植物3-羟基-3-甲基戊二酰辅酶A还原酶基因研究进展

细胞分裂素、赤霉素、脱落酸、叶绿素、萜类等类异戊二烯物质,是植物中广泛存在的一类代谢产物,在植物生长发育过程中起着非常重要的作用。一些萜类化合物作为药物的合成前体或有效的药用成分在工农业及医药生产上具有重要的经济价值。类异戊二烯物质主要通过甲羟戊酸代谢途径中的一系列酶催化合成,其中,3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)是该代谢途径中的第一个关键限速酶,能够将3-羟基-3-甲基戊二酰辅酶A转化成中间代谢产物甲羟戊酸。对植物HMGR基因的克隆、酶结构和功能分析、基因组织表达及调控等方面进行了综述,旨在为其在重要农作物的遗传改良、代谢产物工程植物创制以及植物亲缘关系分析中的应用等研究提供理论依据。     

高等植物的3-羟基-3-甲基戊二酰辅酶A还原酶

介绍了植物3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)的结构和调控,并简略讨论了HMGR调控与植物类异戊二烯途径的关系.     

植物萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的生物信息学分析

采用生物信息学的方法和工具对已在GenBank上注册的橡胶、烟草、辣椒、穿心莲等植物的萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的核酸及氨基酸序列进行分析,并对其组成成分、信号肽、跨膜拓朴结构域、疏水性/亲水性、蛋白质二级及三级结构、分子系统进化关系等进行预测和推断。结果表明该类酶基因的全长包括5′、3′非翻译区和一个开放阅读框,无信号肽,是一个跨膜的亲水性蛋白,包括两个功能HMG-CoA结合motif及两个功能NADPH结合motif,α-螺旋和不规则盘绕是蛋白质二级结构最大量的结构元件,β-转角和延伸链散布于整个蛋白质中,蛋白质的功能域在空间布局上折叠成“V”形,“V”形的两臂由螺旋状的N结构域和S结构域构成,中间部分由L结构域构成。     

菊花等中药水煎剂对离体大鼠肝细胞微粒体羟甲基戊二酰辅酶A还原酶的作用机理

The isolated rat hepatic microsomal preparation was incubated with the aqueous extracts of Flos chryranthemi(菊花), Curcuma aromatica Salisb(郁金), Acan- thopanax senticosus (刺五加), Rhizoma ligustici wallichii (川芎), Radix polygoni multiflori (首乌) and Fructus crataegi pinnatifidae (山楂), respectively (37% 20 min). The activity of hydroxymethlglutaryl coenzyme A reductase was found to decrese about 30 % corresponding to the action of 50mmol/L NaF at similar condition. The mechanism of action of the Chinese drugs was the inhibition of cytosolic protein phosphatase and the activation of cytosolic hydroxymethylglutaryl CoA reductase kinase. Both the results of these two actions suppressed the activity of HMG-CoA reductase.     

京大戟hmgr基因家族3个保守区片段的克隆与分析

采用RT-PCR技术以及两条简并引物,以京大戟嫩叶总RNA反转录的cDNA为模板扩增3-羟基-3-甲基戊二酰辅酶A还原酶基因的保守区片断。序列分析表明,所克隆的cDNA保守区序列长度为458 bp,而且同时得到了三个不同的核苷酸序列,分别命名为hmgr1、hmgr2、hmgr3。与之前得到的京大戟hmgr保守区片段的核苷酸序列的同源性分别为98.03%、96.29%、78.38%,推断的相应氨基酸序列的同源性分别为98.68％、96.71%和85.53%。推断这可能是该基因家族中的三个新的成员。而且同源序列比对发现,由这三个核苷酸序列推断的氨基酸序列与其它植物都有较高的同源性。种系进化树分析表明hmgr3与hmgr1、hmgr2及以前报道的京大戟的hmgr之间有较大的差别。     

细纹豆芫菁3-羟甲基戊二酰辅酶A-还原酶基因全长cDNA克隆及生物信息学分析

3-羟甲基戊二酰辅酶A-还原酶（3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR）是甲羟戊酸途径的关键酶。获得芫菁体内HMGR基因信息是确定甲羟戊酸途径与斑蝥素合成相关性的基础。本研究利用RACE技术从细纹豆芫菁Epicauta mannerheimi (Maklin)体内克隆获得HMGR基因全长cDNA序列, 命名为EmHMGR（GenBank登录号为JQ690539）。该基因全长3 118 bp, 其中5′端非翻译区178 bp, 3′端非翻译区414 bp, 开放阅读框2 526 bp, 编码842个氨基酸。推测的蛋白质分子量为92.8 kDa, 理论等电点为6.0, 预测分子式为C₄₁₃₅H₆₆₀₄N1₀₉₈O₁₂₁₆S₅₀, 不稳定系数为43.37, 总亲水性系数为0.091, 为疏水性不稳定蛋白。序列分析发现该基因编码的蛋白与已报道的其他昆虫HMGR的氨基酸序列一致性达50%以上, 而且包含HMGR_Class I保守功能域、 固醇敏感多肽区及HMGR蛋白的其他保守功能位点。系统进化分析发现该基因与叶甲科昆虫HMGR基因的关系最近。本研究首次从芫菁科昆虫体内克隆获得甲羟戊酸途径的关键酶EmHMGR基因, 为后期芫菁体内斑蝥素生物合成途径的研究奠定了基础。     

大戟甲羟戊酸途径关键酶基因hmgr的克隆与分析

通过比较6种植物8条甲羟戊酸途径关键酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因同源区域,设计简并引物,利用RT-PCR技术成功地从大戟(Euphorbia pekinensis)叶中扩增出458bp的基因片段。通过BlastP比较,所推断的大戟HMGR蛋白序列与杜仲Eucommia ulmoides(AAV54051)、穿心莲Andrographis paniculata(AAP14352)、胡黄连Picrorhiza kurrooa(ABC74565)、橡胶树Hevea brasiliensis(AAU08214)、海岛棉Gossypium barba-dense(ABC71314)、龙胆草Gentiana lutea(BAE92730)的一致性分别达到90%、86%、86%、92%、87%和88%。蛋白质保守区、特征区以及进化树分析,初步证实该基因为hmgr基因,这是首次报道从药用植物大戟中克隆到甲羟戊酸途径关键酶HMGR的基因片段。     

小麦吸浆虫保幼激素酯酶和保幼激素环氧水解酶基因的克隆及在滞育和变态发育过程中的表达动态

【目的】保幼激素(juvenile hormone, JH)在小麦吸浆虫Sitodiplosis mosellana滞育诱导及滞育后静息状态的维持中发挥着重要作用。保幼激素酯酶(hormone esterase, JHE)和保幼激素环氧水解酶(juvenile hormone epoxide hydrolase, JHEH)是调控JH滴度的重要降解酶。本研究旨在探讨JHE和JHEH在小麦吸浆虫滞育和变态发育中潜在功能。【方法】通过RT-PCR和RACE技术从小麦吸浆虫滞育前幼虫克隆JHE和JHEH全长cDNA序列;利用生物信息学软件分析其核苷酸及编码蛋白特性;采用qPCR技术分析其在小麦吸浆虫滞育不同时期(滞育前、滞育期、滞育后静息期和滞育后发育)3龄幼虫及1龄幼虫到成虫不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹、后蛹、雌成虫和雄成虫)中的表达水平。【结果】克隆获得了cDNA全长分别为3 102和1 980 bp的小麦吸浆虫SmJHE和SmJHEH基因(GenBank登录号分别为MG876768和MG876769),其开放阅读框分别长1 740和1 371 bp,分别编码579和45...     

大戟甲羟戊酸途径关键酶基因hmgr的克隆与分析

通过比较6种植物8条甲羟戊酸途径关键酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因同源区域,设计简并引物,利用RT-PCR技术成功地从大戟(Euphorbia pekinensis)叶中扩增出458bp的基因片段。通过BlastP比较,所推断的大戟HMGR蛋白序列与杜仲Eucommia ulmoides(AAV54051)、穿心莲Andrographis paniculata(AAP14352)、胡黄连Picrorhiza kurrooa(ABC74565)、橡胶树Hevea brasiliensis(AAU08214)、海岛棉Gossypium barbadense(ABC71314)、龙胆草Gentiana lutea(BAE92730)的一致性分别达到90%、86%、86%、92%、87%和88%。蛋白质保守区、特征区以及进化树分析,初步证实该基因为hmgr基因,这是首次报道从药用植物大戟中克隆到甲羟戊酸途径关键酶HMGR的基因片段。     

播娘蒿hmgr基因保守区片段的克隆与分析

通过比较6种植物的8条甲羟戊酸途径关键酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因同源区域,设计简并引物,利用RT-PCR技术成功地从播娘蒿叶中扩增出458bp的基因片段。通过BlastP比较,所推断的播娘蒿HMGR蛋白序列与拟南芥(NP_177775)、萝卜(CAA48610)、杜仲(AAV54051)、胡黄连(ABC74565)、喜树(AAB69726)、龙胆草(BAE92730)的一致性分别达到98%、96%、88%、89%、86%和87%。通过对蛋白质保守区、特征区以及进化树分析,证实该片段确为hmgr基因片段,该结果为首次报道。     

麦红吸浆虫幼虫不同滞育时期体内山梨醇含量及山梨醇脱氢酶基因表达水平变化

【目的】本研究旨在明确麦红吸浆虫Sitodiplosis mosellana(Géhin)滞育进程中山梨醇含量及山梨醇脱氢酶基因(SmSDH)mRNA表达水平的变化规律,探讨麦红吸浆虫滞育与山梨醇含量及SmSDH表达水平的关系。【方法】分别采用比色法和实时荧光定量PCR方法,对2013年5月-2014年3月不同时间陕西杨凌养虫圃内麦红吸浆虫滞育前后及滞育期幼虫体内山梨醇含量及SmSDH2a和SmSDH2b mRNA表达水平进行分析。【结果】麦红吸浆虫幼虫滞育前山梨醇含量最高(为9.41μg/mg);滞育期间,12月份含量最高(为8.50μg/mg);滞育解除后山梨醇含量为3.25~3.49μg/mg,显著低于滞育前和整个滞育期(P0.05)。山梨醇脱氢酶基因SmSDH2a及SmSDH2b在滞育进程中mRNA表达水平变化趋势相同,且与滞育强度递减趋势一致,即进入滞育后表达水平显著降低(P0.05)并一直稳定在较低水平,滞育解除后逐渐升高并达到最高。滞育期间,同期不同滞育状态幼虫之间山梨醇含量和SmSDH2a表达水平差异不明显,但裸露幼虫SmSDH2b表达水平始终高于结茧幼虫。【结论】山梨醇和SmSDH参与麦红吸浆虫滞育调控,与滞育维持和解除密切相关。     

麦红吸浆虫蜕皮激素受体（EcR）基因的克隆与表达分析

为了研究蜕皮激素受体（EcR）在麦红吸浆虫Sitodiplosis mosellana (Géhin)滞育活动中的作用, 利用RT PCR和RACE技术克隆得到了麦红吸浆虫蜕皮激素受体基因cDNA全长, 并通过Real-time quantitative PCR研究了其表达情况。该cDNA全长序列被命名为SmEcR （GenBank登录号： KC491135）, 其开放阅读框长1 386 bp, 编码461个氨基酸残基。其蛋白预测分子量52.90 kD, 理论等电点6.24。该蛋白与其他已报道的昆虫EcR蛋白具有很高的同源性, 其中与迟眼蕈蚊Bradysia coprophila中相应蛋白的氨基酸序列一致性高达92%。SmEcR在麦红吸浆虫不同滞育时期和不同虫态中均有表达, 且在不同滞育时期、 不同虫态中的表达量差异很大。在滞育不同时期以11月表达量最高, 12月表达量最低; 在不同虫态以麦穗幼虫中的表达量较低, 而成虫中的表达水平很高。本研究为进一步明确SmEcR在麦红吸浆虫滞育调控中的作用奠定了基础。     

麦红吸浆虫不同滞育期四种糖代谢酶活力分析

海藻糖是麦红吸浆虫Sitodiplosis mosellana (Gehin)滞育期间储藏能量的主要物质,为弄清其在滞育期的积累机理,本文测定了麦红吸浆虫滞育前后和滞育期糖原磷酸化酶（Gpase）、己糖激酶（HK）、磷酸果糖激酶（PFK）和醛缩酶（ALD）这4种糖代谢酶活力的变化。结果表明: 麦红吸浆虫滞育前后这些糖代谢酶活力明显不同,滞育后Gpase 活力显著提高,糖酵解有关酶HK,PFK和ALD活力降低;滞育解除后,Gpase 活力降低,HK,PFK和ALD活力升高。滞育期间,4种糖代谢酶的活力均与滞育发育有关;同时Gpase 和PFK的活力也与环境温度有关,即夏、冬季高于春、秋季;同期不同滞育状态幼虫比较,裸露幼虫HK,PFK和ALD活力总是略高于结茧幼虫,Gpase则相反。滞育当年与第2年同期幼虫4种糖代谢酶的活力无显著差异。     

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.     

Interference with the determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and its properties by a microsomal enzymic activity.

Rat liver microsomes and microsomal extracts contain an enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for 3-hydroxy-3-methylglutaryl coenzyme A. The presence of this activity in enzyme preparations causes errors in the determination of reductase activity and its properties. This contaminant can be removed by gel filtration using Bio-Gel A 1.5m, by washing the microsomes, or by incubating the microsomal extract at 37 °C. The K_m's of the reductase (free of this competing enzymic activity) for d-3-hydroxy-3-methylglutaryl coenzyme A and NADPH are 1.3 and 26 μm, respectively.     

Lack of correlation between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lovastatin resistance in nerve growth factor treated PC-12 cells

Summary 1. The relationships among the mevalonic acid (MVA) forming enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase, cell growth and differentiation, and the cytotoxic effects of the reductase inhibitor lovastatin were studied in PC-12 cells, exposed to growth factors.2. When added individually, nerve growth factor (NGF), basic fibroblast growth factor, and epidermal growth factor induce an increase in HMG-CoA reductase activity in cells grown in serum-containing medium. In the presence of serum, the effect of NGF on HMG-CoA reductase is persistent.3. Short-term serum starvation and long-term NGF treatment, in combination, have an additive effect, resulting in a high reductase activity.4. Unlike serum and MVA, which downregulate levels of HMG-CoA reductase by accelerating its degradation, NGF upregulates reductase by slowing the rate of its degradation. This mechanism, however, appears to operate only in the presence of serum, as after prolonged growth with NGF in serum-free medium, cells have a low reductase activity.5. PC-12 cells grown in the absence of NGF are highly sensitive to lovastatin (25 µM) and more than 70% of the cells die after 48 hr. NGF confers lovastatin resistance on cells grown in the presence or in the absence of serum (only 30–40% cell death after 48 hr with lovastatin).6. NGF-induced resistance on lovastatin develops with time and is apparent only in the well-differentiated PC-12 cells whether or not the cells express a high reductase activity.7. Thus, levels of HMG-CoA reductase activity and lovastatin resistance in PC-12 cells are not directly correlated, though clearly inversed lovastatin cytotoxicity and elevated reductase activities are expressed during the period of cell proliferation.8. These data suggest that fully differentiated neuronal cells may not be affected by prolonged high doses of lovastatin.