蓝藻基因转移系统的选择与建立

蓝藻是一类进行光合放氧的原核生物 ,因其结构的特殊性 ,已成为表达外源基因的理想宿主之一。然而外源基因转化系统的选择与建立一直影响着蓝藻基因工程的快速发展。总结了各类蓝藻基因转移系统的特点、影响因素、各系统间的优缺点、以及不同蓝藻株系最适基因转移系统的选择等 ,为利用蓝藻进行遗传操作提供可能 ,为蓝藻基因工程发展提供信息。     

重组微生物生理学

重组微生物生理学主要研究外源基因与宿主的相互作用以及细胞生理状态对这种相互作用的影响。宿主与外源基因的相互作用可发生在复制、分配、表达、代谢等各种水平,主要的分子机制为核酸-核酸、核酸-蛋白质的相互作用,一些小分子化合物可作为信号或效应子参与大分子的相互作用。重组微生物生理学研究主要包括下列三方面工作:1.噬菌体、质粒与宿主的相互作用以及基因工程菌中目的基因的复制、分配和表达规律。2.微生物响答系统的响答及适应的分子机制及其对外源基因调控的影响。3.细胞生长速率对宿主基因和外源基因调控的影响。这些工作融合了分子水平和细胞水平的研究,模糊了分子生物学和生理学的界限,成为基因工程的理论基础之一。美国微生物学会自1987年起把重组微生物生理学列为年会的一个独立专题。 外源基因与宿主的相互作用 噬菌体和质粒等染色体外基因与宿主基因处于一种依赖、互补和竞争的复杂关系。     

植物质体基因工程调控元件研究进展

随着植物合成生物学的发展,质体逐渐成为许多具有商业价值的次生代谢产物和治疗性蛋白异源生产的理想平台。与核基因工程相比,质体基因工程在外源基因高效表达和生物安全性等方面具有其独特优势。然而,外源基因在质体系统中的组成型表达或对植物生长不利,因此需进一步挖掘、设计调控元件实现对外源基因的精准调控。本文概述了质体基因工程调控元件的研究进展,内容包括操纵子设计与优化思路、多基因共表达调控策略及新型表达调控元件的挖掘等,为植物合成生物学的发展提供参考。     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具,将促进蓝藻作为底盘细胞在合成生物学等领域的发展。     

维生素对转基因鱼腥藻Anabaena sp.PCC7120培养的影响

近年来分子遗传学和基因工程研究证实 ,大肠杆菌的载体和启动子往往可以适用于蓝藻 ,尤其是单细胞蓝藻的转基因 ,这使得蓝藻基因工程得到了较快的发展 ,利用藻类为宿主的基因产物的生产也受到日益关注 ,因此微藻的培养受到广泛重视。刘凤龙等通过三亲结合转移方式将人ＴＮＦ α基因转入鱼腥藻 71 2 0中并得到表达[1 ] ,但转基因鱼腥藻高效培养方面需进一步研究。混合营养培养方式因具有缩短培养周期、实现细胞高密度培养等优点 ,已成为微藻培养新技术。藻类生长除了需要合适的营养源外 ,许多生长因子对生长具有较大影响 ,本文对维生素Ｂ1 、…     

逆转录病毒载体在基因工程疫苗方面的应用

逆转录病毒载体是根据逆转录病毒的特性设计出的一种病毒表达载体,以其能够整合到宿主细胞染色体上并能稳定表达目的基因而被视为基因运载最有效的工具。目前广泛应用于基因治疗、外源基因表达、基因工程疫苗等方面。主要综述了逆转录病毒载体在基因工程疫苗方面的应用现状及前景,为该载体在生物医学领域方面的应用提供有价值的参考。     

杜氏盐藻分子生物学最新进展及展望

杜氏盐藻是一种无细胞壁的单细胞双鞭毛真核藻类,是一种十分重要的藻类资源。过去对杜氏盐藻的研究多集中在形态学、耐盐机理及β-胡萝卜素等方面,近年来,随着藻类基因工程的快速发展,本研究课题组及国内外在杜藻盐藻分子生物学方面做了大量工作,现就杜氏盐藻在这一领域的研究进展进行综述,主要是重要功能基因的克隆与分析、杜氏盐藻调控序列的研究以及杜氏盐藻作为宿主表达外源基因等。     

叶绿体基因工程研究进展

叶绿体是植物细胞和真核藻类执行光合作用的重要细胞器,在叶绿体中表达外源基因比在细胞核中表达具有一些独特优势。叶绿体基因工程涉及叶绿体的基因组特征、转化系统的优点、转化过程及方法等方面,叶绿体基因工程在提高植物光合效率、改良植物特性、生产生物药物及改善植物代谢途径等方面已得到应用。尽管叶绿体基因工程还存在同质化难度高、标记基因转化效率较低、宿主种类偏少等问题,但作为外源基因在高等植物中表达的良好平台其仍然具有广阔的发展和应用前景。     

非随机方法构建酿酒酵母基因工程菌

酿酒酵母是基因工程产品研究和生产的一个重要表达系统,表达载体和宿主细胞是构成表达系统的两大要素,虽然外源基因表达的方式、强度主要由表达载体控制．但宿主细胞的选择对最终获取产品的质量和数量也具有十分关键的作用。酿酒酵母基因工程宿主菌除要求具有高的DNA转化效率、细胞生长密度和稳定性、低的内源蛋白水解酶活性外,还必须具备与表达载体相对应的营养缺陷筛选标记,用传统随机诱变方法得到的营养缺陷变异株,因含有本底和隐性突变,在细胞生长密度和稳定性方面往往不能满足基因工程产品研究和生产的要求,甚至不能有效地表达外源基因。本文报道用重组技术,通过非随机方法构建了酿酒酵母基因工程宿主茁。研究表明用该方法得到的宿主菌在细胞生长密度、稳定性和表达外源基因方面优于用传统随机诱变方法得到的宿主菌。     

哺乳动物细胞高效表达系统研究进展

哺乳动物细胞高效表达系统是基因工程制药研究中的一个重要内容,而表达载体和宿主细胞是哺乳动物细胞表达系统的2个基本组成部分。近年来通过降低目的基因的位置效应、提高目的基因的转录和翻译水平以及目的基因的拷贝数等方面构建哺乳动物细胞高效表达载体。与此同时,研究也对宿主细胞进行了多方面的改造,使之能更好地适应工业化大规模培养的要求,从而降低细胞培养和产品纯化的成本。本就哺乳动物细胞高效表达载体的构建及宿主细胞的改造等方面的最新进展作一简述。     

Pseudo-continuous culture of marine recombinant cyanobacteria under a light dark cycle

Summary Pseudo-continuous culture of recombinant marine cyanobacteria was carried out under a light dark cycle. As a model foreign gene, the structural gene of chloramphenicol acetyl transferase (CAT) was used. Under a light dark alternate conditions, a steady state cell concentration was observed. However, the level of CAT expression did not show a stationary phase, but oscillated with the light dark cycle. This may be due to the lack of energy for foreign peptide synthesis during dark incubation. This result also indicates the possibility of regulation of foreign gene expression by controlling light illumination.     

groESL启动子提高集胞藻6803外源egfp基因表达效率的研究

利用聚球藻7942中热激蛋白基因groESL的启动子(PgroESL)驱动外源egfp基因在集胞藻6803(Syn-echocystis sp.PCC6803)中的表达,通过蛋白免疫印迹技术研究不同温度条件下该外源基因的表达情况。结果表明,42℃诱导30min后,PgroESL启动子能显著提高转基因藻Pg中外源egfp基因的表达,使外源基因的表达量比正常温度条件下提高3.4倍。研究表明,聚球藻7942中groESL操纵子的启动子区域是一类可以受温度诱导的强启动子,能够显著提高宿主细胞中外源基因的表达效率。     

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

Summary Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugants were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 × 10^–4 transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 m NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation. Correspondence to: K. Sode     

Genetic engineering in marine cyanobacteria

Many species of microalgae producing useful materials have been isolated from marine environments. For their industrial application, widely applicable and stable gene expression is required. It is necessary to establish gene transfer methods as an essential first step in genetic manipulation. Although gene transfer techniques for cyanobacteria have been developed, only naturally transformable strains have been used. Here, we describe recent progress made in developing gene transfer methods for marine cyanobacteria. The following are covered: (1) transformation, (2) electroporation, (3) conjugation, (4) particle gun. A plasmid from the marine cyanobacterium, Synechococcus sp., whose copy number is dependent on salinity, was characterized. This plasmid is being used to develop a stable and controllable gene expression system.     

Heterologous assembly and rescue of stranded phycocyanin subunits by expression of a foreign cpcBA operon in Synechocystis sp. strain 6803.

Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membrane-associated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin beta subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin alpha and beta subunits assemble with endogenous phycobilisome components. The phycocyanin alpha subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.     

Digalactosyldiacylglycerol is required for better photosynthetic growth of Synechocystis sp. PCC6803 under phosphate limitation

Digalactosyldiacylglycerol (DGDG) is a typical membrane lipid of oxygenic photosynthetic organisms. Although DGDG synthase genes have been isolated from plants, no homologous gene has been annotated in the genomes of cyanobacteria and the unicellular red alga Cyanidioschyzon merolae. Here we used a comparative genomics approach and identified a non-plant-type DGDG synthase gene (designated dgdA) in Synechocystis sp. PCC6803. The enzyme produced DGDG in Escherichia coli when co-expressed with a cucumber monogalactosyldiacylglycerol synthase. A DeltadgdA knock-out mutant showed no obvious phenotype other than loss of DGDG when grown in a BG11 medium, indicating that DGDG is dispensable under optimal conditions. However, the mutant showed reduced growth under phosphate-limited conditions, suggesting that DGDG may be required under phosphate-limited conditions, such as those in natural niches of cyanobacteria.     

在鱼腥藻7120中建立反义glnA系统

应用反义技术对鱼腥藻7120切的内源glnA基因的表达进行调控,首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶（GS）的基因glnA中取得部分结构基因片段,与表达质粒载体pRL-439及穿梭质粒载体pDC－8相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒pDC－AM,然后应用三亲接合转移法把它转入鱼腥藻对7120.通过新霉素筛选,酶谱鉴定,斑点杂交,质粒的交叉转化以及内源glnA基因表达的GS活性分析,GS相关的胞外泌氨分析及所获藻株的形态学变化,证明已在鱼腥藻7120中建立了人工反义glnA基因的品系。