Cloning of an Arabidopsis thaliana cDNA encoding cystathionine β-lyase by functional complementation in Escherichia coli

Cystathionine -lyase, the second enzyme involved in the methionine biosynthetic pathway in plants, catalyses the synthesis of homocysteine from cystathionine. A cDNA encoding cystathionine -lyase was cloned from an Arabidopsis thaliana expression library by complementation of an Escherichia coli mutant deficient in this enzyme. As deduced from the full-length nucleotide sequence (1.7 kb), the polypeptide contains 464 amino acids and presents a predicted M _r of 50372. A. thaliana cystathionine -lyase exhibits 22% sequence identity with the E. coli corresponding enzyme and contains a 70 amino acid N-terminal additional sequence compared with the bacterial protein. Since the general features of chloroplast transit peptides could be observed in this amino-terminal extension, we propose a chloroplast localization for the cDNA-encoded enzyme. Southern blot analysis suggested that cystathionine -lyase is encoded by a single copy gene in A. thaliana.     

Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development⋆

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1 mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed.     

国内cDNA文库及cDNA克隆的研究概况

本文就笔者所收集到的资料,对国内已报道的5个cDNA文库及9个cDNA克隆的建立进行简要概述,以期对国內cDNA文库及cDNA克隆的研究现状有个大致的了解。     

Characterisation of an Arabidopsis thaliana cDNA encoding a novel thylakoid lumen protein imported by the ΔpH-dependent pathway

An Arabidopsis thaliana (L.) Heynh. cDNA encoding a novel 16-kDa protein (P16) of the chloroplast thylakoid lumen has been characterised. The function of the protein is unknown but it shares some sequence similarity with alpha allophycocyanins. P16 is synthesised with a bipartite, lumen-targeting presequence, and import experiments demonstrated that this protein follows the ΔpH-dependent pathway. Analysis of the thylakoid transfer peptide revealed two unusual features. Firstly, the key targeting determinant is predicted to be a twin-arginine followed by a highly hydrophobic residue two residues later, rather than at the third position as in most transfer peptides. Secondly, the C-terminal domain of the transfer peptide contains multiple charged residues which may help to prevent mistargeting by the Sec-type protein translocase. Received: 16 October 1998 / Accepted: 29 October 1998     

cDNA cloning and bacterial expression of an endo-β-1,4-mannanase, AkMan, from Aplysia kurodai

Previously we isolated an endo-β-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1392bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96Da. Comparison of the primary structure of AkMan with other β-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 β-1,4-mannanases indicated that AkMan together with other molluscan β-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21(DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells.     

cDNA文库构建策略

随着ｃＤＮＡ文库应用的日益广泛,其构建方法也有了很大改进和提高。获得完整和均一的ｍＲＮＡ是构建成功的基础,合成ｃＤＮＡ时可根据不同目的选择相应的反转录酶和方法,用于构建的载体也由质粒发展到噬菌粒并各有其利弊,双链ｃＤＮＡ克隆前应视连接方案进行末端修饰和分级分离,克隆时则需把握ｃＤＮＡ和载体的比例。     

Nucleotide sequence of a cDNA coding for the barley seed protein CMa: an inhibitor of insect α-amylase

The primary structure of the insect -amylase inhibitor CMa of barley seeds was deduced from a full-length cDNA clone pc43F6. Analysis of RNA from barley endosperm shows high levels 15 and 20 days after flowering. The cDNA predicts an amino acid sequence of 119 residues preceded by a signal peptide of 25 amino acids. Ala and Leu account for 55% of the signal peptide. CMa is 60–85% identical with -amylase inhibitors of wheat, but shows less than 50% identity to trypsin inhibitors of barley and wheat. The 10 Cys residues are located in identical positions compared to the cereal inhibitor family with a Pro-X-Cys motif present in all.     

一种新的cDNA末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物。利用一种新的cDNA末端快速扩增方法（SMART RACE）扩增该EST的5′末端,并进行克隆测序,与cDNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA。结果表明：SMAR RACE是一种简便、有效的克隆cDNA5′末端未知序列的技术。     

一种新的cDNA 末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物,利用一种新的cDNA末端快速扩增方法(SMARTRACE)扩增该EST的5′末端,并进行克隆测序,与mRNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA.结果表明,SMARTRACE是一种简便、有效的克隆cDNA5′末端未知序列的技术. Abstract:To clone the full-length cDNAs of genes related to spermatogenesis,ESTs obtained by mRNA differential display were used to design gene-specific primer.Then SMART RACE was performed to obtain the 5′ region of these ESTs.After cloning,sequencing and splicing with ESTs obtained by mRNA differential display,three full-length cDNAs were obtained.The results indicate that SMART RACE is a simple and an effective technique for cloning 5′-end unknown sequence of gene.     

“电子”cDNA文库筛选指导基因的全长cDNA克隆

“电子”ｃＤＮＡ库筛选主要是指通过采用生物信息学的方法延伸表达序列标签（ＥＳＴ）序列,以获得基因的部分及至全长ｃＤＮＡ序列,避免或部分避免构建与筛选ｃＤＮＡ库等烦琐的实验室工作。该方法具体体现了ＥＳＴ数据库的迅速扩张已导致识别与克隆新基因的策略发生革命性的变化。ＥＳＴ序列ＺＡ７３为本实验室克隆到的可能参与辐射致气管上皮细胞恶性转化过程的基因片段,本研究采用“电子”ｃＤＮＡ库筛选的方法对其可能     

cDNA芯片的重复性研究

cDNA芯片正在成为基因表达研究的常用方法,用此法通过一次杂交即可获得大量基因的表达信息[1,2]。目前,一些专业的生物芯片公司已推出部分cDNA芯片产品供研究者应用,然而,由于价格昂贵及受到其他一些条件的限制,国内众多研究者很难对芯片结果进行多次杂交验证,因此cDNA芯片的重复性就成为他们关心的问题,本实验对cDNA芯片用于基因表达研究的重复性进行了研究。１材料与方法１．１探针和cDNA芯片的制作按文献[3]中的方法合成R15Hp35T细胞的探针10μl,以荧光素Cy5-dCTP进行标记。同时制作含有61个基因的cDNA芯片,共10片。…     

人载脂蛋白E7转基因小鼠肾脏cDNA削减文库与cDNA文库

目的　以人apoE7基因制备的高脂血症转基因小鼠模型已建成 ,要用于探查这一突变基因致病的分子机制 ,首先揭示人apoE7在小鼠体内诱发了那些基因的表达异常 ,是重要的环节之一。cDNA削减文库是寻找异常高表达的已知和未知基因有效的手段 ,而cDNA文库更是获取全长基因所必须。方法　自正常小鼠与转基因小鼠肾脏同时提取总RNA ,再分离mRNA ,逆转录制备cDNA后 ,用两次分子杂交削除转基因鼠cDNA中与正常小鼠相同的部分 ,再用PCR与择需PCR法扩增转基因鼠特异的基因 ,克隆入pGEM T载体后 ,制备削减文库。未经削减的转基因小鼠cDNA克隆入λgt11载体 ,制备基因表达文库。结果和结论　自高脂血症转基因小鼠肾脏构建的削减文库得到约 4 0 0个削减克隆 ,测定了部分克隆的序列 ,并进行同源性比较。λgt11 cDNA文库滴度 1 7× 10 7pfu ml,随机测定的克隆其插入片段长约 5 0 0bp以上。     

多巴胺受体的cDNA克隆

通过多巴胺受体的5个cDNA克隆,综述和分析了5个多巴胺受体(D₁R-D₅R)的基因结构,在染色体上的定位及其mRNA在中枢脑区的分布;比较了这5个受体cDNA克隆的结构特征和药理学性质.     

cDNA合成和产量计算

以信使核糖核酸(mRNA)为模板合成互补的DNA链(cDNA),并以此单链DNA合成双链cDNA的技术是基因工程中最初的和重要的环节之一。本文根据研究工作的体会介绍cDNA合成方法,产量计算,影响cDNA合成的因素和解决的方法。一、cDNA合成方法自从1970年Temin和Baltimore 发现鸡骨髓瘤病毒逆转录酶以后,以mRNA为模板合成cDNA的技术很快应用到基因工程研究中。由于大多数合成反应都以polyA-mRNA为模板,因此需要加入寡聚脱氧胸腺核苷酸作为引物。底物是四种脱氧核苷三磷酸,其中一种     

水稻cDNA文库的构建及巯基蛋白酶抑制剂cDNA的分离

取开花后2周左右的水稻未成熟种子提取总RNA,分离mRNA,进而反转录合成cDNA并定向插入λgt22A克隆载体,经体外包装建成水稻未成熟种子的表达型 cDNA文库,经测定库容量达 1.5 × 10⁶ Pfu.进一步人工合成水稻巯基蛋白酶抑制剂(Oryzacystatin)编码基因5＇-端保守的21Nt的寡核苷酸经末端标记作为探针,从 2.1×10⁴Pfu中筛选出 9个阳性克隆,经鉴定其中 8个含有完整的水稻巯基蛋白酶抑制剂编码序列,对 1号克隆的序列分析表明,其除含有 309 bp的水稻巯基蛋白酶抑制剂编码序列外,在 5＇-端及 3＇-端还分别含有 84 bp和 497 bp的非编码序列,其中 3＇-端带有AATAAAA两个加尾信号以及31 Nt的Poly(A)尾,同水稻巯基蛋白酶抑制剂基因组序列比较,编码区碱基序列完全一致,但5＇-端和3＇-端非编码区有明显差异,其中加尾信号以后的cDNA碱基序列中含有大量的不连续插入.