Assay and characterization of a strong promoter element from B. subtilis

A new strong promoter fragment isolated from Bacillus subtilis was identified and characterized. Using the heat stable beta-galactosidase as reporter, the promoter fragment exhibited high expression strength both in Escherichia coli and B. subtilis. The typical prokaryotic promoter conservation regions were found in the promoter fragment and the putative promoter was identified as the control element of yxiE gene via sequencing assay and predication of promoter. To further verify and characterize the cloned strong promoter, the putative promoter was sub-cloned and the beta-Gal directed by the promoters was high-level expressed both in E. coli and B. subtilis. By means of the isolated promoter, an efficient expression system was developed in B. subtilis and the benefit and usefulness was demonstrated through expression of three heterologous and homogenous proteins. Thus, we identified a newly strong promoter of B. subtilis and provided a robust expression system for genetic engineering of B. subtilis.     

不同异源启动子与MDVgB启动子及其复合启动子的活性比较

为了选择适宜的启动子调控外源基因的表达,以改善马立克氏病病毒为载体的重组病毒的免疫保护力。将hCMV立即早期启动子及增强子、SV40早晚期启动子及增强子或hCMV立即早期增强子的部分序列分别与马立克氏病病毒(MDV)自身的囊膜糖蛋白B基因(gB)启动子核心部分在体外杂合,分别构建复合启动子PhCMV-gB、PSV-gB或Pen-gB;将这些启动子与虫荧光素酶报告基因相连,构建表达载体。利用脂质体将以上质粒与内标质粒(pSV-β-LacZ)共转染鸡胚成纤维次代细胞,于转染后48h,将细胞刮下来,利用荧光素酶测定试剂盒和β-半乳糖苷酶测定试剂盒分别测定转染细胞的荧光素酶和β-半乳糖苷酶的活性,通过荧光素酶和β-半乳糖苷酶活性的比值获得虫荧光素酶的相对活性,利用虫荧光素酶的相对活性进行启动子的活性比较。结果表明,复合启动子相对马立克氏病病毒自身的gB启动子,活性有不同程度的提高,其中复合启动子PhCMV-gB的活性最高,而复合启动子PSV-gB和Pen-gB的活性相当;但与商业强启动子相比,复合启动子活性要弱一些或相当。因此,从某种意义上讲,这些复合启动子既具有gB启动子的一些特性,又有商业强启动子的一些特性,为以马立克氏病毒为载体的新兴疫苗的开发奠定了基础。     

昆虫种群遗传控制技术中启动子的研究

利用昆虫遗传转化技术对害虫进行遗传控制是害虫防治研究的新方向,该技术具有物种特异、防效高且对环境友好的特点。启动子是基因表达调控的重要元件,选择合适的启动子是外源基因高效、准确表达的关键,对获得高效、稳定的遗传修饰昆虫品系至关重要。本文简要介绍了昆虫基因启动子的结构特征,重点描述了昆虫种群遗传防治中组成型启动子、性别和组织特异型启动子、特定发育时期启动子和诱导型启动子的研究和应用概况,并对这几类启动子在害虫遗传控制中的应用前景进行了展望。     

A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits

A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.     

Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824.

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of beta-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional beta-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the beta-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the beta-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the beta-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of beta-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.     

Construction of high sensitive detection system for endocrine disruptors with yeast n-alkane-assimilating Yarrowia lipolytica

To construct a highly sensitive detection system for endocrine disruptors, we have compared the activity of promoters with the ALK1, ICL1, RPS7 and TEF1 for heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of lacZ or hERalpha reporter gene, respectively, and the activity was evaluated by beta-galactosidase assay by lacZ or western blot analysis by hERalpha. The expression analysis revealed that the ALK1 and ICL1 promoter were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly high level of expression in the presence of glucose and glycerol, respectively. Particularly, the TEF1 promoter showed the highest beta-galactosidase activity and a significant signal by western blotting with the anti-estrogen receptor compared with the other promoters. Moreover, the detection system was constructed with promoters were linked to the upstream of expression vector for hERalpha gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hERalpha and CXAU1-2XERE was the most effective system for the E2-dependent induction of the beta-galactosidase activity. This system showed the highest beta-galactosidase activity at 10-6 M E2 and the activity could be detected at even the concentration of 10-10 M E2. As the result, we constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity and reproducibility of the system for characterizing environmental estrogens.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Engineering promoter regulation

Systems for easily controlled, conditional induction or repression of gene expression are indispensable tools in fundamental research and industrial-scale biotechnological applications. Both native and rationally designed inducible promoters have been widely used for this purpose. However, inherent regulation modalities or toxic, expensive or inconvenient inducers can impose limitations on their use. Tailored promoters with user-specified regulatory properties would permit sophisticated manipulations of gene expression. Here, we report a generally applicable strategy for the directed evolution of promoter regulation. Specifically, we applied random mutagenesis and a multi-stage flow cytometry screen to isolate mutants of the oxygen-responsive Saccharomyces cerevisiae DAN1 promoter. Two mutants were isolated which were induced under less-stringent anaerobiosis than the wild-type promoter enabling induction of gene expression in yeast fermentations simply by oxygen depletion during cell growth. Moreover, the engineered promoters showed a markedly higher maximal expression than the unmutated DAN1 promoter, under both fastidious anaerobiosis and microaerobisois.     

Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.     

细菌启动子识别及应用研究进展

细菌启动子是细菌中基因表达的必需调控元件,决定了细菌基因表达的强度和时机。通过启动子的插入或缺失,可以改变细菌基因的表达,实现对菌体生长发育以及代谢调控的研究。启动子也是构建各种表达系统、实现异源基因表达的基础。启动子的识别和应用研究,对于实现异源基因的可控表达、有效获得目的产物、促进生物催化和代谢工程研究具有重要的意义。以下对细菌启动子进行了简单的介绍,总结了细菌启动子的识别方法,并对细菌启动子的研究进展和具体应用进行了概述。     

肌肉特异性基因启动子的上游转录调控元件研究进展

真核生物启动子位于基因5’端上游转录起始位点附近,是包含核心启动子以及上游转录调控元件的一段DNA序列,这些转录调控元件控制着基因表达的强度和特异性。肌肉特异性启动子的上游调控元件种类、数量和排列顺序决定着基因在肌肉中的特异性表达。深入研究肌肉启动子的上游调控元件,可以进一步了解肌肉基因表达机制,从而为肌肉性状的改良、增殖分化的机理和疾病的基因治疗等研究提供重要依据。该文回顾了近年来肌肉特异性启动子研究领域中的新发现,包括肌肉特异性启动子转录调控元件的分子机制、建立人工合成肌肉启动子的方法及应用,并探讨该领域中急需解决的问题和发展前景。     

不同启动子控制下Ac转座酶基因的表达对玉米Ds因子在水稻中切离频率的影响

用水稻愈伤组织比较了Ac启动子、35S启动子与Ubi启动子控制下Ac转座酶基因（Ts）的表达对Ds因子切离频率的影响。结果表明Ubi启动子与Ac转座酶编码区嵌合基因（Ubipro-Ts）反式激活Ds因子的切离频率最高,达到了72.9%。通过杂交将Ubipro-Ts基因导入Ds因子转化植株,得到9株Ubipro-Ts基因与Ds因子共存的F1代杂交水稻植株,其中有8株Ds因子发生了切离。用Inverse-PCR的方法从其中一株杂交植株中克隆到Ds因子的旁邻序列,其DNA顺序与亲本中Ds因子原插入位点的序列不同,表明Ds因子转座到了新的基因组位点。     

Influence of plasmid origin and promoter strength in fermentations of recombinant yeast

The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of beta-galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2mu and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average beta-galactosidase specific activity was an order of magnitude below that with the 2mu-based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10-CYC1 promoters. The rate of increase in beta-galactosidase specific activity after induction in batch culture was 3-5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher beta-galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10-CYC1 promoter.