天冬氨酸提高乳酸乳球菌Lactococcus lactis NZ9000酸胁迫抗性的作用机制

【背景】乳酸菌作为重要的发酵微生物在应用过程中面临广泛存在的酸胁迫。【目的】确认天冬氨酸可有效提高乳酸乳球菌的酸胁迫抗性,通过解析天冬氨酸的作用机制,为进一步提高乳酸菌酸胁迫抗性提供可借鉴的思路。【方法】通过荧光定量PCR比较胁迫条件下天冬氨酸对L.lactisNZ9000产能和氨基酸代谢途径中关键基因转录水平的影响,并通过过量表达天冬酰胺酶增加胞内天冬氨酸的含量。【结果】天冬氨酸主要是在转氨酶的作用下生成草酰乙酸和谷氨酸。草酰乙酸参与三羧酸循环,为细胞提供更多的能量;谷氨酸经谷氨酸脱羧酶途径提高细胞的酸胁迫抗性。经pH4.0胁迫处理后,天冬氨酸使糖酵解和三羧酸循环产能途径中关键基因转录上调,胞内ATP含量为对照组的42倍;胞内谷氨酸含量为对照的1.99倍。通过过量表达天冬酰胺酶获得的重组菌株,在pH3.6条件下胁迫0.5h后,存活率约为对照组的11.11倍。【结论】在L. lactis NZ9000中探究了天冬氨酸提高酸胁迫抗性的作用机理,进一步完善了氨基酸代谢提高乳酸菌酸胁迫抗性的理论基础。     

基因工程改善植物生物质能利用的研究进展

利用植物木质纤维资源发酵产乙醇越来越受到人们的重视,但是要达到工业生产仍然存在很多难题。最近在利用植物基因工程技术改善植物自身性状,以利于能源植物的研究方面取得了一定的进展,这些研究包括减少植物自身细胞壁中的木质素含量、细胞中积累表达纤维素酶和木聚耱酶等的方法,使产生的生物质更利于降解利用。     

植物淀粉合成的调控酶

淀粉是植物中最普通的碳水化合物,是人类最主要的食品来源与重要的工业原料。植物淀粉的生物合成主要涉及了4种酶—ADPG焦磷酸化酶、淀粉合成酶、淀粉分支酶和淀粉去分支酶,它们在淀粉的生物合成中发挥着不同作用。近年来,随着基因工程技术的迅速发展及与这些酶有关的众多突变体的发现,使人们对这些酶的结构、特性、功能及表达调控等方面的研究取得了重要进展。并且,人们已开始利用基因工程技术调控植物淀粉的数量与特性,取得了一定成效。在此,文章介绍了调控植物淀粉合成关键酶的生化特性、基因调控及利用基因工程改良植物淀粉等方面所取得进展。     

利用基因工程改良植物脂肪酸和提高植物含油量的研究进展

植物脂肪酸和三酰甘油具有重要生理功能以及巨大的食用和工业价值,其生物合成途径较为复杂。近年来,植物脂肪酸和三酰甘油合成代谢途径的研究取得了很大进展,在多种植物中克隆了脂肪酸和三酰甘油合成代谢途径中的关键酶基因,同时发现一些转录因子在调控植物脂肪酸含量和组成中具有重要作用,并在此基础上开展了利用基因工程改良植物脂肪酸和提高植物含油量的研究,取得了一定的进展。以下系统介绍了植物脂肪酸和三酰甘油合成代谢途径中的关键酶基因以及在调控植物脂肪酸含量和组成中具有重要作用的转录因子,综述了基因工程研究所取得的成果,并对其应用前景进行了展望。     

固定化天冬氨酸酶制备(R)-3-氨基丁酸

从芽孢杆菌Bacillus sp.YM55-1基因组中克隆得到天冬氨酸酶基因,以pET-28a(+)为载体构建天冬氨酸酶基因的表达载体pET-28a(+)-Asp,将天冬氨酸酶基因进行定点突变,在E.coli BL21(DE3)系统中实现了天冬氨酸酶的异源表达。利用重组的天冬氨酸酶,以氨水、(NH_4)_2SO_4为辅料,将底物巴豆酸转化为(R)-3-氨基丁酸。将天冬氨酸酶的工程菌制备成固定化细胞,通过反应条件的优化研究,提高底物的转化率。结果表明:天冬氨酸酶最适pH为9.0,最适反应温度为40℃。在此反应条件下,加入30 g/L固定化细胞,转化22 h,(R)-3-氨基丁酸质量浓度达到220 g/L,对映体过量值e.e._s≥99.95%,底物转化率达到98%,固定化细胞重复使用次数不低于24次。     

植物维生素E合成及其生物技术改良

维生素E是一种抗氧化剂 ,对植物、动物和人类自身都具有十分重要的作用 ,而植物则是人类维生素E的主要来源 ,因此克隆植物中维生素E合成的相关酶基因 ,对维生素E含量进行改良 ,具有重要意义。对植物中维生素E的合成途径 ,相关酶基因的克隆以及用生物技术的方法对维生素E含量进行遗传改良进行了综述。     

异源表达CiRS基因通过生成白藜芦醇增强拟南芥的抗氧化能力

白藜芦醇是一种具有多种医疗保健作用的植物芪类次生代谢产物,在农业、医药、食品和化妆品等领域受到广泛的关注。白藜芦醇合酶是白藜芦醇生物合成中唯一必需的关键酶,决定植物体内白藜芦醇的合成。将中间锦鸡儿中克隆到的CiRS基因(Gen Bank登录号MF678590)转入野生型拟南芥,实验结果显示:野生型的总黄酮含量明显高于转基因株系。HPLC测得转基因拟南芥中有白藜芦醇的生成,并且含量最高达335μg/g FW。紫外照射处理后转基因植物中丙二醛的积累量明显少于野生型。转基因植物提取物DPPH自由基清除能力均高于野生型。这些结果表明,中间锦鸡儿CiRS基因异源表达后利用与黄酮类物质的共同底物合成了白藜芦醇,使得转基因植物的抗氧化性增强。     

高等植物赤霉素2-氧化酶基因的克隆、表达及其调控

赤霉素(GA)是一类重要的植物激素,对高等植物整个生命周期的生长发育起关键作用。调控赤霉素生物合成和代谢途径中的关键酶基因的表达可以控制植物体内赤霉素的含量。GA2-氧化酶是调节赤霉素合成和代谢的关键酶之一,使活性GA失活。本文主要对GA2-氧化酶基因的克隆、表达调控及其在植物基因工程中的应用等方面进行综述,为通过基因工程技术调控植物体内活性赤霉素的含量从而得到改良品种提供思路。     

通过转基因提高β-胡萝卜素生物合成量

在黄花龙胆 (Getianalutea)花瓣中获得了植物类胡萝卜素生物合成途径中的 5个基因GGPS、PSY、ZDS、LycB、LycE ,它们分别位于类胡萝卜素合成途径中生成α 和 β 胡萝卜素的上游 .将其中的主要酶基因PSY、ZDS与 35S启动子和NOS终止子相连 ,通过根癌农杆菌 (Agrobacteriumtumefaciens)转入烟草 ,并通过RT PCR ,Northern分子杂交 ,Western分子杂交等证实这些基因在RNA、蛋白质水平能较好转录及表达 .高效液相层析法分析显示 ,这些基因的翻译产物具有酶的活性 .结果表明 ,PSY可使 β 胡萝卜素含量提高 1 0 8% .     

植物次生代谢途径的遗传操作

植物次生代谢产物种类极其丰富,是人类的宝贵资源,这些产物及其合成途径相关酶具有空间特异性分布的特征。植物次生代谢途径的调控是个复杂的过程,受代谢产物水平、多酶复合物相互作用等多种因素的影响。通过遗传操作改造代谢过程,调控产物在植物体内的含量,是一条切实可行和具有广阔发展空间的途径。目前,改造植物次生代谢途径可以采取单基因操作和多基因操作两种策略进行。     

Capacities and constraints of amino acid utilization in Arabidopsis

Various amino acids, including both L- and D-enantiomers, may be present in soils, and recent studies have indicated that plants may access such nitrogen (N) forms. Here, the capacity of Arabidopsis to utilize different L- and D-amino acids is investigated and the constraints on this process are explored. Mutants defective in the lysine histidine transporter 1 (LHT1) and transgenic plants overexpressing LHT1 as well as plants expressing D-amino acid-metabolizing enzymes, were used in studies of uptake and growth on various N forms. Arabidopsis absorbed all tested N-forms, but D-enantiomers at lower rates than L-forms. Several L- but no D-forms were effective as N sources. Plants deficient in LHT1 displayed strong growth reductions and plants overexpressing LHT1 showed strong growth enhancement when N was supplied as amino acids, in particular when these were supplied at low concentrations. Several D- amino acids inhibited growth of wild-type plants, while transgenic Arabidopsis-expressing genes encoding D-amino acid-metabolizing enzymes could efficiently utilize such compounds for growth. These results suggest that several amino acids, and in particular L-Gln and L-Asn, promote growth of Arabidopsis, and increased expression of specific amino acid transporters enhances growth on amino acids. The efficiency by which transgenic plants exploit D-amino acids illustrates how plants can be engineered to utilize specific N sources otherwise inaccessible to them.     

Role of Amino Acids in Plant Responses to Stresses

Plants subjected to stress show accumulation of proline and other amino acids. The role played by accumulated amino acids in plants varies from acting as osmolyte, regulation of ion transport, modulating stomatal opening, and detoxification of heavy metals. Amino acids also affect synthesis and activity of some enzymes, gene expression, and redox-homeostasis. These roles played by amino acids have been critically examined and reviewed.     

Branched-chain amino acid metabolism in higher plants

Valine, leucine and isoleucine contain short branched carbohydrate residues responsible for their classification as branched-chain amino acids (BCAA). Among the proteinogenic amino acids, BCAA show the highest hydrophobicity and are accordingly the major constituents of transmembrane regions of membrane proteins. BCAA cannot be synthesized by humans and thus belong to the essential amino acids. In contrast, plants are able to synthesize these amino acids de novo and are an important source for these compounds in the human diet. However, BCAA cannot only be synthesized in plants, leucine and probably also valine and isoleucine can also be degraded. Many enzymes operating in turnover are found in mitochondria, while some catabolizing activities are located in peroxisomes. The breakdown of BCAA is physically separated from their biosynthesis in chloroplasts. Additionally, in the order of the Capparales, enzymes of the leucine metabolism seem to be evolutionary related to or may even participate in the methionine chain elongation pathway, the early part of the biosynthesis of aliphatic glucosinolates. In summary, in higher plants a complex network of pathways interferes with the homeostasis of Val, Leu and Ile.     

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.     

Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues,Flowers, and Developing Seeds

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the [beta]-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.     

离体小麦胚胎对10种必需氨基酸的生长反应

试验了小麦离体胚胎对10种必需氨基酸在不同浓度下的生长反应,结果指出,不同氨基酸对小麦胚胎有专一性作用浓度。按照试管植株的生长表现,10种必需氨基酸对试管植株的生长抑制强度(或者说是酶的反馈抑制强度)可分为三种类型,即,强作用型、中等作用型和弱作用型。试验还发现,相同族的氨基酸对试管植株有基本相同的作用强度。这些结果为小麦离体氨基酸抗性选择提供了起始抑制浓度、半生长浓度和临界生长浓度等重要技术参数。     

Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases

Summary We have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidine-containing enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system.