嗜热真菌Thermomyces lanuginosus热稳定几丁质酶基因的cDNA克隆及表达

根据Thermomyces lanuginosus热稳定几丁质酶Chit的N-端氨基酸序列和同源保守序列设计简并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该几丁质酶的编码基因chit,全长cDNA为1500bp,包含一个由442个氨基酸组成的开放阅读框。该基因已在GenBank中注册,登录号为DQ092332。将成熟肽几丁质酶Chit阅读框与酵母表达载体pPIC9K连接,构建重组质粒pPIC9K/chit,转化毕赤酵母GS115,在甲醇的诱导下,成功地分泌出具生物活性的几丁质酶,诱导6d后酶活性达2.261U/mL,酶蛋白表达量为0.36mg/mL。该酶的最适反应温度和pH值分别为60℃和5.5,该酶在50℃以下稳定;65℃的半衰期为40min。     

大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析

为了研究苯丙氨酸解氨酶基因与大蕉(Musa ABB cv. Dongguandajiao)抗枯萎病的关系,利用 RT-PCR 和 RACE技术克隆了大蕉苯丙氨酸解氨酶基因全长 cDNA。此 cDNA 长 1 300 bp,包含一个长为 1 191 bp,编码 397 个氨基酸的完整开放阅读框(ORF),推导的氨基酸序列与水稻 PAL 基因氨基酸序列同源性达 89%,将此基因命名为 M-PAL。Southern杂交结果表明大蕉中存在一个包含 4-5 个 PAL基因的基因家族,将此基因克隆到大肠杆菌表达载体 pET32(a )中,表达的蛋白质分子量大小与推导的相一致,并且表达的蛋白质表现出 PAL 酶活性。对接种香蕉枯萎病菌 4 号生理小种(Fusarium oxysporumf. sp. cubense (FOC) race 4 )后大蕉叶片中 M-PAL基因的转录谱进行研究表明,在接种枯萎病菌后,M-PAL基因在叶片中的转录水平提高,因此推测 M-PAL基因的表达可能与香蕉枯萎病抗性相关。     

水稻胞质核糖体蛋白基因OsRPS7的克隆与序列分析

以已公布的黑麦胞质核糖体蛋白基因ScRPS7的cDNA序列为信息探针,在中国华大水稻基因组数据库中搜索与之高度同源的基因组重叠群。采用计算机拼接和RT-PCR方法克隆了水稻胞质核糖体蛋白基因的全长cDNA序列,命名为OsRPS7。该cDNA序列全长919bp,编码192个氨基酸;其与黑麦、拟南芥和芸薹的S7核糖体蛋白的氨基酸一致率分别为88%、72%和72%。对OsRPS7 的基因组结构和基因的功能进行了分析和预测。Abstract：Using the cDNA of rye cytoplasmic ribosomal protein ScRPS7 as a query probe, a highly homologous rice genomic contig was obtained from Huada rice genome database. The full-length cDNA sequence of rice cytoplasmic ribosomal protein S7 was assembled by informatics based on the contig. Furthermore, with the two primers designed according to this assembled cDNA, the full-length cDNA of rice ribosomal protein was cloned by RT-PCR and named as OsRPS7. The cDNA was 919bp in length and contained a complete Open Reading Frame (ORF) of 576bp, encoding a protein of 192 amino acid residues. The deduced amino acids of OsRPS7 showed 88%、72% and 72% identity with those from Secale cereale、Arabidopsis thaliana and Brassica oleracea, respectively. The genome structure of OsRPS7 was analyzed, and its function was predicted in this paper.     

茶树肌动蛋白基因（CsActin1）全长cDNA克隆与生物信息学分析

以茶树（Camellia sinensis）萌动芽为材料,根据茶树萌动芽芽抑制消减杂交文库中分离得到的肌动蛋白（actin）基因的5′-片段设计引物,利用3′-RACE技术克隆了其cDNA全长序列,该基因cDNA全长1 470 bp,命名为CsActin1（GenBank登录号HQ235647）。序列分析表明,CsActin1开放阅读框长1 134 bp,编码377个氨基酸,5′非编码区100 bp,3′非编码区236 bp。推测的蛋白质分子量为41.70 kD,等电点约为5.31,具有肌动蛋白家族的特征信号序列（YVGDEAQs.KRG和WIAKaEYDE）和肌动蛋白相关蛋白的特征信号序列（LLTEApLNPkaNR）。CsActin1与GenBank中注册的其它植物肌动蛋白核苷酸序列的相似性在80%以上,氨基酸序列相似性在95%以上。与其它植物肌动蛋白的进化树分析结果表明,茶树肌动蛋白与杨树的两个肌动蛋白间的亲缘关系最为密切。并对推导的蛋白结构进行了分析。     

金龟子绿僵菌羧基转运蛋白基因MaJEN1及其启动子的克隆与分析

用YADE法扩增了球孢白僵菌T—DNA插入突变体T12中与T—DNA左边界相连的基因组序列。在此基础上得到了金龟子绿僵菌的羧基转运蛋白的全长cDNA,MaJEN1。MaJEN1全长1695bp,其中含有长为1524bp的开放阅读框(0RF),编码508个氨基酸的蛋白。氨基酸序列与粗糙脉孢霉和啤酒酵母菌的羧基转运蛋白JEN1相似性分别为69％和31％。采用PCR扩增得到了MaJEN1的基因组序列GMaJEN1,序列分析发现,GMaJEN1含有两个内含子。Southern杂交发现GMaJEN1在金龟子绿僵菌基因组上为单拷贝。利用RT—PCR法对MaJEN1的表达特性进行了分析,结果表明MaJEN1在蟑螂壳诱导培养基中表达,在该培养基中的表达受葡糖糖抑制。进一步采用YADE法得到了长为1626bp的GMaJEN1上游序列,其中含有可能的葡萄糖抑制调控序列。     

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca(2+)-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a 5'-terminal untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7- and 4.9-fold at 6h after injury and 8h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury healing and the immune response in A. irradians.     

疏棉状嗜热丝孢菌脂肪酶基因的克隆及其在毕赤酵母中的高效表达

疏棉状嗜热丝孢菌Thermomyces lanuginosus可产生具有重要工业生产价值的脂肪酶。根据已报道的相应序列设计特异引物,综合运用PCR、RT-PCR技术克隆到脂肪酶基因的全长DNA和cDNA序列。其中DNA序列长1071bp,包含876bp的开放阅读框以及3段内含子;cDNA序列长885bp。结构基因编码蛋白包含292个氨基酸,前17个氨基酸构成信号肽。序列提交GenBank,登录号分别为EU022703和EU370914。将脂肪酶基因cDNA序列的开放阅读框克隆到酵母分泌型表达载体pPIC9K中,转化毕赤酵母GS115得到重组子且实现了分泌表达。将重组子诱导产酶,在培养温度30℃、甲醇添加量1%的情况下,小规模发酵量达0.93mg/mL,其分泌表达的最高酶活为7.2U/mL。重组酶最适反应温度和pH分别是60℃和8.0。表达蛋白在60℃保温1h后仍有完全酶活,具有较高的热稳定性。     

Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta

We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.     

球孢白僵菌热休克蛋白基因Bbhsp70的cDNA及上游序列克隆与分析

运用SMART RACE RT-PCR技术与DNA步移技术,首次从球孢白僵菌中克隆出完整的热休克蛋白基因Bbhsp70编码区序列及上游序列。该基因cDNA全长2405bp,5′端非翻译区171bp,3′端非翻译区263bp,开放阅读框(ORF)1971bp,编码656个氨基酸。成熟蛋白理论分子量为71.3kDa,理论等电点为4.92。上游序列长度3559bp,其中有305bp序列与cDNA序列重叠。分析表明,上游序列中没有明显的TATA-盒和CAAT-盒,但含有CCAAT-bindingfactor、GC-box等重要的转录因子结合位点,以及热激应答元件(HSE)和GATA元件等启动子顺式调控元件。     

Cloning and characterization of ribonuclease T2 gene (RNHe30) from the basidiomycete,Hericium erinaceum

A gene encoding a ribonuclease T2 (RNase T2) family enzyme, RNHe30, was cloned from Hericium erinaceum by PCR. The deduced amino acid sequence from the complimentary DNA (cDNA) (1074 bp) encodes a 302-aa protein (RNase He30) that has the consensus amino acid sequences of RNase T2 family enzymes including the putative signal peptide. The presence of five introns in the genomic DNA was confirmed by comparison of the cDNA and genomic DNA sequences. The promoter region contains a putative CAAT box and a consensus TATA box. Genes coding homologous enzymes were also identified in various other basidiomycetes. A phylogenetic tree of RNase T2s from these fungi was constructed from a multiple alignment of the deduced amino acid sequences. The tree showed that the enzymes were divided into two main groups.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

The 44-kDa Regulatory Subunit of the Paramecium cAMP-Dependent Protein Kinase Lacks a Dimerization Domain and May Have a Unique Autophosphorylation Site Sequence

ABSTRACT. The 44-kDa regulatory subunit (R₄₄) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R₄₄ cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R₄₄ amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R₄₄ sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N -terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R₄₄ clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R₄₄ contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.     

Cloning and molecular analysis of cDNA encoding a carboxymethylcellulase of the yeast Cryptococcus flavus.

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.     

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.     

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.     

Molecular cloning of Na(+)-ATPase cDNA from a marine alga, Heterosigma akashiwo

We cloned novel Na(+)-ATPase (HANA) cDNA from marine alga Heterosigma akashiwo. The full-length HANA cDNA was 4467 bp long and coded for a 1330 amino acid protein with a molecular weight of 146,306. The deduced product exhibited around 40% identity in amino acids with Na(+)/K(+)-ATPase alpha-subunits. A hydrophilic sequence of 285 amino acid residues that showed no homology with any sequence listed in databases existed in the M7--M8 junction of HANA. This is the first report on the primary structure of putative Na(+)-transporting ATPase from plant cells.     

大熊猫Sjogren综合症/硬皮病自身抗原1基因(SSSCA1)的克隆及比较

硬皮病或称系统性硬化症(systemic sclerosis,SSc),又名sjogren's综合症,是一种以局限性或弥漫性皮肤及内脏器官结缔组织纤维化或硬化,最后发展至萎缩为特点的疾病.根据受累范围、程度、病程分为局限性SSc和弥漫性SSc两类,累及的内脏器官为肺脏、食管,患者常死于肺部感染、肾衰竭、心力衰竭等.     

野生茄子（Solanum torvum）抗黄萎病相关基因Sto Vel的克隆与分析

采用RT—PCR和RAcE技术从野生茄子中扩增克隆到一个抗黄萎病相关基因,命名为StoVel,其cDNA全长3400bp,含有3153bp的完整开放阅读框,编码1051个氨基酸,该基因编码的蛋白序列与刚果野茄、类番茄和番茄Vel编码的氨基酸序列同源性分别为82％、81％和80％,且有很高的功能区段保守性。将该cDNA全长序列提交Gen Bank,登陆号为DQ020574。半定量PCR表明该基因为组成型表达,在根中表达最多,叶中最少。