链霉菌和大肠杆菌穿梭质粒载体的构建

本文报道了链霉菌和大肠杆菌穿梭质粒载体pSE-3的构建;把具有双启动子的大肠杆菌的质粒pGEM-3与新霉素抗性基因启动子缺失的链霉菌的探针质粒pIJ486分别用BamHI和BglⅡ酶切,T4 DNA连接酶连接后转化到E.coli HB101(Amp(?),Neo(?)),所得重组质粒能强启动pIJ486质粒上的氨基糖苷磷酸转移酶基因(aph),并使新霉素抗性基因在大肠杆菌中得到强表达。此重组质粒被命名为pSE-3,当其转化到变青链霉菌TK54(Tsr(?),Neo(?))的原生质体前,新霉素抗性基因亦能得到强表达。酶切结果表明,构建的具有两个启动子的穿梭质粒载体pSE-3上有HindⅢ和EcoRI的单酶位点,拷贝数约为39。经再转化和传代50代等研究表明,穿梭质粒载体pSE-3在链霉菌和大肠杆菌中均是稳定的。为某些有应用价值的目的基因在大肠杆菌和链霉菌中的克隆与表达提供了一个有价值的穿梭质粒载体。     

以thyA基因为选择压力非抗性质粒载体的构建

以干酷乳杆菌L.casei34103染色体DNA为模板,利用PCR技术扩增胸苷酸合成酶（Thymidylatesynthase,thyA)基因,回收纯化,选择以红霉素抗性为选择压力的可以在大肠杆菌和乳酸菌中穿梭表达的质粒pW425e为基本质粒,以thyA基因取代红霉素基因,获得重组载体并鉴定,此重组载体可以对thyA基因缺陷的大肠杆菌E.coli X51和嗜酸乳杆菌DOMLaS 107进行功能弥补,进而构建了以thyA基因为地选择压力的非抗生素抗性穿梭表达载体,其大小为3716bp,并命名为pW425t。     

灵芝-8基因的番茄果实特异性启动子植物表达载体的构建

构建含有灵芝-8（LZ-8）基因和番茄果实特异性E8启动子的重组载体,并将其转化到根瘤农杆菌中。通过PCR法获取LZ-8基因和E8启动子序列,将目的基因和E8启动子序列构建到植物表达载体pBI121中,获得果实特异性表达LZ-8蛋白的重组质粒。并采用PCR、限制性内切酶酶切和序列测定分析法,对重组质粒进行鉴定,将其转入根瘤农杆菌GV3101中。PCR法、限制性内切酶酶切图谱和序列测定分析均表明番茄果实特异性表达LZ-8蛋白的重组质粒构建成功。获得了含有LZ-8基因和E8启动子的重组质粒,并成功转化根瘤农杆菌,为下一步LZ-8蛋白在番茄果实中特异表达奠定基础。     

短短小芽孢杆菌启动子筛选载体的构建

为了筛选短短小芽孢杆菌强启动子元件,应用PCR技术从枯草杆菌168中分离出α-淀粉酶基因,用其作为报告基因与质粒pUB110和pKF3一起构建了启动子筛选载体pKB/A.将短短小芽孢杆菌细胞壁蛋白基因启动子引入该载体构建成重组质粒pKB/PA,电穿孔法转化短短小芽孢杆菌50后发现α-淀粉酶以活性形式分泌表达.结果表明短短小芽孢杆菌启动子筛选载体构建成功.     

mCherry红色荧光标记乳酸菌的融合表达系统构建及应用

为开发新型荧光蛋白标记乳酸菌以填补国内研究空白,利用pSIP载体,构建了以红色荧光蛋白mCherry为标记,并以乳酸菌胆盐水解酶基因bsh为报告基因的乳酸菌融合表达系统。在4种不同启动子(P_(sppA)、P_(ldhL)、P_(32)和P_(slpA))调节下,相继实现了融合基因的诱导型和组成型表达,表达的融合重组蛋白mCherry-BSH同时检测出红色荧光活性和胆盐水解酶BSH活性。mCherry红色荧光标记的乳酸菌融合表达系统的成功构建不仅为研究乳酸菌在生物体内的分布、定植及存活情况从而揭示其益生功能的作用机理提供有利条件,也为更多活性蛋白在乳酸菌宿主中的表达、细胞定位、功能鉴定的研究奠定基础。     

变形链球菌F-ATPase启动子荧光蛋白载体的构建及表达

目的构建由质子移位膜ATP酶(membrane-bound proton-translocating ATPase,F-ATPase)启动子启动的绿色荧光蛋白报告基因穿梭表达载体,观察其在大肠埃希菌中的表达同时鉴定表达产物。方法以变形链球菌(UA159)基因组为模板,扩增F-ATPase启动子片段,构建由F-ATPase启动子启动的绿色荧光表达载体pFgfp,酶切F-ATPase启动子及绿色荧光蛋白编码基因,连接到穿梭质粒pDL276,构建重组载体pLFgfp。结果重组质粒pLFgfp酶切及基因序列分析证实目的片段成功插入,重组载体转化后的大肠埃希菌有绿色荧光蛋白的表达,并能随着细菌传代继续表达。结论 F-ATPase启动子启动的绿色荧光蛋白穿梭表达载体pLFgfp构建成功,为研究生物膜环境中耐酸菌F-ATPase毒力因子的表达奠定基础。     

乳酸菌表达CRISPR gRNA载体的构建与鉴定北大核心

[目的]构建并筛选重组gRNA(向导RNA)表达质粒p SJCR-LDH和p SJCR-C9PX,并对gRNA在乳酸菌中的表达进行鉴定。[方法]通过PCR技术扩增复制子SH71、氯霉素抗性基因CM和gRNA表达盒片段。将CM片段分别与gRNA表达盒进行融合,再与SH71复制子进行无缝克隆。通过转化,构建重组质粒p SJCR-LDH和p SJCR-C9PX。应用PCR、RT-PCR、Real-Time PCR对重组质粒进行鉴定,gRNA表达验证及拷贝数检测。[结果]PCR及RT-PCR结果显示获得169 bp大小目的条带,表明获得重组质粒载体且该gRNA表达载体在植物乳杆菌WCFS1株、CGMCC1.557株、乳酸乳球菌MG1363株中均成功表达,绝对荧光定量PCR检测获得LDH-gRNA标准曲线为y=-3.480log X+45.34,扩增效率为93.8%;C9PX-gRNA标准曲线为y=-3.327log X+37.07,扩增效率为99.8%。[结论]为利用CRISPR基因编辑技术在乳酸菌中进行基因操作奠定基础,为乳酸菌载体构建提供方法。     

乳酸菌表达CRISPR gRNA载体的构建与鉴定

[目的]构建并筛选重组gRNA(向导RNA)表达质粒p SJCR-LDH和p SJCR-C9PX,并对gRNA在乳酸菌中的表达进行鉴定。[方法]通过PCR技术扩增复制子SH71、氯霉素抗性基因CM和gRNA表达盒片段。将CM片段分别与gRNA表达盒进行融合,再与SH71复制子进行无缝克隆。通过转化,构建重组质粒p SJCR-LDH和p SJCR-C9PX。应用PCR、RT-PCR、Real-Time PCR对重组质粒进行鉴定,gRNA表达验证及拷贝数检测。[结果]PCR及RT-PCR结果显示获得169 bp大小目的条带,表明获得重组质粒载体且该gRNA表达载体在植物乳杆菌WCFS1株、CGMCC1.557株、乳酸乳球菌MG1363株中均成功表达,绝对荧光定量PCR检测获得LDH-gRNA标准曲线为y=-3.480log X+45.34,扩增效率为93.8%;C9PX-gRNA标准曲线为y=-3.327log X+37.07,扩增效率为99.8%。[结论]为利用CRISPR基因编辑技术在乳酸菌中进行基因操作奠定基础,为乳酸菌载体构建提供方法。     

以绿色荧光蛋白为报告基因的原核启动子检测体系构建

以质粒pMUTIN-GFP 扩增获得的目的gfp 基因为报告基因,将其克隆到大肠杆菌-枯草芽孢杆菌穿梭载体pBE2,构建成一个具有启动子活性检测功能的重组质粒pBE2-GFP .将组成型启动子P43和诱导型启动子Pspac克隆入pBE2-GFP ,得到重组表达载体pBE-GFP-P43和pBE-GFP-Pspac,转化至大肠杆菌和枯草芽孢杆菌.荧光显微镜检测GFP 蛋白的表达情况.结果 表明,2种不同类型的启动子均能在大肠杆菌BL21和枯草芽孢杆菌1A751中启动gfp 基因的表达.     

一种含双歧杆菌种属特异性复制子的新型大肠杆菌-双歧杆菌穿梭质粒的构建

目前,双歧杆菌的转化是一个技术难题,与大肠杆菌等宿主菌的高转化效率不同,采用普通的原核质粒无法转化双歧杆菌.为此,本文提出双歧杆菌转化对质粒复制子具有"种属特异性"要求,并通过构建含有双歧杆菌特异复制子的新型穿梭质粒,以求解决这一难题.首先从GenBank获取长双歧杆菌隐性质粒pMB1的序列信息,采用Overlap-PCR方法获得其全长DNA,作为拟构建质粒的复制子;继而采用重组技术,将其与pMK4质粒片段(含大肠杆菌复制子pUC和抗氯霉素基因Cat)重组,构建大肠杆菌-双歧杆菌穿梭质粒;用电穿孔法将重组质粒转化双歧杆菌,通过观察不同电转参数下的转化效率,选择双歧杆菌转化的最佳条件.结果,成功获得全长1899bp的pMB1复制子并构建成功含有pMB1和pUC双复制子的原核重组质粒,经酶切和测序鉴定正确,命名为pCMB1.以重组质粒成功转化了长双歧杆菌NCC2705和NQ1501,而其它3种野生型双歧杆菌(包括1株长双歧杆菌)未能转化成功.结论:质粒中含有双歧杆菌种属特异的复制子是实现双歧杆菌转化的必要条件;即使是含有特异复制子的质粒也只能转化有限数量种型甚至有限数量种株的双歧杆菌;选择最佳电转化条件能显著提高转化效率.     

General and specialized vectors derived from pBM02, a new rolling circle replicating plasmid of Lactococcus lactis

This paper reports the construction of several general cloning vectors and a specialized depurative vector based on a new lactococcal plasmid that replicates by the rolling circle mechanism [pBM02; Plasmid 49 (2003) 118]. Most vectors are shuttle vectors for Escherichia coli-Lactococcus lactis and carry replicons of both ColE1 and pBM02 plasmids (ColE1 is used even though the pBM02 replicon is fully active in both Gram-positive and Gram-negative organisms). Segregational and structural studies indicated that the new vectors were stable enough for the majority of applications. Further, since the basic replicon is compatible with plasmid derivatives of pWV01 and pSH71, they can be maintained in the same cell with members of the two largest vector series for L. lactis and other lactic acid bacteria, the pGK, and the pNZ series.     

Use of Long-Term Stored Vector Information in the Neotropical Ant <Emphasis Type="BoldItalic">Gigantiops destructor</Emphasis>

We investigated how the formicine ant Gigantiops destructor can use vector information to navigate within the cluttered environment of the rain forest. Displaced foragers use skylight information to move in the theoretical feeder-to-nest direction, whether they are prevented from updating their path-integrator during foraging or captured at the departure from their nest, i.e. with a current accumulator state very close to zero. Only ants that have collected food are able to download a long-term stored reference vector pointing in the nest direction, irrespective of the current accumulator state of their path-integrator stored in a working memory and independent of familiar landmarks. Depending on the release sites, ants that became lost at a maximum distance of 50 cm could still hit and recognize their familiar route, or they engaged in a systematic search for it centered on the release sites. In contrast to Cataglyphis desert ants, Gigantiops ants do not rely primarily on the current accumulator state of their egocentric path integrator. Such a long-term vector-based navigation primed by food capture is well adapted for a tropical ant foraging during periods spanning several hours. This could prevent the numerous cumulative errors in the evaluation of the angles steered that might result from a continuously running path-integrator operating during complex foraging patterns performed at ground or arboreal levels and during passive displacement in response to heavy rain.     

在无天然游离质粒产甘油假丝酵母中非整合表达合成咖啡酸

【背景】咖啡酸（3,4-二羟基肉桂酸）是一种有多种生物活性和药用价值的天然酚类化合物,产甘油假丝酵母（Candida glycerinogenes）具有咖啡酸前体代谢途径,高耐酸且生长与发酵速率快,是潜在高产咖啡酸的底盘细胞,但无游离载体将影响咖啡酸合成的深入研究。【目的】探索在无天然游离质粒的C. glycerinogenes中构建操作更简便、表达能力更强的游离载体合成咖啡酸的可行性。【方法】筛选自主复制序列（autonomously replicating sequence,ARS）,构建适用于C. glycerinogenes合成咖啡酸的游离载体,并通过改造其ARS位置、标记基因URA5启动子长度、基因表达元件和利用Kozak序列优化表达并合成咖啡酸。【结果】构建的5个分别含不同ARS的载体中,pTGAPU-CA-AOX1t-KLARS在C. glycerinogenes中能自我复制并表达合成咖啡酸的基因,而且当ARS位于目的基因表达元件上游、URA5启动子截短250 bp,或分别采用Kozak序列与终止子URA5t后,咖啡酸产量较改造前均有明显提升,最高产量为初始产量的3.73倍,达29.1 mg/L,高于前期整合表达产量。【结论】在C. glycerinogenes中非整合表达合成咖啡酸且优于整合表达,为今后利用游离载体改造咖啡酸合成代谢途径提供了新工具,同时为其他无游离质粒菌株构建非整合表达体系提供参考。     

The genetics and expression of an esterase locus inAnopheles gambiae

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain ofAnopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.     

A Modular Vector for Agrobacterium Mediated Transformation of Wheat

Wheat (cv Chinese Spring) tissues were transformed using Agrobacterium tumefasciens and a new plasmid modular vector, pMVTBP. We constructed pMVTBP with unique restriction sites connecting (1) the CaMV 35S promoter, (2) a Kozak sequence, (3) the FLAG epitope, (4) the (His)₆ epitope, (5) a coding region (for wheat TATA Binding Protein, wTBP) and (6) the CaMV 35S 3′UTR. This vector thus allows easy exchange of different regulatory or coding sequences. Explants of either germinating mature seeds, or immature embryos, were induced to callus for up to two weeks, treated with virulence-induced bacteria for one hour, then regenerated into plantlets. Transient expression of a GUS reporter gene, assayed at about one week, occurred in 10–12% of calluses. Expression of the FLAG-tagged wTBP was also detected, by immunostaining. Stable expression, by selective growth on geneticin, and by GUS expression at about six weeks, occurred in 1–2% of calluses, quite comparable to that achieved by other methods. An erratum to this article is available at .     

Cloning Vectors for Rice

We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc, respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning of full-length cDNA clones into our binary vectors.     

Development of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> as a vector modifiable for different host species to produce therapeutic proteins

We have developed Cucumber mosaic virus (CMV) as a plant virus vector especially for production of pharmaceutical proteins. The CMV vector is a vector modifiable for different host plants and does not require further engineering steps. CMV contains three genomic RNA molecules (RNAs 1–3) necessary for infectivity. With this system, instead of creating different vector constructs for each plant we use, we take advantage of the formation of pseudrecombinants between two CMV isolates by simply reassembling a vector construct (RNA 2 base) and an RNA molecule containing the host determinant (mostly RNA 3). In this study, the gene for acidic fibroblast growth factor (aFGF), one of the human cytokines, was cloned under the control of the subgenomic promoter for RNA 4A of the CMV-based vector, C2-H1. Infected Nicotiana benthamiana plants produced aFGF at levels up to 5–8% of the total soluble protein. The tobacco-produced aFGF was purified, and its biological activity was confirmed. Using this system, which provides a versatile and viable strategy for the production of therapeutic proteins in plants, we also demonstrated a high level of aFGF in Glycine max (soybean) and Arabidopsis thaliana.     

Comparison of the mutations induced by p-benzoquinone, a benzene metabolite, in human and mouse cells

Benzene is one of the chemicals widely contaminating the environment. Benzene is suggested to be a human leukemogen. When benzene is absorbed in the human body, it is metabolized firstly in the liver and subsequently in the bone marrow where it provokes initiation of leukemia. In the present study, we analyzed mutations induced by p-benzoquinone (p-BQ), a benzene metabolite, in human cells using a shuttle vector plasmid pMY189, and compared frequencies, types and spectra of the mutations with those of the mutations previously revealed in mouse cells using a similar plasmid pNY200. We found that p-BQ induces mutations in human and mouse cells at similar frequencies but with different types of mutagenesis. The proportion of tandem base mutations was significantly lower in human cells than in mouse cells. Most base substitutions were induced in G:C base pairs in both human and mouse cells. However, the proportion of G:C→C :G transversion is significantly higher in human cells. These findings indicate that the p-BQ-induced DNA damage in human and mouse cells is processed in a different manner, and that extrapolation of mice findings on experimental benzene carcinogenesis to human cancer risk assessment should be conducted carefully.     

Short communication: Construction of a cloning vector for Paenibacillus polymyxa and P. azotofixans

A cloning vector that could replicate in Paenibacillus polymyxa, P. azotofixans and Bacillus subtilis was constructed using two Staphylococcus aureus plasmids. The recombinant plasmid confers chloramphenicol and erythromycin resistance and contains unique restriction sites for PvuII and BclI. The stability of pRJ45 was analysed.