斜纹夜蛾中肠糖代谢相关基因的克隆及表达模式分析

斜纹夜蛾Spodoptera litura是一种世界性分布的重要农业害虫, 在生长发育过程中要经历幼虫 蛹的变态发育过程。由于变态发育前后昆虫的食性发生了明显的改变, 作为食物消化吸收的中肠也发生了解体和重建。与此相适应, 昆虫中肠的各种物质和能量代谢也可能会相应地发生改变。为研究斜纹夜蛾中肠变态发育过程中糖代谢途径的变化情况, 我们从斜纹夜蛾中肠EST文库中鉴定出了12个糖代谢相关基因, 克隆了其中3个基因的全长cDNA, 并应用半定量PCR和定量PCR的方法检测了其在幼虫 蛹变态发育期中肠组织的转录表达以及对激素和饥饿等因素的响应情况。结果表明： 这3个基因（α-L-岩藻糖苷酶、 N-乙酰葡萄糖胺-6-磷酸去乙酰酶和烯醇化酶基因）的开放阅读框分别为1 461, 1 200和1 299 bp, 预测的分子量分别为56.3, 43.3和46.7 kDa。这12个糖代谢相关的基因在变态发育期的中肠组织中具有5种不同的mRNA表达模式： （Ⅰ）只在幼虫期高表达（唾液麦芽糖酶前体蛋白、 糖基水解酶31家族成员蛋白、 线粒体乙醛脱氢酶、 β-1,3 葡聚糖酶基因）; （Ⅱ）只在预蛹期高表达（β-葡萄糖醛酸酶、 β-N-酰基氨基葡萄糖苷酶3基因）; （Ⅲ）只在蛹期高表达（葡萄糖胺-6-磷酸异构酶基因）; （Ⅳ）在预蛹期和蛹期高表达（α-葡萄糖苷酶、 α-淀粉酶、 N-乙酰葡糖胺 6 磷酸脱乙酰酶和α-L-岩藻糖苷酶基因）; （Ⅴ）在变态发育期恒定表达（烯醇化酶基因）。这说明, 为适应变态发育斜纹夜蛾中肠糖代谢途径发生了明显的改变。保幼激素对这些基因的表达没有明显的影响, 但蜕皮激素对Ⅰ类基因（如糖基水解酶31家族成员蛋白基因）具有一定的抑制作用, 对Ⅲ类基因（如葡萄糖胺-6-磷酸异构酶基因）有显著的上调作用。此外, 我们还发现饥饿对几乎所有这些基因的表达都有显著的抑制作用。这些结果说明, 昆虫中肠变态发育过程中糖代谢相关基因的动态变化可能受到蜕皮激素以及饥饿相关因素的共同调控。这一研究对从代谢角度揭示昆虫变态发育的分子机理具有重要意义。     

川楝素对斜纹夜蛾幼虫取食行为的影响

斜纹夜蛾5龄幼虫的取食活动是非连续进行的。在每两次取食活动之间存在一个取食间间隔．在每次取食活动期间,也存在一非常短暂的取食内间隔。经川楝素处理后的斜纹夜蛾5龄幼虫．其21小时内发生的取食活动和取食间间隔的次数减少．平均每次取食活动和取食间间隔持续的时间延长;可是在每次取食活动中,用于取食的时间明显减少。这是因为．取食内间隔的次数增加了．取食内间隔的时间也延长了。所有上述指标,处理虫与对照之间均存在着显著或极显著的差异。     

斜纹夜蛾3种SlitPBPs的组织表达模式分析

性信息素结合蛋白(pheromone binding proteins,PBPs)能够与性信息素分子结合,从而启动昆虫的寻偶及交配行为。本研究采用半定量RT-PCR技术分别对斜纹夜蛾3种性信息素结合蛋白SlitPBP1、SlitPBP2和SlitPBP3基因在雌、雄虫不同组织的基因表达模式进行了比较分析。组织表达模式结果表明,SlitPBP1基因只在雌、雄虫触角和雌虫前足中表达而在其他组织中无表达;SlitPBP2基因在雌虫的触角、喙、前足、中足、后足、翅膀和雄虫的触角、喙、前足、翅膀中均有表达,且在同一部位,SlitPBP2基因在雌虫的表达量均高于雄虫相应部位的表达量;除雌、雄虫的胸部外,SlitPBP3在雌、雄虫的触角、喙、前足、中足、后足、腹部和翅膀中均有较高水平的表达。可见,SlitPBP1、SlitPBP2和SlitPBP3基因在斜纹夜蛾不同组织的表达模式各不相同。本研究结果可为深入研究不同SlitPBPs蛋白在斜纹夜蛾生长发育过程中的不同生理功能提供科学参考。     

斜纹夜蛾铁硫亚基蛋白的表达及功能鉴定

铁硫亚基蛋白(rieske iron-sulfur protein, RISP) 是线粒体复合物Ⅲ的关键蛋白亚基之一, 在呼吸链电子传递过程中起到重要作用。本研究通过RT-PCR克隆得到斜纹夜蛾Spodoptera litura RISP基因SlitRISP的ORF, 构建了pET32a-SlitRISP原核表达载体, SDS-PAGE和Western blot检测结果显示, SlitRISP原核表达蛋白以包涵体的形式存在于菌体沉淀中, 且RISP抗体可成功用于该蛋白的免疫印迹检测。为了进一步鉴定SlitRISP在斜纹夜蛾离体细胞系SL-1中的功能, 通过向细胞内转染siRNA, 利用RNAi技术沉默SL-1中的SlitRISP。qRT-PCR结果表明, 分别经50 nmol/L和100 nmol/L siRNA处理48 h后, SL-1中SlitRISP的表达均几乎完全被抑制; Western blot结果显示, SL-1中SlitRISP含量显著低于CK。当SL-1 SlitRISP被成功沉默后, 通过检测SL-1线粒体膜电位、 细胞ATP含量和细胞增殖抑制率鉴定RISP在线粒体电子传递过程中的重要作用。流式细胞仪测定结果表明, 经50 nmol/L和100 nmol/L siRNA处理24 h后, SL-1线粒体膜电位相对于CK分别降低23.52%和11.32%, 而处理48 h后, SL-1线粒体膜电位则分别升高5.58%和27.66%; siRNA处理24 h和48 h后, SL-1 ATP含量相对于CK分别降低82.71%和84.50%, 最终导致SL-1细胞增殖抑制率分别为53.64%和67.94%。这些结果表明SlitRISP在SL-1中参与线粒体膜电位的形成和细胞ATP的合成。介于RISP在线粒体电子传递链中的重要作用, 其可能成为新型杀虫作用靶标, 这可为研制新型呼吸抑制剂提供参考。     

Action of the Protease from Streptomyces cellulosae on l-Leu-Gly-Gly

We introduced efficient incorporation of unsaturated fatty acids into volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine, insect-derived elicitors of plant volatiles, in the common cutworms Spodoptera litura by the incubation of larval gut tissues with unsaturated (linolenic, linoleic, and oleic acids) or saturated fatty acids (palmitic and stearic acids) sodium salt, and L-[α-¹⁵N]glutamine.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Associations of protein 4.2 with band 3 and ankyrin

It was shown previously that the intestinal fatty acid binding protein (I-FABP) is not essential for the absorption of dietary fat. One notable feature of I-FABP deficiency was the enhancement of body weight gain in male mice but not in female mice. To explore a possible cause for this gender dimorphic effect, we examined the changes in expression of genes that encode liver fatty acid binding protein (L-FABP) and ileal lipid binding protein in the small intestine resulting from I-FABP deficiency. The results indicate that both L-FABP and ilbp levels are modestly increased in the small intestine of chow-fed mice lacking I-FABP. There was no discernible alteration of overall morphology or histology in the small intestine but changes in liver histology were evident in I-FABP deficient male mice. Glucose tolerance was also investigated in aged mice. I-FABP deficiency had no effect on glucose tolerance in male mice but it appeared to be improved in female mice. Thus, male and female mice clearly respond differently to the loss of I-FABP from the small intestine but the observed changes in the abundance of L-FABP and ilbp protein do not readily account for this phenomenon. (Mol Cell Boichem xxx: 1–8, 2005)     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

杆状病毒SpltMNPVSl136基因的克隆、表达及其产物功能

计算机分析斜纹夜蛾核多角体病毒（SpltMNPV)基因组序列,发现第136个读码框基因表达产物具有病毒囊膜蛋白的基本特征,计算机预测的SL136蛋白氨基酸序列N端具有信号肽,C端具有跨膜区,N端区还有一个许多病毒融合蛋白共有的卷曲螺旋结构。通过PCR扩增,我们克隆了Sl136基因并分别构建了原核表达载体pBVSl136及重组病毒表达载体,SDS-PAGE结果表明Sl136基因在大肠杆菌和昆虫细胞中均获得了较高的表达,另外,我们还构建了一个瞬时表达载体pUCSl136。单独转染Sl-zsu-1细胞后,低pH值环境可诱导细胞发生膜融合并形成合胞体,这些实验结果表明,Sl136基因表达的产物是一个病毒膜融合蛋白。     

斜纹夜蛾普通气味结合蛋白GOBP1基因的表达定位分析

气味调控斜纹夜蛾Spodoptera litura（鳞翅目,夜蛾科）的交配和产卵行为,而嗅觉气味结合蛋白（OBP）是昆虫与外界环境进行化学信息交流中的一种重要蛋白。本研究基于已报道的斜纹夜蛾普通气味结合蛋白GOBP1基因cDNA序列（GenBank登录号为EF159978）设计引物,通过RT-PCR法分析了GOBP1 mRNA的组织特异性表达情况,并对GOBP1基因条带进行了克隆、测序和Blast比对。此外,再通过RNA原位杂交的方法进一步分析了GOBP1 mRNA在触角中的表达定位。结果表明:GOBP1只在斜纹夜蛾的触角组织中表达,而且GOBP1转录本较多分布于靠近嗅觉感受器即触角边缘部位。这些结果进一步说明GOBP1是斜纹夜蛾嗅觉过程中的重要蛋白,同时为深入研究GOBP1与其他蛋白的相互空间定位关系、GOBP1的功能等奠定了基础。     

A comparison of heart and liver fatty acid-binding proteins: interactions with fatty acids and possible functional differences studied with fluorescent fatty acid analogues

Summary Fatty acid-binding proteins (FABP) are distinct but related gene products which are found in many mammalian cell types. They are generally present in high abundance, and are found in those tissues where free fatty acid (ffa) flux is high. The function(s) of FABP is unknown. Also not known is whether all FABP function similarly in their respective cell types, or whether different FABP have unique functions. The purpose of these studies was to assess whether different members of the FABP family exhibit different structural and functional properties. Two fluorescent analogues of ffa were used to compare the liver (L-FABP) and heart (H-FABP) binding proteins. The propionic acid derivative of diphenylhexatriene (PADPH) was used to examine the physical properties of the ffa binding site on L- and H-FABP, as well as the relative distribution of ffa between FABP and membranes. An anthroyloxy-derivative of palmitic acid, 2AP, was used to monitor the transfer kinetics of ffa from liver or heart FABP to acceptor membranes, using a resonance energy transfer assay. The results demonstrate that the ffa binding sites of both FABP are hydrophobic in nature, although the L-FABP site is more nonpolar than the H-FABP site. Equilibration of PADPH between L-FABP and phosphatidylcholine (PC) bilayers resulted in a molar partition preference of > 20: 1, L-FABP : PC. Similar studies with H-FABP resulted in a PADPH partition preference of only 3:1, H-FABP : PC. Finally, the transfer of 2AP from H-FABP to acceptor membranes was found to be 50-fold faster than transfer from L-FABP. These studies demonstrate that important structural and functional differences exist between different members of the FABP family, and therefore imply that the roles of different FABP may be unique.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Liver FABP - H-FABP Heart FABP - SUV Small Unilamellar Vesicle - PADPH 3-[p-(6-Phenyl)-1,3,5-Hexatrienyl]-phenylpropionic acid - 2AP 2-(9-Anthroyloxy)Palmitic acid - Q Quantum yield - _F Fluorescence lifetime     

性信息素对斜纹夜蛾雄蛾嗅觉相关基因 abp,pbp 和 or 表达的影响

【目的】信息素是个体之间传递信息的重要分子,研究性信息素对斜纹夜蛾 Spodoptera litura 嗅觉相关基因表达的影响对于增加性信息素作用机理的认识及其应用有重要的意义。【方法】本研究通过实时定量PCR（qRT-PCR）技术探究在性信息素刺激处理条件下,斜纹夜蛾成虫嗅觉相关基因 abp, pbp 和 or 表达水平的变化;利用性信息素在田间诱捕斜纹夜蛾雄蛾,并通过自动计数器记录每小时诱虫量,从而间接显示其交配行为的节律性。【结果】斜纹夜蛾雄虫触角中嗅觉相关基因abp, pbp 和 or 的表达具有节律特性。经性信息素化合物(Z9, Z11-14:OAc+Z9, Z12-14:OAc)刺激处理后,abp, pbp 和 or 表达量也发生了显著的改变。通过记录田间性信息素诱捕器在一天中不同时间段内诱捕的雄蛾数量,发现诱捕到的斜纹夜蛾也具有节律特性。【结论】基因表达水平上的节律特性可能与雄虫交配活动的节律相关联,说明性信息素处理也在一定程度上改变了其节律及其对性信息素的神经反应。这一结果也首次从基因水平证明性信息素的刺激处理提高了周缘神经系统对性信息素反应的敏感性,有助于我们理解性信息素作用的分子机理,对迷向及性诱和测报应用具有指导意义。