Transformation of Acetobacter polyoxogenes with Plasmid DNA by Electroporation

Acetobacter polyoxogenes was transformed with plasmid DNA by electroporation. The following points were essential for transformation: (i) dilution of the culture broth with cold water and air bubbling of the culture broth for transformation at discharging from a jar fermentor, and (ii) selection of transformants by liquid cultivation. For shortening of the lag time in cultivation for selection of transformants, the following treatments were useful: (i) addition of sucrose to the cell suspension during transformation and to the broth for cultivation, and (ii) addition of 1 mm MgCl₂ to a mixture of cells and DNA during electroporation.     

灵芝发酵液的营养保健成分及其在化妆品中的应用

用液体发酵法制取的灵芝发酵液至少含有17种氨基酸、多种矿物质、维生素B_2和多糖,用于生产灵芝洗发剂,对脂溢性皮炎主要临床症状的治疗效果总有效率均在84％以上。     

产灵芝胞外多糖培养基的优化及发酵指标变化

经单因素和正交试验优化,灵芝胞外多糖最佳发酵培养基各成分质量分数为:麦芽糖2%,黄豆粉1%,FeSO4·7H2O0.02%,KH2PO40.1%,土豆汁体积分数30%,pH自然,产量可达到86.36g·L-1(湿重)。灵芝胞外多糖产量受发酵过程各因素的影响,发酵过程中pH、总糖、还原糖和氨基氮有一定的相关性。灵芝多糖整个发酵过程需要144h左右,第6d达到发酵终点。     

红花花青素合成酶基因的克隆及表达分析

根据红花转录物测序结果中得到的中间序列,采用R11-PCR和RACE方法从红花花瓣中克隆到1个4嬲基因的全长cDNA,该基因全长序列1226bp,具有完整的开放阅读框（ORF）,共1050bp,编码349个氨基酸。生物信息学软件分析显示,该基因编码的蛋白理论分子量约为82．27kDa,等电点为5．09,序列里含有典型的加尾信号序列AATAA和Poly（A）。保守结构域预测表明,该基因编码的蛋白具有典型的ANS蛋白功能结构域,其保守结构域中含有铁离子及2．0-酮戊二酸结合位点。结合其他物种的臌因构建系统树表日月,红花ANS蛋基因与其他物种氨基酸具有一定的同源性,其中与芍药的亲缘关系最近。应用实时荧光定量PCR分析表明,ANS基因在红花的初花期和盛花期的表达量最高。     

Co-immobilization of d-hydantoinase and d-carboamylase on Chitin: Application to the Synthesis of p-hydroxyphenylglycine

The immobilization of a multi-enzyme extract is an excellent method for multi-step biotransformations. This paper describes how a multi-enzyme extract from Agrobacterium radiobacter, rich in d-hydantoinase and N-carbamyl-d-amino acid amidohydrolase was immobilized on chitin for its application on the synthesis of p-hydroxyphenylglycine. The adsorption derivative showed a higher activity than the covalent one. Compared to the soluble multi-enzymatic extract, the adsorption derivative showed greater pH-stability in the pH range under study. Its optimum pH ranged from 7–8. Furthermore, it showed high activity at low temperature.     

盾叶薯蓣环阿屯醇合酶全长基因的克隆与分析

环阿屯醇合酶(cycloartenol synthase,CAS)是薯蓣皂甙元生物合成途径中的第一个关键酶.以基因组DNA为模板,利用染色体步行和长距离PCR方法首次克隆了盾叶薯蓣CAS全长基因.序列分析比较结果表明,盾叶薯蓣CAS全长基因为7 192 bp,由18个外显子和17个内含子组成.外显子总长为2 280 bp,编码759个氨基酸,最长的外显子为198 bp,最短的为47 bp;内含子总长4 912 bp,最长的内含子为1 551 bp,最短的为68 bp.Southern blot杂交分析表明,CAS基因在盾叶薯蓣基因组中为单拷贝.     

花生花斑种皮花青素生物合成相关miRNA及其靶基因鉴定与分析

花生在世界粮油食品中占有重要地位,其种皮颜色有白色、红色、紫色、粉红色及花斑等多种颜色,花斑种皮花生是其独特的成员之一,具有便于区分的优良性状。本研究以花斑种皮花生VG-02为研究材料,结果表明与花斑种皮颜色合成相关差异表达的miRNA富集的代谢途径有苯丙烷生物合成、类黄酮生物合成、异黄酮的生物合成、昼夜节律植物。miRNA测序结果中共筛选出86个差异表达miRNA,其中20个差异表达的miRNA与花生花斑种皮颜色合成相关。其中包括4个共同靶向花青素、类花青素和IFS2靶基因的miRNA,即miR₈、miR₅0、miR₅1和miR₂39;5个靶向花青素生物合成中结构基因的miRNA,即调控CHS靶基因的miR₃98-x,调控4CL靶基因的miR₄82-z,调控F3′H靶基因的miR₂66和miR₁82以及调控花青素3-O-葡萄糖苷-6靶基因的miR₅;1个靶向花青素生物合成调节基因的miRN...     

The biosynthetic genes encoding for the production of the dynemicin enediyne core in Micromonospora chersina ATCC53710

Dynemicin is a novel anthraquinone-fused member of the 10-membered enediyne antitumor antibiotic family. The development of a genetic system for the dynemicin producer Micromonospora chersina confirmed, for the first time, the requirement of the putative enediyne core biosynthetic genes (dynE8, U14 and U15) and a tailoring oxidase gene (orf23) for dynemicin production. Cloning and sequence analysis of a 76 kb of genomic sequence region containing dynE8 revealed a variety of genes conserved among known enediyne loci. Surprisingly, this fragment and flanking chromosomal DNA lacked any obvious genes encoding for the biosynthesis of the anthraquinone, suggesting that the location of genes encoding for the biosynthesis of the dynemicin enediyne core and the dynemicin anthraquinone are chromosomally distinct. The demonstrated trace production of a shunt product from mutant strain QGD23 (Deltaorf23) also sets the stage for subsequent studies to delineate the key steps in enediyne core biosynthesis and tailoring.     

乙烯生物合成基因工程在果蔬保鲜中的应用

水果和蔬菜的成熟、衰老与乙烯密切相关。乙烯生物合成过程受到多种因素的综合调控。通过基因工程调节乙烯生物合成相关酶的含量或活性以阻断或减少果蔬中乙烯的产生,从而延缓果蔬成熟或衰老,是果蔬保鲜最重要的策略之一。多种果蔬ACC合成酶、ACC氧化酶与微生物ACC脱氨酶、SAM水解酶的基因已被克隆;采用基因工程调控这些酶基因在果蔬中的表达,可能延长果蔬的贮藏保鲜时间。乙烯生物合成基因工程在果蔬保鲜中具有良好的应用前景,少数耐贮藏转基因果蔬已经实现商品化生产。     

西伯利亚蓼PsGRX基因的克隆及其在NaHCO_3胁迫下的表达

采用cDNA末端快速扩增（RACE）技术从西伯利亚蓼叶cDNA文库中克隆到谷氧还蛋白基因（PsGRX）的完整编码区cDNA序列（GenBank注册号为GU139794）,长度为465bp,编码106个氨基酸。根据与其他植物谷氧还蛋白的氨基酸序列的比对以及系统进化分析的结果,初步确定此基因为谷氧还蛋白基因家族成员。实时定量PCR的结果显示,PsGRX在西伯利亚蓼的叶、茎、地下茎中均有表达,叶中表达量最高,地下茎和茎中较低。在NaHCO3胁迫的过程中,此基因在叶、茎和地下茎中的表达模式也有较明显的差异。     

猕猴桃6个LOX基因家族成员实时定量PCR引物特异性的检测与应用

从基因家族成员的视角开展基因表达研究是阐明基因功能的重要组成内容,实时定量PCR（QPCR）技术是分析基因表达的有效手段.以猕猴桃脂氧合酶（LOX）基因家族6个成员为对象,分析了引物特异性的检测方法.该方法整合了熔点曲线分析、琼脂糖电泳、交叉PCR扩增和PCR产物测序等分析手段,有效消除其他成员的交叉扩增干扰,为利用QPCR检测基因家族成员表达提供了特异、准确与可行的途径.     

Red重组系统及在微生物基因敲除中的应用

在完成了对各种微生物基因组的测序以后,功能基因学的研究变得尤为重要。研究基因功能最直接的方法便是将待研究的基因失活。最初构建基因突变体是采用大肠杆菌的RecA系统,但是RecA重组系统操作复杂,重组效率低。最近建立了Red重组系统,该系统由3个蛋白组成：α蛋白(即λ核酸外切酶),β蛋白,Gam蛋白。应用Red系统进行基因敲除,可以直接利用线性打靶DNA,两侧同源臂长度在35~60 bp即可发生同源重组,且重组效率高。 Abstract:Since many DNA-sequencing projects of varied microorganisms have been completed,studies on their functional genomics become more important.Inactivation of an interesting gene is a direct method to characterize its function.Though the Esherichia coli RecA recombination system can be used to produce gene mutants,it needs a complex manipulation process.Furthermore,its efficiency is very low.Recently a Red recombination system was developed.This recombination system consists of three proteins:α protein（λ exonuclease）,β protein and Gam protein.In this system,the linear targeting DNA which contains a selectable marker flanked with a homologous region as short as only 35~60 bp can be directly targeted for gene knock-out with a higher efficiency.     

PCR直接测序方法及其在肿瘤研究中的应用

PCR直接测序技术是PCR扩增与核酸测序技术相结合的一种方法.根据此技术的原理,建立了一种以PCR扩增引物为测序引物,α-³⁵S dATP直接掺入,Taq DNA聚合酶直接测序PCR扩增产物的方法.实验表明:该方法简便、快速、稳定.用此方法对人食管癌组织中的抗癌基因p53进行了突变测序分析,发现食管癌组织中p53存在点突变,插入、丢失移码突变.并用此方法对人和恒河猴的p53内含子序列进行了测定,发现猴第5内含子为81个核苷酸,第8内含子为92个核苷酸.     

The Microbiology and Biochemistry of Anaerobic Bioreactors with Relevance to Domestic Sewage Treatment

Anaerobic granular and fixed-film reactors have been successfully operated for wastewater treatment at full scale for over two decades and represent a sustainable, energy-producing approach, which is increasingly being directed towards treatment of domestic sewage. Research over the past two decades, and significant operational experience, has demonstrated that there are no fundamental microbiological barriers to the implementation of AD for domestic sewage treatment in regions with warm and temperate climates. Despite this, the underlying microbiology of methanogenesis is not fully understood and novel groups of microbes have been identified in sludge, with unknown functions. The methanogenic process has recently been subject to systematic investigation using newly developed analytical and microbiological approaches. A combination of process monitoring, physiological, molecular microbiological and microscopic methods are beginning to generate a comprehensive, integrated data set at micro-organism, granule and reactor level and the current state of knowledge is reviewed here. Information on the formation of granules, on the relationship between reactor operating conditions and microbial consortia and on the impact of process changes on the microorganisms in reactors will, in future, enable the link between the processes occurring at microorganism level (scale ca. 1 μm–1 mm) and the processes occurring within reactors (scale >1 m), which will enhance the efficiency and applicability of anaerobic sewage treatment.     

促髓细胞分化的锌指基因单向PCR扩增直接测序的研究

锌指基因是一种造血调节基因,编码锌指结构蛋白,主要在髓细胞中表达,促进髓细胞分化,在急性早幼粒白血病维甲酸治疗中,促使病情缓解。本文报道了我们从基因分子上研究锌指基因作用中,探索并建立了单向聚合酶链反应（ＰＣＲ）扩增特定单链ＤＮＡ,直接测序的新方法。它能产生质和量均佳的单链ＤＮＡ,无需纯化即可直接用于测序,使复杂的测序研究简便易行,可在２,３天内完成。这种单向ＰＣＲ扩增特定单链ＤＮＡ直接测序的方法,经对锌指基因的ｃＤＮＡ测序,得到验证。此法不仅适用于疾病研究中的ＤＮＡ测序,还可制各单链ＤＮＡ探针,更利于基因结构组成的研究。     

病毒诱导的基因沉默及其在植物基因功能研究中的应用

RNA介导的基因沉默是近年来在生物体中发现的一种基于核酸水平高度保守的特异性降解机制.病毒诱导的基因沉默（virus induced gene silencing, VIGS）是指携带植物功能基因cDNA的病毒在侵染植物体后,可诱导植物发生基因沉默而出现表型突变,进而可以研究该目的基因功能.至今,已经建立了以RNA病毒、DNA病毒、卫星病毒和DNA卫星分子为载体的VIGS体系,这些病毒载体能在多种寄主植物（包括拟南芥、番茄和大麦）上有效抑制功能基因的表达.VIGS已开始应用于N基因和Pto基因介导的抗性信号途径中关键基因的功能研究、抗病毒相关的寄主因子研究以及植物代谢和发育调控研究.在当前植物基因组或EST序列大量测定的情况下,VIGS为植物基因功能鉴定提供了有效的技术平台.     

Development of a PCR method for the detection and quantification of benzoyl-CoA reductase genes and its application to monitored natural attenuation

Benzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10–40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.     

紫背天葵叶和花中花青素合成相关转录组基因分析

为获取紫背天葵(Gynura bicolor D C.)花青素合成代谢相关调控基因信息,该试验以紫背天葵叶片为材料,以其花朵为对照,进行转录组测序,并进行CHS、CHI、F3H等8类合成酶基因以及MYB、bHLH及WD40等3类转录因子检索,从中选取8个相关差异表达显著调控基因进行qRT-PCR验证分析。结果显示:(1)在紫背天葵中共获得72个花青素合成酶信息,其中差异表达明显的有1个F3′H和2个3GT下调,9个F3H基因中有上调基因4个和下调基因5个。(2)在紫背天葵中获取到238个MYB、113个bHLH和219个WD40转录因子,这3类转录因子中差异表达明显的分别为22个、16个和7个。(3)qRT-PCR结果显示,所选取的8个花青素合成相关调控基因,在紫背天葵叶及花朵中的下调表达趋势与转录组测序结果完全一致,但不同基因差异表达趋势略有不同。研究表明,在紫背天葵叶片和花朵中所存在的大量花青素合成代谢调控基因中,只有少量差异表达显著,但转录因子相比合成酶的调控更为复杂。