美洲大蠊3-磷酸甘油醛脱氢酶基因的克隆及表达

旨在通过5’-RACE获得美洲大蠊3-磷酸甘油醛脱氢酶(GAPDH)基因的全长cDNA序列,进行生物信息学分析,构建原核表达载体,诱导重组蛋白表达,为进一步研究其功能奠定基础.通过3’-RACE技术,PCR扩增获取编码美洲大蠊GAPDH蛋白的全长cDNA序列;采用生物信息学方法推导出该序列编码的氨基酸序列及其理化性质;预测信号肽、蛋白疏水性、可溶性、跨膜区结构、二级结构、三级结构,并构建系统发育树;构建原核表达载体pET28a-GAPDH,IPTG诱导重组蛋白表达,并用Histag抗体Western blotting验证.结果显示,美洲大蠊GAPDH基因,其完整阅读框含999个碱基,编码332个氨基酸.序列分析显示,该蛋白与家蚕GAPDH相似性为89％,具有GAPDH保守功能域,经IPTG诱导获得重组蛋白.     

Purification,properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults

A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.     

Cloning of vitellogenin cDNA of the American cockroach, Periplaneta americana (Dictyoptera), and its structural and expression analyses

A cDNA expression library constructed from poly (A)(+) RNA prepared from vitellogenic female fat body cells of the American cockroach, Periplaneta americana (Dictyoptera) was screened using a polyclonal antiserum against the 100-kD polypeptide(s) from the egg extract. A partial Vg cDNA clone was obtained and sequenced. The 5' end portion of the cDNA was then obtained by the RACE method, cloned, and sequenced. The combined complete Vg cDNA was 5,854 bp long and contained a single ORF encoding 1,896 amino acids. The entire deduced amino acid sequence was aligned confidently with those of the known insect Vgs. A GL/ICG motif, a number of cysteines at conserved locations following this motif, and a DGXR motif upstream of the GL/ICG motif were present near the C-terminal. The chemically determined N-terminal amino acid sequence of the 170-kD polypeptide from the egg extract completely matched the deduced sequence starting from just after one of the consensus (RXXR) cleavage sites, indicating the occurrence of post-translational cleavage in the fat body cells. The Vg gene begins to be expressed in the 2-day-old adult female fat body cells but is never expressed in ovaries or in male fat body cells. Hemolymph Vg was first detected by immunoblotting in 4-day-old adult females, 2 days after the beginning of gene expression. Western blot analysis of major yolk polypeptides in nine cockroach species belonging to the two superfamilies, Blattoidea and Blaberoidea, using the antisera against P. americana major yolk polypeptides showed that the similarities in Vn antigenicity are basically limited to within a superfamily.     

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.     

Tubular bodies in dendritic outer segments projecting to second embryonic cuticle from anlagen of contact chemoreceptors in Locusta migratoria L. (Orthoptera : Acrididae) and Periplaneta americana (L.) (Dictyoptera : Blattidae)

Bundles of dendritic outer segments project to the 2nd embryonic cuticle from anlagen of contact chemoreceptors in the antennae of 9-day embryos (80–85% stage) of Locusta migratoria L. (Orthopteroidea) and in the maxillary palps of 24-day embryos (80% stage) of Periplaneta americana (L.) (Blattodea). The dendrite sheaths fuse with irregular nodular thickenings in the 2nd embryonic cuticle in Locusta; in Periplaneta the sheaths are joined to it by filaments. In both species one of the dendritic outer segments shows a dilation within which a tubular body is contained. This tubular body is located in Locusta at the nodular thickening where the dendrite sheath is connected to the 2nd embryonic cuticle. In Periplaneta, the tubular body was observed near the junction between the dendrite sheath and the cuticle in most cases, but at some distance to the cuticle in others. The occurrence of a tubular body is discussed with reference to mechanoreceptive capacities of the sensory cells and to the phylogeny of the 2nd embryonic cuticle.     

Fate of a bacteriophage in Aedes aegypti, Anopheles quadrimaculatus (Diptera: Culicidae), and Periplaneta americana (Orthoptera: Blattidae)

The viability of a bacteriophage of Escherichia coli was unaffected by injection into the hemocoel of the mosquitoes, Aedes aegypti and Anopheles quadrimaculatus, but was reduced by injection into the cockroach, Periplaneta americana. Treatment of the cockroach with India ink, known to be phagocytized in the cockroach hemocoel, did not block the reduction of phage viability. Phage viability was unaffected by incubation with gut homogenates of A. aegypti but was possibly affected by homogenates of P. americana.     

Molecular Cloning, Expression and Subcellular Localization of a BiP Homolog from Rice Endosperm Tissue

The ER luminal binding protein, BiP, has been linked to prolamineprotein body formation in rice. To obtain further informationon the possible role of this chaperone in protein body formationwe have cloned and sequenced a BiP cDNA homolog from rice endosperm.The rice sequence is very similar to the maize BiP exhibiting92% nu-cleotide identity and 96% deduced amino acid sequenceidentity in the coding region. Substantial amino acid sequencehomology exists between rice BiP and BiP homo-logs from severalother plant and animal species including long stretches of conservationthrough the amino-terminal ATPase domain. Considerable variation,however, is observed within the putative carboxy-terminal peptide-bind-ingdomain between the plant and nonplant BiP sequences. A singleband of approximately 2.4 kb was visible when RNA gel blotsof total RNA purified from seed tissue were probed with radiolabeledrice BiP cDNA. This band increased in intensity during seeddevelopment up to 10 days after flowering, and then decreasedgradually until seed maturity. Protein gel blots indicated thatBiP polypeptide accumulation parallels that of the prolaminepolypeptides throughout seed development. Immunocytochemicalanalysis demonstrated that BiP is localized in a non-stochasticfashion in the endoplasmic reticulum membrane complex of developingendosperm cells. It is abundant on the periphery of the proteininclusion body but not in the central portion of the proteinbody or in the cisternal ER membranes connecting the proteinbodies. These data support a model which proposes that BiP associateswith the newly synthesized prolamine polypeptide to facilitateits folding and assembly into a protein inclusion body, andis then recycled. (Received October 21, 1996; Accepted January 20, 1997)     

粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究

目的:克隆粘质沙雷氏菌脂肪酶基因(lipA)使其在大肠杆菌B121(DE3)中实现高效表达,并对重组酶进行酶学性质研究.方法:以产脂肪酶粘质沙雷氏菌总DNA为模板,PCR扩增脂肪酶基因lipA,构建重组表达载体pET-lipA,并将其导入大肠杆菌进行诱导表达,对表达产物进行SDS-PAGE和酶学性质的测定.结果:经过优化培养条件,脂肪酶活力最高能达到104U/mL.重组脂肪酶的最适反应温度为40～45℃,最适pH为7.0～7.5,在50℃保温1h下仍能保持80%的酶活力,Ca2+、Sr2+、Mn2+和Mg2+对脂肪酶酶活有较强的激活作用,尤其是Ca2+使脂肪酶酶活提高了1倍多,而Ni2+、Fe2+、Fe3+、Cu2+、Zn2+和Al3+对酶活具有较强的抑制作用,尤其是Zn2+和Al3+使酶活力几乎完全丧失.该酶对一些有机溶剂有较好的耐受性,与50%甲醇混合24h,仍能保持84%的酶活力.结论:该脂肪酶具有较好的热稳定性和甲醇耐受力,作为生产生物柴油的催化剂具有很大的应用价值,为基因工程酶法生产生物柴油打下良好的基础.     

Characterization and purification of polymorphic arylalkylamine N-acetyltransferase from the American cockroach, Periplaneta americana.

We separated two forms of arylalkylamine N-acetyltransferase (AANAT) from various organs of the American cockroach, Periplaneta americana. Both forms of the enzyme had an equivalent molecular mass of 28 kDa. One form isolated from the testicular accessory glands had high enzyme activity at acidic pHs. The isoelectric point was 5-6 and the substrate specificity was wider than the other type. The other isolated form from female midguts had a higher level of enzyme activity at basic pHs. These findings suggested that P. americana contains polymorphic AANAT, as is the case in Drosophila melanogaster. These forms differed not only in pH specificity, and substrate specificity but in chromatographic behavior and kinetic properties. Most of the organs we examined contained a mixture of the two forms since two types of AANAT activity were separated in different chromatographic fractions when two pH conditions were used for activity measurement.     

Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum

A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K _m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V _max value of 46 μmol min⁻¹ mg⁻¹.     

小麦EDR1基因的克隆、鉴定和表达

为了研究普通小麦(Triticum aestivum L.)中是否有EDR1途径存在,根据拟南芥EDR1基因及其同源物设计了一对兼并性引物,用来分离小麦的EDR1同源物.以用小麦叶片RNA合成的cDNA为模板进行RT-PCR扩增,获得了代表小麦EDR1基因(命名为TaEDR1)的627 bp长的cDNA片段(GenBank登录号:AY743662).此后,通过RACE技术成功地获得了编码959个氨基酸的全长TaEDR1基因的cDNA序列.TaEDR1的氨基酸序列与大麦EDR1(标记为HvEDR1)有92%的相同.在TaEDR1的羧基末端有一个高度保守的丝氨酸/苏氨酸激酶催化功能域.因为存在一个推测的核定位基序,这个蛋白可能在细胞核中起作用.首次提供了证明普通小麦中存在EDR1同源物的分子生物学证据.用半定量RT-PCR方法研究了接种小麦白粉病菌[Blumeria graminis(DC.)E.O.Speer f.sp.tritici Em.Marchal,Bgt]后叶片中TaEDR1基因的转录谱.结果表明,在接种白粉病菌后TaEDR1基因在叶片中的转录水平提高.组织特异性表达谱分析证明,小麦TaEDR1基因在叶片、茎、穗、根中均有表达.研究提示TaEDR1可能在小麦防卫应答反应中起作用.