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.     

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.     

A Method Suitable for Extracting Genomic DNA from Animal and Plant——Modified CTAB Method

[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.     

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.）.     

茶薪菇基因组DNA的RAPD反应体系优化(英文)

[Objective] The research aimed to screen out optimum RAPD reaction system on genomic DNA of Agrocybe chaxingu Huang.[Method] The single factor experiment was adopted to select the required Mg2+ concentration, template DNA concentration,primer concentration,dNTPs concentration,Taq enzyme concentration and anneal temperature initially.[Result] The optimum reaction system for RAPD amplification of Agrocybe chaxingu Huang was listed as follows:2.5 μl Buffer, 2 mmol/L Mg2+, 75 ng DNA, 0.5 μmol/L primer, 150 μmol/L dNTPs and 2.0 Taq enzyme.The reaction process was also listed as follows: denaturation for 5 min at 92 ℃,35 cycles(1 min at 92 ℃, 1 min for 35.5 ℃ and elongation for 2 min at 72 ℃),10 min at 72 ℃.[Conclusion] The research provided reference for conducting RAPD analysis of and studying genetic relationship and genetic diversity of Agrocybe chaxingu Huang.     

Regulation of Development and Endogenous Polyamine Level in Chinese Cabbage (B. Campestris ssp. Pekinensis)

The endogenous polyamine levels were tested at every developmental stage in different ecotypes of the Chinese cabbage. The results showed that polyamincs (PAs) were decreased in the process of flower bud initiation and fornfation, but the floral stem formation and elongation were accompied by PAs increase. The level of Spm was related to the bolting date of Chinese cabbage. During the inducing of flower bud initiation and elongation of floral stem, and the changes of Spm were reversed between early bolting date and late bolting cultivars, but they have the same requirment for Spd for the starting of flower bud initiation and the starting of bolting. The level of Spd changed little during entire developmental stage for early bolting cultivars, but a lot for late bolting cultivars. The time that the highest level of Put appeared is related to the bolting date of Chinese cabbage.     

Genetic Polymorphism of Kappa Casein （K-CN）in Iraqi Buffalo Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

This study was carried out the animal production department, genetic engineering lab, college of agriculture, （UoB）, Iraq. The aim of this study was to use the polymerase chain reaction restriction fragment length polymorphism （PCR-RFLP） as a fast, efficient and low cost method to detect the genetic variants of kappa-casein gene （k-CN） in Iraqi buffalo using three different primers specific for bovine k-CN to amplify the gene segment, followed by digestion using restriction enzyme （Hind III） for genotyping. DNA from 50 Iraqi buffaloes was extracted by phenol chloroform method. PCR was carried out in a final reaction volume of 25 μL and the reaction mixture was subjected to standard PCR protocol. The results of this work show that among the examined 50 Iraqi Buffalo were homozygous for the K-CN and genotyped as BB for all three primers but gave different bands. Thus PCR-RFLP using Hind III revealed all the samples to be monomorphic for this locus. The restriction digestion analysis of 397 bp PCR product of k-CN indicates the presence of two fragments of 154 bp and 225 bp for BB-genotype. A 437 bp fragment of the bovine genomic K-CN gene was amplified. One Hind III restriction site is found in position 346 of the amplified fragment of allele k-CN B, yielded 91 bp and 346 bp. Amplified products from Iraqi buffalo （530）, after being digested with Hind III, yielded two separate DNA fragments of different sizes i.e., 160 bp and 370 bp. For the first time completed research such specifications in Iraq, for the first time using molecular biology in genetic identification. Our objectives of this study have been to aid in understanding domestication, Buffalo origin and their history and evolution, to identify genetically unique breeds, to provide an objective basis for conservation decisions and to aid the formulation of breeding plans.     

Sequence related amplified polymorphism （SRAP） reaction system optimization and its application

In this study, three methods such as CTAB,SDS and Shanlichun methods were used to extract genomic DNA from the seedling of rape to find the best method. The principle, characters and application of SRAP were introduced. In order to obtain the optimal SRAP reaction system, the factors including concentrations of DNA, dNTP, etc. of reaction system were modified to better the system of rape. Th9 result showed that the optimum concentrations were 15ng DNA template, 0.2mM dNTP, 1.0μM primer and 2.0U Taq enzyme in this 25μL SRAP-PCR system.     

AFLP标记在蔷薇遗传检测中的应用

采用单酶切和双酶切AFLP技术分别对5种不同蔷薇品种基因组DNA的遗传多态性进行了检测,比较了两种方法的多态性比率.单酶切采用10条人工设计的与接头序列相识别的AFLP选择性引物,用Pst Ⅰ酶切,共获得108个AFLP标记,片段大小在100 2 000 bp,得到33个多态位点,多态性位点百分率为30.56%;双酶切采用25对双酶切AFLP引物,在50-600bp扩增出清晰条带658条,得到182个多态位点,占总扩增带的27.6%.对多态性片段进行统计,计算出不同品种间的相似系数和遗传距离.结果表明:硕苞蔷薇同其它4种蔷薇的遗传距离偏远,且其它4种蔷薇的遗传距离均较近,这与蔷薇品种来源和培育史相符.该研究表明两种AFLP标记均可用于蔷薇品种基因组DNA的遗传多态性的检测,准确评价尚待和其它品种对比研究后确定.

Abstract：

Single and double restriction digestion methods were used to detect the genetic polymorphism of the genomic DNA of five rose varieties, and the results of the two methods were compared. In single digestion, 10 human-designed AFLP selective primers different from the adaptor sequence were cut by Pst Ⅰ enzyme and 108 AFLP markers were obtained with a fragment size of 100-2 000 bp. Meanwhile, 33 polymorphic loci were gained and the proportion of polymorphic loci was 30.56%. In double digestion, 25 pairs of double enzyme primers were adopted, 658 clear bands were amplified in 50-600 bp and 182 polymorphic loci were gained, which were 27.6% of the total amplified bands. Based on the statistical analysis of polymorphic bands, the similarity coefficient and genetic distance among the different varieties were estimated.The results indicated that macartney rose (Rosa bracteaea) was genetically distant from the other four rose varieties, which were genetically close among themselves, a fact which is in agreement with the origins of the varieties and their cultivation history. Thus, the research results showed that the two types of AFLP markers can be used in detecting the genetic polymorphism of the genomic DNA of rose varieties, but more accurate evaluation will depend on comparison with more varieties.     

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.     

大白菜基因组DNA的提取及AFLP反应体系的建立

1材料与方法1．1材料供试大白菜种质材料32份,分别由北京市农林科学院蔬菜研究中心品种资源库及蔬菜研究中心大白菜育种课题组和中国农业科学院蔬菜花卉研究所国家种质资源中期库提供。材料编号、品种名称、来源地见表1。     

水杨酸对Cd胁迫下两种大白菜主要营养品质的影响

以对Cd抗性不同的2个大白菜品种超级夏秋王(抗Cd品种)和丰抗70(非抗Cd品种)为试验材料,采用盆栽试验方法,研究了在Cd胁迫的情况下,水杨酸(SA)对2种大白菜主要营养品质的影响。结果表明,SA提高了抗性品种叶片的Vc含量和可溶性蛋白质含量,降低了其可溶性糖含量;而对于非抗性品种,SA提高了其叶片中的可溶性糖含量和可溶性蛋白质含量,降低了Vc含量。     

不同类别白菜傅立叶变换红外光谱判别研究

[目的]利用傅立叶变换红外光谱（FTIR）技术对不同类别的白菜进行判别区分分析.[方法]将不同类别的白菜打浆处理,经冷冻真空干燥后结合FTIR技术对白菜溴化钾压片扫描,对中红外光谱数据进行DA聚类判别分析,并对模拟甲醛白菜样品做预测判别分析.[结果]试验得出,30个普通白菜、78个绿色白菜及30个模拟甲醛处理白菜建模样品能有效地聚类分布在不同区域;预测4个模拟甲醛处理的白菜样品都能准确判别分布在甲醛白菜范围内,准确率超过95％.[结论]利用FTIR技术结合光谱数据的DA判别分析,能较准确地对普通白菜、绿色白菜、模拟甲醛处理白菜进行区分分析,并对抽样验证甲醛白菜的类别进行准确预测判别.     

乌鲁木齐市再生水灌溉对小白菜内源微生物的影响

[目的]研究乌鲁木齐市再生水用于农业灌溉的可行性。[方法]以小白菜为研究对象,分别用清水对照、浓度50%的再生水和浓度100%的再生水浇灌,测定小白菜内部微生物菌群数。[结果]浓度50%的再生水增加了小白菜68.94%的湿重,但是随着外源微生物内化程度的加深,小白菜体内菌群总数和大肠菌群数都在0.05水平显著高于其他。[结论]再生水在提高小白菜质量的同时能增加内部细菌含量,不宜用于小白菜等可食用性作物的浇灌。若在污水处理时加强消毒措施,则可以解决该问题。     

大白菜延伸DNA纤维的制备和检测(英文)

[目的]制备和检测大白菜延伸DNA纤维。[方法]以大白菜幼叶为材料,采用刀切分离细胞核、SDS释放DNA、盖玻片滑动牵拉DNA的方法,首次获得大白菜DNA延伸纤维。[结果]以基因组DNA和25SrDNA为探针进行原位杂交检测,该DNA纤维长度大约为1.93Mb,25SrDNA在大白菜基因组中的拷贝数在258-687之间。该方法获得的DNA延伸纤维平行、清晰、适合FISH分析。[结论]该研究将会促进大白菜基因图谱的构建和组织分析。     

白菜和酸菜中亚硝酸盐含量的变化规律

[目的]研究白菜和酸菜中亚硝酸盐含量的变化规律,为人们健康饮食提供依据。[方法]分别测定生、熟白菜在不同时间、不同条件下亚硝酸盐含量的变化,同时测定酸菜不同腌制时间亚硝酸盐的含量。[结果]切开的大白菜中亚硝酸盐含量在8 h内变化不大,在放置1 d后含量快速增加,到第4天达到高峰。而熟白菜放置到24 h后就达到高峰,而且煮熟的白菜中亚硝酸盐含量明显高于生白菜。在常温、冷藏、冷冻3种储存方法中,冷冻的白菜亚硝酸盐含量变化最小。酸菜在腌制第5天时亚硝酸盐含量出现高峰,20 d后亚硝酸盐含量较低、变化较小。[结论]白菜最好现做现吃;腌制的酸菜最好在腌制20 d后食用。     

洁利康对有机磷农药残留的去除效果

[目的]明确洁利康对有机磷农药残留的去除效果。[方法]以洗涤灵、洁利康和清水为供试清洗剂,采用高效液相色谱法,研究洁利康对大白菜叶片表面的对硫磷、水胺硫磷残留的去除效果。[结果]洁利康对大白菜叶片水胺硫磷残留的去除率为82.3%,而洗涤灵和清水的去除率分别为78.7%和62.2%。洁利康对大白菜叶片对硫磷残留的去除率达90.8%,洗涤灵和清水的去除率分别为81.2%和76.9%。[结论]洁利康能有效去除大白菜叶片表面的有机磷农药残留,是很有发展前途的专用农药残留清洗剂。     

不同贮藏方式对白菜中亚硝酸盐和Vc含量的影响

[目的]寻找白菜食用前的最佳贮藏方式。[方法]采用20、4℃、非包装(自然状态)和密封包装4种方式贮藏白菜,研究白菜亚硝酸盐和Vc含量的变化。[结果]白菜在贮藏过程中亚硝酸盐的含量先升后降,但均高于新鲜白菜中的含量,4℃贮藏的白菜亚硝酸盐含量明显低于20℃贮藏的。密封与否对白菜的亚硝酸盐含量有一定影响,室温条件下非包装和冰箱中密封的方式均有利于抑制亚硝酸盐的生成。随着时间的延长,白菜在贮藏过程中Vc含量不断减少,低温和密封均有利于Vc的保存。[结论]低温有利于抑制亚硝酸盐的生成,减少Vc的损失,密封包装有利于减少Vc的损失,可选择冰箱密封的方式贮藏白菜,亚硝酸盐含量最低,Vc含量最高。     

不同施氮水平对小油菜产量及叶片硝酸盐含量的影响

[目的]研究施氮量与小油菜叶片硝酸盐含量的关系,控制小油菜叶片硝酸盐含量。[方法]利用盆栽试验,研究不同施氮水平对小油菜叶片中硝酸盐含量的影响,采用比色法测定小油菜叶片中的硝酸盐含量。[结果]随着氮肥施用量的增加,小油菜叶片中硝酸盐含量增加。[结论]为减少硝酸盐含量的积累,应适当控制氮肥用量。     

沼肥与化肥施用对大白菜品质的影响研究

[目的]为沼肥应用于无公害蔬菜生产提供理论依据。[方法]用沼肥和化肥种植白菜,对这些白菜进行品质对比分析,测定其可溶性糖、蛋白质、氨基酸的含量和亚硝酸的残留量。[结果]施用沼肥的白菜糖含量提高11.2%,蛋白质含量提高20.0%,氨基酸含量提高38.0%,而亚硝酸盐的残留量却降低43.0%。[结论]该研究表明沼肥是无公害蔬菜生产用肥的最佳选择。