烟草NtGSH2基因的克隆及表达特性分析

谷胱甘肽(glutathione,GSH)是生物体内的非蛋白巯基类化合物,具有抗氧化、抗衰老和重金属解毒等重要的生理功能。本研究从烟草(Nicotiana tabacum L.)中克隆到谷胱甘肽合成酶基因NtGSH2,该基因编码555个氨基酸残基的蛋白质。氨基酸序列比对和进化树分析发现,NtGSH2与其他植物同源蛋白具有较高的一致性,且与枸杞(Lycium chinense)LcGSHS/LcGS和番茄(Solanum lycopersicum)SlGSH2归为一类,亲缘关系最近。组织表达模式分析表明,NtGSH2基因在烟草的根、茎、叶、花和种子中均表达,其中在花中的表达量最高,在种子中的表达量最低。实时荧光定量PCR检测表明,NtGSH2受CdCl_2和ZnCl_2诱导表达。将NtGSH2连接到原核表达载体pET32a上,转化获得含有NtGSH2基因的大肠杆菌BL21(DE3)工程菌并成功表达目的蛋白。该研究结果为进一步分析NtGSH2基因功能及谷胱甘肽的生物合成提供了一定的理论依据。     

一个新的二色补血草金属硫蛋白基因LbMT2的克隆及其表达分析

从二色补血草cDNA文库中分离出一个新的金属硫蛋白基因LbMT2全长cDNA序列。该基因全长518 bp, 其中5′非翻译区(UTR)64 bp, 3′非翻译区205 bp, 开放阅读框(ORF)249 bp, 编码由82个氨基酸组成的蛋白质, 编码蛋白的分子量为8.1 kDa, 理论等电点(pI)为4.72。利用实时定量PCR方法研究了二色补血草在CuSO4、CdCl2、NaCl、低温和PEG胁迫下不同时间该基因的表达模式。结果表明, CuSO4、CdCl2、NaCl和低温处理均能诱导LbMT2基因在二色补血草的根和叶中的表达, 而PEG处理则抑制了LbMT2在根和叶中的表达。构建LbMT2基因的原核表达载体pGEX-LbMT2, 通过IPTG诱导在大肠杆菌(Escherichia coli) BL21中融合表达, SDS-PAGE 电泳获得35 kDa 的蛋白条带, 大小与预期相符。     

甘薯非特异性脂质转移蛋白基因克隆与表达分析(英文稿)

非特异脂质转移蛋白（nsLTP）是植物界普遍存在的一类涉及多种胁迫反应的可溶性蛋白。为了阐明甘薯中非特异性脂质转移蛋白基因IbLTP1和IbLTP2在盐胁迫反应中的功能,本研究运用PCR技术,对IbLTP1和IbLTP2基因进行了克隆,并通过生物信息学方法分析了序列结构、蛋白质保守结构域和系统进化关系;利用qRT-PCR方法检测了这两个基因在不同组织中的表达模式以及盐胁迫条件下的表达差异;将IbLTP1和IbLTP2基因克隆到大肠杆菌的原核表达载体pET32a中,对重组菌BL21（pET32a-LTP）的耐盐性进行分析。序列分析表明：IbLTP1和IbLTP2编码区均不含内含子并都具有等位基因。IbLTP1和IbLTP2基因的蛋白质序列分别包括114和94个氨基酸残基并且不含色氨酸残基,蛋白序列N端含有信号肽序列。保守结构域和系统进化分析结果表明：IbLTP1和IbLTP2均含有nsLTP蛋白的保守结构域,IbLTP1属于Type Ⅰ而IbLTP2属于Type Ⅱ。实时荧光定量PCR分析表明：IbLTP1在幼叶中表达量最高,根中表达量最低;而IbLTP2在茎中表达量最高,成熟叶中表达量最低。在NaCl胁迫条件下,IbLTP1和IbLTP2表达量在根中基本无变化而在茎和叶中上调。大肠杆菌BL21（DE3）中异源表达IbLTP1和IbLTP2基因能够提高转基因菌株对NaCl的耐受性。因此,本研究推测IbLTP1和IbLTP2基因可能在甘薯盐胁迫反应中发挥了作用。     

棉花GhVHA A基因的原核表达及其重组大肠杆菌的抗逆性分析

为进一步验证棉花GhVHA-A基因的功能,该研究将棉花GhVHA-A基因构建到原核表达载体pET28a上,利用IPTG诱导其在大肠杆菌BL21(DE3)中高效表达,同时对重组大肠杆菌BL21(pET28a-GhVHA-A)进行抗逆性分析。结果表明:(1)半定量RT-PCR分析发现,棉花幼苗液泡膜H+-ATPase基因(GhVHA-A)表达水平受脱水和高盐胁迫诱导。(2)将1 872bp长的编码区序列连接至原核表达载体pET28a上,成功构建了原核表达载体pET28a-GhVHA-A;SDS-PAGE电泳检测结果表明,在70kD左右处有1条特异表达的蛋白质条带,与预期的目的产物大小一致。(3)重组菌BL21(pET28a-GhVHA-A)的抗逆性分析发现,重组菌对PEG6000(20%)和NaCl(0.5mol/L)的抗性明显高于对照菌株BL21(pET28a),表明GhVHA-A基因在大肠杆菌中表达后能够增强菌株的抗性。本研究结果为GhVHA-A基因在植物抗逆基因工程中的应用提供了理论依据。     

猪源戊型肝炎病毒基因IV型ORF3编码蛋白的表达及鉴定

通过PCR从已构建的猪源戊型肝炎病毒全基因克隆扩增ORF3全基因,将扩增产物插入到pMD18-T载体中,亚克隆至原核表达载体pET28a（+）,构建pET28a-ORF3表达载体,转入E.coli BL21 (DE3),IPTG诱导表达。Ni-NTA层析柱纯化表达蛋白,用SDS-PAGE、免疫印迹、ELISA等方法分析鉴定表达产物。结果成功扩增到345 bp的目的基因;构建了重组表达载体pET28a-ORF3;转化宿主菌E.coli BL21 (DE3)后表达产物的相对分子质量在6.50~16.5 kDa之间,与预期表达的目的蛋白相对分子质量相符;表达的目的蛋白能与阳性猪源和人源血清发生特异性反应,证实其具有较好的反应原性。     

蓖麻RcTUBβ1基因的克隆和原核表达

微管蛋白基因(Tubulin)具有高度的保守性,常作为目标基因相对表达量研究时的内参基因.本研究旨在通过构建蓖麻(Ricinus communis L.)RcTUBβ1基因的原核表达载体pET32a(+)-RcTUBβ1并优化表达工艺以表达融合蛋白His-RcTUBβ1.以蓖麻雌花为材料,通过RT-PCR技术,扩增得到含酶切位点BamHⅠ和Xho Ⅰ 的 RcTUBβ1 基因(GenBank Accession number:XM_002509734.3)的ORF 序列,经双酶切构建原核表达载体pET32a(+)-RcTUBβ1,将pET32a(+)-RRcTUBβ1转化到大肠杆菌表达菌株BL21(DE3)中诱导表达,并考察诱导时间、诱导温度、诱导IPTG浓度对表达的影响.结果表明,所获得的蓖麻RcTTUBβ1 ORF序列与GenBank中的序列一致,长度为1 341 bp,未发生移码突变.IPTG可诱导产生分子量约为68kDa的融合蛋白,最优表达条件为诱导温度28℃、诱导时间7 h和IPTG 0.5 mmol/L,融合蛋白主要以包涵体的形式存在.这为纯化融合蛋白以制备其抗体,用以研究RcTUBβ1的免疫组织化学分布及时空表达模式提供了基础.     

FASN基因的原核表达及生物信息学分析

脂肪酸合成酶(FASN)在生物体内起着重要的作用,主要参与恶性肿瘤的数量调控。本研究旨在构建pET28a-FASN原核表达载体,并表达重组His-FASN蛋白,对该基因进行结构与功能的生物信息学分析。设计FASN基因特异性引物,通过PCR扩增获得的目的基因与原核表达载体pET28a连接,经IPTG诱导表达His-FASN蛋白。获得基因片段大小为1 320 bp,编码440个氨基酸;成功构建至pET28a原核表达载体,通过优化表达,确定在温度为35℃、IPTG浓度0.5 mmol/L、诱导时间为6 h的条件下融合蛋白表达量较高,获得蛋白大小约为53 kD;生物信息学分析结果表明FASN基因编码的蛋白是一个不稳定且具有亲水性的蛋白,不存在信号肽及跨膜区,可成为蛋白激酶磷酸化位点有12个Ser、5个Thr、3个Tyr。此外,从蛋白相互作用网络中发现,相互作用的蛋白包括主要酰基辅酶A合成酶长链家族成员及乙酰辅酶A羧化酶家族成员,为开发抑制剂药物提供了理论依据。     

南极衣藻谷胱甘肽-S-转移酶基因的表达及其对大肠杆菌低温耐受性的增强作用

谷胱甘肽-S-转移酶(Glutathione-S-transferase, GST, EC2.5.1.18)是生物体内一种重要的抗氧化酶, 为阐明GST在南极衣藻(Chlamydomonas sp. ICE-L)中的具体地位, 采用实时荧光定量PCR对不同温度下南极衣藻的GST基因的表达进行了分析; 并构建了原核表达载体pET28a(+)-GST, 转化至大肠杆菌BL21(DE3)中进行诱导表达, 通过平板培养法探讨了重组菌E. coli BL21(pET28a(+)-GST)对低温胁迫的耐受性。结果显示, GST在0℃时表达量最高, 最高可达对照组的两倍多; pET28a(+)-GST重组表达载体在E. coli BL21中实现了高效表达, 且主要以包涵体形式存在, 经HisTrap HP柱分离纯化获得高纯度的GST融合蛋白, 并通过SDS-PAGE及Western blot分析得以验证; 对低温胁迫实验发现南极衣藻GST蛋白的表达可以提高重组菌E. coli BL21对低温的耐受性, 说明GST基因对南极衣藻适应南极低温环境具有重要作用。     

柽柳翻译起始因子(eIF-5A)基因的克隆及原核表达

根据柽柳cDNA文库中获得的eIF-5A基因片段,用RACE技术克隆出其全长cDNA序列.cDNA长度为799 bp,编码159个氨基酸.将该cDNA序列克隆到原核表达载体pET28a中,获得重组质粒pET28a-eIF5A.不同浓度NaCl胁迫下大肠杆菌(Escherichia coli)BL21(pET28a-eIF5A)比E.coli BL21(pET28a)有明显的抗盐性,前者菌株存活率在1.0 mo1·L-1NaCl盐胁迫下是后者的9.3倍,据此认为E.coliBL21(pET28a-eIF5A)的耐盐性可能与eIF-5A基因的表达相关.该基因的GenBank登录号为AY587771(基因)、AAT01416(蛋白).     

玉米FAD2基因的克隆及序列分析

高等植物中的A12脂肪酸脱饱和酶是将油酸转化为亚油酸的酶。根据已发表的其他高等植物的FAD2基因的保守序列设计同源引物,通过RT—PCR从玉米幼胚中扩增得到一个特异的cDNA基因片段。通过生物信息学分析,从玉米幼胚cDNA和基因组中均扩增得到1164 bp FAD2基因（GenBank登陆号：DQ496227）,它编码387个氨基酸,含有完整的ORF框,在ORF框内无内含子。序列联配与树状分析结果表明,FAD2推导的氨基酸序列与其他物种的A12脱饱和酶基因具有同源性。它含有3个组氨酸保守域和2段很长的疏水区,是一个跨膜4次的膜结合蛋白。半定量RT—PCR分析显示FAD2基因在玉米幼胚中表达量最高,在叶、茎、根中亦有低水平表达。     

香芹酚和丁香酚对腐皮镰刀菌的抑菌活性及抑菌机理

【背景】腐皮镰刀菌(Fusarium solani)是世界上最具破坏性的土传病原菌之一,严重影响作物的产量及品质。因此寻找并开发广谱的可持续生物防治药剂迫在眉睫,而植物次级代谢物为自然界筛选生物源天然杀菌剂提供了丰富的原料。【目的】研究香芹酚和丁香酚对腐皮镰刀菌的抑菌活性,探究其可能涉及的抑菌机理。【方法】采用菌丝生长速率法、十字交叉法和孢子萌发法分析香芹酚和丁香酚对菌丝和孢子的抑制活性,通过扫描电镜(scanning electron microscope,SEM)观察菌丝微观形态的变化,利用荧光染料碘化丙啶(propidium iodide,PI)观察细胞膜的损伤情况,并测定其对腐皮镰刀菌细胞膜胞外电导率、蛋白质含量和麦角固醇合成的影响。【结果】香芹酚和丁香酚对腐皮镰刀菌菌丝生长和孢子萌发均有显著的抑制效果,并呈现剂量依赖效应,EC50值分别为92.39μL/L和263.00μL/L。SEM结果表明,2种精油处理腐皮镰刀菌后,其细胞壁和细胞膜均遭到破坏从而不能维持菌丝正常的线性形态,表现出不同程度的弯曲、褶皱和凹陷。PI染色结果发现,2种精油处理严重破坏了腐皮镰刀菌细胞膜的完整性和...     

Composition of the essential oils of Ocimum urticifolium (Lamiaceae) chemotypes grown in Rwanda

NTEZURUBANZA, L., SCHEFFER, J. J. C. & SVENDSEN, A. B., 1988. Composition of the essential oils of Ocimum urticifolium (Lamiaceae) chemotypes grown in Rwanda. Leaves and flowers of 48 specimen of Ocimum urticifolium (synonym: O. suave), Lamiaceae, were collected in different parts of Rwanda. The volatile components of each specimen were isolated by hydrodistillation under standardized conditions, and the 48 essential oils analysed by thin-layer chromatography. This preliminary analysis showed six different oil compositions. Thereafter, six oil samples were fractionated by liquid—solid chromatography. The fractions thus obtained, and all other oil samples were then analyzed by capillary gas chromatography. In this way 46 compounds were identified in the various oil samples. Eight components, which amounted to more than 10% in at least one oil sample, were used to characterize the oils. Twelve oil types were distinguished, three of which were dominated by one component only: eugenol, methyleugenol and transmethylisoeugenol respectively. The other types were more complex. In some parts of Rwanda almost one oil type seemed to occur, whereas in other areas a more heterogeneous collection of types was found. Eugenol was quantitatively most important in 31 of the 48 oil samples, in 22 of which this component amounted to more than 50%.     

In vitro activity of eugenol against <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms

Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices. Candida albicans is the most frequently isolated causative pathogen of candidiasis, and the biofilms display significantly increased levels of resistance to the conventional antifungal agents. Eugenol, the major phenolic component of clove essential oil, possesses potent antifungal activity. The aim of this study was to investigate the effects of eugenol on preformed biofilms, adherent cells, subsequent biofilm formation and cell morphogenesis of C. albicans. Eugenol displayed in vitro activity against C. albicans cells within biofilms, when MIC₅₀ for sessile cells was 500 mg/L. C. albicans adherent cell populations (after 0, 1, 2 and 4 h of adherence) were treated with various concentrations of eugenol (0, 20, 200 and 2,000 mg/L). The extent of subsequent biofilm formation were then assessed with the tetrazolium salt reduction assay. Effect of eugenol on morphogenesis of C. albicans cells was observed by scanning electron microscopy (SEM). The results indicated that the effect of eugenol on adherent cells and subsequent biofilm formation was dependent on the initial adherence time and the concentration of this compound, and that eugenol can inhibit filamentous growth of C. albicans cells. In addition, using human erythrocytes, eugenol showed low hemolytic activity. These results indicated that eugenol displayed potent activity against C. albicans biofilms in vitro with low cytotoxicity and therefore has potential therapeutic implication for biofilm-associated candidal infections.     

Attraction of feral Bactrocera dorsalis males (Diptera: Tephritidae) to natural versus commercial sources of methyl eugenol

Males of Bactrocera dorsalis (Hendel) are strongly attracted to methyl eugenol (ME), which occurs in over 450 plant species. Given this powerful attraction, ME is commonly used in surveillance and eradication programs against this invasive agricultural pest. Preliminary observations revealed that B. dorsalis males visited ME-bearing flowers of the fruit fly orchid (FFO) Bulbophyllum cheiri subsp. cheiri even when these occurred near traps baited with far greater quantities of ME. Based on this evidence, we undertook field experiments to assess the attraction of feral B. dorsalis males to FFO flowers relative to commercial sources of ME. At the edge of a secondary forest, an FFO flower was placed midway between two ME sources located 20 m apart, and attracted flies were collected over an entire day. When the ME sources were unenclosed (not in traps), the relative attractiveness of FFO flowers to B. dorsalis males varied with the amount of ME placed nearby. The FFO flower (i) attracted a similar proportion of males when 1 g ME was placed at the flanking sites but (ii) captured significantly smaller proportions when the nearby sites had 6 or 10 g commercial ME sources. Similar tests with the commercial sources enclosed in traps showed that (i) 6 g ME sources in Steiner traps attracted significantly more B. dorsalis males than FFO flowers but (ii) 10 g sources in Clear traps (1 L buckets with 4 entrance holes) did not outperform the FFO flowers, presumably owing to limited dispersion of volatiles from the trap. Implications of these results for surveillance programs are discussed.     

丁香酚对大鼠弓状核温度敏感神经元电活动的影响

本实验采用微电极细胞外记录技术,在大鼠弓状核记录到单一放电单位６８例,其中热敏神经元１１例,占１６．２％,冷敏神经元２５例,占３６．８％;温度不敏感神经元３２例,占５３％。本实验同时观察了皮下注射丁香酚（２０μｇ／１００ｇ）对温度敏感神经元放电活动的影响,其结果为１０例冷敏神经元的放电频率均减慢;４例热敏神经元的放电频率均增快;而１４例温度不敏感神经元的放电活动则无明显改变。以上结果表明弓状核内存在以冷敏神经元居多的温度敏感神经元,并探讨了丁香酚的降温作用与它能够抑制弓状核的冷敏神经元,激活热敏神经元的电活动有关,亦成为弓状核参与体温调节的有力佐证     

Sexual maturity and intraspecific mating success of two sibling species of the Bactrocera dorsalis complex

Bactrocera carambolae Drew & Hancock and B. papayae Drew & Hancock (Diptera: Tephritidae), two closely related sibling species in the B. dorsalis (Hendel) complex were shown to have different rates of sexual maturity. The response of B. carambolae males to methyl eugenol was observed to begin 10 days after emergence and increased with age. The attractancy peaked at 28 days after emergence and above. The males were also found to require a higher dosage of methyl eugenol (1 mg) for optimal response. These results are in contrast with those of B. papayae reported previously. The response to methyl eugenol was also observed to correspond with the age when first mating was performed.After eclosion, both sexes of B. carambolae mated approximately 2 weeks later and showed significantly lower matings/individual when compared with B. papayae in a 60-day observation period. Both sexes of B. papayae and males of B. carambolae were able to remate on the next day. However, B. carambolae females required a minimum refractory period of 8 days in order to be sexually receptive again.During the 60-day period, the mating propensity in B. papayae (100% success) was higher than B. carambolae (71–87% success) (P < 0.05). This observation was substantiated by a higher intraspecific mating success of B. papayae as compared to B. carambolae in a field cage (P < 0.001).     

Differentiation between the two subspecies of Atherosperma moschatum Labill. (Atherospermataceae) from their leaf oils

The leaf oils of both subspecies of Atherosperma moschatum have been investigated to determine if there were any chemical differences between the two subspecies. While both subspecies of A. moschatum contained methyl eugenol (55–87%), safrole (5–20%) and linalool (1–6%), camphor (5–10%) was found to be only in the oils of A. moschatum subsp. moschatum and to be either absent or, at most, less than 0.5% in the oils of A. moschatum subsp. integrifolium. There was also a range of monoterpene hydrocarbons and 1,8-cineole, all in the range of 0.1–5%, that were present in subspecies moschatum but virtually absent in subspecies integrifolium.     

丁香酚抑制日本沼虾应激反应效果的研究

本实验以丁香酚和日本沼虾为对象,研究了丁香酚对日本沼虾因温度的急剧变化和形态测量所造成的应激反应的抑制效果.结果显示,丁香酚浓度为500 mg/L时,对照组和实验组的存活率开始呈显著下降趋势的温差幅度分别为10和14℃;当温差为18℃时,对照组存活率为0,实验组存活率为0.653±0.033;当温差达到10℃后,实验组存活率明显高于对照组存活率(P＜0.05).形态测量结果显示,实验组的断肢率显著低于对照组,而存活率显著高于对照组(P＜0.05).研究表明,丁香酚可明显提高日本沼虾存活率,并显著抑制日本沼虾的应激反应.