Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.     

Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability

A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. Imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.     

碱茅（Puccinellia tenuifolra）Put-Cu／Zn—SOD基因的克隆及在酵母中的表达

从碱茅根cDNA文库分离得到Put－Cu／Zn—SOD全长cDNA,其序列全长为2700bp,该基因的开放读码框为长615bp,所编码的蛋白由204个氨基酸组成,其预测分子量约为20．6kD、理论等电点约为5．63。Put－Cu／Zn—SOD氨基酸序列与水稻Cu／Zn-SOD序列有较高的同源性为87％。将Put-Cu／Zn—SOD基因构建到酵母表达载体pYES2,并转化至酵母（INVSc1）。对pYES2-Put-Cu／Zn—SOD转化酵母进行盐碱、氧化胁迫实验。结果表明：重组酵母的抗盐碱、氧化能力明显高于对照（pYES2转化酵母）,结果显示了碱茅的Cu／Zn—SOD基因在酵母表达中,具有提高酵母抗盐碱和耐氧化能力。     

Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio)

A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequence showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.     

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.     

棉花肉桂醇脱氢酶基因GhCAD3的克隆及原核表达

肉桂醇脱氢酶(CAD)是木质素生物合成过程中的一个关键酶类。本研究从棉花中克隆了一个CAD基因,命名为GhCAD3(GenBank登录号为FJ376601)。GhCAD3全长1573 bp,具有1个1080 bp的开放阅读框,5′非编码区为35 bp,3′非编码区为458 bp,编码359个氨基酸,预测分子量约为39.116kD,等电点为7.48。氨基酸同源性分析发现,GhCAD3与其他CAD一致性为64.13%。为了进一步研究GhCAD3基因的功能,构建了该基因的原核表达载体pET-28a-CAD3,经酶切鉴定后转化到大肠杆菌BL21(DE3)中。SDS-PAGE电泳分析表明,最佳诱导表达条件为0.5 mmol/L IPTG在37℃下诱导7 h。     

甘薯茎线虫乙酰胆碱酯酶基因ace-3全长cDNA的克隆和序列分析

利用RT- PCR结合RACE技术克隆了甘薯茎线虫（Ditylenchus destructor）乙酰胆碱酯酶基因cDNA,Dd-ace-3,提交GenBank登录号EF583057。该cDNA序列5’端存在反式剪接引导序列SL1,全长2517bp ,包含一个1836bp的开放阅读框;在推导出的611个氨基酸残基的前体蛋白中, N 端的前21个氨基酸残基为信号肽, C-28的位置存在糖基磷脂酰肌醇(GPI)锚定位点,除此之外的562个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为64396.81D。在一级结构中,形成催化活性中心的3个氨基酸残基 (Ser232 ,Glu360 和His479) ,以及在亚基内形成二硫键的6个半胱氨酸完全保守;在电鳐乙酰胆碱酯酶分子的催化功能域中存在14个保守的芳香族氨基酸残基,其中11个在甘薯茎线虫乙酰胆碱酯酶中完全保守。该酶的氨基酸序列与秀丽小杆线虫（Caenorhabditis elegans）ACE-3和ACE-4的同源性达56.1%和50.0%。与其它线虫和物种乙酰胆碱酯酶的聚类分析显示,甘薯茎线虫的乙酰胆碱酯酶与Ш型乙酰胆碱酯酶ACE-3同属一个支系。     

Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.     

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.     

编码日本梨树(Pyrus pyrifolia )花粉类β-1,3-葡聚糖酶蛋白的cDNA克隆与核苷酸序列

构建了日本梨树(Pyrus pyrifolia)花粉cDNA文库,发现了一个编码花粉管分泌的类β-1,3-葡聚糖酶蛋白(BGN-1)的cDNA(bgn-1)并测定了其序列.bgn-1由1408 bp组成,包括47 bp的5'-非翻译区,由1194bp组成并编码了397个氨基酸的ORF和167 bp的3'-非翻译区.3'-非翻译区含有1个NUE(AATTAA)和3个似FUE的基序,分别位于1369～1374(AATTAA)、1320～1325、1327～1332(TTTTTA)和1363～1368(TTTGGA).bgn-1编码的蛋白(BGN-1)与DDBJ中的植物β-1,3-葡聚糖酶的序列相似性范围为37%～59%,与3D结构已知的大麦β-1,3-葡聚糖酶(GⅡ)的序列相似性为39.7%.对GⅡ与BGN-1进行的疏水簇分析(HCA同源分为87.1%,＞75%的一般标准)和使用3D-PSSM软件进行的分析暗示,BGN-1的3D结构与大麦GⅡ同源性最大(≥95%的肯定度).BGN-1的信号肽长度为22 aa,成熟的BGN-1(375 aa)的理论分子量为4 0723 D,理论pI值为9.59,诊断氨基酸残基模式(D,L,S,L)与禾谷类的与生长相关的亚家族D的诊断氨基酸残基模式(D,L,S,Q)极相似.在BGN-1的C-端延伸区的位点352～367之间发现了1个功能不清,重复出现的ProXXPro结构.     

总状毛霉(Mucor racemosus)甲壳素脱乙酰酶全长cDNA的克隆及序列分析

用保守的特异引物进行PCR扩增,结合RACE技术,分离和克隆到了总状毛霉(Mucorracemosus)甲壳素脱乙酰酶(CDA)的全长cDNA,并进行了全序列测定,提交GenBank登陆号DQ538514。研究结果表明:(1)总状毛霉CDA基因全长为1506bp,包括67bp5'非翻译区,1344bp阅读框以及95bp3'非翻译区,3'非翻译区包含Poly(A)加尾信号AATAAA。总状毛霉的CDA基因共编码448个氨基酸,在该基因中部还包含一个144氨基酸的多糖脱乙酰酶结构域,约占CDA基因全长的32%。(2)总状毛霉CDA基因与其它相近种米根霉(Rhizopusoryzae)、卷柄根霉(Rhizopuscircinans)的CDA1和CDA2、鲁氏毛霉(Mucorrouxii)、卵形孢球托霉(Gongronellabutleri)、匍枝根霉(Rhizopusstolonifer)、布拉克须霉(Phycomycesblakesleeanus)和酿酒酵母(Saccharomycescerevisiae)的CDA1和CDA2的基因序列同源性分别为:75%、58%、56%、56%、48%、39%、39%、17%和16%;相应的氨基酸序列的同源性分别为:69%、57%、59%、55%、47%、30%、32%、18%和21%。表明CDA基因在不同的真菌中有着不同的亲缘关系。(3)根据总状毛霉CDA基因的氨基酸序列构建的不同真菌的系统树,与采用经典分类法构建的系统树基本一致。(4)通过生物信息学的方法,预测该基因所编码的蛋白质三级结构,验证了该蛋白质具有甲壳素脱乙酰酶完整的功能性结构,并包含一个多糖脱乙酰酶结构域,两者具有相似的空间结构。     

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.     

杨梅铜锌超氧化物歧化酶基因（MrCu／Zn-SOD1）的原核表达及活性鉴定

为获得高表达杨梅（Morellarubra）铜锌超氧化物歧化酶（MrCu／Zn-SOD1）的工程菌,本实验采用RT-PCR技术从杨梅果实中分离扩增了MrCu／Zn．SOD1的cDNA序列（456bp）,将该基因重组到原核表达载体pGEX-2T中,酶切、测序分析表明,重组质粒pGEX-MrCu/Zn-SOD1结构正确。重组质粒转化大肠杆菌BL21（DE3）进行诱导表达。IPTG诱导表达分子量约41kD融合蛋白GST．MrCu／Zn．SOD1。诱导表达后的菌体超声裂解液经谷胱甘肽亲和层析纯化,得到高纯度的GST—MrCu／Zn—SOD1。采用氯化硝基四氮唑蓝法和黄嘌呤氧化法分析其活性。结果表明,GST-MrCu／Zn-SOD1具有特异性SOD酶活性。     

Expression and characterization of sweet potato invertase in Pichia pastoris

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.     

紫杆柽柳谷胱甘肽硫转移酶基因的克隆及功能鉴定

根据GenBank已公布的植物谷胱甘肽硫转移酶基因EST序列，设计引物利用3’和5’RACE方法，获得紫杆柽柳GST基因(TaGST)全长cDNA序列，全长为1175 bp，开放读码区672 bp, 编码224个氨基酸。其所编码的蛋白与大豆的GST10同源性最高为48%。利用pET-28a（+）构建了紫杆柽柳GST基因的原核表达载体，转入BL21大肠杆菌进行了原核表达。诱导表达蛋白的SDS-PAGE检测表明, 所表达的蛋白分子量约为26 kD，与预测的分子量大小一致。初步的酶学活性的检测表明所克隆的基因编码的肽段具有催化GST蛋白通用底物CDNB和GSH反应的活性。     

诸葛菜OvCYP86MF基因的克隆及其特性分析

为进一步阐明细胞色素P450基因CYP86MF在诸葛菜发育中的分子机理,本文根据已知同源基因保守序列设计特异引物,利用RT-PCR和RACE技术从诸葛菜中获得一个细胞色素P450基因(OvCYP86MF)全长cDNA序列,该序列全长为1876bp,含有1605bp的完整开放阅读框,可编码534个氨基酸,分子量和等电点分别为61.4kDa和6.90,具有细胞色素P450蛋白的典型特征,即保守结构域FNAGPRLCIG;原核表达显示该基因的融合蛋白在体外可以诱导表达;DANSTAR和Clustal W软件分析表明该基因的全长cDNA序列及其编码氨基酸序列与十字花科物种拟南芥相似性很高,达到80%以上,亲缘关系最近;与CYP86C亚家族成员在氨基酸水平上的相似性均高于50%,因此推断该基因属于CYP86C这个亚家族。Northern杂交分析表明该基因在花蕾中特异表达。