番茄GDP-D-甘露糖焦磷酸化酶cDNA的克隆、表达及定位

GDP-D-甘露糖焦磷酸化酶催化GDP-D-甘露糖的合成,是植物抗坏血酸生物合成途径中上游的关键酶。以马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列为信息探针,在GenBank dbEST数据库中找到65条高度同源的番茄EST序列,通过序列拼接及RACE-PCR得到了番茄该基因的全长cDNA序列,命名为LeGMP。LeGMP与马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列一致率为96％,推导的氨基酸序列与马铃薯、烟草、紫苜蓿、拟南芥的GDP-D-甘露糖焦磷酸化酶基因的一致率分别为99％、97％、91％、89％。经Northern杂交分析,LeGMP在番茄根、茎、叶、花、果实中都有表达,但表达水平有差异。利用75个番茄远缘杂交重组系（IL系）将LeGMP定位在番茄第3染色体上的D区段（3-D）。     

Purification and properties of phosphofructokinase from fruits of Lycopersicon esculentum

A procedure is described using affinity chromatography on Blue Sepharose and on an immobilized ATP column by which phosphofructokinase has been purified by 260-fold from tomato fruits. The properties of the enzyme are affected by the pH at which the preparation is made and maintained. At the pH optimum, pH 8.0, the enzyme is very heterogeneous with up to three forms present differing in MW. At pH 7.5 a single major form of MW 180 000 is present, and evidence that raising the pH to 8.0 promotes dissociation of the enzyme is discussed.     

Carotene epoxides of Lycopersicon esculentum

A series of oxygenated carotenoids has been isolated from tomatoes. Two of these compounds have been identified, by comparison of their chromatographic and spectroscopic properties with those of semisynthetic samples, as epoxides of lycopene (1,2-epoxy-1,2-dihydro-ψ,ψ-carotene and 5,6-epoxy-5,6-dihydro-ψ,ψ-carotene). The other related compounds have been identified by their chromatographic, spectroscopic and chemical properties as mutatochrome (5,8-epoxy-5,8-dihydro-β,β-carotene) and epoxides of phytoene (1,2-epoxy-1,2,7,8,11,12,7′,8′,11′,12′-decahydro-ψ, ψ-carotene), phytofluene (1,2-epoxy-1,2,7,8, 11,12,7′,8′-octahydro-ψ,ψ-carotene and 1,2-epoxy-1,2,7,8,7′,8′,11′,12′-octahydro-ψ,ψ-carotene) and ξ-carotene (1,2-epoxy-1,2,7,8,7′,8′-hexahydro-ψ,ψ-carotene). The presence in tomatoes of apo-6′-lycopenal (6′-apo-ψ-caroten-6′-al), 8′-apo-lycopenal (8′-apo-ψ-caroten-8′-al) and lycoxanthin (ψ,ψ-caroten-16-ol) has been confirmed by comparison with authentic samples.     

Electrophoretic investigation of enzymes from developing Lycopersicon esculentum fruit

Acetone powders were prepared from tomato fruit tissue sampled during development and the proteins were separated by polyacrylamide disc gel electrophoresis. The gels were stained to show up general proteins, lipoproteins, glycoproteins and certain enzymes. Minor changes in protein and glycoprotein patterns accompanied development. Most enzymes exhibited more than one active band, with maximum diversity and specific activities usually appearing in extracts from mature green tissue and least with over-ripe tissue. The results support the view that enzyme synthesis accompanies the climacteric respiration rise at the expense of non-metabolic protein.     

Some properties of pyruvate kinase extracted from Lycopersicon esculentum

Pyruvate kinase was extracted from Me₂CO-dried tissue of various parts of tomato plants. Recovery of the enzyme was improved by the inclusion of thiols in the extraction medium, and its stability was increased considerably in the presence of glycerol and to a lesser extent tetramethylammonium chloride. A phosphatase was present in the tissue extracts which hydrolyses phosphoenolpyruvate in the absence of added ADP. ATP inhibited pyruvate kinase but stimulated the phosphatase, while Mg²⁺ stimulated both enzymes. Data obtained suggest that tomato leaf pyruvate kinase has an absolute dependence on monovalent cations for activity, K⁺ being the principal activator. The phosphatase was inhibited non-selectively by monovalent cations. The total activity of pyruvate kinase and its concentration on a tissue fresh weight basis was greatest in the leaves, activity increasing with the maturity of the tissue. Less enzyme was present in roots, and least in the fruit.     

cDNA Cloning and Structural Analysis of Rice ADP-Glucose Pyrophosphorylase Gene

ADP-glucose pyrophosphorylase is a key enzyme in the pathway of starch synthesis. The gene encoding this.important enzyme was cloned by PCR amplification from immature seeds of a Chinese rice cultivar-Oryza sativa var. japonica cv. Zhonghua 10. It was then sequenced and compared with the same gene reported from other rice cultivar. The gene obtained in this study is composed of 1461 bp and encodes 483 amino acid residues. Its nucleotide sequence and deduced amino acid sequence shared 99.6% and 99.7% homology with those of published data respectively. The analyses on the structure and evolution of this gene have been conducted.     

过表达GDP-D-甘露糖焦磷酸化酶(GMP)基因提高灵芝多糖的生产

灵芝多糖是灵芝的主要生物活性成分之一,具有多种药理活性,但灵芝多糖的低产量限制了其广泛应用.相关研究表明,灵芝中过表达多糖生物合成相关基因能够提高多糖产量,但过表达GDP-D-甘露糖焦磷酸化酶(GMP)基因提高灵芝多糖产量未有报道.本研究克隆获得了灵芝gmp基因,并成功构建出了过表达gmp基因的工程菌株(GMP菌株),...     

番茄低温响应转录因子SlNAC41克隆及表达分析

NAC转录因子在调控植物生长发育、生物及非生物逆境应答中发挥着重要作用。前期,我们通过对番茄幼苗在低温胁迫下的基因表达谱进行分析,发现Unigene SGN-U212711受低温诱导表达强烈。本研究从番茄中克隆了该基因,命名为Sl NAC41,其开放阅读框（ORF）1 173 bp,编码390个氨基酸,蛋白N端具有典型的NAM结构域,属于NAC转录因子家族成员。预测Sl NAC41蛋白分子量为43.5 k Da,等电点为5.2。实时荧光定量PCR分析表明,Sl NAC41在番茄各组织均有表达,在花中的表达量最高,在红熟果中的表达量最低。低温、干旱、高盐、甲基紫精（MV）、脱落酸（ABA）及乙烯利（ETH）处理均能诱导该基因的表达,其中,以低温和干旱诱导表达最为强烈。利用PLACE和Plant CARE对启动子序列进行预测分析发现,Sl NAC41启动子区含有大量响应光、病原菌侵染、激素、低温、脱水及盐胁迫的顺式作用元件。这些结果表明,Sl NAC41可能在番茄生物及非生物胁迫应答中发挥重要调控作用。     

Pigment evolution in Lycopersicon esculentum fruits during growth and ripening

During growth and subsequent maturation, the distribution and formation of pigments in the inner pulp and in the outer region of the pericarp of ‘che     

番茄花粉特异表达的高赖氨酸蛋白基因(tsb)的克隆与特性分析

赖氨酸是人和单胃动物必需的八种氨基酸之一,食物蛋白中的必需氨基酸种类不全或数量不足,都会影响其它氨基酸的利用.玉米等主要禾谷类作物由于缺少赖氨酸,种子蛋白利用率还不到50%.在长期以玉米为口粮的地区,人们往往患有营养不足或糙皮病.     

Effect of potassium nutrition on some enzymes from ripening Lycopersicon esculentum fruit

The maximum respiration rate of tomato fruit during the climacteric period was markedly increased when the plants were grown under potassium-deficient conditions. Whereas potassium deficiency had no effect on cytoplasmic glutamate-oxoloacetate transaminase, there was a significant increase in the activity of this enzyme from mitochondria once the fruit began to change colour. Malate dehydrogenase was reduced in activity by potassium deficiency. It is suggested that the augmented mitochondrial transiminase levels, coupled with reduced malate dehydrogenase activity in low potassium fruit, result in reduced levels of oxaloacetic acid which is a potent inhibitor of Krebs cycle oxidations, thus leading to higher respiration rates for the intact fruit.     

牦牛白细胞介素2(IL2)基因cDNA 的分子克隆和表达研究

目的克隆牦牛免疫基因并研究其免疫特性。方法以伴刀豆球蛋白A（ConA）激活的体外培养的牦牛血液淋巴细胞,提取激活淋巴细胞的总RNA,用RT—PCR方法,从中克隆出白细胞介素2cDNA,连接到T载体上测序,并亚克隆到pC,EX4T-1表达质粒,在大肠杆菌进行了重组表达,纯化融合表达YIL2产物,MTT比色法测定其体外刺激牦牛血液淋巴母细胞增殖的免疫活性。结果YIL2cDNA序列分析显示：在其编码区442位点的一个碱基发生突变（由C突变为T）,从而导致终止密码子（TAA）出现,使YIL2蛋白表达提前终止,与其它牛IL2蛋白相比少了8个氨基酸。MTT比色法测定结果表明重组牦牛IL2蛋白体外能显著促进牦牛淋巴母细胞的增殖。结论本实验成功克隆了牦牛IL2基因,其原核表达产物具有显著的免疫活性,这为研制新型免疫增强剂来提高牦牛对各种疾病的抵抗力,增强牦牛疫苗的免疫保护率奠定了基础。     

西瓜防御素基因ClPDF2.1的克隆及表达分析

该研究从西瓜中克隆了西瓜防御素基因ClPDF2.1(GenBank登录号为KC481267)。序列分析结果表明,ClPDF2.1基因开放阅读框为225bp,编码75个氨基酸,预测蛋白质分子质量为8.237kD,等电点为9.375。蛋白质结构分析表明,ClPDF2.1蛋白含有防御素特有的8个半胱氨酸结构域。进化分析显示,ClPDF2.1与拟南芥PDF2归为一类,与五彩椒的亲缘关系最近(59%)。荧光定量PCR分析表明,ClPDF2.1在西瓜根、茎、叶器官中都有表达,叶中表达量高于根,茎中表达量最低。ClPDF2.1基因的表达也受到外源植物激素水杨酸、茉莉酸甲酯、乙烯利和西瓜枯萎病菌的诱导上调,表明ClPDF2.1基因通过信号传导途径参与对西瓜枯萎病菌株的防御反应。西瓜防御素基因的克隆及表达分析为其功能鉴定奠定了基础。     

人Mn—SOD cDNA的克隆及高效表达

用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）,以人肝细胞株（Ｌ０２）总ＲＮＡ为模板,扩增了人锰超氧化物歧化酶（ｈＭｎ－ＳＯＤ）的ｃＤＮＡ。重组到Ｔ７启动子控制下的表达载体ｐＥＴ－２４ａ（＋）中,构建表爱质粒ｐＥＴ－ＭｎＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳ－ＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代－β－Ｄ－半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为２２ｋＤ的蛋白质,与抗人     

Biosynthesis and distribution of tomatine in cultured excised Lycopersicon esculentum roots

Growth of cultured excised tomato roots in the presence of ¹⁴C-mevalonic acid lactone results in labelling of tomatine. In the main axis of the root, incorporation was greatest in the apical meristematic region. Tomatine was present in equal concentrations in all parts of the cultured root system.     

甜椒细胞质小分子量热激蛋白基因(CaHSP18)的cDNA克隆与表达

用RT-PCR和RACE-PCR技术,从热激处理的甜椒叶片总RNA中扩增出了细胞质小分子量热激蛋白(sHSP)全长779 bp的cDNA基因序列,包含一个480 bp开放阅读框,编码159个氨基酸.Southern杂交结果表明在甜椒基因组中有该基因的小的多基因家族.Northern结果显示该基因在甜椒根、茎、叶中的表达受热激和低温的诱导.原核表达分析表明该基因在高温以及低温条件下可以提高大肠杆菌的生存能力.     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。