磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响

【目的】L-缬氨酸生物合成的前体物质是丙酮酸。为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1(Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-Δpepc,并研究pepc敲除后菌株生理特性的改变。【方法】运用交叉PCR方法得到pepc基因内部缺失的同源片段Δpepc,并构建敲除质粒pK18mobsacB-Δpepc。利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-Δpepc。采用摇瓶发酵对C.glutamicum V1-Δpepc进行发酵特性的研究。对谷氨酸棒杆菌模式菌株C.glutamicum ATCC 13032、出发菌株C.glutamicum V1和敲除菌株C.glu-tamicum V1-Δpepc的丙酮酸激酶(Pyruvate kinase,PK)、丙酮酸脱氢酶(Pyruvate dehydro-genase,PDH)、丙酮酸羧化酶(Pyruvate carboxylase,PC)分别进行测定和分析。【结果】PCR验证以及PEPC酶活测定都表明筛选到pepc缺陷的突变菌株C.glutamicum V1-Δpepc,摇瓶发酵结果表明,突变菌株C.glutamicum V1-Δpepc不再积累L-缬氨酸而是积累L-精氨酸达到7.48 g/L。酶活测定结果表明出发菌株的PDH和PC酶活均低于模式菌株C.glu-tamicum ATCC13032和重组菌株C.glutamicum V1-Δpepc,出发菌株的PK与PEPC酶活与模式菌株没有较大的差异。【结论】研究表明,通过切断PEPC参与的三羧酸循环的回补途径,增加磷酸烯醇式丙酮酸向丙酮酸的流向使丙酮酸向TCA循环的流量增加,精氨酸的累积量提高。同时,以丙酮酸为前体的L-缬氨酸和丙氨酸的积累量降低。     

蓝莓磷酸烯醇式丙酮酸羧化酶基因cDNA克隆及表达分析

磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPCase,EC4.1.1.31)在植物代谢过程中发挥重要作用。本研究从蓝莓果实中克隆到磷酸烯醇式丙酮酸羧化酶编码基因VcPEPC。该基因开放阅读框全长为2 907 bp,可编码968个氨基酸,分子量为110.59 kD。由于该蛋白具有磷酸烯醇式丙酮酸羧化酶结构特征,认为VcPEPC属于PEPcase家族(pfam00311)。二级结构预测表明,VcPEPC蛋白序列含有双螺旋(54.44%)、扩展链(11.67%)、β转角(6.71%)以及无规则卷曲(27.17%)。进化树分析发现VcPEPC与瓜、蓖麻和葡萄遗传距离较近。反转录PCR分析表明,VcPEPC基因在蓝莓根、茎、叶、花、果实中均有表达。在奥尼尔叶片中的表达量高于蓝丰、布里吉塔和夏普兰,在夏普兰绿色大果中表达量较高,成熟后基因表达下调。     

磷酸烯醇式丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶介导的草酰乙酸回补途径对谷氨酸棒杆菌V1氨基酸代谢的影响

【目的】谷氨酸棒杆菌是工业生产氨基酸的主要菌株,以缬氨酸高产菌株谷氨酸棒杆菌V1为研究对象,探讨磷酸烯醇式丙酮酸羧化酶(PEPC)和磷酸烯醇式丙酮酸羧激酶(PCK)介导的草酰乙酸回补途径对菌株生理特性以及主要氨基酸代谢流量的影响。【方法】通过基因工程手段,在谷氨酸棒杆菌V1中过表达pepc(编码PEPC)和pck(编码PCK),比较重组菌与出发菌关键酶活性、发酵特性以及主要氨基酸积累量变化。【结果】构建两株重组菌V1-pepc(强化草酰乙酸回补途径)和V1-pck(弱化草酰乙酸回补途径),重组菌生长均较出发菌延缓,总生物量、葡萄糖和硫酸铵消耗基本不变;过表达pck,PCK活性提高22.8%,丙氨酸、缬氨酸、谷氨酸、精氨酸积累量分别提高了11.8%、17.2%、27.8%和19.5%;过表达pepc,PEPC活性提高27.5%,同时PC活性降低12.9%,天冬氨酸族和谷氨酸族氨基酸的整体流量变化不大,丙氨酸族氨基酸的整体流量降低了14.7%。【结论】丙氨酸族氨基酸受此回补途径影响较大,天冬氨酸族氨基酸受此影响较小。     

蔓花生PEPC基因家族的生物信息学分析

为了解花生中磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC)的功能,对二倍体祖先种野生蔓花生(Arachis duranensis)基因组数据库进行分析,发现存在9个Ad PEPC基因家族成员,这些基因的序列长度为3 584~12 956 bp,开放阅读框(ORF)长度为702~3 168 bp,分布在3、5、7、8、9、10号染色体上。蔓花生Ad PEPC家族蛋白的氨基酸序列中均含有HCO3-结合位点和PEP结合位点等保守结构域,根据序列特征可分为植物型、细菌型和序列较短的PEPC等3类,同类蛋白序列的同源性较高,基因结构中的内含子与外显子的数目也较相似。基因表达分析表明,多数成员在花或茎中的表达量较高,Ad PEPC1;2和Ad PEPC4;2在茎中的表达量最高,其他家族成员尤其是Ad PEPC2、Ad PEPC1;5和Ad PEPC1;3在花中的表达量明显高于其他组织,Ad PEPC1;5基因在叶中不表达。Ad PEPC3在根、茎、叶和花中均不表达,推测该基因为假基因。这为深入研究Ad PEPC家族基因的功能奠定了基础。     

香格里拉水韭磷酸烯醇式丙酮酸羧化酶基因(PEPC)的克隆及其表达载体构建

以中国特有植物香格里拉水韭（Isoetes shangrilaensis X.Liu）为材料,通过转录组测序数据分析筛选出磷酸烯醇式丙酮酸羧化酶基因（IsPEPC）,根据该基因序列,从香格里拉水韭cDNA中克隆获得磷酸烯醇式丙酮酸羧化酶（PEPCase）的编码基因IsPEPC,并将此基因插入pCAMBIA-2300-N-eGFP及pMD质粒载体上,再采用农杆菌介导的花序浸染法将2个重组载体分开转入野生型拟南芥（Arabidopsis thaliana（L.）Heynh.）中。结果显示：IsPEPC基因蛋白编码序列长度为2928 bp,编码975个氨基酸;同源性检索分析结果表明,该蛋白与其近源物种江南卷柏（Selaginella moellendorffii Hieron.）的PEPC基因蛋白序列同源性为79.8%。对转基因的T₁代拟南芥通过抗性筛选并在gDNA水平上阳性鉴定,初步鉴定得到pC2300-N-eGFP-IsPEPC转基因株系26个和pMD-IsPEPC转基因株系32个。     

棉花PEPC基因种子特异性ihpRNA表达载体的构建及鉴定

磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC)是控制植物体中蛋白质和脂肪酸含量比例的关键酶。本研究从棉花中克隆得到PEPC基因,长度为433bp,并将该基因的正反义片段分别和种子特异性启动子napin启动子(1123bp)、α球蛋白B基因启动子(1149bp)连接,插入到植物表达载体pCADS1341中。经酶切和PCR鉴定,成功的构建了PEPC基因的种子特异性ihpRNA表达载体pCADSNPSPA和pCADSBPSPA,为后期高含油量棉花材料的选育打下了基础。     

不同发育时期追施氮肥对冬小麦旗叶中光合酶活性的影响

小麦开花后,随着旗叶的衰老,旗叶中1,5-二磷酸核酮糖羧化酶（RuBPC）、磷酸烯醇式丙酮酸羧化酶（PEPC）和乙醇酸氧化酶（GO）活性呈下降趋势。随着追施氮肥时期的推迟,光合酶活性呈增加趋势,这意味着氮肥追施时间后移有利于提高小麦光合速率。在旗叶衰老后期,大穗型品种小麦旗叶中光合酶活性略高于多穗型品种小麦。     

镉胁迫对旱柳光合作用和内肽酶变化的影响

通过水培方法,添加不同浓度CdCl2(0、5、25、50 μmol·L^-1)处理14 d,测定叶绿素含量、核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)和磷酸烯醇式丙酮酸羧化酶(PEPC)活性、游离氨基酸含量及内肽酶活性,调查Cd对旱柳光合作用和内肽酶变化活性影响。结果发现：Cd处理降低了总叶绿素、叶绿素a、b含量; Rubisco活性随着介质中Cd浓度增加而降低;Cd抑制根和叶PEPC活性;同对照相比,根中游离氨基酸含量没有显著变化,而叶中游离氨基酸含量增加;不同浓度Cd处理降低根的内肽酶活性,高浓度Cd使叶内肽酶活力增加。这些结果表明,Cd通过降低叶绿素含量,促进叶内肽酶活性和抑制了CO₂羧化酶活性来影响旱柳光合作用。     

植物磷酸烯醇式丙酮酸羧化酶的多生理功能

磷酸烯醇式丙酮酸羧化酶(简称PEPC)(正磷酸:草酰乙酸羧化酶,EC4.1.1.31)进行下列催化反,这一反应首先在C_3植物菠菜叶片中发现。后来的研究证明PEPC广泛存在于高等植物的所有组织、藻类及细菌中,但在动物组织中未测出此酶。一般认为,PEPC是一个胞质酶。但也有证据指出PEPC可能与叶绿体有联系。C_3植物组织的PEPC活性是C_4植物叶片的2～5％。从不同来源的植物以及组织中得到的纯化PEPC来     

油菜烯醇酶基因的克隆与分析

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP).我们通过RACE-PCR方法从油菜(Brassica napus L.)中克隆到了编码烯醇酶的全长基因.序列分析表明该基因全长cDNA为1 624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38 kD,等电点为5.78.比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性.Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在.RT-PCR和Northern分析表明烯醇酶基因在100 mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降.该研究表明所克隆基因是植物烯醇酶基因家族的新成员.     

Coccystes undoxylophus

Ohne Zusammenfassung     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

UeberAquila pennata undminuta

Ohne Zusammenfassung     

Aquila pennata undminuta

Ohne Zusammenfassung     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.