血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～ 70bp     

一类依赖于NAD的玉米胞质型苹果酸脱氢酶基因的克隆及其序列分析

苹果酸脱氢酶普遍存在于各种生物中,它负责催化草酰乙酸和苹果酸之间的相互转换.根据其辅酶的特异性和在细胞内的分布及其生理功能的不同,苹果酸脱氢酶在高等植物中可以区分出不同的类型,依赖于NAD的细胞质型苹果酸脱氢酶是其中研究较少的一类.根据已发表的其他高等植物的依赖于NAD的胞质型苹果酸脱氢酶基因的保守序列,运用SMART RACE RT-PCR技术,从玉米叶片中分离了cyMDH 的1 264 bp全长cDNA序列,通过生物信息学分析发现,该序列含有一个999 bp的完整的开放阅读框,其共编码332个氨基酸(GenBank登陆号 EU625276).序列联配与树状分析结果表明,该玉米cyMDH 序列与多个物种的cyMDH 基因具有高度的同源性.组织特异性表达分析显示MDH基因在玉米叶片中表达量最高,在茎、根中亦有低水平表达.本研究将为更深入的研究玉米cyMDH 基因的分子调控机理奠定基础.     

Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from <Emphasis Type="Italic">Enteromorpha prolifera</Emphasis> (Ulvophyceae,Chlorophyta)

Heat shock protein 70 (Hsp70) is one of the important members of heat shock protein (Hsp) families and plays essential roles in folding nascent protein, translocation, refolding denatured protein, protein degradation, adverse stress resistance, and so on. In this study, homologous cloning coupled with the rapid amplification of cDNA ends was used to clone full-length cytosolic heat shock protein 70 of Enteromorpha prolifera (designed as EPHsp70). Bioinformatics was used to analyze structural feature, homologous relationship, and phylogenetic position of EPHsp70. The full length of EPHsp70 cDNA was 2,265 bp, with a 5′ untranslated region of 65 bp, a 3′ untranslated region of 217 bp, and an open-reading frame of 1,983 bp encoding a polypeptide of 660 amino acids with an estimated molecular weight of 71.39 kDa and an estimated isoelectric point of 5.03. EPHsp70 had five degenerate repeats of tetrapeptide GGMP and three typical Hsp70 signature motifs. The C-terminus amino acid sequence of cytosolic EPHsp70 was EEVD, and the conservation of EPHsp70 of N-terminus was higher than that of C-terminus. The homology between EPHsp70 and the cytosolic Hsp70s of other algae and land plants was more than 70%.     

Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma

The 70-kD heat shock proteins (hsp70s) are a group of ubiquitous, highly conserved molecular chaperones that have been implicated in a variety of processes, ranging from DNA replication to protein folding and transport. To learn more about the evolution and possible functions of higher plant chloroplastic hsp70s, we isolated a cDNA clone encoding the major stromal hsp70 of pea chloroplasts, which we term CSS1 (Chloroplastic Stress Seventy). This cDNA clone encodes a 75,490-D protein that is very closely related to an hsp70 from the cyanobacterium, Synechocystis. CSS1 is nuclear encoded and synthesized as a higher molecular mass precursor with a chloroplastic transit peptide approximately 65 amino acids long. CSS1 mRNA was detected in RNA samples from leaves and roots of pea (Pisum sativum) plants grown at 18°C but increased 9- and 6-fold, respectively, after brief exposure of the plants to elevated temperature. We discuss the possible role(s) of CSS1 in chloroplastic protein transport and other processes.     

CaMBP-10的cDNA克隆和表达及钙调素结合活性分析

采用RT PCR法 ,从中国大白菜中分离了编码CaMBP 1 0的cDNA克隆 .该cDNA全长 4 96bp ,编码 92个氨基酸 ,3′端含有 2 1 6bp的非编码区和poly A尾 .将此BP 1 0cDNA的成熟蛋白序列导入表达质粒pET1 5b并转化至大肠杆菌E .coliBL2 1 (DE3)condonplus RIL进行表达 .以免疫印迹和钙调素结合分析法对重组BP 1 0进行鉴定 ,证明其保持了与天然BP 1 0相同的钙调素结合活性 .氨基酸和核苷酸序列分析结果显示 ,它与植物转脂蛋白高度同源 ,特别是含有 8个保守半胱氨酸 .BP 1 0与转脂蛋白之间具极为相似的理化性质如分子量、等电点、热稳定性等 .据此认为 ,CaMBP 1 0是转脂蛋白家族的新成员 ,Ca2 + CaM信号系统可能参与植物转脂蛋白功能的调节     

Structure and expression of a human gene coding for a 71 kd heat shock ''cognate'' protein.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.     

Identification of <Emphasis Type="Italic">DUF538</Emphasis> cDNA clone from <Emphasis Type="Italic">Celosia cristata</Emphasis> expressed sequences of nonstressed and stressed leaves

DUF538 domain-containing protein family consists of several plant proteins of unknown functions. This protein family has already been discovered by genome annotation tools and cloned as an inducible gene product under various environmental stress conditions. For the first time, we presented a full length DUF538 cDNA (encoding 170 amino acid residues) clone, which was randomly isolated from Celosia cristata leaf cDNA library constructed under normal growth conditions and consistently amplified from leaf cDNA populations prepared from nonstressed and drought-stressed leaves. We predicted that a DUF538 gene product can be a putative candidate for common stress-related protein (regulatory factor) in the plant system. The nucleotide and deduced amino acid sequences of the isolated clone have been submitted to EMBL data bases under accession no. AJ535713.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein.

We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a lambda gt10 cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and an M(r) of 25,839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carboxyl-end (213-229) consistent with an integral membrane protein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NH2-terminus (amino acids 1-33) and within the last 150 amino acids of these proteins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mitochondria or their ability to block programmed cell death.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

金黄色葡萄球菌特异性PCR检测靶点的自动化筛选

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。     

水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。     

水母雪莲黄酮合成酶FNSII基因克隆及其在3种细胞系中的表达

水母雪莲是中国传统珍稀药材,其主要活性成分之一木犀草素具有预防和治疗癌症的功效。以水母雪莲绿色细胞系为试材,采用RT-PCR和RACE-PCR方法获得1个水母雪莲FNSII基因cDNA全长,命名为SmFNSII(GenBank登录号为KF170286)。序列分析结果表明:SmFNSII全长1 710 bp,包含34 bp的5?非编码区、125 bp的3?非编码区和1个长度为1 551 bp编码516个氨基酸的开放阅读框。氨基酸序列分析表明SmFNSII属于细胞色素P450 CYP93B亚家族单氧化酶。序列比对和系统进化分析表明,SmFNSII氨基酸序列与同科植物毛山柳菊的亲缘关系最近,相似性达87%以上。实时荧光定量PCR分析表明,SmFNSII在白色、绿色和红色细胞系均有表达,红色系中的表达量最高,白色系中表达量最低,此结果与高效液相色谱分析3种颜色细胞系中木犀草素含量结果一致。构建pET-SmFNSII原核表达重组质粒,并在大肠杆菌中诱导表达,诱导蛋白大小与预期一致。通过筛选FNSII基因表达水平高、高产木犀草素的水母雪莲细胞系和植株,可培育抗炎抗癌活性较高、具有高保健功能的雪莲生药新品种。     

Characterization and SNP variation analysis of a HSP70 gene from miiuy croaker and its expression as related to bacterial challenge and heat shock

Heat shock proteins (HSPs) play crucial roles in the immune response of vertebrates. In order to study immune defense mechanism of heat shock protein gene in miiuy croaker (Miichthys miiuy), a cDNA encoding heat shock protein 70 (designated Mimi-HSP70) gene was cloned from miiuy croaker. The cDNA was 2195?bp in length, consisting of an open reading frame (ORF) of 1917?bp encoding a polypeptide of 638 amino acids with estimated molecular mass of 70.3?kDa and theoretical isoelectric point of 5.55. Genomic DNA structure analysis revealed that the Mimi-HSP70 gene contain no introns in coding region and four SNPs with 373?C/T, 789?G/A, 1005?C/T, and 1185?G/A were detected by direct sequencing of 20 samples from six different populations. BLAST analysis, structure comparison and phylogenetic analysis indicated that Mimi-HSP70 should be an inducible cytosolic member of the HSP70 family. The deduced amino acid sequence of Mimi-HSP70 had 82.4%-92.2% identity with those of vertebrate. A real-time quantitative RT-PCR demonstrated that the HSP70 gene was ubiquitously expressed in ten normal tissues. Under different temperature shock stress, the expression of Mimi-HSP70 gene in miiuy croaker increased at first and then decreased with the rise of temperature, finally, reached a maximum level in liver, spleen and kidney tissues. Infection of miiuy croaker with Vibrio anguillarum resulted in significant changes expression of Mimi-HSP70 gene in the immune-related tissues. These results indicated that expression analysis of Mimi-HSP70 gene provide theoretical basis to further study the mechanism of anti-adverseness in the miiuy croaker.     

水母雪莲Myb转录调控因子SmP基因的克隆及序列分析

采用TDPCR(Touch down PCR)法从水母雪莲(Saussurea medusa Maxim)红色系愈伤组织cDNA文库中筛选并克隆了雪莲Myb转录调控因子SmP (S.medusa Maxim Mybrelated P gene)基因。序列分析表明该基因全长969bp,包括一个771bp的完整开放阅读框架（ORF）,编码一个256氨基酸残基的蛋白质。氨基酸序列的同源性分析表明在N-端具有两个典型的R2R3-Myb-DNA结合结构域。C-端富含亲水的丝氨酸S(18.38%),且以寡聚体的形式存在,具有转录调控因子激活结构域常见的特征。     

锯缘青蟹(Scylla serrata)Hsp70基因cDNA的克隆及序列分析

采用RT-PCR及RACE技术克隆锯缘青蟹(Scylla serrata)的热休克蛋白Hsp70基因并进行序列分析。克隆测序后拼接得到一条长2482 bp的cDNA序列,该序列ORF(Open reading frame,开放阅读框)为1950 bp,编码649个氨基酸,分子量约为71.06 kD,理论等电点为5.24。3'UTR(untranslated region,非编码区)为158 bp,5'UTR为40 bp。通过antheprot分析发现2个Hsp70家族的签名序列:IFDLGGGTFDVSIL,IVLVGGSTRIPKIQK;Dnak特征基序DLGTT-S-V;非细胞器基序:RARFEEL;核定位信号标签:KKDPSESKRALRRL;胞质Hsp70特征基序EEVD。同源性分析表明,锯缘青蟹Hsp70编码区核苷酸序列与凡纳滨对虾(Litopenaeus vannamei)、斑节对虾(Penaeus monodon)、罗氏沼虾(Macrobrachium rosenbergii)的相似性分别为84.02%、83.87%和79.60%;核苷酸序列所推导出的Hsp70氨基酸序列,与凡纳滨对虾、斑节对虾和罗氏沼虾的相似性分别为92.79%、92.17%和96.47%。本研究克隆了锯缘青蟹Hsp70基因,为进一步深入研究锯缘青蟹的抗逆机理及其遗传改良奠定了基础。