and the template ismore while the template concentration is too high , and the chance of collision between the primer and the relative will reduce . The results showed that the best effect is40ng.2.3 TaqDNA polymerase dosage on PCR amplificationThe amount of Taq DNA polymerase in PCR is restricting by enzyme activity, enzyme heat resistance and other factors. Use high concentration of Taq enzyme not only wasteful, but also leads to the accumulation of non-specific amplification products. The low concentration of Taq enzyme will decrease the efficiency of the new strand synthesis and reduced amplification product. The gradient, 0.5U 1U, 2.0U are fully illustrate this point, the reaction results shown in Figure 11B. When 0.5U Taq polymerase, the amplified bands less and unstable , When the amount of 1U Taq polymerase can amplify a certain number of bands, but some of the bands are obscure ; When Taq enzyme dosage is 2.0U , the non-specific bands generated, resulting in bands get together and confusion. Therefore, the amount of Taq polymerase1.5U is appropriately.2.4 Primer concentration on PCR amplificationThis test provided a gradient from 0 to 50ng to augment, the reaction results shown in Figure 11C. When the amount of primer is 10ng, the amplification of polymorphic bands vague and the polymorphic bands reduce (second band did not get amplified). And 20ng, 30ng have been very good amplification. When 40ng, 50ng, some bands are blur. Thus, when the low concentration of the primer, the efficiency of the primer bound to the template will be low , and resulting polymorphic less . As the concentration of primers increase , the polymorphism increased , but the too high concentration of primers lead to the non-specific amplification and primer dimer appear . And impact on the yield of target sequence . Therefore the optimum content of each primer 30ng is the best.2.5 dNTP on PCR amplificationWhen dNTP concentration is too high , it will cause the error incorporation of the polymerase , and the concentration is too low, will affect the efficiency of the synthesis, or due to dNTP premature consumed so that the product will single stranded . There are three gradient provided 100,200,300μmol amplification in this study , the reaction results shown in Figure 11D. The results showed that the amplified products were 100 ~ 300μmol can have product, and 200μmol is the best.2.6 Mg2 + concentration on PCR amplificationMg2+ concentration affects the activity of enzyme, but also affects primer annealing, solution templates and intermediate products from specific temperature, the product, primer dimer formation[13]. Therefore , it is necessary to optimize the Mg2+concentration in the reaction . In this experiment, we used Mg2+ concentration from 0 to 8.0mmol for amplification, the reaction results shown in Figure 11E. No PCR product without Mg2+, so Mg2+is essential factor to the DNA polymerase activity. When the Mg2+ concentration is too high, some obscure bands appeared. At 0.5~4mmol, amplification product appeared, and it can appear the most clear bands at 2.0mmol.Figure 1 Different SRAP primers gradient PCR on tobacco in the Jade Bi No.1 amplification plot Figure1.A: primer combinations m7e6 template DNA concentration gradient (Table 2 is set low - high) of amplificationFigure1.B: primer combinations m6e3 in TaqDNA polymerase concentration gradient (Table 2 is set low - high) of amplificationFigure 1.C: primer combinations m7e8 primer concentration gradient (Table 2 is set low - high) of amplification Figure 1.D: primer combinations m6e3 in dNTP concentration gradient (Table 2 is set low - high) of amplification Figure 1.E: primer combinations m7e6 in Mg2 concentration gradient (Table 2 is set low - high) of amplification2.7 Annealing temperature on PCR amplificationAnnealing temperature determines the specificity of PCR, the low temperature annealing increases non-specific amplification, the high-temperature annealing to improve the specificity of amplification [13]. When the annealing temperature in strict accordance with Quiros and Li etc. [6] for the reaction to expand tobacco DNA , the yield is always low , and the band is not clear. This study is based on Parmaksiz [14]research and Tm=4(G+C)+2(A+T) on the annealing temperature to set a gradient test. The first annealing temperature was set at 38℃,37℃, 36 ℃, 35℃, 34℃, 33 ℃, 31℃.and the second annealing temperature was set at 52℃ 51℃, 50℃ , 49℃, 48℃, 47℃. Finally lower the first annealing temperature to 33℃and raise the second annealing to 53℃，but obtaining a higher yield obtained and not affecting the specific product, and the stability of the reaction strengthened. Therefore , it is necessary to adjust the appropriate annealing temperature depending on the species.In summary, the improved CTAB method can complete DNA SRAP-PCR analysis successfully . Optimization of the reaction system for tobacco SRAP: in 25μL reaction system, Tris-HCl(pH8.0)10mmol, KCl 50mmol, MgCl 2.0mmol, dNTP200μmol, the upper and lower primers 30ng, DNA templates 40ng, DNA polymerase 1.5U. The reaction procedure: pre-denaturation at 94℃5min, before the reaction run in 5 cycles 94℃1min, 33℃1min, 72℃1min , subsequent 30 cycles the annealing temperature increased to 53℃, 72℃ extending for 5min. Different primers in 10 tobacco cultivars of amplification shown in Figure 2, showing good stability and polymorphism.Figure 2 A, B, C, respectively SRAP primer m2e9, m2e10, m2e11 table 10 tobacco cultivars in the amplification3 Discussion and Prospect3.1 Tobacco Germplasm basic research and molecular breedingTobacco as a special-purpose crops, improve the quality and aroma of resistance is the core task of the current tobacco breeding. In order to a breakthrough, we must be strengthened broaden the use of germplasm resources, mining valuable germplasm materials. At present, many research institutions have adopted physical and chemical mutagenesis or aerospace-specific mutagenesis to create new means of germplasm. The research units showed that tobacco has a beneficial wild species in disease resistance, insect resistance and quality aspects genes are available, and there is a wealth of genetic polymorphism. paying attention to the study of the use of wild species and genetic improvement of tobacco wild relatives , but also application of biotechnology methods will wild genes into crops, improvement of existing varieties, in order to improve the resistance of the varieties and quality. So the application of modern biotechnology combined with traditional routine technology, but for germplasm innovation and molecular assisted breeding to lay the good foundation.3.2 The application of RAPD molecular marker technology in tobacco breedingExisting RAPD technology is mainly used in genomic DNA genetic polymorphism on tobacco, exogenous gene detection and tracking, disease-resistant properties tag, genetic differences between varieties and genetic variation of tobacco was more reported [3 ~ 5], RAPD technology has the characteristics of easy operation, fast, it have higher application value in genetic polymorphism and seed purity testing. But RAPD is susceptible to the influence of experimental conditions, easy to produce false-positive belt, repeatability is poor. Sometimes the results are not comparable between laboratories, thus limits its broad application.3.3 ISSR molecular marker technology in tobaccoThe writer wang tao and others is the first time to use the ISSR molecular marker to detection and clustering analysis three tobacco cultivated wild, the results showed that ISSR molecular markers can beclear to the flue-cured tobacco wild and cultivated species, drying, mahogany, burley tobacco, tobacco smoke, then according to its genetic differences in different types. Wild genetic polymorphism is very rich, and the genetic polymorphism in the cultivation of relatively poor, only different kinds of tobacco showed a certain degree of genetic diversity, reflects the tobacco varieties narrow genetic basis. Therefore, to strengthen resources and wild germplasm innovation and development of tobacco varieties in favor of the use of genetic research, is the urgent task in tobacco genetic breeding and biotechnology research.3.4 The application of the SRAP markers in tobacco3.4.1 Stability and polymorphism of a molecular marker Compared with RAPD technology , new type of SRAP markers using the higher annealing temperature and longer primer, not only guarantee the stability of amplification, and product sequencing easily, which can provide more genetic information. As a result, the defect of a can make up for RAPD and ISSR, makes possible the stable positioning of excellent genes, which can make the molecular marker assisted breeding true implementation in tobacco.Because of a special primer design, SRAP can detect the translatability of genes box area, so that thus reflects the polymorphisms of genes between species, the testing results of the reflected more species genetic diversity and genetic relationship. Budak etc. [16] using ISSR and SSR, RAPD and SRAP mark 30 primer to detect genetic polymorphisms of 15 primers buffalo grass (Buffalograss) at the same time . And the results of the four molecular marker detection comparison found that SRAP is the molecular marker only can distinguish the 15 kinds of genotypes , draw of a more suitable for the detection of close relatives, the differences between varieties. Therefore, this method is more suitable for the establishment of tobacco cultivation of fingerprint, which can be more effective in tobacco seed patent protection. Ferriol studies [7]about melon showed that SRAP can better reflect the history of variation and evolution of the morphology of melon than AFLP . In addition , using SRAP technique to analysis the clustering of 19 copies Cucurbita (C. maxima) germplasm showed the material can be objectively grouped [15]according to the type (food, fodder, ornamental). Thus we could put the tobacco accurately dividing in group by using type (flue-cured tobacco, mahogany, burley tobacco, air) on genetic, making tobacco germplasm resources research more scientific and purpose.3.4.2 Construct genetic linkage map of tobaccoSince SRAP polymorphic bands generated more specific, the domestic cotton has been successfully applied to construct a genetic map [8]. Li and Quiros used kale×cauliflower (cauliflower×kale) formed to constructed high-density RIL population genetic map[6]. Li, who constructed the B. oleracea by transcription patterns SRAP[17]. Because of SRAP has advantage in the aspect of building map , so choice of tobacco hybrid combinations scientifically, establish tobacco parents, F1, F2 and F3 genetic group, and genetic linkage map of tobacco can be expected to build in the near future .Reference1 王彦亭. 我国烟草育种工作发展思路[ J] . 中国烟草科学,2001( 4) : 1~ 512 方宣钧, 吴为人, 唐纪良. 作物DNA 标记辅助育种[ J] . 北京:科学出版社, 2001.3 肖炳光, 卢江平, 卢秀萍等. 烤烟品种的RAPD 分析[ J] . 中国烟草学报, 2000, 6( 2) :10~ 15.4 Bai D, Brandle J E, Reeleder R. Identification of RAPD markers linked with a gene for resistance to black root rot of tobacco [ J] .Theor Appl Genet, 1996, 91: 1184~ 11895 Yi Y H, Rufty R C,Wernsman E A. Mapping the rootknot nematode resistance gene ( Rk) in tobacco with RAPD markers[ J] . Plant disease,1998, 82: 1319~ 132216 Li G, Quiros C F. Sequence - related amplified polymorphism(SRAP) . A new marker system based on a simple PCR reaction: its application to mappingand gene tagging in Brassica[ J] .Theor Appl Genet, 2001, 103: 455~ 46117 Ferriol M, Pico B, Nuez F. Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers[ J] . Theor Appl Genet, 2003, 107: 271~ 28218 Lin Z, Zhang X, Nie Y, He D , Wu M. Construction of a geneticlinkage map for cotton based on SRAP[ J] . Chinese Science Bu-lletin, 2003, 48( 19) : 2063~ 206719 Li G, Quiros C F. Analysis expression and molecular characterization of BOGSL- ELONG. a major gene involved in the Aliphatic Glucosinolate pathway of Brassica species[ J]. Genetics, 2002, 162:1937~ 1943110 王关林. 植物基因工程原理与技术[M] . 北京: 科学出版社,1998: 307~ 31211 吴冠芸, 潘华珍, 吴羽. 生物化学与分子生物学实验常用数据手册[M] . 北京: 科学出版社, 1999112 钟文庆等. 植物化学基本知识[M] . 轻工业出版社, 1985:100~ 1131 13 林万明. PCR 技术操作和应用指南[M] . 北京: 人民军医出版社, 1993: 7~ 14114 Budak H, Shearman R C, Parmaksiz I, Gaussoin R E, Riordan TP, Dweikat I. Theor Appl Genet, 2004, 108: 328~ 334115 Ferriol M, Pico B, Nuez F. Genetic diversity of some accessions of Cucurbita maxima from Spain using RAPD and SBAP markers[ J].Genetic Resources and Crop Evolution, 2003, 50( 3) : 227~ 238116 Budak H, Shearman R C, Parmaksiz I, Dweikat I. Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs, SSRs, RAPDs, and SRAPs[J] . Theor Appl Genet, 2004, 109: 280~ 2881 17 Li G, Gao M, Yang B, Quiros C F. Gene for gene alignment betweenthe Brassica and Arabidopsis genomes by direct transcriptome mapping[ J] . Theor Appl Genet, 2003, 107: 168~ 1801。