柞蚕14-3-3基因的鉴定及表达分析

14-3-3蛋白是一种可以改变其结合蛋白构象的酸性蛋白质.柞蚕14-3-3 cDNA序列全长1 220 bp,包括一个126 bp的5'非编码区和一个350 bp的3'非编码区.该基因的开放读码框长度为744 bp,编码247个氨基酸.序列比对结果表明,柞蚕14-3-3蛋白与家蚕的14-3-3蛋白具有高度同源性.此外对柞蚕14-3-3基因进行了原核表达和重组蛋白纯化.SDS-PAGE和免疫印迹结果表明,分子量大小约32 kD的重组蛋白在大肠杆菌中得到了成功表达.     

粘虫甘油醛-3-磷酸脱氢酶基因cDNA的克隆、序列分析及表达量检测

运用RT-PCR和RACE技术,以粘虫(Mythimna separata(Walker))cDNA为模板,对甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因进行克隆获得全长cDNA序列,并利用生物信息学方法,对GAPDH全长cDNA序列及推测得到的GAPDH蛋白序列进行分析.结果表明,获得的粘虫GAPDH基因cDNA序列长度为1 317 bp,其中包括80 bp的5′非编码区、238 bp的3′非编码区和999 bp的开放阅读框(Open Reading Frame,ORF),编码一个332个氨基酸蛋白,具有GAPDH蛋白家族的两个功能结构域.该GAPDH蛋白理论相对分子质量为35.498 6 kDa,等电点为7.63,富含6种类型的特定功能位点.该蛋白序列与其他动物GAPDH蛋白序列具有77.4%～92.9%高度同源性.GAPDH基因表达量检测结果显示GAPDH在粘虫6种不同组织间表达量无显著差异(P0.05),表明GAPDH可作为研究粘虫功能基因表达量分析的可靠内参基因.该基因的cDNA序列已经递交GenBank并获得登录号为HM055756.     

梭梭14-3-3蛋白基因HaFT-5、HaFT-6克隆及表达分析

14-3-3蛋白是生物体内重要的调节蛋白,对细胞代谢及循环、信号传导、转录调节、蛋白跨膜转运、以及生物胁迫和非生物胁迫等多种生理过程均有重要的调节作用。本研究用RT-PCR从梭梭中克隆2个14-3-3蛋白基因,分别命名为Ha FT-5、Ha FT-6。用q RT-PCR分析Ha FT-5、Ha FT-6基因在不同处理后的表达。研究显示,Ha FT-5全长501 bp,编码139个氨基酸;Ha FT-6全长647 bp,编码226个氨基酸。在干旱、高盐、ABA、IAA处理后梭梭Ha FT-5和Ha FT-6基因后呈现不同的表达模式。综上所述,这2个基因可以响应逆境胁迫和2种激素信号,本研究可为进一步探索梭梭的抗逆分子机制提供了理论基础。     

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.     

黄花苜蓿LEA3基因片段克隆与生物信息学分析

根据紫花苜蓿LEA3-1 mRNA(EU665182)的cDNA序列,设计1对特异引物,以经过ABA干旱胁迫处理的黄花苜蓿为材料,提取总RNA.采用RT-PCR方法克隆获得了747 bp的cDNA片段,命名为MfLEA3-1(GenBank登录号:HQ327439).推测该序列编码了248个氨基酸,这些氨基酸主要由10.1％苏氨酸(Thr)、24.6％丙氨酸(Ala)、17.7％赖氨酸(Lys)和12.9％天冬氨酸(Asp)组成,不含有半胱氨酸和色氨酸,这与其他物种LEA蛋白成分非常接近.该氨基酸序列中存在13个11-mer重复序列,根据第3组蛋白分类标准,MfLEA3-1蛋白比较接近于type Ⅰ,但是11-mer重复中残基分布与type Ⅰ有所不同,推测MfLEA3-1蛋白属于新的第3组LEA蛋白类型.黄花苜蓿中成功获得了LEA3基因片段,为最终克隆黄花苜蓿LEA3基因序列全长奠定基础.     

麦红吸浆虫3-羟基-3-甲基戊二酰辅酶A还原酶基因SmHMGR的克隆及在滞育与发育变态过程中的表达动态

【目的】3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是保幼激素(JH)合成途径的限速酶。麦红吸浆虫Sitodiplosis mosellana是一种典型的专性幼虫滞育昆虫。本研究旨在探讨HMGR基因在麦红吸浆虫滞育和发育变态过程中的作用。【方法】通过RT-PCR和RACE技术克隆麦红吸浆虫滞育前幼虫HMGR基因全长cDNA序列;利用生物信息学软件分析HMGR基因核苷酸和其编码的蛋白氨基酸序列特性;采用qPCR技术测定其在麦红吸浆虫滞育不同时期3龄幼虫及不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹和后蛹以及雌雄成虫)中的mRNA表达水平。【结果】克隆获得一条麦红吸浆虫HMGR基因全长cDNA序列,命名为SmHMGR(GenBank登录号: MG876766)。该基因全长2 548 bp,其中开放阅读框长2 328 bp,编码775个氨基酸,预测的蛋白分子量为84.16 kD,理论等电点为8.29。序列分析发现该基因编码的蛋白具有HMGR蛋白家族典型的HMG-CoA-reductase-classⅠ催化功能域及其他保守功能基序;序列比对和系统发育分析表明,SmHMGR与达氏按蚊Anopheles darling等长角亚目(Nematocera)昆虫HMGR的相似性最高、亲缘关系最近。SmHMGR在麦红吸浆虫滞育前的3龄早期幼虫中表达量显著升高,进入滞育后一直维持较高水平,并在滞育后静息阶段的当年12月至翌年1月达到最高。SmHMGR在蛹期表达量低于幼虫期,预蛹期表达量最低;在雌成虫中表达量显著高于在蛹和雄成虫中的表达量。【结论】SmHMGR的表达与麦红吸浆虫发育密切相关,可能在滞育诱导、维持及滞育后静息状态的维持及生殖中发挥作用,其表达量的降低可能参与了幼虫到蛹的变态。     

棕色棉F3'H-羟化酶基因的克隆与生物信息学分析

根据葡萄的类黄酮3′-羟化酶(F3'H)基因全长cDNA序列Blast所得棉花的EST序列设计引物,以开花后16 d(DPA16)的新彩棉5号(xC-5)纤维为材料,利用RACE和RT-PCR技术分离得到了2个类黄酮3′-羟化酶基因cDNA序列,此2个序列编码区完全相同,仅在3'UTR区存在片段长短的差异,推测可能是基因转录后加工方式不同所造成.克隆所获得的棉花F3'H基因编码区全长1 533 bp,编码510个氨基酸,氨基酸序列分析预测表明,该基因所编码蛋白含有一个跨膜结构域,是一种分泌蛋白,定位于内质网上,并含有一段与细胞色素P450功能区相匹配的保守功能域;序列比对结果表明,棉花F3'H基因与其他多个物种的F3'H基因在氨基酸序列上有较高的同源性;聚类分析结果表明,棉花F3'H蛋白与双子叶植物大豆的F3'H亲缘关系较为接近,而与单子叶植物高梁等作物则较远.     

小花草玉梅正常和自然变异植株的AP3-3基因研究

野生小花草玉梅(Anemone rivularis var.flore-minore)正常植株和花被片自然变异植株的外观形态差异很大,该研究以二者为材料,利用常规PCR和高效热不对称PCR(Hi-Tail PCR)技术从其正常和变异植株的基因组中各分离得到1个B类基因。序列分析证明,二者隶属于B类MADS-box基因AP3家族的旁系同源基因AP3-3分枝,分别命名为NArAP3-3(正常植株)和VArAP3-3(变异植株)。NArAP3-3基因全长3 795bp,VArAP3-3基因全长3 898bp,二者均含有1个666bp的开放阅读框(ORF),可编码221个氨基酸,具有典型的植物MADS-box基因结构,其编码肽链包含了MADS区、K区、Ⅰ区和C区。对比NArAP3-3和VArAP3-3基因的全长序列,发现VArAP3-3基因比NArAP3-3多了1段49bp的插入,且在ORF序列与NArAP3-3基因相比有4个碱基突变。对二者的全长序列、所编码的221个氨基酸及插入序列的生物信息学分析显示,二者在基因启动子、蛋白质基本性质、结构功能域、二级三级预测结构等方面均有差异,推测这些差异可能是花被片变异产生的原因之一。该研究结果为进一步探索其变异机制奠定了基础。     

黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因克隆及序列分析

根据6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6PGDH)基因的保守氨基酸序列设计简并引物,应用RT-PCR技术从黄瓜栽培种品种'北京截头'(Cucumis sativus 'Beijingjietou')叶片中获得了640 bp的特异片段,以该序列在EST数据库进行同源检索筛选,发现甜瓜EST序列AM715537.2与之高度一致,据此设计引物经RT-PCR扩增、分子克隆和序列拼接,获得了黄瓜6-磷酸葡萄糖酸脱氢酶基因全长序列,命名为Cs6PGDH(GenBank登录号FJ610345).序列分析表明,该基因全长1 829 bp,其中开放读码框(ORF)长1 488 bp编码495个氨基酸组成的多肽,编码区内无内含子存在,5'、3'端非翻译区长度各为70 bp和271 bp.Blast同源性分析显示该基因编码的氨基酸序列与拟南芥、大豆、水稻、玉米、菠菜等物种6PGDH 基因有74%以上的一致性.由于与其他物种胞质6PGDH相类似氨基酸N端都缺少长度约为40aa的转运肽,推断Cs6PGDH为黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因.     

草莓ξ-胡萝卜素脱氢酶基因ZDS的克隆及特征分析

用RT-PCR和RACE技术从草莓(Fragaria ananassa Duchesne)果实中克隆到ξ-胡萝卜素脱氧酶基因ZDS,命名为FaZDS,其cDNA全长2148 bp,具有1个1710 bp的完整开放阅读框(ORF),编码569个氨基酸.序列分析表明,FaZDS编码的氨基酸序列与其它植物的ZDS蛋白有很高的相似性.系统进化树分析表明,草莓与苹果的ZDS蛋白亲缘关系较近.原核表达结果表明FaZDS基因在大肠杆菌中能高效表达.用半定量RT-PCR技术进行组织表达模式分析表明,FaZDS基因在草每的花、叶片和果实中均有表达,表达量为花＞红果＞粉红果＞白果＞青果＞叶.     

Denitrification kinetics of simulated fish processing wastewater at different ratios of nitrate to biomass

Denitrification was studied in anoxic batch cultures of a simulated fish processing wastewater at 37 r C and pH 7.5, using a denitrifying enrichment culture from fishery wastewater. Different initial nitrate to biomass ratios (So/Xo) were used: nitrate and biomass varied from 7.5 to 94.7 mg NO₃-N l^–1, and from 20 to 4300 mg volatile suspended solids l^–1, respectively. The specific maximum denitrification rate (r _m) and the cell yield (Y _{X / S}) depended on the So/Xo ratio under anoxic conditions: r _m increased from 1.2 to 1584 mg NO₃-N g^–1 VSS h^–1 and Y _{X / S} decreased from 42 to 0.03 mg VSS mg^–1 NO₃-N when So/Xo varied from 5.5 10^{– 3} to 9.3 mg NO₃-N/mg VSS. Nomenclature C_{NO3 – N} nitrate concentration, mg NO₃-N l^–1 K _S saturation constant, mg NO₃-N l^–1 r _m specific maximum denitrification rate, mg NO₃-N g^–1 VSS h^–1 So initial substrate concentration, mg l^–1 t time, h TOC total organic carbon VSS volatile suspended solids x biomass concentration, g VSS l^–1 Xo initial biomass concentration, g VSS l^–1 Y _X/S substrate to biomass cell yield, mg VSS/mg N Greek symbols: _m maximum specific growth rate of the anoxic microbial population, 1 h^–1     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

Nitrate availability and calcite production in Emiliania huxleyi Lohmann

High-calcifying cells of Emiliania huxleyi were grown on a synthetic seawater medium and the effect of nitrate (NO^- ₃) concentration on growth, calcite accumulation, calcification rate and DIC (dissolved inorganic carbon) utilisation determined. The stoichiometry between NO^- ₃ utilisation and calcite production was 1:6·5 (mol/mol). Calcification and growth were tightly coupled: calcite production ceased when cultures entered the stationary phase due to NO^- ₃ depletion, but by adding a pulse of NO^- ₃ growth and calcification were restored. The initial C/N ratio in the medium was important in relation to calcification rate. At 20 µM NO^- ₃ the total DIC (2 mM) was rapidly depleted, the calcification rate subsequently declining, whereas at 5 and 10 µM NO^- ₃ rates of calcification were constant at 20 g carbon cell^-1 × 10¹⁴·h^-1 throughout culture growth, excess DIC being present relative to the available NO^- ₃. Calcite production per unit NO^- ₃ was similar for isolates of E. huxleyi from neritic, oligotrophic and nitrate-rich waters. In laboratory cultures, where the photon flux density is optimised for growth, the initial NO^- ₃ concentration is a reliable indicator of final calcite yield.     

Nitrate reductase activity and growth in Paul's Scarlet rose suspension cultures in relation to nitrogen source and molybdenum

Growth and nitrate reductase activity were measured in Paul's Scarlet rose cell suspensions, cultured in media purified from molybdenum and containing nitrate or urea as sole nitrogen source with or without added Mo. Urea could replace nitrate to yield 80% of the fresh weight in nitrate medium. Nitrate reductase activities were compared by in vivo and in vitro assays. The latter varied due to inactivation during extraction. Compared with activities in cells in complete NO₃ ^- medium, activity in NO₃ ^--Mo cells was reduced to 30% and, in urea-grown cells, to trace amounts. Increases in nitrate reductase activity were found when NO₃ ^- alone was added to NO₃ ^- or urea+Mo cultures. In NO₃ ^--Mo cultures, Mo alone or with NO₃ ^- caused a similar increase in activity, whereas urea-Mo cultures required both NO₃ ^- and Mo for enzyme induction.Abbreviations FAD flavin adenine dinucleotide - Mo molybdenum - NADH reduced nicotinamide adenine dinucleotide - NO₃ ^-+Mo standard MX₁ culture medium - NO₃ ^--Mo MX₁ medium purified of Mo and used for continuous subculture with nitrate - NR nitrate reductase - PSR Paul's Scarlet rose - PVP polyvinylpyrrolidone - U urea - U+Mo MX₁ medium containing urea instead of nitrate - U-Mo MX₁ medium containing urea instead of nitrate and also purified of Mo     

Effect of NH4 +:NO3 - ratios on growth and nitrogen uptake by onions

The modelling of ion uptake by plants requires the measurement of kinetic and growth parameters under specific conditions. The objective of this study was to evaluate the effect of nine NH _inf4 ^sup+ :NO _inf3 ^sup− ratios on onions (Allium cepa L.). Twenty-eight to 84 day-old onion plants were treated with NH _inf4 ^sup+ :NO_f3/sup− ratios ranging from 0 to 100% of each ionic species in one mM solutions in a growth chamber. Maximum N influx (I_max) was assessed using the N depletion method. Except at an early stage, ionic species did not influence significantly I_max, the Michaelis constant (K_m) and the minimum concentration for net uptake (C_min). I_max for ammonium decreased from 101 to 59 pmole cm^-2 s^-1 while I_max for nitrate increased from 26 to 54 pmole cm^-2 s^-1 as the plant matured. On average, K_m and C_min values were 14.29 μM, and 5.06 μM for ammonium, and 11.90 μM and 4.54 μM for nitrate, respectively. In general, the effect of NH₄ ⁺:NO₃ ^- ratios on root weight, shoot weight and total weight depended on plant age. At an early stage, maximum plant growth and N uptake were obtained with ammonium as the sole source of N. At later stages, maximum plant growth and N uptake were obtained as the proportion of nitrate increased in the nutrient solution. The was no apparent nutrient deficiency whatever NH₄ ⁺:NO₃ ^- ratio was applied, although ammonium reduced the uptake of cations and increased the uptake of phosphorus. The research was supported by the Natural Sciences and Engineering Research Council of Canada.     

EFFECTS OF NITRATE CONCENTRATION ON THE GROWTH AND PHYSIOLOGY OF LAMINARIA SACCHARINA (PHAEOPHYTA) IN CULTURE1,2

Laminaria saccharina Lamour. sporophytes were grown in enriched and synthetic media through a range of nitrate concentrations, There was an approximately linear relationship between growth and nutrient concentration up to 10 μ substrate concentration. The half-saturation constant (K_{2) was ca. 1.4 μ NO3-. The internal levels of NO3- increased at substrate concentrations above 10 μM b>3- and reached levels several thousand times higher than the surrounding medium. Thus there is evidence for luxury consumption of NOsb>3}^-. The chlorophyll content and photosynthetic capacities of plants also increased with increasing external NO₃^- The ecological implications of this work are considered.     

NITRATE UPTAKE BY LAMINARIA LONGICRURIS (PHAEOPHYCEAE)1,2

Laminaria longicrucis De la Pylaie took up exogenous nitrate under both summer and winter conditions. During July and August no NO₃^- was detected in the ambient water or in algal tissues although it was present in both in February. Discs (2.3 cm diam.) of thin blade tissue were incubated with NO₃^- at four temperatures, with and without illumination. Similar values Jor NO₃^- uptake were found for both summer and winter collected plants when measured in light at 0 C. An apparent K of 4–6 μM was recorded for both types of plants; the V_max ranged from 7 to 10 μmol h^-1 g^-1 dry wt measured in ca. 1800 μW cm^-2 of cool-white fluorescent light. Uptake rates at 5 C were 66%, and at 0 C 30% of those for controls run at 15 C. The alga scavenged NO₃- from solutions <0.5 μM. Ammonia did not inhibit NO₃- uptake. Antibiotic pretreatment reduced NO₃- uptake by a maximum of 12%. Nitrite uptake was inhibited in proportion to the concentration of NO₃- in the medium.     

Cytosolic glutamine synthetase and not nitrate reductase from the green alga Chlamydomonas reinhardtii is phosphorylated and binds 14-3-3 proteins

 The nitrate reductase activity from Chlamydomonas reinhardtii was not altered when extracts were incubated with yeast 14-3-3 proteins in the presence of Mg-ATP. However, the C. reinhardtii extracts contained 14-3-3 proteins capable of inhibiting the spinach nitrate reductase, raising the question of their physiological substrates. Two C. reinhardtii proteins of about 48 and 35 kDa were eluted from 14-3-3 affinity chromatography columns and bound to 14-3-3s in overlay assays. The 48-kDa protein corresponded to the cytosolic isoform of glutamine synthetase (GS1). The GS1 was phosphorylated by a Ca²⁺- and calmodulin-dependent protein kinase partially purified from the alga. However, neither phosphorylation nor 14-3-3 binding seemed to change GS catalytic activity. Received: 3 February 2000 / Accepted: 6 May 2000     

Molecular characterization of a 14-3-3 zeta gene from Plodia interpunctella: A potential marker for phylogenetic inference

The 14-3-3 proteins are a large family of approximately 30 kDa acidic proteins and acting in the regulation of many biological processes. In this study, a 14-3-3 zeta (Pi14-3-3z) gene from the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae) was isolated and characterized. The full-length cDNA of Pi14-3-3z is 1382 bp, including a 5'-untranslated region (UTR) of 141 bp, 3′-UTR of 497 bp and an open reading frame (ORF) of 744 bp encoding a polypeptide of 247 amino acids which contains a 14-3-3 homologues domain (PF00244). The deduced Pi14-3-3z protein sequence has 81%–100% identity with the homologues in comparison to with other individuals. qPCR analysis revealed that Pi14-3-3z was expressed at the four developmental stages and in all tissues tested. Based on the amino acid of 14-3-3z, phylogenetic analysis demonstrated a similar topology with the traditional classification, suggesting 14-3-3z protein has the potential value in phylogenetic inference.