谷氨酸棒状杆菌乙酸盐代谢PTA、AK、ICL和MS酶活性研究

从谷氨酸棒状杆菌乙酸盐代谢中涉及的磷酸转乙酰酶PTA、乙酸盐激酶AK、异柠檬酸裂解酶ICL和苹果酸合成酶MS 4种酶入手,建立了一套稳定的酶活性测定方法,并对这些酶在葡萄糖和乙酸盐不同碳源代谢中的酶活性特征进行了比较分析,发现碳代谢中存在葡萄糖效应;乙酸盐对谷氨酸棒状杆菌PTA、AK、ICL和MS酶活性有明显的诱导作用.     

单体异柠檬酸脱氢酶的研究进展

异柠檬酸脱氢酶是三羧酸循环的关键酶之一,依赖金属离子Mg2＋（或Mn2＋）和辅酶NAD（P）,催化异柠檬酸氧化脱羧生成α-酮戊二酸及CO2,为细胞新陈代谢提供能量和生物合成的前体物质。根据亚基的组成,原核生物IDH可分为单体IDH和同源二聚体IDH。本文主要对单体IDH的空间结构、催化机制及分子进化机制等研究进展进行了综述。     

Direct demonstration of an adaptive constraint

The role of constraint in adaptive evolution is an open question. Directed evolution of an engineered beta-isopropylmalate dehydrogenase (IMDH), with coenzyme specificity switched from nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide phosphate (NADP), always produces mutants with lower affinities for NADP. This result is the correlated response to selection for relief from inhibition by NADPH (the reduced form of NADP) expected of an adaptive landscape subject to three enzymatic constraints: an upper limit to the rate of maximum turnover (kcat), a correlation in NADP and NADPH affinities, and a trade-off between NAD and NADP usage. Two additional constraints, high intracellular NADPH abundance and the cost of compensatory protein synthesis, have ensured the conserved use of NAD by IMDH throughout evolution. Our results show that selective mechanisms and evolutionary constraints are to be understood in terms of underlying adaptive landscapes.     

谷氨酸棒状杆菌在乙酸盐和葡萄糖基质上的蓝白生长筛选(英文)

在谷氨酸棒状杆菌乙酸盐代谢调控关键酶-异柠檬酸裂解酶aceA基因两段启动子区域后面组装了无启动子的β-半乳糖苷酶报道基因,并且带上四环素抗性标记基因构建了两个检测质粒pROH7 和pROHO.通过该两个质粒转化了谷氨酸棒状杆菌野生型菌株Corynebacterium glutamicum ACTT 14752,分别获得了含有两个检测质粒的两种谷氨酸棒状杆菌,由此建立了谷氨酸棒状杆菌在乙酸盐和葡萄糖基质上的两个蓝白生长筛选系统.结果显示当两种菌株生长在含有葡萄糖的基本培养基上时呈现白色菌落,而生长在含有乙酸盐的基本培养基上时呈现蓝色菌落;其中菌落颜色还随菌株中检测质粒pROH7 和pROHO的不同而呈现淡蓝色和深蓝色的变化.含有检测质粒pROH7 和pROHO菌落的颜色变化反映了细胞在葡萄糖和乙酸盐不同和碳源上的调控状态,该两个质粒在鉴定乙酸盐代谢中的调控变化方面是有积极意义.     

脐橙果实发育过程中有机酸代谢相关酶的研究

以罗伯逊脐橙为试材,研究了脐橙果实有机酸代谢相关酶的活性变化动态,及其与果实有机酸积累关系,旨在探索影响脐橙果实有机酸含量的重要酶。结果表明:果实有机酸的积累期,CS、PEPC活性随有机酸积累而增大,并呈显著正相关;有机酸降解期,IDH的活性与有机酸含量呈极显著负相关;而MDH和ACO对酸含量的影响小。因而可认为PEPC和CS是影响果实有机酸合成的重要酶,而影响有机酸分解的重要酶则是IDH。     

异柠檬酸裂解酶抑制剂对L-谷氨酸合成的影响

[目的]研究琥珀酸和苹果酸对L-谷氨酸合成的影响。[方法]以谷氨酸棒杆菌的典型茵株Corynebacterium glutamicum ATCC 13032为供试菌株,研究琥珀酸和苹果酸对细胞生长、L-谷氨酸的合成、发酵液中的残糖量以及细胞中异柠檬酸裂解酶活性的影响。[结果]琥珀酸对异柠檬酸裂解酶的活性具有部分抑制作用。在0-5．0g／L范围内,琥珀酸的添加提高了L-谷氨酸发酵的糖酸转化率,细胞生长和L-谷氨酸分泌受到抑制,残糖量增大。苹果酸的添加降低了异柠檬酸裂解酶的活性和L-谷氨酸的产量,残糖量和细胞生长没有明显的规律性变化。同时添加琥珀酸和苹果酸各2．0g/L时,异柠檬酸裂解酶的活性和细胞生长都有所下降,L-谷氨酸的产量和残糖量没有明显变化。[结论]添加琥珀酸增加了L-谷氨酸的产量,添加苹果酸则降低了L-谷氨酸的产量。     

ICL、ICDH、ODH和GDH酶与L-谷氨酸的合成

通过对对数生长期细胞的异柠檬酸裂解酶（ICL）、异柠檬酸脱氢酶（ICDH）、α-酮戊二酸脱氢酶（ODH）和谷氨酸脱氢酶（GDH）的分析以及对细胞L-谷氨酸产生量和发酵残糖的综合分析,发现由于乙醛酸循环关键酶ICL的缺失,L-谷氨酸产生量大幅下降,说明乙醛酸循环是谷氨酸棒杆菌中的主要回补途径,L-谷氨酸合成需要乙醛酸循环。在保证乙醛酸循环回补功能的前提下,提高ICDH活性,减弱ODH活性,有利于L-谷氨酸的合成。     

An integrated genomic analysis of human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.     

Metabolic diversification--independent assembly of operon-like gene clusters in different plants

Operons are clusters of unrelated genes with related functions that are a feature of prokaryotic genomes. Here, we report on an operon-like gene cluster in the plant Arabidopsis thaliana that is required for triterpene synthesis (the thalianol pathway). The clustered genes are coexpressed, as in bacterial operons. However, despite the resemblance to a bacterial operon, this gene cluster has been assembled from plant genes by gene duplication, neofunctionalization, and genome reorganization, rather than by horizontal gene transfer from bacteria. Furthermore, recent assembly of operon-like gene clusters for triterpene synthesis has occurred independently in divergent plant lineages (Arabidopsis and oat). Thus, selection pressure may act during the formation of certain plant metabolic pathways to drive gene clustering.     

Microbodies in germinating fern spores: evidence for glyoxysomal activity

Enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, are active during the germination of spores of the fern Dryopteris filix-mas. Increases in activity of both enzymes are correlated with the breakdown of lipid reserves. The occurrence of these enzymes suggests that the microbodies previously described in these spores are glyoxysomes.     

Adk1过表达和柠檬酸钠补料促进酵母S-腺苷甲硫氨酸的合成

S-腺苷甲硫氨酸（SAM）是参与生物体众多生化反应的生理活性物质，酵母胞内ATP的水平是限制胞内SAM合成的因素之一。在酿酒酵母CGMCC 2842中克隆并过表达Adk1基因，发现Adk1基因的过表达使得酵母胞内ATP水平提高47.1%，SAM积累量提高47%；发酵16 h向发酵液中添加6 g·L-1柠檬酸钠，异柠檬酸脱氢酶基因mRNA水平和IDH酶活显著提高，在发酵24 h时胞内ATP水平提高39.4%，与对照相比，SAM积累量和对生物量得率分别提高79%和78.8%，表明胞内ATP水平的提高促进Met转化为SAM，这为ATP代谢调控而改善SAM合成提供有力的理论依据。     

Adk1过表达和柠檬酸钠补料促进酵母S-腺苷甲硫氨酸的合成

S-腺苷甲硫氨酸（SAM）是参与生物体众多生化反应的生理活性物质,酵母胞内ATP的水平是限制胞内SAM合成的因素之一。在酿酒酵母CGMCC 2842中克隆并过表达Adk1基因,发现Adk1基因的过表达使得酵母胞内ATP水平提高47.1%,SAM积累量提高47%;发酵16 h向发酵液中添加6 g·L-1柠檬酸钠,异柠檬酸脱氢酶基因mRNA水平和IDH酶活显著提高,在发酵24 h时胞内ATP水平提高39.4%,与对照相比,SAM积累量和对生物量得率分别提高79%和78.8%,表明胞内ATP水平的提高促进Met转化为SAM,这为ATP代谢调控而改善SAM合成提供有力的理论依据。     

马铃薯卷叶病毒CP基因的突变及其原核表达

通过人工合成DNA的方法,对马铃薯卷叶病毒外壳蛋白(CP)基因第52～177核苷酸(126bp)这段序列进行了突变,将其中12个AGA、AGG、CGA等精氨酸稀有密码子变成了原核高效表达的同义密码子CGT与CGC,将另外两个AGA精氨酸密码子变成了错义密码。构建了突变基因的原核表达载体pBAD-LRCP2,Bsp1407Ⅰ与MssⅠ酶切及DNA测序结果表明,基因突变符合要求,表达载体的构建正确。在37℃,大肠杆菌工程株TOP10(pBAD-LRCP2)用0.2%L-阿拉伯糖诱导培养4h,SDS-PAGE显示蛋白图谱上有一条36ku的诱导表达的融合蛋白条带,结果表明突变基因在PBAD启动子驱动下在大肠杆菌中实现了表达。     

Organic Acid Concentrations and the Relative Enzymatic Changes During the Development of the Citrus Fruits

Seasonal changes in enzyme activities and citrate concentration during the development of citrus fruits were investigated. The result showed that the organic acid concentrations reached a peak in the 100 - 130days after anthesis and gradually declined during later stages of fruit maturation in most varieties of citrus,but declined only slightly thereafter in lemon [Citrus lin on (L.) Burm]; there is no relation between the activity of citrate synthetase(CS) and the different acid concentration in different citrus fruits; the increase of the activity of the cytosolic aconitase in the late period of the development of citrus fruits accelerated the degradation of citric acid in citrus fruits; the higher the activity of phosphoenolpyruvate carboxylase(PEPC) and the ratio of NAD-dependent isocitrate hydrogenase(PEPC/NAD-IDH- ), the more the concentrations of organic acids in citrus fruit.     

Mechanisms involved in biocontrol and plant induced resistance by richoderma. asperellum (T. harzianum T-203)

Growing awareness of the environmental damage caused by the use of chemical substances for plant disease control in agriculture has raised the need to study biological alternatives, such as activating the defense response of plant crops by inducers not toxic to the environment. Trichoderma spp. are effective biocontrol agents for a number of soilborne pathogens, and are also known for their ability to enhance plant growth and to induce systemic resistance (ISR) in plants. In our laborator…     

CRISPR/Cas9基因组定向编辑技术的发展与在作物遗传育种中的应用

CRISPR/Cas9系统是近年发展起来的、由导向RNA介导的基因组定向编辑技术。总结了CRISPR/ Cas9基因组定向编辑技术的发展历程,并综述了其在作物遗传育种研究中的多方面应用。CRISPR/Cas系统是存在于大多数细菌与所有古生菌中的一种后天免疫系统,以消灭外来质体或者噬菌体。 根据Cas蛋白组分及氨基酸序列不同,已发现的CRISPR/Cas系统可以分为3种不同类型,Ⅰ型、Ⅱ型和Ⅲ型。其中,Ⅱ型是以Cas9蛋白及导向RNA为核心组份,组成较为简单,是目前经过改造用于开发基因组定向编辑技术的主要类型。自CRISPR/Cas9技术体系首先在人类与动物细胞系中建立后,经过改造的CRISPR/Cas9系统被迅速地应用于拟南芥、烟草、高粱、水稻、小麦、玉米等不同植物基因组的定向编辑研究中。CRISPR/Cas9与ZFNs或TALENs一样都是通过自身的核酸内切酶活性引起靶位点DNA序列双链断裂,然后通过非同源末端连接或同源重组介导的修复2种方式引入突变。至今,在多种作物中已实现诱导产生多种定点突变（包括插入、缺失或修饰等）,并可获得较高的突变诱导率和可稳定遗传的基因组编辑后代植株。与ZFNs或TALENs技术相比,CRISPR/Cas9技术可以实现对基因组中多个靶基因同时进行编辑,从而可以用来修饰同一基因家族中的不同成员或同一代谢途径中的不同调控基因,为其一大优势。由于CRISPR/Cas9技术具有突变诱导率高、成本低、易于操作及可以多重基因编辑等特点,已成为具有广阔应用前景的作物遗传改良与育种研究的分子操作系统。CRISPR技术除了可以对基因组中不同靶基因进行定向编辑以外,还可以广泛地应用于基因表达调控研究、细胞定位运输系统研究及新型RNA沉默系统构建等方面。基因组编辑技术是继转基因技术之后人类对生物进行遗传操作的又一个革命性技术。但是,与转基因技术相比,CRISPR/Cas9基因组编辑技术操作更加简单、快捷。应用CRISPR/Cas9基因组编辑技术进行育种可以不引入外源基因,在进行基因组编辑之后可以不留下转基因的痕迹,从而导致定义转基因生物的不明确性,因此,政府监管部门是否应该按照转基因的管理办法来监管CRISPR/Cas9技术的应用尚有待决定。     

50 million years of genomic stasis in endosymbiotic bacteria

Comparison of two fully sequenced genomes of Buchnera aphidicola, the obligate endosymbionts of aphids, reveals the most extreme genome stability to date: no chromosome rearrangements or gene acquisitions have occurred in the past 50 to 70 million years, despite substantial sequence evolution and the inactivation and loss of individual genes. In contrast, the genomes of their closest free-living relatives, Escherichia coli and Salmonella spp., are more than 2000-fold more labile in content and gene order. The genomic stasis of B. aphidicola, likely attributable to the loss of phages, repeated sequences, and recA, indicates that B. aphidicola is no longer a source of ecological innovation for its hosts.     

Protein crystal growth in microgravity

The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. space shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on Earth.     

γ-氨基丁酸代谢旁路阻断对苏云金芽胞杆菌芽胞形成和晶体蛋白产量的影响

γ-氨基丁酸旁路(GABA shunt)普遍存在于原核和真核生物体中,对于动物、植物和微生物具有不同的生理功能.苏云金芽胞杆菌gab基因簇中gabT和gabD基因所编码的γ-氨基丁酸转氨酶和琥珀酸半醛脱氢酶参与GABA shunt.在前期研究获得的苏云金芽胞杆菌HD-73菌株gabT、gabR(gab基因簇中的调控基因)和gabD基因缺失突变菌株的基础上,从菌株生长、芽胞产量和晶体蛋白产量等方面研究GABA shunt阻断对Bt芽胞和晶体形成的影响.结果表明,GABA shunt阻断对芽胞产量有一定影响,但对Bt生长和晶体蛋白产量无明显影响     

银杏HDR基因的克隆与功能分析

[目的]获得银杏HDR基因的克隆并研究HDR基因的功能。[方法]采用RT-PCR技术获得银杏HDR的全长cDNA,命名为Gb-HDR（GenBank登录号：DQ364231）,该基因cDNA全长为1827bp,包含1425bp的开放阅读框,编码474个氨基酸残基的蛋白;并构建GbHDR的原核表达载体pTrcGbHDR,与原核表达载体pAC-BETA共转入大肠杆菌XL1-Blue,获得β-胡萝卜素工程菌。[结果]克隆获得实际长度为1441bp的GbHDR基因,该序列包含1425bp的HDR基因ORF,编码474个氨基酸残基的蛋白,其预测分子量为53.2kD,等电点预测为5.76。功能互补分析表明,GbHDR能推动工程菌XL1-Blue＋pTrcGbHDR＋pAC-BETA超量表达β-胡萝卜素,在颜色互补平板上呈现β-胡萝卜素特有的橘黄色,证实GbHDR具有典型的HDR基因功能。[结论]获得1株可高量积累β-胡萝卜素的大肠杆菌工程菌,为最终实现β-胡萝卜素代谢工程提供侯选基因和作用靶点。