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1.
3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-coenzyme A reductase,HMGR)是天然橡胶生物合成途径中的关键酶之一,在先前的研究中本课题组已克隆了与Hmgr1启动子互作的转录因子基因HbCZF1。本研究在此基础上,构建了HbCZF1的原核表达载体PET-28a(+)-HbCZF1,将其转化E.coli Rosetta(DE3)菌株。经优化条件后,在20℃,0.1 mmol/L IPTG诱导4 h的条件下,获得了分子量约47 ku的HbCZF1融合蛋白。  相似文献   

2.
牻牛儿基牻牛儿基焦磷酸合酶(Geranylgeranyl diphosphate synthase,GGPS)是萜类合成途径的结构酶,对植物生长发育具有重要意义。本研究通过RACE和RT-PCR方法克隆得到5条潜在的茶树GGPS序列,分别命名为CsGGPS1-4和CsGGPS9,其中CsGGPS9存在3条等位基因,分别是CsGGPS9-1、CsGGPS9-2和CsGGPS9-3,在系统进化树上与其他基因分成两支。蛋白质序列分析表明,茶树GGPS家族成员都具有polyprenyl_synt结构域,不存在信号肽序列。亚细胞定位预测结果显示,CsGGPS1、CsGGPS2和CsGGPS4定位在叶绿体上,CsGGPS3和CsGGPS9定位在线粒体上。通过Swiss Model进行三维建模,结合"three-floor"模型对茶树GGPS家族成员的功能进行预测,预测结果显示,CsGGPS1、CsGGPS2和CsGGPS4是GGPS;CsGGPS3是异源二聚体形式的牻牛儿基焦磷酸合酶的小亚基;CsGGPS9的催化主产物是碳链数大于30的异戊烯基焦磷酸。q RT-PCR分析表明,CsGGPS1整体表达丰度较低,仅在一芽二叶中表达量稍高;CsGGPS2在茶树各个组织中均有表达,在花中表达量最高,且花发育过程中表达量先上升后下降;CsGGPS3在叶和幼根中的表达量高于花,花发育过程中表达平稳;CsGGPS4在茶树各个组织中表达量数值相近,在花发育过程中表达量变化趋势与CsGGPS2相同;CsGGPS9的表达量在成熟叶中显著低于幼嫩叶片。  相似文献   

3.
类黄酮7-O-甲基转移酶(F7-OMT)具有甲基转移酶功能,能催化底物类黄酮甲基化产生植物抗毒素,提高植物的抗菌性。为了给进一步研究该酶蛋白的功能奠定基础,以青稞"94-19-1"为材料,采用同源克隆方法得到青稞F7-OMT基因编码片段。生物信息学分析表明,青稞F7-OMT开放阅读框(ORF)全长为1173bp,编码390个氨基酸,蛋白分子质量为42 207.8Da,等电点pI为5.36,无信号肽序列,具有甲基转移酶2(Methyltransf_2,pfam00891)和二聚化(Dimerization,pfam08100)保守域。通过亚克隆将F7-OMT连接到表达载体pET-32a上,构建pET-32a-F7-OMT融合表达载体,在E.coli BL21(DE3)pLysS中诱导表达,并采用亲和层析纯化法得到纯化蛋白。  相似文献   

4.
焦磷酸酶对橡胶的生物合成具有重要的调控作用.将已克隆的3个可溶性无机焦磷酸酶基因的编码区插入到原核表达载体pGEX-6P-1上,构建3个焦磷酸酶基因表达载体(pGEX-HbSIPl、pGEX-HbSIP2和pGEX-HbSIP3),再将表达载体转人大肠杆菌表达菌株进行诱导表达.结果表明:pGEX-HbSIP1、pGEX-HbSIP2表达载体在大肠杆菌BL21(DE3)中获得高效表达,pGEX-HbSIP3只在补充稀有密码子的大肠杆菌Rosetta (DE3)中表达,表达率分别为35.4％、43.7％和18.5％.对原核表达的蛋白质进行纯化,获得3种可溶性重组蛋白GST-HbSIP1、GST-HbSIP2和GST-HbSIP3,总回收率分别约为7％、8％、3％.该结果为进一步研究这3种焦磷酸酶蛋白的分子特性、抗体制备及功能研究奠定基础.  相似文献   

5.
采用RT-PCR和RACE(Rapid amplification of cDNA ends)技术,从艾纳香(Blumea balsamifera L.DC)的叶片中克隆到单萜化合物合成的关键酶牻牛儿基焦磷酸合成酶(BbGPPS)基因。研究结果显示,BbGPPS基因的cDNA全长1 692 bp,包含开放阅读框(ORF)1 083 bp,编码361个氨基酸;亚细胞结构定位于叶绿体,既非膜蛋白也非分泌性蛋白。疏水性分析显示,BbGPPS是亲水性蛋白。同源性比对结果显示,BbGPPS蛋白与其他植物中GPPS蛋白具有高度的相似性,且具有异戊烯基结构域。系统发育分析表明,所有序列被聚为5大类,BbGPPS与其他菊科植物聚类一类,与万寿菊(Tagetes erecta)TeGPPS亲缘关系最近,其次是甜菊(Stevia rebaudiana)SrGPPS。  相似文献   

6.
从Agrobacteriumtumefaciens(strainAKE10)中分离其质粒并以质粒DNA为模板进行多聚酶链式反应,特异扩增出一条长约720bp的DNA片段,经克隆及序列测定表明,为根癌农杆菌AKE10质粒异戊烯基转移酶基因的完整编码序列,与Genebank中公布的核苷酸序列具有100%的同源性。与A.tumefaciensPO22、Tm-4、BO542菌株和A.vitisCG474、Tm4菌株质粒中异戊烯基转移酶基因进行序列比较,同源性分别为90%、89%、86%、89%和89%;推导氨基酸序列的同源性分别为90.42%、88.23%、90.10%、89.58%和89.12%。成功构建了异戊烯基转移酶基因植物表达载体pBT,为探索植物细胞分裂素生物合成的分子机制、作用机理及激素互作等研究打下了基础。   相似文献   

7.
利用PCR方法克隆香蕉束顶病毒海南分离物(Banana bunchy top virus,BBTV-HN)DNA2编码区,将其插入到原核表达载体pGEX-6p-1谷胱苷肽-S-转移酶(GST)基因下游.所得克隆pGEX-HN-B2测序结果表明,插入的DNA2片段为267 bp,且表达相位和理论上设计的完全一致.把此重组质粒转化到E.coli Rosetta(DE3)中,经过异丙硫代半乳糖苷(IPTG)诱导后出现约33 Ku融合蛋白质表达条带.为获得特异的血清,切下目的表达条带并免疫兔子.经Western-blotting检测,获取的血清可以和表达的33 Ku融合蛋白特异结合,表明已成功获得DNA2编码蛋白的血清.  相似文献   

8.
利用同源克隆和RACE(Rapid amplification of cDNA ends)技术从甘蓝型油菜中克隆到精氨酸脱羧酶(Arginine decarboxylase,ADC)基因cDNA全长序列,命名为BnADC。BnADC全长为2 649bp,包含344bp的5′非翻译区(5′ Untranslated region,5′-UTR)、226bp的3′非翻译区(3′ Untranslated region,3′-UTR)和2 079bp的完整开放阅读框(open reading frame,ORF),编码75.1kD的蛋白质。5′-UTR中含有12bp的uORF (Upstream open reading frame),编码MIRE 4个氨基酸。氨基酸同源性比对表明,BnADC蛋白与其他植物ADC蛋白具有很高的相似性,与芥菜的同源性高达93%;系统进化树分析表明,BnADC与芥菜、拟南芥的ADC亲缘关系较近。在获得全长cDNA的基础上,构建融合表达载体pET30a(+)-BnADC,转化大肠杆菌（Escherichia coli）表达菌株BL21(DE3),SDS-PAGE检测到一个约81.1kD的融合蛋白被E. coil表达,且融合蛋白主要存在于菌体沉淀中。  相似文献   

9.
应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆NSP (Nuclear shuttle protein)基因的编码区,并通过Gateway技术定向重组到原核表达载体pDEST~(TM)-17中的6xHis标签下游,经菌落PCR和测序鉴定结果表明,成功构建了原核表达载体pDEST~(TM)-17-NSP.阳性克隆转化Ecoli BL21(DE3),经IFFG诱导表达、SDS-PAGE分析和western blot检测,结果显示,融合蛋白以包涵体的形式稳定表达,分子量约为20 ku,与预计值相符.通过诱导条件的优化,确定了最佳的融合蛋白表达条件为25℃、0.1 mmol/L IPTG条件下诱导4h.从而为制备NSP多克隆抗体和进一步研究NSP的功能奠定了基础.  相似文献   

10.
小麦TaMBD3基因的克隆和表达   总被引：1，自引：0，他引：1  
DNA甲基化(m5C)在基因表达调控过程中发挥重要作用,而甲基结合域蛋白(MBD)是能特异识别甲基化位点的反式作用因子.为了探讨小麦中MBD基因的结构与功能,以拟南芥MBD基因的EST为基础,通过电子克隆结合RT-PCR方法分离克隆了包含ORF的小麦甲基结合域蛋白基因TaMBD3.序列分析显示,TaMBD3具有典型的甲基结合域,其中包含了能与甲基化DNA相结合的保守氨基酸残基.组织表达特性分析表明,TaMBD3在幼穗和茎中的表达量高于其它组织器官.采用电子定位的方法,将TaMBD3基因初步定位在6AS1-0.35-0.65和C-6BL3-0.36两个区域.  相似文献   

11.
本研究以冰菜（Mesembryanthemum crystallinum L.）为实验材料,运用多种柱色谱和波谱技术,从冰菜乙醇提取物的乙酸乙酯和正丁醇相中共分离鉴定得到了11个单体化合物,其中苯丙素类化合物2个,分别是顺式对羟基肉桂酸（化合物4）、阿魏酸（化合物3）;生物碱类化合物2个,分别是烟酸（化合物6）、ethane-1,2-diyldinicotinate（化合物7）;糖苷类化合物5个,分别是2-ethoxy-5-(hydroxymethyl)tetrahydrofuran-3,4-diol（化合物5）、3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl(E)-3-(4-hydroxy-3-methoxyphenyl)acrylate（化合物8）、(E)-2-(hydroxymethyl)-6-(4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenoxy)tetrahydro-2H-pyran-3,4,5-triol（化合物9）、6-(1R,2S-2-hydroxy-4-(S,E-3-hydroxybut-1-en-1-yl)-3,5,5-trimethylcyclohex-3-en-1-yl)oxy-5-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4-triol（化合物10）、E-4-hydroxy-3,5,5-trimethyl-4-(3-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)but-1-en-1-yl)cyclohex-2-en-1-one（化合物11）;苯丙呋喃酮类化合物1个:黑麦草内酯（化合物2）;甾体类化合物1个:β-谷甾醇（化合物1）。本研究丰富了冰菜中的化学成分,也为更好地开发利用冰菜的功能性成分提供理论依据,有利于冰菜的推广种植。  相似文献   

12.
Two new isocoumarin derivatives, including a new 5-hydroxy-8-methyl-2H, 6H-pyrano[3,4-g]chromen-2,6-dione (1) and 6,8-dihydroxy-3,7-dimethylisocoumarin (2b), a new chevalone derivative, named chevalone E (3), and a new natural product pyripyropene S (6) were isolated together with 6, 8-dihydroxy-3-methylisocoumarin (2a), reticulol (2c), p-hydroxybenzaldehyde, chevalone B, chevalone C, S14-95 (4), and pyripyropene E (5) from the ethyl acetate extract of the undescribed marine sponge-associated fungus Aspergillus similanensis KUFA 0013. The structures of the new compounds were established based on 1D and 2D NMR spectral analysis, and in the case of compound 3, X-ray analysis was used to confirm its structure and the absolute configuration of its stereogenic carbons. Compounds 1, 2a–c and 3–6 were evaluated for their antimicrobial activity against Gram-positive and Gram-negative bacteria, Candida albicans ATCC 10231, and multidrug-resistant isolates from the environment. Chevalone E (3) was found to show synergism with the antibiotic oxacillin against methicillin-resistant Staphylococcus aureus (MRSA).  相似文献   

13.
为了解新疆小麦面粉挥发性成分组成,用顶空固相微萃取气相色谱-质谱联用技术(headspace solid phase microextraction gas chromatography-mass spectrometry,HS-SPME-GC-MS)测定12个小麦品种的挥发性物质构成,结合相对气味活度值(relative odor activity value,ROAV)、主成分分析(principal component analysis,PCA)和聚类分析,分析新疆不同小麦品种面粉中的关键香气成分构成差异。结果表明,12个小麦面粉样品中共鉴定出143种挥发性化合物,其中烷烃类50种,烯烃类8种,醛类13种,醇类28种,酮类16种,酯类7种,酸类4种,苯类9种,其他化合物8种。ROAV分析发现不同小麦品种面粉主要挥发性物质有12种：己醛、壬醛、反-2-辛烯醛、癸醛、(E,E)-2,4-壬二烯醛、1-辛烯-3-醇、2-正戊基呋喃、正己醇、1-壬醇、6-甲基-5-庚烯-2-酮、3-辛烯-2-酮、γ-壬内脂。主成分和聚类分析表明,春小麦、冬小麦分别聚为一类。  相似文献   

14.
Zhang H  Wang H  Cui H  Li Z  Xie Z  Pu Y  Li F  Qin S 《Marine drugs》2011,9(9):1502-1509
A new anthracene derivative, 3-hydroxy-1-keto-3-methyl-8-methoxy-1,2,3, 4-tetrahydro-benz[α]anthracene, was isolated from the marine strain Streptomyces sp. W007, and its structure was established by spectroscopic analysis including mass spectra, 1D- and 2D-NMR ((1)H-(1)H COSY, HMBC, HSQC and NOESY) experiments. 3-hydroxy-1-keto-3-methyl-8-methoxy-1,2,3,4-tetrahydro-benz[α]anthracene showed cytotoxicity against human lung adenocarcinoma cell line A549.  相似文献   

15.
Two strains of Azadinium poporum, one from the Korean West coast and the other from the North Sea, were mass cultured for isolation of new azaspiracids. Approximately 0.9 mg of pure AZA-36 (1) and 1.3 mg of pure AZA-37 (2) were isolated from the Korean (870 L) and North Sea (120 L) strains, respectively. The structures were determined to be 3-hydroxy-8-methyl-39-demethyl-azaspiracid-1 (1) and 3-hydroxy-7,8-dihydro-39-demethyl-azaspiracid-1 (2) by 1H- and 13C-NMR. Using the Jurkat T lymphocyte cell toxicity assay, (1) and (2) were found to be 6- and 3-fold less toxic than AZA-1, respectively.  相似文献   

16.
Four ferulic acid dehydrodimers were isolated from rye bran and purified by preparative HPLC after alkaline hydrolysis. The identity of the compounds were confirmed by UV,1H and13C-NMR and mass spectroscopy. The content of the four identified dimers corresponded to a total dimer concentration of 307 μg/g (dry matter) of the whole grain. The concentrations in the bran fraction were 10–20 times higher than in the starchy endosperm. The four dimers were in decreasing amounts: ((Z)- β -{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-3-methoxy-cinnamic acid (8-O-4′-DiFA);trans -5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (8-5′-DiFA benzofuran form), (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid (5-5′-DiFA) and (E,E)-4,4′-dihydroxy-3,5′-dimethoxy-β,3′-bicinnamic acid (8-5′-DiFA). The ferulic acid dehydrodimers were also synthesised by a simple procedure from ethyl ferulate using peroxidase and hydrogen peroxide.  相似文献   

17.
为了研究原始热带雨林鹦歌岭土壤放线菌（Streptomyces sp.）YG-7的次生代谢产物及其α-葡萄糖苷酶抑制活性,采用多种柱色谱方法对土壤放线菌YG-7的发酵产物进行分离纯化得到9个化合物,经过波谱数据分析分别鉴定为：(1) 2-acetamido-5-chlorobenzamide, (2) cyclo (L-Pro-L-Leu), (3) 3,6-dibenzylidene-2,5-piperazinedione, (4) albonoursin, (5) (3Z,6S)-3-benzylidene-6-isobutylpiperazine-2,5-dione, (6) 3-hydroxy-2-methyl-4-pyrone, (7) isophthalic acid, (8) methyl 3-carbamoylbenzoate, (9) 2,3-dihydroxypropyl hexadecanoate. 其中,化合物1、7和8为新天然产物。活性测试结果表明化合物1、3~5和7~8对α-葡萄糖苷酶具有明显的抑制活性。  相似文献   

18.
采用固相微萃取法(SPME)萃取紫苏子的挥发性物质，经气相色谱－质谱联用仪(GC-MS)检测，结果表明，紫苏子的挥发性成分主要由石竹烯、紫苏醛、a-石竹烯、柠檬烯、临氨基苯甲酸沉香酯、4-乙烯基－4-甲基－3-异丙烯基－1-异丙基－环己烯、(E,E)-1-甲基-5-亚甲基-8-异丙基-1,6-环癸二烯、2,6-二甲基-6-(4-甲基-3-戊基)双环[3.1.1]己-2-烯、1,4-二乙基-1,4-二甲基-2,5-环己二烯等组成，其中石竹烯相对含量最高（41.2 %），紫苏醛次之(12.38 %)。  相似文献   

19.
A polyoxygenated and halogenated labdane, spongianol (1); a polyoxygenated steroid, 3β,5α,9α-trihydroxy-24S-ethylcholest-7-en-6-one (2); a rare seven-membered lactone B ring, (22E,24S)-ergosta-7,22-dien-3β,5α-diol-6,5-olide (3); and an α,β-unsaturated fatty acid, (Z)-3-methyl-9-oxodec-2-enoic acid (4) as well as five known compounds, 10-hydroxykahukuene B (5), pacifenol (6), dysidamide (7), 7,7,7-trichloro-3-hydroxy-2,2,6-trimethyl-4-(4,4,4-trichloro-3-methyl-1-oxobu-tylamino)-heptanoic acid methyl ester (8), and the primary metabolite 2’-deoxynucleoside thymidine (9), have been isolated from the Red Sea sponge Spongia sp. The stereoisomer of 3 was discovered in Ganoderma resinaceum, and metabolites 5 and 6, isolated previously from red algae, were characterized unprecedentedly in the sponge. Compounds 7 and 8 have not been found before in the genus Spongia. Compounds 1–9 were also assayed for cytotoxicity as well as antibacterial and anti-inflammatory activities.  相似文献   

20.
The crusts of two white breads (French-type), differing in the flavour profiles owing to differences in both the amount of yeast and the dough-making process, were analysed by using instrumental and sensory methods. 2-Acetyl-1-pyrroline, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 2,3-butanedione,methional, (E)-2-nonenal, methylpropanal, and 2- and 3-methylbutanal were identified as key odorants of the baguette crusts. Quantitative measurements indicated that the more intense, roasty odour note of the baguette prepared with a higher amount of yeast was caused by a higher concentration of pyrroline. In contrast, the more intense malty note of the baguette prepared with pre-fermented dough was correlated with an increase in the malty smelling aldehydes methylpropanal, and 2- and 3-methylbutanal.  相似文献   

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