Isolation and characterisation of a cDNA encoding a novel cytosolic ascorbate peroxidase from potato plants (Solanum tuberosum L.)

Ascorbate peroxidase (APX) is one of the key enzymes of the plant antioxidant system playing, along with catalase, a central role in hydrogen peroxide scavenging. An approach to further increase the knowledge about cytosolic APX gene organization can be achieved by isolating and characterisating new cDNAs, thus providing new insights about the physiological roles and regulation of these enzymes. A partial cDNA clone (corresponding to the 3’ untranslated region), cytosolic ascorbate peroxidase-related, was isolated from potato sprouts by RT-PCR. Database analysis retrieved several expressed sequence tags (ESTs) coding potato cytosolic ascorbate peroxidase, that were used to infer the complete cDNA sequence. The deduced amino acid sequence revealed high homologies with other plant cytosolic ascorbate peroxidases, confirming the reliability of the virtual cDNA. Northern blot analysis revealed the existence of a single band related to the isolated cDNA and the southern blotting results allowed the elaboration of a possible gene organization.     

戊型肝炎病毒（HEV）开读框架2中1.3kb cDNA的克隆与序列分析

以戊型肝炎（Ｈｅｐａｔｉｔｉｓ）病人高热期血清为原材料,设计特定引物,经ＲＴ－ＰＣＲ扩增获得开放阅读框２（ＯＲＦ２）１．３ｋｂ基因片段,用ＥｃｏＲＩ和ＢａｍＨＩ插入克隆载体ｐＵＣ１８,测定了该克隆基因片段的核苷酸序列并进行了比较分析。其片段长度为１３０９ｂｐ,其Ａ＝２４９（１９．２％）,Ｇ＝３１８（２４．２９％）,Ｔ＝３３９（２５．９％）,Ｃ＝４０３（３０．７９％）;与印度株和缅甸株的同源性在９０     

链霉菌酪氨酸酶基因的克隆与表达

本文综述了近年来对链霉菌酪氨酸酶基因的研究。由酪氨酸酶合成黑色素是链霉菌属各个种的共同特性,并受到酪氨酸酶基因的控制。链霉菌几个种的酪氨酸酶基因已克隆到,其产黑色素的特性使之作为重要的标记基因得以广泛应用,同时,该基因在链霉菌的不同种及不同属的微生物中得到了高水平的表达。链霉菌酪氨酸酶基因表达调控的分子机制也得到较深入的研究。     

Isolation and expression of cDNA clones for a rat liver asialoglycoprotein receptor

cDNA clones for the major rat liver asialoglycoprotein (ASGP) receptor were isolated from a phage gtl 1 library using synthetic oligonucleotide probes corresponding to two regions of the protein sequence. The longest clone obtained encoded all but the first 11 codons of the receptor. The cDNA was completed with synthetic oligonucleotides and was used to direct the synthesis of mRNA for the receptorin vitro. Subsequent translation in a wheat germ lysate produced authentic ASGP receptor which assembled correctly into microsomal membranes.     

玉米苹果酸脱氢酶基因的分离与结构分析

以一个玉米（ＺｅａｍａｙｓＬ．）杂种一代超亲表达的ｃＤＮＡ片段为探针,从玉米幼苗期ｃＤＮＡ文库中筛选到一个全长１２８７ｂｐ的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ编码细胞质苹果酸脱氢酶,推导的氨基酸序列与龙须海棠（Ｍｅｓｅｍｂｒｙａｎｔｈｅｍｕｍ　ｃｒｙｓｔａｌｌｉｕｍ　Ｌ．）及拟南芥（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）同一编码基因的氨基酸序列同源性分别为９０％和８４％。这是禾谷类作物中首次克隆的编码细胞质苹果酸脱氢酶的完整基因。     

鸡肌球蛋白轻链激酶表达载体的构建

鸡肌球蛋白轻链激酶（ＭＬＣＫ）在调节平滑肌细胞收缩中具有重要作用．用ＰＣＲ产生构建所需的限制性内切酶ＳａｌⅠ位点,将鸡ＭＬＣＫｃＤＮＡ插入质粒ｐＢＫｒｓｖ中,构建成ｐＢＫｒｓｖ－ＭＬＣＫ,并通过酶切图谱、ＳａｌⅠ位点序列分析得到证实．重组质粒在大肠杆菌中获得表达．     

cDNA Cloning and Characterization of Rat C5a Anaphylatoxin Receptor

Activation of the complement cascade plays an esssential role in the early stages of inflammation. C5a and its receptor are particularly active in anaphylaxis. To determine the pathological roles played by C5a and C5a receptor (C5aR) in rats, we cloned C5aR cDNA and analyzed distribution of its mRNA in various organs including lung from an LPS-stimulated rat. Furthermore, we generated a polyclonal antiserum which specifically recognizes rat C5aR, as confirmed by its specific interaction with cells transfected with rat C5aR cDNA.     

Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit

Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin is a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present in larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3′ end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5′-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. © 1995 Wiley-Liss, Inc.     

Cloning and Characterization of a Caenorhabditis elegans cDNA Encoding a New Insulin/IGF-Like Peptide

A Caenorhabditis elegans cDNA encoding a new insulin/IGF-like peptide was cloned and examined. The predicted peptide shows significant sequence similarity with the peptide Ceinsulin-1 reported previously and contains a characteristic insertion consisting of three residues in the putative B domain as with the Ceinsulin-1. The gene expression pattern during development is almost identical to that of Ceinsulin-1. The predicted tertiary structure of the peptide is quite similar to that of Ceinsulin-1, and their predicted receptor-recognition surfaces also closely match. These facts suggest that both peptides could recognize the same receptor.     

Isolation and Characterization of a cDNA Encoding Maize Cyto solic Malate Dehydrogenase

A cDNA fragment derived from a gene over-expressing in hybrid maize ( Zea mays L. ) was isolated with RT-PCR and used as probe to screen cDNA library of hybrid maize seedlings. A positive cDNA clone ZH02 corresponding to the full-length mRNA sequence was obtained, which was shown to have an open reading frame encoding 332 a.a. DNA and proteinase database search revealed that the deduced amino acid sequence of ZH02 has high similarity with the cMDH of Mesembryaathemum crystallium L. and Arabidopsis thaliana (L.) Heynh. up to 90% and 84%, respectively. This is the first report of the full-length gene coding for the cereal cMDH.     

从中国病人克隆丙型肝炎病毒基因组C区基因及其在大肠杆菌中的表达

本文通过逆转录（ＲＴ）－聚合酶链式反应（ＰＣＲ）,从两份分别来自湖南省娄底地区丙型肝炎病人和河北省秦皇岛市职业献血员丙型肝炎病毒（ＨＣＶ）ＲＮＡ打点杂交阳性的血清中,扩增并克隆到１段５６３ｂｐ的ＨＣＶ基因组Ｃ区抗原基因Ｃ２６９／８３１,并通过ＰＣＲ得到了３个表达片段Ｃ８３１、Ｃ８０１和Ｃ５８７。测定Ｃ２６９／８３１基因的全序列后发现,中国人ＨＣＶ湖南分离株与ＨＣＶ－Ⅰ型株ＨＣＶ－ＵＳ和Ⅱ型株ＨＣＶ－ＢＫ在该基因区段的核苷酸／氨基酸序列的同源性,分别为９０．３％／９４．６％和９５．２％／９４．６％。利用原核高效表达载体ｐＢＶ２２０在大肠杆菌中有效地表达了非融合的Ｃ区抗原基因重组蛋白ＣＬ、ＣＭ和ＣＳ。通过免疫筛选法及Ｗｅｓｔｅｍ印迹法对约占菌体可溶性蛋白１１％的表达产物进行了鉴定。采用ＴｒｉｔｏｎＸ－１００和盐析处理表达产物,再进行离子交换层析纯化,得到可用于检测ＨＣＶ血清抗体的核壳蛋白（Ｃ）抗原。通过不同分子量抗原的表达,发现由Ｃ区Ｎ端８９个氨基酸组成的多肽ＣＳ其抗原性与由１５８或１６８个氨基酸组成的多肽ＣＭ或ＣＬ相同,但抗原的稳定性和表达量显著优于后两种抗原。本研究为研制ＨＣＶ抗体诊断试剂盒奠定了重要基础。     

西瓜八氢番茄红素合成酶基因片段的克隆和序列分析

以西瓜品种ZXG00152为材料,提取瓜瓤总RNA,进行反转录,依据植物八氢番茄红素合成酶(PSY)的氨基酸保守序列设计引物,以cDNA第一链为模板,扩增得到长约750 bp的cDNA片段。将该片段克隆到pMD19-T载体,测序结果表明该基因片段长748 bp,编码249个氨基酸,Blast搜索结果表明,由该片段推导出的氨基酸序列与其它植物的八氢番茄红素合成酶有较高的同源性,其中与甜瓜的一致性达到97.8%。该片段已在GenBank中登录(登录号:DQ494214)。     

大鼠FMR1同源基因cDNA的克隆、测序与选择剪接的初步分析

应用ＲＴ－ＰＣＲ方法,从新生大鼠脑组织总ＲＮＡ扩增大鼠ＦＭＲ１同源基因的ｃＤＮＡ片段,克降至ｐＵＣ１８质粒中进行序列分析．获得从终止密码子起共１６８１ｂｐ的编码序列,尚缺少约２００ｂｐ的５′序列．所克隆的这部分大鼠ＦＭＲ１ｃＤＮＡ,不含有对应于人ＦＭＲ１基因的外显子１２及外显子１７第一和第三剪接受点之间的序列,提示大鼠ＦＭＲ１基因也有选择剪接表达．同源性分析显示,大鼠ＦＭＲ１与小鼠ＦＭＲ１基因的同源性为９７．７％,与人ＦＭＲ１基因的同源性为９４．９％;与小鼠ＦＭＲＰ（ＦＭＲ１蛋白）的氨基酸序列同源性为９８．４％,与人ＦＭＲＰ的氨基酸序列同源性为９７．９％．以大鼠ＦＭＲ１ｃＤＮＡ片段为探针检测到大鼠不同组织中ＦＭＲ１基因的选择剪接表达．上述结果为以大鼠为动物模型深入研究ＦＭＲ１基因功能奠定了基础．     

Isolation and Characterization of Low-Molecular-Weight Chromium-binding Substance (LMWCr) from Chicken Liver

Chromodulin (also known as low-molecular-weight chromium-binding substance, LMWCr) is a chromium-binding oligopeptide proposed to play a role in insulin signaling and chromium transport in mammals. This laboratory has isolated and purified this material from a non-mammalian source, an avian. Spectroscopic and physical characterization of the isolated material suggests the material is an oligopeptide with a multinuclear chromium assembly bridged via asparatate and glutamate residues very similar to its mammalian counterparts. The isolated material also possesses a biological activity similar to other LMWCr isolates.     

互补DNA杂交分析和血清学方法对香石竹环斑病毒分离物的研究

在联帮德国奥卡河中分离到一种病毒分离物(W),通过鉴别寄主反应的测定,病毒粒子的电子显微镜观察,cDNA斑点杂交和血清学的研究,鉴定出这一病毒分离物为香石竹环斑病毒。其外壳蛋白亚基的分子量为3.8×10~4。cDNA吸印转移杂交分析表明,香石竹环斑病毒的两个RNA组份(RNA~1、RNA_2)之间没有核苷酸序列同源性。RNA_1和RNA_2分别由3.7和1.5仟碱基组成。     

小麦Mlo及NBS—LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因MlocDNA序列及一个来源于栽培一粒小麦（Triticum monococcumL.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选。结果获得两个表达基因的cDNA克隆。其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%。另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类。白粉菌诱导前后,该片段RT-PCR扩增产物存在差异。表明该片段可能与小麦抗病性相关。利用“中国春”缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上。     

一种快速、简便、高效cDNA合成及克隆策略

以鱼呼肠孤病毒双链ＲＮＡ为模板,建立一种快速、简便、高效的ｃＤＮＡ合成及克隆策略。在一定量模板条件下,采用随机六聚体为引物合成ｃＤＮＡ第一链。以退火方式形成双链ｃＤＮＡ,直接通过琼脂糖凝胶电泳检测其ｃＤＮＡ合成产量。双链ｃＤＮＡ经两端补齐后,平头连接于具有阳性选择标记的载体中,以高效电转化的方式进行快速克隆。     

烟叶脉坏死病毒原鉴定及cDNA克隆与序列分析

田间采集辽宁地区烟叶脉坏死病标样所得分离物,在测定的１２个科３５种植物中只侵染茄科的一些烟草品种及洋酸浆（ｐｈｙｓａｌｉｓｆｌｏｒｉｄａｎａ）,可由桃蚜（Ｍｙｚｕｓｐｅｒｓｉｃａｅ）传播。病叶汁液稀释限点（ＤＥＰ）为１０￣（－２）－v１０￣（－３）;失毒温度（ＴＩＰ）为５５一６０℃;体外保毒期（Ｌ）为４８－７２小时。病毒粒体形态呈线条状７２０×１２ｎｍ,病叶脉坏死部细胞质中含风轮状内含体。病毒提取物的紫外最大吸收为２６５ｎｍ,最小吸收为２４５ｎｍ,Ａ＿（２８０）／Ａ＿（２６０）为０．８２。该病毒分离物与ＰＶＹ￣０抗血清呈阳性反应。以病毒ＲＮＡ为模板,按国外报道的ＰＶＹ￣Ｎ序列合成引物经逆转录合成ｃＤＮＡ。用ＰＣＲ扩增出约０．８０ｋｂ的ＣＰ基因片段,将这一片段插入载体ｐＧＥＭ７Ｚ－ｆ（＋）中转化Ｅ．ｃｏｌｉＤＨ５ａ菌株得到了ＣＰ基因的克隆。ｃＤＮＡ序列分析表明,和国外报道的ＰＶＹ￣Ｎ序列同源性极高,初步表明引起辽宁地区烟叶脉坏死病的毒原为ＰＶＹ￣Ｎ。