一种适用于动物与植物总DNA提取的方法——改良CTAB法(英文)

[Objective] The study aimed to introduce a rapid and effective method that is suitable for extracting genomic DNA from animal and plant. [Method] The genomic DNAs were extracted from tender leaves of 24 peanut cultivars and from the liver,lung and kidney of white mouse through the specifically modified CTAB method. The DNAs were run on agarose gel,next detected by DNA/Protein analyzer. Finally PCR amplification was conducted to detect the quality of DNAs extracted using the modified CTAB method. [Result] The clear and orderly bands were observed in gel detection,and the values of OD260/OD280 for DNAs extracted via modified CTAB method were between 1.77-1.83. The DNAs performed well in PCR amplification. [Conclusion] The DNAs extracted by modified CTAB method could satisfy the requirement of PCR amplification.     

拮抗链霉菌AFLP分析技术体系的研究(英文)

[Objective] The aim of this study was to investigate the preparation method and amplification system of antagonistic streptomyces DNA templates based on AFLP assays, and also provide a basis for the application of AFLP technology in the analysis of streptomyces or even actinomyces. [Method] The DNAs were extracted by the modified CTAB method and amplified by the Pst Ⅰ/Mse Ⅰ AFLP kit and its reaction system. The amplified products were analyzed by the denatured polyacrylamide gel electrophoresis. [Result] The genomic DNAs of ten antagonistic strains of Streptomyces were extracted and tested. The result of 0.8% agarose gel electrophoresis showed that the major DNA bands were clear without degradation and RNA residue, with the fragment sizes ranging from 37.64 to 40.86 Kb. By ultraviolet spectrophotometry, the OD260/OD280 values varying from 1.625 to 1.833 were obtained. Furthermore, the agarose gel electrophoresis of DNA products digested by Pst Ⅰ/Mse Ⅰ presented the dispersed fluorescent long band, which indicated that the enzymatic hydrolysis was fully carried out. The amplified bands of DNA templates by the screened three pairs of primers were clear with rich polymorphism. [Conclusion] The preparation method and amplification system of DNA template established in this study can be used in the AFLP analysis of Streptomyces.     

青蒿组织中快速有效DNA提取方法的研究(英文)

[Objective] The objective of this study is to explore a rapid and efficient method of extracting genomic DNA from Artemisia abrotanum. [Method] Three methods of Cutting [Method],Liquid Nitrogen [Method] and Quartz Sand [Method] based on SDS method were employed to extract Artemisia abrotanum genomic DNA from tender leaf at seedling stage,tender spike and old leaf at heading stage. The obtained DNAs were detected by absorbance detection,agarose gel and PCR amplification. [Result] Cutting [Method] performed better than the other two methods compared in purity,extracting cycle and cost,accordingly more suitable for PCR amplification. The results also show that young spike is the best material for extracting genomic DNA from Artemisia Annua.     

An Improved Method of Extracting Artemisia abrotanum Genomic DNA

[Objective] The objective of this study is to explore a rapid and efficient method of extracting genomic DNA from Artemisia abrotanum. [Method] Three methods of Cutting [Method],Liquid Nitrogen [Method] and Quartz Sand [Method] based on SDS method were employed to extract Artemisia abrotanum genomic DNA from tender leaf at seedling stage,tender spike and old leaf at heading stage. The obtained DNAs were detected by absorbance detection,agarose gel and PCR amplification. [Result] Cutting [Method] performed better than the other two methods compared in purity,extracting cycle and cost,accordingly more suitable for PCR amplification. The results also show that young spike is the best material for extracting genomic DNA from Artemisia Annua.     

大白菜基因组DNA的提取及AFLP反应体系的建立(英文)

[Objective] The obtained clear AFLP fingerprint of Chinese cabbage provided basis for studies on the molecular markers of Chinese cabbage cultivars and the phylogenetic relationship among Chinese cabbage cultivars. [Method] With the test materials of leaves of Chinese cabbages, the high-quality total DNA from leaves of Chinese cabbages was extracted by the modified CTAB method. DNA restriction-ligase reaction, pre-amplification and selective amplification were optimized, and the AFLP silver-staining reaction system for Chinese cabbage was established. [Result] The quality of DNA template influenced restriction enzyme digestion and the subsequent ligase amplification reaction, while the modified CTAB extraction method could be used in AFLP analysis of Chinese cabbage to obtain a clear AFLP fingerprint. The optimum conditions for restriction enzyme digestion of genomic DNA from Chinese cabbage were as follows: 150 g DNA template, 12.5 μl reaction volume, 1.25 U Eco R Ⅰ, 1.25 U Mse Ⅰ and 5×Reaction Buffer with 4 h at 37 ℃. The ligation reaction with 2.5 h at 20 ℃ was the optimum condition. Six pairs of primers including E-AAC/M-CAG, E-AAG/M-CAC, E-ACA/M-CTG, E-ACT/M-CAC, E-ACT/M-CTT and E-ACT/M-CTC all had its own stable and clear patterns. [Conclusion] With abundant bands and high polymorphism, AFLP selective amplification is an efficient molecular marker for genomic polymorphism of Chinese cabbage.     

小球藻的分离及其DNA提取方法的研究(英文)

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.     

小球藻的分离及其DNA提取方法的研究(英文)

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.     

Isolation of Chlorella vulgaris and Its DNA Extraction Methods

[Objective] The aim of this study is to isolate Chlorella vulgaris(chlorella)and extract its genomic DNA.[Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples;the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method.[Result] The proper conditions for chlorella culture were as following:temperature 20-25 ℃,illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min;improved CTAB method was suitable for extracting genomic DNA from chlorella.[Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.     

枸杞nrDNA ITS测序鉴定的初步研究（英文）

[Objective] The study aimed to identify wolfberry(Lycium Linn.)germplasm resources at molecular level by analyzing the nrDNA ITS sequence.[Method] Genomic DNA from wolfberry leaves extracted by modified CTAB method were regarded as templates for PCR amplification by specific primer,clone and sequencing.[Result] The nrDNA ITS sequences were obtained and then differentiated among three tested materials.[Conclusion] PCR amplification and sequencing on nrDNA ITS is a feasible approach to identify different wolfberry germplasm resources.     

基于nrDNA ITS序列的18份宁夏枸杞资源的遗传多样性(英文)

[Objective] The study aimed to investigate the genetic polymorphism of eighteen Lycium barbarum resources via nrDNA ITS sequencing. [Method] The genomic DNAs from Lycium barbarum leaves were isolated by modified CTAB method for PCR amplification on the nrDNA ITS region using specifically synthesized primers; the amplified fragments were cloned and sequenced, then the sequencing results were clustered. [Result] nrDNA ITS sequences of the tested eighteen Lycium barbarum were firstly obtained in the present study. For all eighteen tested materials, the variation range of whole ITS region was 559-634 bp, with an average of 612 bp; alignment analyses showed that the whole length of internal transcribed spacer (ITS1+ITS2) was 480 bp, within which there are 194 variation sites (accounting for 40.4%) and 286 conserved sites (accounting for 59.6%). The cluster results showed that the eighteen tested materials could be grouped into three classes. [Conclusion] Analysis of nrDNA ITS sequence may avail to identify the Lycium barbarum germplasm resources.     

PCR-based Assay for the Detection of Xanthomonas campestris pv. Mangiferaeindicae Causing Bacterial Black Spot in Mango

[Objective] This study aimed to develop a PCR assay for detecting Xanthomonas campestris pv. mangiferaeindicae(Xcm) in culture and in planta. [Method] Primers(Xcm HF and Xcm HR) were designed based on the partial sequence of hrp B gene from xanthomonads to develop a PCR assay for Xcm. Furthermore, specificity and sensitivity of the primer pairs were analyzed in detection of genomic DNA and cell from Xcm. [Result] Amplication was positive only with genomic DNA from positive control ATCC11637 and 12 Xcm strains; no PCR products were amplified with genomic DNA from ten other xanthomonads and seven other bacterial species. The sensitivity of detection was 2.4 pg/μl genomic DNA, and 1.8 × 104CFU/ml cells. The primers also worked well for pathogen detection in direct PCR assays of Xcm colonies grown on liquid medium and in PCR assays of total DNA from leaf, branch and fruit lesions. [Conclusion] A PCR assay was successfully established for rapid detection of Xcm in culture and in planta.     

西川红景天nrDNA ITS序列初步研究(英文)

[Objective] The study aimed to analyze the ITS sequences of nrDNA from Rhodiola alisa and investigate the difference of evolution rate between nrDNA and trnS-trnG and rpl20-rps12 sequences of cpDNA(chloroplast DNA).[Method]Total DNA was extracted from silica-dried leaves of R.alsia by using modified CTAB method.With the extracted DNA sample as template,nrDNA ITS region was amplified,then purified and sequenced.In addition,the yielded ITS sequences were also compared with the known trnS-trnG and rpl20-rps12 sequences of cpDNA from R.alsia.[Result]The ITS sequence of nrDNA from R.alsia was 701 bp in length,of which 13 variable sites were found with a percentage of 1.85%.Of the 13 variable sites,8 were caused by point mutations,5 were the results of insertions or deletions.The(A+T)content and(G+C)content were 46.9% and 53.1%,respectively.The nucleotide diversity(π)was 0.004 27.[Conclusion]The ITS region of nrDNA from R.alsia was more conservative and evolved more slowly than the trnS-trnG and rpl20-rps12 sequences of its cpDNA.     

Rapid Amplification of Flanking Sequences of a Known DNA Region by Partial Restriction Digestion and Hot Start PCR

A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.     

Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.     

新疆红花主要栽培品种遗传多样性的RAPD分析(英文)

[Objective] Study on the genetic diversity in main cultivars of safflower distributing in Xinjiang Uighur Autonomous Region by means of RAPD makers.[Method] Genomic DNAs of 29 safflower accessions from Xinjiang Uighur Autonomous Region were extracted for PCR amplification using 20 RAPD primers.[Result] Totally 156 bands were amplified,among which 144 bands were polymorphic(accounting for 92.31%),indicating that safflower is endowed with plentiful genetic diversity.Based on the DNA fingerprint,the 29 safflower accessions were grouped into four populations,the classification results may be not related with ecological regionality.[Conclusion] RAPD technique is an available tool to analyze the genetic diversity of safflower germplasm at molecular level.     

Genetic Diversity in Main Cultivars of Safflower in Xinjiang Uighur Autonomous Region Based on RAPD Analysis

[Objective] Study on the genetic diversity in main cultivars of safflower distributing in Xinjiang Uighur Autonomous Region by means of RAPD makers.[Method] Genomic DNAs of 29 safflower accessions from Xinjiang Uighur Autonomous Region were extracted for PCR amplification using 20 RAPD primers.[Result] Totally 156 bands were amplified,among which 144 bands were polymorphic(accounting for 92.31%),indicating that safflower is endowed with plentiful genetic diversity.Based on the DNA fingerprint,the 29 safflower accessions were grouped into four populations,the classification results may be not related with ecological regionality.[Conclusion] RAPD technique is an available tool to analyze the genetic diversity of safflower germplasm at molecular level.     

新疆红花主要栽培品种遗传多样性的RAPD分析(英文)

[Objective] Study on the genetic diversity in main cultivars of safflower distributing in Xinjiang Uighur Autonomous Region by means of RAPD makers.[Method] Genomic DNAs of 29 safflower accessions from Xinjiang Uighur Autonomous Region were extracted for PCR amplification using 20 RAPD primers.[Result] Totally 156 bands were amplified,among which 144 bands were polymorphic(accounting for 92.31%),indicating that safflower is endowed with plentiful genetic diversity.Based on the DNA fingerprint,the 29 safflower accessions were grouped into four populations,the classification results may be not related with ecological regionality.[Conclusion] RAPD technique is an available tool to analyze the genetic diversity of safflower germplasm at molecular level.     

Preparation of high molecular weight （HMW） genomic DNA from tea plant （Camellia sinensis） for BAC library construction

A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.     

Combining Phytate/Ca~（2＋） Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat （Triticum aestivum L.） Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.