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4-香豆酸辅酶A连接酶(4-coumarate:CoA ligase, 4CL)在苯丙烷类代谢途径中具有重要调控作用。该研究利用转录组数据在甘薯品种QS48中鉴定了11个4CL基因,并将这些基因命名为Ib4CL1~Ib4CL11。序列比对和功能域分析发现, 11个Ib4CL蛋白均具有典型的4CL结构特征,包含保守的BoxⅠ和BoxⅡ的结构域。进化和保守基序分析显示,Ib4CL1和Ib4CL2属于Ⅰ类4CL,Ib4CL3属于Ⅱ类4CL,而Ib4CL4~Ib4CL11属于4CL类似蛋白。转录组数据和荧光定量PCR分析显示,Ib4CL3和Ib4CL10在甘薯QS48的叶中转录表达水平显著高于茎和块根,与绿原酸的积累呈正相关关系;Ib4CL4和Ib4CL8在嫩叶中表达较高,与花青素的积累密切相关。研究结果为进一步揭示甘薯4CL基因在苯丙烷类代谢途径中的功能奠定了基础。 相似文献
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棉花4-香豆酸辅酶A连接酶基因克隆及原核表达 总被引:4,自引:0,他引:4
本研究从棉花中克隆了一个4CL基因,命名为Gh4CL2(GenBank登录号为FJ848870)。研究结果表明:Gh4CL2基因cDNA全长2332bp,具有一个1725bp的开放阅读框,5′非编码区为64bp,3′非编码区为543bp,编码574个氨基酸,预测分子量约为62.106kD,等电点为5.94。氨基酸同源性分析发现,Gh4CL2与来自白杨、大豆和紫草的4CL一致性较高。进一步研究Gh4CL2基因的功能,构建了该基因的原核表达载体pET-28a-4CL2,经酶切鉴定后转化到大肠杆菌BL21(DE3)中。SDS-PAGE分析表明,最佳诱导表达条件为0.5mmol/LIPTG在37℃下诱导4h,重组蛋白主要以包涵体形式出现。 相似文献
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以从光皮桦茎叶组织提取的mRNA为模板,根据其他已克隆到的阔叶类树种中4-香豆酸辅酶A连接酶(4CL)基因的同源序列设计兼并引物,进行RT-PCR扩增,获得部分基因片段,然后结合5’,3’RACE方法从光皮桦中扩增出1个4CL基因的全长cDNA序列,命名为Bl4CL。该基因cDNA全长为1983bp(GenBank登录号FJ410448),具有完整的开放阅读框架(69~1697bp),编码蛋白为542个氨基酸,包含一个AMP结合功能域和一个含有12个氨基酸的功能基序。与其他植物中的4CL进行同源性比对的结果显示,Bl4CL蛋白与东北白桦的同源性最高,达到了98%。该基因在光皮桦的根和茎中表达量较高,而在花和叶中的表达量低。 相似文献
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以从光皮桦茎叶组织提取的mRNA为模板,根据其他已克隆到的阔叶类树种中4-香豆酸辅酶A连接酶(4CL)基因的同源序列设计兼并引物,进行RT—PCR扩增,获得部分基因片段,然后结合5’,3’RACE方法从光皮桦中扩增出1个4CL基因的全长cDNA序列,命名为B14CL。该基因cDNA全长为1983bp(GenBank登录号FJ410448),具有完整的开放阅读框架(69—1697bp),编码蛋白为542个氨基酸,包含一个AMP结合功能域和一个含有12个氨基酸的功能基序。与其他植物中的4CL进行同源性比对的结果显示,B14CL蛋白与东北白桦的同源性最高,达到了98%。该基因在光皮桦的根和茎中表达量较高,而在花和叶中的表达量低。 相似文献
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以苦荞栽培种‘西荞2号’为材料,利用同源克隆和RT-PCR技术获得Ft4CL保守片段,采用RACE技术获得Ft4CL基因的3'末端及5'末端序列,并进一步采用生物信息学方法进行序列分析。结果表明:从苦荞花蕾总RNA中获得一条苦荞麦(Fagopyrum tatarium)4-香豆酸辅酶A连接酶基因(4-coumarate:Coa ligase,Ft4CL)的cDNA全长序列。生物息学分析结果显示,Ft4CL基因ORF全长1 602 bp,可编码553个氨基酸,理论标准分子质量为58.02kDa,等电点(pI)为5.23。该研究首次从苦荞中获得Ft4CL基因的cDNA全长序列,该基因具有植物4CL同源基因的典型特征,推导的氨基酸序列具有4CL的所有活性位点并归属于黄酮代谢支路。该研究结果可为深入研究苦荞黄酮代谢途径奠定基础,为采用代谢工程技术提高苦荞黄酮含量提供候选靶基因。 相似文献
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烟草4CL蛋白免疫荧光定位研究 总被引:1,自引:0,他引:1
4-香豆酸辅酶A连接酶(4CL)是维管植物木质素生物合成途径的关键酶,应用原核表达系统获得了毛白杨可溶性4CL1融合蛋白,以Ni2 -Agrose亲和柱层析纯化得到的SDS-PAGE电泳纯的毛白杨4CL1融合蛋白为抗原,免疫家兔获得毛白杨4CL1多克隆抗体,Western blotting鉴定表明兔抗毛白杨4CL1多克隆抗体具有高度特异性,免疫荧光定位发现普通烟草4CL1蛋白特异性地在木质部表达.为进一步应用木质部特异表达启动子定向调控木质素生物合成奠定了理论基础. 相似文献
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肉桂酸-4-羟化酶(Cinnamic acid-4-hydroxylase,C4H,EC 1.14.13.11)是苯丙烷途径中第二步反应酶,同时也是花色素苷前体生物合成途径中关键酶。该研究根据植物C4H的同源序列设计引物,通过RTPCR结合RACE的方法,在紫色甘薯中获得了与其相应的C4H基因,命名为Ib C4H(Gen Bank登录号GQ373157)。结果表明:(1)序列分析表明Ib C4H长1 668 bp,编码505个氨基酸,该氨基酸序列与其c DNA序列与Ib C4H蛋白与马铃薯C4H蛋白序列最为接近,与苹果、黑莓、大阿米芹、油菜一致性很高,均在70%以上。(2)二级结构预测表明α-螺旋和无规则卷曲是Ib C4H蛋白最大量的结构元件,而延伸链则散布于整个蛋白中。(3)三维结构建模预测,Ib C4H蛋白具备细胞色素P450氧和铁离子结合位点等典型的C4H结构。该研究结果为进一步了解花色素苷生物合成途径中的作用奠定了基础,也为花青素生物合成分子机理和代谢调控提供了靶位点和理论参考。 相似文献
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为揭示大麦中黄酮合成的分子调控机制,利用反转录PCR结合同源克隆和RACE技术首次从青稞(裸大麦)叶片中克隆获得肉桂酸-4-羟化酶基因(HvC4H)的全长cDNA序列(Genbank登录号:KF927086),总长度1951 bp,ORF为1518 bp,编码505个氨基酸,等电点PI=9.01,平均亲水指数(GRAVY)为-0.170,属于亲水性碱性蛋白,高级结构分析表明其具有细胞色素P450家族保守域及C4H特异的功能性活性位点。利用实时荧光定量PCR分析胚乳发育5个时期不同组织(茎、叶及子粒)的表达情况,结果显示HvC4H基因在青稞胚乳发育期的表达情况存在着明显的组织差异性,在茎中的表达量最高。本研究为通过调控C4H基因的表达从而提高大麦黄酮的含量奠定了分子生物学基础,对于改良大麦的品质、抗性、生长发育等性状具有重要意义。 相似文献
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Dae Hwa Yang Byung Yeoup Chung Jae -Sung Kim Jin -Hong Kim Pil -Yong Yun Young Ki Lee Yong -Pyo Lim Myung Chul Lee 《Journal of Plant Biology》2005,48(3):311-318
Plant cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of phenylpropanoids,
terpenes, lipids, and alkaloids. We isolated a cDNA clone for cinnamate-4-hydroxylase (C4H) from a Japonica type rice(Oryza sativa L. cv. llpumbyeo). This C4H has a deduced amino acid sequence that is 85% identical tothat ofSorghum bicolor. Our phylogenetic analysis also showed that theOsC4HL gene is closely related toC4H fromS. bicolor. A putative genomic DNA sequence corresponding toOsC4HL contained cis-elements (boxes P, A, L, and TCA motifs), AT-rich elements, and wound-response elements that control gene expression
in its promoter region.OsC4HL expression was detected in all the tissue types, with the highest level being measured in the roots. It was also apparently
up-regulated by wounding stress. These data suggest that theOsC4HL gene isC4H member in theCYP73 subfamily. 相似文献
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Shinya T Gális I Narisawa T Sasaki M Fukuda H Matsuoka H Saito M Matsuoka K 《Plant & cell physiology》2007,48(10):1404-1413
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Köhle Annegret Sommer Susanne Li Shu-Ming Schilde-Rentschler Lieselotte Ninnemann Helga Heide Lutz 《Molecular breeding : new strategies in plant improvement》2003,11(1):15-24
The bacterial gene ubiC encodes chorismate pyruvate-lyase (CPL), which converts chorismate to 4-hydroxybenzoate (4HB). The ubiC gene was expressed in tobacco (Nicotiana tabacum L., Solanaceae) and potato (Solanum tuberosum L., Solanaceae) under the control of the very strong constitutive plant promotor (ocs3) mas. High accumulation of 4HB glucosides as new, artificial secondary metabolites was observed in the transgenic plants. 4HB glucoside content reached 5.1% of dry weight in tobacco cell cultures and 4.0% of dry weight in the leaves of potato shoots. This is the highest content of an artificial secondary metabolite produced by genetic engineering of plants reported so far. Surprisingly, no growth retardation and no phenotypical changes were observed in the transgenic cell cultures and plants. Glucosylation of 4HB was achieved by endogeneous, constitutively expressed glucosyltransferases. The total amount of 4HB glucoside acccumulated showed a strict linear dependence on the expression level of ubiC. 相似文献
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The effect of exogenous adenine nucleotides on CO2 fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C4 plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate + oxalacetate induced CO2 fixation, as well as reverse the inhibition of CO2 fixation by carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO2 fixation varied from 40 to 70 μmol CO2 fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80–140 μmol/mg chlorophyll per h, since 2 ATP are required for each CO2 fixed. Fixation of CO2 could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts. This suggests that external ATP is exchanging for internal ADP. In contrast, ADP and AMP were found not to traverse chloroplast membranes, on the basis that neither nucleotide inhibited CO2 fixation or stimulated oxygen evolution that was limited by available ADP for phosphorylation. Further evidence that ATP can enter the chloroplasts was obtained by direct measurements of the increase in ATP in the chloroplasts due to addition of exogenous ATP in the dark. These studies yielded minimal rates of ATP uptake on the order of 30–40 μmol/mg chlorophyll per h. It is suggested that a membrane translocator exists that specifically transports ATP into the chloroplasts in exchange for ADP. The significance of these findings are considered with respect to the C4 pathway of photosynthesis. 相似文献
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通过构建红色亚栖热菌(Meiothermus ruberCBS-01)的基因组DNA文库,克隆得到该嗜热菌海藻糖合成途径中的磷酸海藻糖合成酶(TPS)和磷酸海藻糖磷酸酯酶(TPP)基因。以pET21a为表达载体,将磷酸海藻糖合成酶和磷酸海藻糖磷酸酯酶在大肠杆菌中进行表达并纯化,利用薄层层析的方法验证了这两个酶的活性。同时,本研究检测了红色亚栖热菌在各种环境压力下细胞内含物成分的变化情况,发现在高渗环境压力的诱导下,该菌会在胞内积累大量的6-磷酸海藻糖,而并非海藻糖,这为进一步研究TPS/TPP和TreS途径在细胞体内的作用奠定了基础。 相似文献
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Zhou XR Robert SS Petrie JR Frampton DM Mansour MP Blackburn SI Nichols PD Green AG Singh SP 《Phytochemistry》2007,68(6):785-796
The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants. 相似文献
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Darius J.R. Lane Dong-Hun Bae Aritee R. Siafakas Yohan Suryo Rahmanto Lina Al-Akra Patric J. Jansson Robert A. Casero Des R. Richardson 《生物化学与生物物理学报:疾病的分子基础》2018,1864(9):2793-2813
Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N1-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced 3H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation. 相似文献