mRNA差异显示

分离差异表达的基因是研究生命调节过程的重要手段,mRNA差异显示技术是一种能成功分离差异表达基因的方法,文章对该方法的基本原理、方法步骤及其应用作了较为详细的介绍。     

信使RNA差异显示技术的最新进展

各种疾病，如肿瘤、遗传性疾病都是一种或多种基因异常表达的结果。寻找、定位疾病相关基因在目前分子生物学研究中占有十分重要的地位，Ｌｉａｎｇ等１９９２年首创了信使ＲＮＡ差异显示技术（ｍｅｓｓｅｎｇｅｒＲＮＡｄｉｆｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ，ｍＲＮＡ...     

降低mRNA差异显示技术假阳性率的一种方法

为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 .     

荧光标记mRNA差异显示技术

目的：应用荧光标记的ｍＢＮＡ差异显示技术。方法：提取未经过／经过ＩＦＮ－ＬＰＳ处理的三组人单核细胞系Ｕ９３７的总ＲＮＡ并以此为模板,采用荧光标记的锚定引物,通过逆转录、差异显示ＰＣＲ反应,经５．６％变性聚丙烯酰胺凝胶电泳分离差异条带,回收后将其再扩增。结果：三组样本的ＤＤ－ＰＣＲ产物电泳显示长３００ｂｐ￣２．０ｋｂ不等的扩增片段,条带清晰、明亮,背景低,各样本相互间的差异不仅呈有无的变化,亦表现出     

原核细胞mRNA差异显示技术的引物设计

由于原核细胞mRNA3'端不存在ploy(A)结构,因而原核细胞DDRT-PCR引物设计不同于真核细胞。尽管不能根据oligo(dT)设计引物,但利用全基因中高度分散重复的短序列或回文序列却能有效克服mRNA分子结构的影响,最大限度地扩增全长cDNA;并且这2种引物设计方法还可以提高RNA指纹图谱的重复性,降低反应的假阳性,从而为原核细胞DDRT-PCR引物设计提供新的思路。     

利用mRNA差异显示技术研究香菇发育相关基因

以香菇子实体不分化突变株和对照菌株L98411为材料,利用mRNA差异显示技术研究二者的基因表达差异,共分离到21条差异片段,其中12个分别与泛素、ATP合成酶、锌指蛋白、GTP结合蛋白、延伸因子g1线粒体前体、过氧化物酶、硫酯酶、类TrpB酶、2,4-二烯酰辅酶A还原酶、糖苷水解酶、热激蛋白、疏水蛋白等已知基因有较高的同源性,这表明香菇子实体分化发育是一个复杂的过程,涉及到胞内转运、转录调控、细胞分化、蛋白合成、各种代谢途径中的酶、抗逆反应等诸多途径的协同调控。     

mRNA差异显示技术克隆中国葡萄属野生种核糖体RNA基因

克隆功能基因是研究植物基因结构及功能的重要途径,利用mRNA差异显示技术,筛选获得了中国葡萄属野生种华东葡萄白河-35-1在人工接种白粉菌前后差异表达基因的cDNA片段Vprdrf4。经Northern杂交验证,该片段为正调控片段,Blaset检索表明与真核生物核糖体RNA基因的同源性高达90%以上,GenBank登录号为CD347664。     

mRNA差异显示技术及其应用

mRNA差异显示技术是国外最近发展起来的一种分离表达水平出现差别的基因的新技术。目前已广泛用于人类及其他动物生长发育基因调控、基因克隆研究,也开始用于植物基因分析、杂种优势机理等方面研究。本文综述这一新技术原理、实验步骤、应用及问题与展望。     

mRNA差异显示技术中特异条带回收方法的比较

目的：建立简化的mRNA差异显示技术中特异条带的回收方法。方法：用单个小鼠早期发育的胚胎构建mRNA差异显示技术,用一步法和煮沸法分别从银染的聚丙烯酰胺凝胶中回收差异显示的特异条带,并进行再扩增、回收、克隆及酶切鉴定。结果：两种不同的回收方法经过mRNA差异显示技术程序环节,证明其具有相同的实验效果。结论：应用简化的一步法和煮沸法对单胚构建的mRNA差异显示技术中的特异条带进行回收,具有有效性和可靠性。     

柑橘mRNA差异显示技术体系建立

明柳甜橘是从春甜桔自然芽变中选育出来的新品种,本研究以春甜橘和明柳甜橘的叶片为试验材料,首先提取春甜橘和明柳甜橘叶片的总RNA,经3种锚定引物(HT11A,HT11C,HT11G)反转录成3类cDNA,再利用相应的锚定引物和34个随机引物(HAP1-HAP34)组成引物对,反转录cNA进行差异显示PCR扩增,将扩增产物经6%的变性聚丙烯酰胺凝胶电泳后进行银染,获得了比较清晰的差异条带,然后回收差异条带并进行克隆,初步分离获得一些春甜橘和明柳甜橘差异基因,建立了柑桔mRNA差异显示技术,为进一步分析导致柑橘芽变的分子机理奠定基础.     

Methylation Pattern of Radish (Raphanus sativus) Nuclear Ribosomal RNA Genes

The methylation pattern of radish Raphanus sativus nuclear rDNA has been investigated using the Hpa II, Msp I, and Hha I restriction enzymes. The presence of numerous target sites for these enzymes has been shown using cloned rDNA fragments. A large fraction of the numerous rDNA units are heavily methylated, being completely resistant to Hpa II and Hpa I. However, specific sites are constantly available in another fraction of the units and are therefore unmethylated. The use of different probes allowed us to demonstrate that hypomethylated sites are present in different regions. Major hypomethylated Hha I sites have been mapped in the 5′ portion of 25S rRNA coding sequence. Among the hypomethylated fraction, different methylation patterns coexist. It has been possible to demonstrate that methylation patterns are specific for particular units. The Hha I pattern of rDNA in tissues of different developmental stages was analyzed. Evidence for possible tissue specific differences in the methylation pattern is reported.     

Characterization of Radish (Raphanus sativus) Storage Proteins

Radish (Raphanus sativus cv Rond rose à bout blanc Vilmorin) seeds, as other cruciferae oil seeds, contain two major types of storage protein aggregates which can be separated by gel filtration into 12 and 1.7 Svedberg fractions. These two fractions have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, and two bidimensional gel electrophoresis systems. These results were compared with those obtained with rapeseed storage proteins. Radish 12 Svedberg particles are made of a series of nine major polypeptides ranging from 33 to 30 kilodaltons. These polypeptides present charge heterogeneity. The 12 Svedberg particle is made of six subunits 55 kilodaltons. Each subunit is a couple of two polypeptides linked by a disulfide bridge. The 1.7 Svedberg particle has a simpler composition. It is made of two polypeptides of 10 and 12 kilodaltons and smaller peptides of 7 kilodaltons. Twelve and 1.7 Svedberg particles also differ in their amino acid composition, the 1.7 Svedberg being particularly rich in glutamic acid and proline. Its components are basic. The organization of the rapeseed storage protein is similar but more complex.     

萝卜品种指纹图谱SRAP与AFLP分析

应用SRAP与AFLP两种分子标记技术进行了萝卜品种鉴定分析。对萝卜基因组DNA的SRAP-PCR反应体系中引物、Mg2 、dNTPs浓度进行优化,确定最优体系为引物0.3μmol.L-1,dNTP 0.2 mmo.lL-1,Mg2 3.0 mmo.lL-1。对SRAP-PCR中的退火温度(50℃)设置了12个梯度处理,以em2-me2为引物时带型无明显差异。7个供试萝卜材料的SRAP和AFLP指纹图谱分析表明,供试材料均可被SRAP和14个AFLP引物准确鉴定,每对引物组合都产生独特的指纹图谱。11个SRAP引物组合共产生155条带,多态性条带84条。聚类分析与相对遗传距离(GD)表明,供试材料聚为4类,CB-03-2与SHCB-02-1亲缘关系最近(GD=0.054 9);齐虹大连和Heiseng的亲缘关系最远(GD=0.203 4)。基于16个AFLP标记引物组合分析结果表明,供试材料聚为3类,CB-03-2和SHCB-02-1的亲缘关系最近。SRAP与AFLP综合分析结果表明,供试材料可聚为3类,其中CB-03-2与SHCB-02-1亲缘关系最近(GD=0.047 6)。     

萝卜品种指纹图谱SRAP与AFLP分析

应用SRAP与AFLP两种分子标记技术进行了萝卜品种鉴定分析。对萝卜基因组DNA的SRAP-PCR反应体系中引物、Mg²⁺、dNTPs浓度进行优化,确定最优体系为引物0.3 μmol·L^-1,dNTP 0.2 mmol·L^-1,Mg²⁺ 3.0 mmol·L^-1。对SRAP-PCR中的退火温度（50℃）设置了12个梯度处理,以em2-me2为引物时带型无明显差异。7个供试萝卜材料的SRAP和AFLP指纹图谱分析表明,供试材料均可被SRAP和14个AFLP引物准确鉴定,每对引物组合都产生独特的指纹图谱。11个SRAP引物组合共产生155条带,多态性条带84条。聚类分析与相对遗传距离（GD）表明,供试材料聚为4类,CB-03-2与SHCB-02-1亲缘关系最近(GD=0.054 9);齐虹大连和Heiseng的亲缘关系最远(GD=0.203 4)。基于16个AFLP标记引物组合分析结果表明,供试材料聚为3类, CB-03-2和SHCB-02-1的亲缘关系最近。SRAP与AFLP综合分析结果表明,供试材料可聚为3类,其中CB-03-2与SHCB-02-1亲缘关系最近(GD=0.047 6)。     

alpha-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

An α-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding α-l-arabinopyranoside and β-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg²⁺, Ag⁺, l-arabino-(1→4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off α-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of α-l-arabinofuranosidase and β-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.     

A beta-Galactosidase from Radish (Raphanus sativus L.) Seeds

A basic β-galactosidase (β-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pl values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on p-nitrophenyl β-d-galactoside and β-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg²⁺ and 4-chloromercuribenzoate. d-Galactono-(1→4)-lactone and d-galactal acted as potent competitive inhibitors. Using galactooligosaccharides differing in the types of linkage as the substrates, it was demonstrated that radish seed β-Galase specifically split off β-1,3- and β-1,6-linked d-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the β-galase alone but could be partially degraded by the enzyme after the treatment with a fungal α-l-arabinofuranosidase leaving some oligosaccharides consisting of d-galactose, uronic acid, l-arabinose, and other minor sugar components besides d-galactose as the main product.     

Adenosine Phosphates in Germinating Radish (Raphanus sativus L.) Seeds

Changes in concentrations of adenosine phosphates (AMP, ADP, and ATP), oxygen utilization, and fresh weights were measured during the first 48 hours after imbibition of water by quiescent radish seeds (Raphanus sativus L.) at 22.5 C. The changes in ATP concentrations, oxygen utilization, and fresh weights followed a triphasic time course, characterized by a rapid initial increase, which extended from 0 to approximately 1.5 hours, a lag phase from 1.5 to 16 hours, and a sharp linear increase from 16 to 48 hours. In unimbibed seeds, the concentrations of ATP, ADP, and AMP were <0.1, 0.9, and 2.2 nmoles/seed, respectively. After imbibition of water by the quiescent seeds, for 1 hour, the ATP concentration had increased to 2.5, and ADP and AMP concentrations had decreased to 0.3 and 0.1 nmole/seed, respectively. These early changes occurred also in seeds maintained under anaerobic conditions (argon), or when treated with either 5 mm fluoroacetate, or 5 mm iodoacetate. The concentrations of ADP and AMP did not change significantly from 1 to 48 hours. The termination of the lag phase at 16 hours correlated with radicle emergence. Cell division in the radicles was initiated at approximately 28 hours. ATP concentrations in seeds maintained under argon or treated with fluoroacetate remained relatively constant from approximately 2 to 48 hours. In contrast, the ATP concentration of iodoacetate-treated seeds decreased curvilinearly from 4 to 48 hours. Oxidative phosphorylation was estimated to have contributed 15, 20, and 65% of the pool ATP at 1.5, 16, and 48 hours, respectively.     

Non-Random Mating in a Wild Radish, Raphanus sativus

Abstract When mating is non-random among several, compatible donors, the fitness of pollen donors, maternal plants, and offspring may be affected. Although this process may be important, it is much less studied than other forms of non-random mating such as incompatibility and avoidance of inbreeding. Therefore, the amount and consequences of non-random mating were investigated in greenhouse studies with wild radish, Raphanus sativus . Six compatible donors differed in the number, position, and weight of seeds sired, so mating was non-random at the level of mate identity. Mate number also affected mating patterns; fruits with more fathers were allocated more resources. This keeps mate number per fruit high. In contrast, other processes appear to keep mate number below the maximum so that mate number per fruit is regulated at an intermediate level. Mate identity had clear consequences as offspring with different fathers were of different sizes after 11 weeks. The effects of mate number on offspring success were less clear. These and other data suggest that non-random mating among compatible donors is a relatively common process in wild radish. It may occur through mechanisms controlled by the pollen tubes, the maternal plants or the embryos. While this non-random mating is the raw maternal for sexual selection in plants, whether sexual selection actually occurs and how important it may be is still nuclear.     

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.     

利用氟乐灵进行同源四倍体萝卜种质创制研究

本研究应用除草剂氟乐灵处理两叶一心幼苗生长点,进行同源四倍体萝卜种质诱导,对变异植株进行形态、细胞学鉴定和花粉母细胞染色体数目鉴定。结果表明,应用 0.2 mmol/L 和 1.0 mmol/L 氟乐灵处理,6个萝卜品种都获得同源四倍体植株,10 mmol/L 处理仅在 Nau-zhqh 得到同源四倍体;其中 0.2 mmol/L处理 Nau-dy 和 1.0 mmol/L 处理 Nau-xbch 获得四倍体最高诱导率(40%);四倍体种质与二倍体种质相比,形态性状、气孔大小、保卫细胞内叶绿体数目、花器官大小、花粉粒大小及花粉萌发率都存在显著差异,将形态、气孔鉴定和染色体计数结合可以准确确定变异株的倍性。研究表明利用氟乐灵诱导是进行萝卜同源四倍体种质创新的有效途径之一。本研究应用除草剂氟乐灵处理两叶一心幼苗生长点,进行同源四倍体萝卜种质诱导,对变异植株进行形态、细胞学鉴定和花粉母细胞染色体数目鉴定。结果表明,应用 0.2 mmol/L 和 1.0 mmol/L 氟乐灵处理,6个萝卜品种都获得同源四倍体植株,10 mmol/L 处理仅在 Nau-zhqh 得到同源四倍体;其中 0.2 mmol/L处理 Nau-dy 和 1.0 mmol/L 处理 Nau-xbch 获得四倍体最高诱导率(40%);四倍体种质与二倍体种质相比,形态性状、气孔大小、保卫细胞内叶绿体数目、花器官大小、花粉粒大小及花粉萌发率都存在显著差异,将形态、气孔鉴定和染色体计数结合可以准确确定变异株的倍性。研究表明利用氟乐灵诱导是进行萝卜同源四倍体种质创新的有效途径之一。