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。     

14-3-3蛋白与植物细胞信号转导

14-3-3蛋白通过直接蛋白质-蛋白质相互作用对植物代谢关键酶、质膜H^＋ -ATP酶等发挥广泛调节作用。越来越多证据显示14-3-3蛋白通过与转录因子和其他信号分子结合参与调控植物细胞信号转导。对植物细胞中14-3-3蛋白调控信号转导途径,尤其是植物细胞对胁迫响应的调控机制进行了综述。     

14-3-3蛋白对植物发育的调控作用

14-3-3蛋白是高度保守并在真核生物中普遍存在的一类调节蛋白。不同的14-3-3蛋白同工型具有不同的细胞特异性, 并通过识别特异的磷酸化序列与靶蛋白相互作用, 被称为蛋白质与蛋白质相互作用的桥梁蛋白。在植物生长发育过程中, 14-3-3蛋白通过与其它蛋白的相互作用参与多种植物激素信号转导、各种代谢调控、物质运输和光信号应答等调控过程。该文主要对近年来有关14-3-3蛋白在植物生长发育中的调控作用, 特别是14-3-3蛋白参与调控植物激素信号转导等方面的研究进展进行综述。     

植物中14-3-3蛋白的主要功能

14-3-3蛋白家族广泛存在于真核生物中,序列高度保守。主要以同源或异源二聚体形式存在,可以同时与两个靶蛋白或者与一个靶蛋白的两个结构域相互作用,通过与靶蛋白上的一小段共有序列的磷酸化丝氨酸/苏氨酸残基结合来发挥其调控功能。本文综述了植物中的14-3-3蛋白及其主要功能,并重点综述了14-3-3蛋白对植物基本碳、氮代谢的调控。     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

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。     

南京椴HMGR基因的克隆和功能验证

3-羟基-3-甲基戊二酰辅酶A还原酶（3-hydroxy-3-methylglutaryl-CoA reductase,HMGR）是植物萜类代谢中甲羟戊酸途径的关键酶,本研究运用cDNA末端快速扩增（RACE）技术,首次从珍稀植物南京椴中克隆出HMGR的全长基因TmiHMGR,其长度为2 160 bp,包含一个1 758 bp的开放阅读框,其推导蛋白TmiHMGR编码585个氨基酸残基,相对分子量为62.9 kD,pI为6.11。将TmiHMGR与其他植物HMGR氨基酸序列构建进化树,结果显示TmiHMGR与苹果的HMGR聚为一枝。采用半定量RT-PCR分析TmiHMGR在根、茎和叶中的表达情况,结果表明该基因在茎中的表达量最高,根和叶中的表达量相对较弱。验证功能的颜色互补实验结果显示,TmiHMGR能够使代谢流明显朝类胡萝卜素合成的方向进行,说明TmiHMGR在萜类产物生物合成中是一个重要因子。     

苹果中α-法尼烯的代谢途径及其分子调控

概述了苹果中α-法尼烯在虎皮病诱导和昆虫诱导中的作用、代谢途径及其分子调控机制,尤其是3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)对其合成的调控.     

甘草过表达HMGR、SQS1、β-AS基因再生植株的诱导

目的:诱导过表达3-羟基-3-甲基戊二酰CoA还原酶(HMGR)、鲨烯合成酶1(SQS1)和β-香树脂醇合成酶(β-AS)基因的甘草再生植株。方法:以植物双元表达载体pCAMBIA1305.1为载体骨架,HMGR、SQS1、β-AS基因为目的基因,利用基因重组试剂盒eFusion CloningKit分别构建含有3个外源基因的重组载体,并将其转入根癌农杆菌EHA105,采用根癌农杆菌介导法将目的基因转入甘草外植体。结果与结论:获得了过表达HMGR、SQS1和β-AS基因的甘草愈伤组织及试管苗,为进一步研究这3个功能基因过表达对甘草酸合成的影响奠定了基础。     

14-3-3蛋白在细胞周期调节中的作用

14-3-3是一个在真核细胞中广泛表达、功能复杂的蛋白家族,主要通过磷酸化依赖的方式与靶蛋白结合,从而发挥其调控作用。细胞周期的调节对维持基因组的稳定性至关重要。近年来的研究发现,14-3—3蛋白可以和越来越多的细胞周期调节蛋白相互作用,调节G2／M期和G1／S期转换,从而对细胞周期起调控作用。简要综述了14—3—3蛋白在细胞周期调节中的作用。     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Ether-linked lipids of Balb/c3T3, SV3T3 and concanavalin A-selected SV3T3 revertant cells

Ether-linked lipids were analyzed in Balb/c3T3, SV3T3 and Concanavalin A-selected SV3T3 revertant cells. The three cell lines were found to contain significant quantities of alk-1-enyl- and alkyl-linked phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and small amounts of alkyldiacylglycerols. Compared to 3T3 cells, SV3T3 cells contain a higher amount of alk-1-enyl-linked PC, while in SV3T3 revertant cells the concentrations of the various ether lipids are similar to those of 3T3 cells. The major difference in the composition of ether groups of SV3T3 cells, compared to 3T3 cells, is an increase of 18:0 accompanied by a decrease of 18:1 in the alk-1-enyl-linked PE and PC. Alk-1-enyl-linked PC of SV3T3 revertant cells also shows an increase of 18:0, while the decrease of 18:1 was not statistically significant.     

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

3-Bromo-3-deazaneplanocin and 3-bromo-3-deazaaristeromycin: Synthesis and antiviral activity

As an outgrowth of our program to explore 3-deazaadenine carbocyclic nucleosides, 3-bromo-3-deazaneplanocin (5) and 3-bromo-3-deazaaristeromycin (6) have been synthesized from a readily available cyclopentenol and cyclopentanone and either 4-amino- or 4-chloro-1H-imidazo[4,5-c]pyridine (6-amino- or 6-chloro-3-deazaadenine) in 5 steps and 7 steps, respectively. Antiviral analysis found 5 to display significant activity towards a number of (-)-ssRNA and a few dsDNA viruses. Compound 6 was less active than 5 against selected examples of those viruses affected by 5.     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.