鬼针草属六种植物的同工酶电泳实验研究

目的:建立鬼针草6个品种过氧化物酶同工酶(POD)电泳指纹图谱,探讨鬼针草种内变异规律.方法:聚丙烯酰胺凝胶电泳分析.结果:鬼针草6个品种过氧化物酶同工酶PAGE指纹图谱差异显著.结论:聚丙烯酰胺凝胶同工酶电泳图谱可用于鉴别鬼针草6个种质.     

不同居群生姜呈香部位的气相色谱指纹图谱研究

采用气相色谱分析技术对代表性产地的生姜水蒸气蒸馏-乙醚萃取物进行了比较,确定了气相色谱分析条件,分析评价了生姜水蒸气蒸馏-乙醚萃取物化学成分指纹图谱,在本文采用的条件下,生姜气相色谱指纹图谱具有较好的特征性,可作为生姜蒸馏法挥发油的专属性指纹图谱。     

人肺腺癌细胞A—549和正常细胞HBE的蛋白质组差异分析

为了研究人肺腺癌细胞A 5 49和正常细胞HBE的蛋白质组差异 ,用固相pH梯度双向凝胶电泳分离人肺腺癌细胞系A 5 49和正常细胞HBE的总蛋白质 ,银染显色 ,PDQuest 2 DE软件分析 ,对部分差异蛋白质点进行基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)测定其胶内酶解后的肽质指纹图谱 ,用PeptIdent软件查询SWISS PROT数据库。结果获得了分辨率和重复性均较好的双向电泳银染图谱 ,图象分析探测到A 5 492 DE图谱的平均蛋白质点数为 (890± 38)个 ,HBE的平均蛋白质点数为 (75 7± 2 7)个 ,不同胶间蛋白质点的位置偏差在IEF方向为 (2 .85± 0 .48)mm ,在SDS PAGE方向为 (2 .6 9± 0 .37)mm。差异表达分析发现A 5 49和HBE图谱有5 35个蛋白质点相互匹配 ,其中A 5 49有 35 5个未被匹配 ,HBE中有 2 2 2个未被匹配 ;对A 5 49和HBE中的 18个差异蛋白质点分别进行肽质指纹分析 ,经数据库查询 ,初步鉴定为一些与物质代谢、细胞因子、信号转导有关的蛋白质。提示人肺腺癌细胞A 5 49和正常细胞HBE的蛋白质组具有差异 ,这种蛋白质组的差异分析有助于进一步研究肺腺癌的相关蛋白质及分子标记物     

不同产地麦冬’H—NMR指纹图谱研究

目的：建立不同产地中药麦冬1H—NMR指纹图谱,为麦冬及其提取物质量控制提供依据。方法：采用1H-NMR建立不同产地麦冬甲醇提取物的指纹图谱。结果：7个样品的主要化学成分糖、脂肪族、皂苷和高异黄酮的氢信号峰有高度的相似性,但不同麦冬品种、不同产地,1H—NMR指纹图谱有较大的差异,可以此区分。结论：1H-NMR指纹图谱不但可以鉴定不同麦冬品种,还可以区分不同产地的麦冬。     

白芷醇溶蛋白质提取工艺优化及其指纹图谱研究

目的：基于响应面设计优化白芷醇溶蛋白质提取工艺，并研究其指纹图谱特征。方法：首先考察液料比、提取时间、乙醇浓度、提取温度四个单因素对白芷醇溶蛋白质提取率的影响，再通过Box-Behnken方法对影响提取率的工艺因素进行响应面优化，最后对白芷及易混淆药材的醇溶蛋白质指纹图谱进行对比分析。结果：最佳提取工艺条件为：液料比13∶1(mL/g)，提取时间8.75 h，乙醇浓度42%，提取温度45℃，在此条件下提取率为0.294%。SDS-PAGE分析表明白芷醇溶蛋白质样品分子量范围介于15～95 kDa,91.4 kDa与65.6 kDa分子为高丰度蛋白质。此外，白芷与天花粉、粉葛、山药的醇溶蛋白质指纹图谱有显著差异。结论：利用响应面法优化了白芷醇溶蛋白质的提取工艺，并建立其专属性SDS-PAGE指纹图谱，为其分子鉴定及资源开发提供实验依据。     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

Bt Cry1Ac毒素筛选的粉纹夜蛾离体抗性细胞与敏感细胞蛋白质组的差异分析

采用双向电泳技术和肽质量指纹图谱技术分析了Bt Cry1Ac毒素筛选的粉纹夜蛾Trichoplusia ni抗性BTI-Tn-5B1-4细胞与同源敏感细胞的蛋白质组的差。用Melanie ViewerⅡ软件在抗性细胞的双向电泳图谱上检测到的平均蛋白质点数为707±25个（n=3）,在敏感细胞的双向电泳图谱上检测到的平均蛋白质点数为637±19个（n=3）,其中分辨率高、重复性良好的显著差异点有10余个。对其中的一个敏感细胞特有的显著差异斑点进行了肽质量指纹图谱分析,经数据库 查询表明该蛋白质与胞质外周蛋白具有同源性。     

僵蚕药材蛋白质分级指纹图谱建立及产地鉴定相关分子研究

建立云南与四川僵蚕药材的总蛋白质指纹图谱及分级指纹图谱,对比分析以发现其中的差异表达蛋白质分子并进行鉴定及生物信息学分析,探索其用于产地鉴定的可行性。提取两种僵蚕的酸溶、碱溶、水溶及醇溶性总蛋白质,对其进行丙酮分级沉淀,对所得样品进行SDS-PAGE指纹图谱对比分析,对所得差异条带进行LC-MS/MS分析,数据库检索后进行生物信息学分析。两种僵蚕的总蛋白质指纹图谱相似度很高,难以据此进行产地鉴定;两种僵蚕的分级指纹图谱则有较显著差异,有望用于产地鉴定;依据分级指纹图谱差异共鉴定出β-葡萄糖苷酶、抗胰凝乳蛋白酶、抗胰蛋白酶、含脂肪酶结构域蛋白等273种蛋白质。云南与四川僵蚕的蛋白质组成及其分级指纹图谱存在显著差异,有望用作产地鉴定的分子依据;醇溶蛋白质构建分级指纹图谱可以提供更加丰富的信息及更好的分辨率;分级指纹图谱技术具有简便易行、分辨率较高的优点,为其他中药材的产地鉴定研究提供了方法学思路。     

开唇兰属植物的种间蛋白质组差异

目的:通过差异蛋白峰的比较和指纹图谱的对照,分析开唇兰属五种金线莲的种间蛋白质组差异,初步探索了这种差异在品种鉴定中的应用并推断出品种之间的亲疏关系。方法:采用SELDI-TOF-MS方法研究了开唇兰属五种金线莲的种间蛋白质组差异。结果:通过SELDI-TOF-MS方法对开唇兰属五种金线莲种间蛋白质组谱图进行的分析结果表明,属内种间存在蛋白质组差异和特征蛋白质峰,据此建立了属内五种植物的蛋白质组指纹图谱,并依据蛋白质组的差异推测了属内五种金线莲的亲疏关系。结论:依据SELDI-TOF-MS鉴定结果,找到了开唇兰属五个物种间的蛋白差异,并基于差异蛋白数据分析了品种遗传变异性,结果表明地域和气候会对品种的变异产生一定的影响。     

基于HPLC指纹图谱和多成分定量评价不同品种百合鲜品质量

目的：建立3种不同品种百合鲜品HPLC指纹图谱，并同时测定其中5种成分。利用聚类（CLA）、主成分分析（PCA）等方法考察不同来源百合质量，为其质量控制评价提供参考。方法：HPLC梯度洗脱法建立了20批百合鲜品的指纹图谱，通过中药指纹图谱相似度评价软件进行相似度评价，采用聚类、主成分分析等进行分析。并同时测定5种成分的含量。结果：标定15个色谱共有峰，20批药材共有峰相似度为0.930～0.997；通过聚类分析分为2类；PCA筛出累积方差贡献率达86.70%的2个主成分；偏最小二乘法-判别分析（OPLS-DA）结果表明不同来源百合药材存在明显差异，对样品有显著影响的色谱峰分别为3号、6号（王百合苷C）和10号。三种分析方法所得结果基本一致。结论：所建立的HPLC指纹图谱和多指标成分定量方法可行，可为系统评价不同品种百合提供一些参考。     

沙梨8个栽培品种的DNA指纹鉴定

利用RAPD标记技术,对8个沙梨(Pyrus pyrifolia)主栽品种进行RAPD分析。结果显示,从33个10碱基随机引物中筛选出了2个多态性高的引物S2075和S1296,扩增位点分别为11个和10个,以此为基础建立了8个品种的DNA指纹图谱;根据这2个引物中任何一个的扩增图谱均可以将这8个品种区分。研究结果为沙梨品种的区别与鉴定提供了一种有效方法。     

Development of a novel and efficient strategy for practical identification of Pyrus spp (Rosaceae) cultivars using RAPD fingerprints

Accurate and reliable cultivar identification of crop species is essential to guarantee plant material identity for purposes of registration, cultivar protection and production. To facilitate identification of plant cultivars, we developed a novel strategy for efficient recording of DNA molecular fingerprints in genotyped plant individuals. These fingerprints can be used as efficient referential information for quick plant identification. We made a random amplified polymorphic DNA (RAPD) marker analysis of 68 pear cultivars. All pear genotypes could be distinguished by a combination of eight 11-mer primers. The efficiency of the method was further verified by correct identification of four cultivars randomly chosen from the initial 68. The advantages of this identification include use of fewer primers and ease of cultivar separation by the corresponding primers marked on the cultivar identification diagram. The cultivar identification diagram can efficiently serve for pear cultivar identification by readily providing the information needed to separate cultivars. To the best of our knowledge, this is the most efficient strategy for identification of plant varieties using DNA markers; it could be employed for the development of the pear industry and for the utilization of DNA markers to identify other plant species.     

Polymorphisms in grapevine DNA detected by the RAPD PCR technique

A sensitivie, reproducible technique is described for detecting polymorphisms between the nuclear DNA of grapevine isolates using the RAPD PCR technique. Unique fingerprints of a number of cultivars were readily distinguished by using either single primers or mixtures of two primers. The method will be used to provide a databank of fingerprints for the rapid identification of grapevine cultivars, and to develop phylogenetic relationships. It will also be extended and modified in an attempt to detect polymorphisms between DNAs of clonal selections of individual cultivars.     

Evaluation of genetic diversity and chemical profile of ginger cultivars in north-western Himalayas

Eighteen ginger cultivars from Northwest Himalayan region, showing significant differences in rhizome size, texture and pungency, were selected and characterized both by chemical and genetic analyses. The genetic analysis was undertaken utilizing molecular markers (ISSR and SSR) while chemical characterization was done through HPLC of four chemical markers (gingerol homologues and shogaol). The data revealed moderate to high diversity in the collection, clustering them broadly into two groups. Both ISSR and SSR techniques were efficient in distinguishing all the 18 ginger cultivars, however, SSR markers were observed to be better in displaying average polymorphism (77.8%) than ISSR (66.7%). Based on statistical analysis, one ISSR and two SSR primers could be identified which effectively distinguished closely related ginger cultivars. Chemical profiling and subsequent multivariate analysis distinguished five lines which were distinct from rest of the collection. The study has contributed in understanding the genetic and chemical diversity of the region, characterization of lines for commercial exploitation and ginger gene pool conservation.     

A novel strategy for identification of 47 pomegranate (Punica granatum) cultivars using RAPD markers

DNA marker can be used for precise plant cultivar identification. However, DNA markers have often not been used effectively for the identification of plant cultivars due to a lack of an effective analysis strategy. We used a novel strategy for effective identification of plant individuals based on a new way of recording DNA fingerprints of the genotyped plants; a cultivar identification diagram can be manually generated and used as key reference information for quick identification of plant and/or seed samples. Forty-seven pomegranate varieties popularly cultivated in various provinces of China were subjected to RAPD marker analysis. Using the cultivar identification diagram strategy, they were clearly separated by the fingerprints of 11 RAPD primers. The utility and accuracy of the cultivar identification diagram analysis results were confirmed by the identification of three randomly chosen groups of cultivars among the 47 varieties.     

Fractionation and identification of proteins by 2-DE and MS: towards a proteomic analysis of Plasmodium falciparum

Since completion of genome sequencing of the malarial parasite Plasmodium falciparum, proteomic tools for the identification of parasite proteins have become particularly attractive as they allow a more thorough interpretation of these data. Recent advances in 2-D PAGE, MS, and bioinformatics have created great opportunities for mapping and characterization of protein populations. We employed these improvements in a proteomic approach for the analysis of proteins detected in two blood stages of P. falciparum, (i) in the schizont stage and (ii) in the merozoite stage. For the isolation of merozoites, we introduced a new protocol based on the preparation of clustered structures of merozoites upon treatment of cultures with the common cysteine proteinase inhibitor E64. Peptide mass fingerprints of excised and trypsinated protein spots, acquired by MALDI-TOF MS were generated to identify a variety of proteins. Moreover, prefractionation procedures were used to enrich and map low-abundance proteins in protein samples. The data demonstrate that classic proteomic analyses using 2-D PAGE are now feasible for P. falciparum and represent the first step in the direction of creating 2-D reference maps for this medically most relevant protozoon.     

A new strategy employed for identification of sweet orange cultivars with RAPD markers

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.     

Characterization of subrepeat regions within rDNA intergenic spacers of the edible basidiomycete Lentinula edodes

For 16 commercial cultivars of Lentinula edodes, DNA fragments for the nuclear rDNA intergenic spacers IGS1 and IGS2 were amplified and analyzed. IGS1 contained a subrepeat region, named SR1, and IGS2 contained a pair of direct repeats and a subrepeat region, named SR2. Three and five types of subrepeats were found in SR1 and SR2, respectively. Heterogeneity in the lengths of IGS1 and IGS2 arose mainly from the number of different kinds of subrepeats within SR1 and SR2. The DNA fingerprints from the PCR products targeting SR1 and SR2 were specific for each of the 16 cultivars, and had enough variation for discrimination among the cultivars. This result suggests that the DNA fingerprints targeting SR1 and SR2 are useful for investigations of L. edodes cultivars.     

Bean lectins IV: genetic variation in the non-denatured structure of lectins from different Phaseolus vulgaris L. cultivars

Summary Variation in the native conformation of bean lectins was examined using electrophoresis of non-denatured total protein extracts and purified albumin and globulin lectin. The observed variation was related to the genetic variation reported previously for lectin polypeptide composition as revealed by two-dimensional isoelectricfocusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE). When eleven cultivars with different IEF-SDS/PAGE lectin polypeptide compositions were compared, eight had unique non-denatured lectin patterns and three had identical patterns. For some cultivars differences in non-denatured lectin patterns were observed between the purified albumin and globulin lectin preparations.     

Identification of closely related citrus cultivars with inter-simple sequence repeat markers

 Inter-simple sequence repeat (ISSR) markers generated by 22 primers were tested for their ability to distinguish among samples from 94 trees of 68 citrus cultivars. Within each of the six cultivar groups studied, most of these cultivars are so closely related that they are difficult to distinguish by other molecular-marker techniques. ISSR markers involve PCR amplification of DNA using a single primer composed of a microsatellite sequence anchored at the 3′ or 5′ end by 2–4 arbitrary, often degenerate, nucleotides. The amplification products were separated on non-denaturing polyacrylamide gels and detected by silver staining. ISSR banding profiles were very repeatable on duplicate samples. Different citrus species had very different fingerprint patterns. Within Citrus sinensis (L.) Osbeck and C. paradisi Macf., in which all cultivars have originated by the selection of mutants, ISSR markers distinguished 14 of 33 sweet orange and 1 of 7 grapefruit cultivars. Five of six lemon cultivars were discriminated by ISSR markers. Many differences were found among mandarin cultivars; however, all five satsuma cultivars analyzed had identical ISSR fingerprints. Four of five citrange cultivars were distinguishable, but ‘Troyer’ and ‘Carrizo’ had identical ISSR fingerprints. ‘Kuharske Carrizo’ citrange, which has better citrus nematode resistance than other ‘Carrizo’ citrange accessions, had unique ISSR fingerprints. Three ISSR markers that differentiated certain sweet orange cultivars were hybridized to Southern blots of sweet orange DNA digested with different restriction endonucleases. The sweet orange cultivars tested could be distinguished by these ISSR-derived RFLP markers. Moreover, one ISSR marker unique to ‘Ruby’ blood orange was observed in its progeny trees. Received: 9 September 1996 / Accepted: 4 April 1997