GDA2基因的克隆、原核表达与融合蛋白的纯化

摘要：GDA2（G2 pea dark accumulated gene）是与G2豌豆（Pisum sativum L.）短日照条件下不衰老现象紧密相关的基因之一。实验用cDNA差示分析法（representational difference analysis, RDA）得到一个短日照下特异表达的G2豌豆cDNA，并命名为 GDA2。核酸序列分析表明，该cDNA全长1 120 bp，最大的开放阅读框为642 bp，编码一个由213个氨基酸构成的分子量约为24 kD的蛋白质，与GDA1的同源性为36%。在GDA2的cDNA两端分别引入Bgl Ⅱ与XhoⅠ的酶切位点，用PCR方法将其克隆到原核表达载体pGEX-4T-1中，经过酶切筛选和测序鉴定，得到所需的表达载体pGEX-GDA2。将pGEX-GDA2导入E.coli BL21菌株，经IPTG诱导，得到分子量约51 kD的 GST-GDA2融合蛋白，并利用Glutathione Sepharose 4B亲和柱对该融合蛋白加以纯化。GDA2-GST融合蛋白的表达和纯化工作，为深入研究GDA2蛋白的结构与功能以及该基因同衰老的关系打下良好的基础。     

甘薯肌醇-1-磷酸合成酶基因的克隆及序列分析

甘薯(Ipomoea batatas (L.) Lam.)新品系农大603是从感茎线虫(Ditylenchus destructor)病品种徐薯18的辐照后代中获得的一个抗茎线虫病的突变体。以农大603和徐薯18块根的mRNA为模板，根据植物抗线虫病基因NBS保守氨基酸序列设计引物，进行 RT-PCR分析，发现农大603的肌醇-1-磷酸合成酶(Myo inositol-1-phosphate synthase , MIPS)基因的表达量高于徐薯18。采用3'RACE技术扩增出MIPS基因的3'末端cDNA。根据植物MIPS基因 5'端一段保守的氨基酸序列设计兼并引物，并与3'端的特异引物组合，扩增出该基因的5'端cDNA序列。DNA序列比对表明，甘薯MIPS基因与大豆(Glycine max)、番茄(Lycopersicon esculentum )的MIPS基因同源性较高，分别达83.63 %和83.89 %。甘薯MIPS基因全长cDNA的克隆，有利于进一步研究该基因与抗甘薯茎线虫病的关系。     

cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.     

Jug r 4, a legumin group food allergen from walnut (Juglans regia Cv. Chandler)

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.     

Evaluation of the phenolic content in the aerial parts of different varieties of Cichorium intybus L

Fresh aerial parts of different chicory varieties: green chicory (c.v. "Catalogna"), two red chicory varieties ("radicchio rosso di Chioggia" and "radicchio rosso di Treviso"), and Witloof or Belgian endive were analyzed by HPLC/DAD/MS. The chromatographic fingerprint was diagnostic for each variety. A monocaffeoyl tartaric acid, chlorogenic acid, and chicoric acid were detected in all the varieties, while cyanidin 3-O-glucoside, delphinidin 3-O-(6' malonyl) glucoside, and cyanidin 3-O-(6' malonyl) glucoside were the main phenolic compounds in the red varieties. The flavonoidic compounds, quercetin 3-O-glucuronide and luteolin 7-O-glucuronide, were absent in the Witloof sample. The phenolic compounds from total leaves were the same as those obtained from only the colored parts; nevertheless, the total amount was remarkably lower with a decrease of up to 80% for Belgian endive. Chemical stability at high temperature was observed for the phenolic fraction from the green variety after decoction at 100 degrees C for 30 min.     

Marine sponge: a potential source for methoxylated polybrominated diphenyl ethers in the Asia-Pacific food web

Marine sponges collected in Palau, Micronesia, were investigated for hydroxylated or methoxylated analogues of brominated diphenyl ethers (BDEs), brominated dibenzo-p-dioxin (BDD), and brominated biphenyls. The neutral fractions of Haliclona sp. and Callyspongia sp. contained 2'-methoxy-2,3',4,5'-tetraBDE, 6-methoxy-2,2',4,4'-tetraBDE, 2',6-dimethoxy-2,3',4,5-tetraBDE 2,2'-dimethoxy-3,3',5,5'-tetrabromobiphenyl, several methoxy-triBDEs, and dimethoxy-penta-/hexaBDEs. The methoxylated BDEs in sponges were strikingly similar to those of local fish living in the western Pacific Ocean. The total concentrations of these compounds (ΣMeO-PBDE) in both sponges were 63.5 μg/g extractable organic matter (EOM) for Haliclona sp. and 36.5 μg/g EOM for Callyspongia sp., which were about 2 orders of magnitude higher than the levels seen in tropical coral reef fish (unicornfish or surgeonfish) (280-290 ng/g lipid) and groupers (550 ng/g lipid) from Okinawan coastal waters. The phenolic fractions of both sponges contained hydroxy-methoxy tetra-/pentaBDEs as well as hydroxy-tetraBDD, in addition to the corresponding phenolic tetraBDE analogues. Although the total concentrations of phenolic products (27-80 μg/g EOM) in both sponges fell within a range comparable to the methoxylated products, ΣOH-PBDE in local fish were trace level (less than 10 ng/g lipid of) or undetectable. This survey indicates that marine sponges are a possible source of the MeO-PBDE analogues that biomagnify via the food chain to the higher trophic organisms in the western Pacific, whereas the distribution of the corresponding hydroxylated analogues is limited.     

Characterization of a glutenin-specific serine proteinase of Sunn bug Eurygaster integricepts Put

Glutenin hydrolyzing proteinases (GHPs) have been purified, by affinity chromatography, from wheat seeds damaged by the Sunn bug Eurygaster integriceps (Hemiptera, Scutelleridae). A 28 kDa protein was partially sequenced by mass spectrometry and Edman degradation which showed homology to serine proteases from various insects. Three full length clones were obtained from cDNA isolated from Sunn bug salivary glands using degenerate PCR based on the sequences obtained. The cleavage site of the protease was determined using recombinant and synthetic peptides and shown to be between the consensus hexapeptide and nonapeptide repeat motifs present in the high molecular weight subunits of wheat glutenin (PGQGQQ∧GYYPTSLQQ). Homology models were generated for the three proteinases identified in this study using the high resolution X-ray structure of a crayfish (Pontastacus leptodactylus) trypsin complexed with a peptide inhibitor as template (PDB accession 2F91). The novel specificity of this protease may find applications in both fundamental and applied studies.     

Identification and antioxidant potential of flavonoids and low molecular weight phenols in olive cultivar chemlali growing in Tunisia

Increasing interest in phenolic compounds in olives is due to their antioxidant and health-enhancing properties. In this study the phenolics in fruits of the Tunisian olive cultivar Chemlali were extracted by methanol-water and fractionated using Sephadex LH-20 column chromatography. The identification of phenolic monomers and flavonoids was based on separation by high-performance liquid chromatography equipped with a diode array detector followed by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analysis. Oleuropein, a secoiridoid glycoside esterified with a phenolic acid, was the major compound. Eight phenolic monomers and 12 flavonoids were also identified in Chemlali olives. Five flavonoids were isolated and purified using Sephadex LH-20 column chromatography and preparative paper chromatography. The antioxidant activity of the extract and the purified compounds was evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and by using the beta-carotene-linoleate model assay. Acid hydrolysis of the extract enhanced its antioxidant activity. Hydroxytyrosol and quercetin showed antioxidant activities similar to that of 2,6-di-tert-butyl-4-methylphenol. A hydroxyl group at the ortho position at 3' on the B ring of the flavonoid nucleus could contribute to the antioxidant activity of the flavonoids.     

茶树黄酮醇合成酶基因的克隆与原核表达

本研究采用EST测序技术和RT—PCR技术,获得了一个茶树茶多酚代谢中的重要基因——黄酮醇合成酶（FLS）基因,在GenBank登录（GenBank accession No．EF205150）,其序列全长1317bp,其中开放阅读框长996bp,编码331个氨基酸,3]端有一个明显的多聚腺苷酸加尾信号,推测的蛋白分子量约为37．5kD,理论等电点为5．80。序列分析表明它与葡萄FLS基因序列的亲缘关系比较近。将该基因重组到表达载体pET-32a（＋）中进行原核表达,经IPTG诱导、SDS-PAGE检测,结果表明茶树黄酮醇合成酶基因能在大肠杆菌BL21中表达,电泳检测到一条大约61kD的外源蛋白,与预测的融合蛋白分子量相符。用Ni-NTA亲和层析柱对融合蛋白进行纯化,得到了纯度在90%以上的纯化蛋白,为进一步研究PET-FLS融合蛋白的活性及功能奠定了基础。     

高羊茅硝态氮转运蛋白基因NRT1.1的克隆及表达模式分析

为探究高羊茅(Festuca arundinacea)硝态氮转运蛋白基因(NRT1.1)的表达模式,本研究以黔草1号高羊茅为试验材料,采用RACE和RT-qPCR技术对高羊茅NRT1.1基因的cDNA全长序列进行扩增,并对其不同胁迫处理下的表达情况进行分析。生物信息学分析发现,高羊茅NRT1.1的理论等电点为4.81,平均亲小性为0.919,含有约32.63% α-螺旋、7.63% β-转角和53.73%不规则卷曲。结果表明,NRT1.1基因的cDNA序列全长为2 328 bp,编码606个氨基酸,预测蛋白质分子量为193.9 kDa,且高羊茅NRT1.1与黑麦草NRT1.1氨基酸序列的相似性最高。RT-qPCR表达分析发现,高羊茅叶片NRT1.1受低氮处理0.5~1 h时表达量达到峰值,显著(P< 0.05)高于对照组;在干旱和热处理下,NRT1.1表达量分别在6 h和12 h时达到峰值,且显著(P< 0.05)高于对照组;在盐处理下,仅在6 h时NRT1.1表达量高于对照组,其余时间均受显著(P< 0.05)抑制。本研究结果为解析高羊茅NRT1.1基因的表达模式提供了分子生物学基础。     

茶树生长素抑制蛋白基因CsARP1的克隆与表达分析

从茶树休眠芽抑制消减杂交文库中分离得到生长素抑制蛋白基因的3＇-片段,以休眠芽为材料,利用RACE技术克隆了其cDNA全长,并利用荧光定量PCR研究了该基因在不同休眠阶段芽的相对表达量。结果从茶树休眠芽中获得一个全长为711bp的生长素抑制蛋白基因CsARP1（GenBank登录号为HQ225758）。该基因开放阅读框...     

猪肌肉生长抑制素(MSTN)前肽定点突变载体的构建及其原核表达

肌肉生长抑制素(MSTN)能够限制肌纤维的增粗增大,而突变的肌肉生长抑制素前肽(Pro-MSTN-D75A)能阻止MSTN与相应受体的结合,从而解除MSTN对肌纤维的抑制作用。猪是我国最重要的肉用家畜,通过操作MSTN提高其瘦肉产量,将对我国生猪生产具有特殊意义。本研究用通城猪妊娠3个月的胚胎为材料,提取背最长肌RNA并以此为模板,用反转录PCR扩增猪MSTN前肽cDNA序列(不含信号肽),并将其连接到原核表达载体pGEX-6p-1上。通过定点突变将编码天冬氨酸的密码子变为丙氨酸密码子(D75A),构建了原核表达载体pGEX-ProM(SP-)和pGEX-ProM(SP-)D75A。重组质粒转化大肠杆菌(Escherichia coli)BL21感受态细胞并经IPTG诱导表达,结果表明,转化pGEX-ProM(SP-)质粒的E.coliBL21工程菌用终浓度为0.6mmol/L的IPTG诱导5h表达情况最好;而转化pGEX-ProM(SP-)D75A的工程菌在1mmol/L的IPTG诱导6h的条件下表达情况最好。用GST-Trap蛋白纯化系统纯化回收总分子量大小约为51kD融合蛋白(GST蛋白标签分子量为26kD,MSTN前肽蛋白分子量为25kD)。纯化蛋白的浓度ProM(-SP)为1.87mg/mL,ProM(-SP)D75A为1.21mg/mL。本研究建立了大肠杆菌表达猪MSTN前肽的最佳条件,提高了生产效率,并为研究MSTN前肽在动物体内的生物学功能以及制备单克隆抗体提供基础资料。     

甘薯茎线虫(Ditylenchus destructor)β-1,4内切葡聚糖酶基因(Dd-eng-1b)cDNA全长的克隆与序列分析

β-1,4内切葡聚糖酶是植物寄生线虫口针分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用。本文以甘薯茎线虫为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b (GenBank登录号为FJ430142)。此cDNA全长序列为1640bp,包括一个1443bp的完整ORF,编码一含481个氨基酸的蛋白,其理论分子量与等电点分别为50.89KD,pI为6.94。序列比对分析表明含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBDⅡ)。cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36bp、76bp、187bp和344bp,切割点符合5‘-GT...AG-3’的规律。系统进化树分析表明,该基因与细菌 Bacillus.subtilis和Erwina carotovora 分泌的纤维素酶属于同一支。     

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.     

麦长管蚜嗅觉相关蛋白Gqα基因的克隆及真核表达

根据已报道无脊椎动物嗅觉相关蛋白Gqα的氨基酸序列保守区设计简并引物,以麦长管蚜(Sitobion avenae)有翅成蚜cDNA为模板,结合RT-PCR及RACE技术扩增编码麦长管蚜Gqα蛋白的cDNA序列,其cDNA序列开放阅读框长为 1 062 bp,编码352个氨基酸,预测蛋白分子量为40.8 kD,与GenBank多个物种Gqα cDNA序列及氨基酸序列均有很高相似性(≥82.17%)(GenBank登录号为EF638906),并具有Gα亚基q类蛋白的典型特征。利用TOPO及Gateway技术,通过体外重组反应,构建苜蓿丫纹夜蛾核型多角体杆状病毒(Autographa californica nuclear polyhedrosis virus, AcMNPV)重组病毒。将N端融合了V5-His6标记的V5-His6Gqα序列整合到AcMNPV病毒基因组DNA中, 得到具有独立转染活性的重组杆状病毒。转染粉纹夜蛾(Trichoplusia ni)离体细胞系Tn-5B1-4(Tn细胞),进行Gqα蛋白的真核表达。SDS-PAGE检测到预期分子量约42 kD的蛋白带,Western blot检测到表达产物。表明含有6个组氨酸标签及V5抗体结合位点的Gqα融合蛋白V5-His6Gqα在昆虫离体细胞系中成功地表达。     

Anti-inflammatory effects of phenolic compounds isolated from the fruits of Artocarpus heterophyllus

Artocarpus heterophyllus Lam is a large evergreen tree cultivated throughout Southeast Asia for its fruits. Its leaves and roots have been used for medicinal purposes. The aim of this work was to study the in vitro anti-inflammatory effects of phenolic compounds isolated from the ethyl acetate extracts of the fruits of Artocarpus heterophyllus. Three phenolic compounds were characterized as artocarpesin [5,7,2',4'-tetrahydroxy-6-(3-methylbut-3-enyl) flavone] ( 1), norartocarpetin (5,7,2',4'-tetrahydroxyflavone) ( 2), and oxyresveratrol [ trans-2,4,3',5'-tetrahydroxystilbene] ( 3) by spectroscopic methods and through comparison with data reported in the literatures. The anti-inflammatory effects of the isolated compounds ( 1- 3) were evaluated by determining their inhibitory effects on the production of proinflammatory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. These three compounds exhibited potent anti-inflammatory activity. The results indicated that artocarpesin ( 1) suppressed the LPS-induced production of nitric oxide (NO) and prostaglandin E 2 (PGE 2) through the down-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions. Thus, artocarpesin ( 1) may provide a potential therapeutic approach for inflammation-associated disorders.