大肠杆菌海藻糖合成酶基因的克隆和表达

利用Ｍｕ转座子细胞内克隆了大肠杆菌海藻糖合成酶 ｏｔｓＢＡ基因,克隆频率为１．４５ ｘ １０(－３)／ Ｋａｎ(ｒ)转导子。经遗传互补、酶切和部分序列分析表明ｏｔｓＢＡ基因位于克隆质粒。亚克隆 ２．８７ｋｂ ＤＮＡ片段至不同拷贝数表达质粒并分别转化大肠杆菌ｏｔｓＢＡ基因缺失株,转化株恢复 在０．５ｍｏｌ／Ｌ ＮａＣｌ培养基上生长的功能,高渗透压诱导实验表明,转化株能够合成克隆基因 产物海藻糖,但合成量不受克隆质粒拷贝数影响。海藻糖良好的抗高渗能力可能在农作物育 种方面发挥重要作用。为构建含有海藻糖合成酶基因的植物表达载体,并在农杆菌的介导下 转入植物,赋予其抗高渗、耐干旱能力奠定了重要的研究基础。     

大肠杆菌\%otsA\%基因的克隆和表达

用PCR方法扩增了15kb的otsA基因片段,将该片段连接到多拷贝克隆载体后转化otsBA缺失和otsA缺陷的大肠杆菌菌株,使转化株重新获得otsA基因功能。生长曲线表明转化株在高渗培养基中生长良好,薄层层析法(TLC)检测海藻糖实验说明转化株细胞经诱导后合成海藻糖,otsA基因的克隆和表达为赋予转基因植物抗高渗、耐干旱能力提供了实验依据和材料。     

大肠杆菌海藻糖的代谢调控

海藻糖是一种重要的抗逆物质。大肠杆菌中ｏｔｓＢＡ操纵子编码的两种酶负责海藻糖合成。ｏｔｓＢＡ基因的表达受渗透压诱导和σ＾ｓ因子的调节。细胞的周质海藻糖酶（ｔｒｅＡ）将外源海藻藻分解成两个葡萄糖分子。尽管大肠杆菌中渗透压诱导合成的海藻糖并不能保护细胞抗干燥,我们将ｏｔｓＡ单个基因通过农杆菌转入烟草时,转基因株提高了耐盐和抗干燥特性,同时在转基因烟草提取物中检测到海藻糖,证明ｏｔｓＡ基因在烟草中表达并合成海藻糖。我们认为若将ｏｔｓＡ基因转入其它植物,可望改善这些植物的抗干旱、耐盐碱特性和延长采摘后的保鲜期。     

酿酒酵母海藻糖合成酶基因的克隆和在大肠村菌中的表达

用PCR方法克隆了1．5kb的酿酒母Sacchromyces cerevisiae海藻糖合成酶基因TPSI,将该片段连接到pUC19载体,通过转化分别引入海藻糖合成酶基因缺失和缺陷的大肠杆菌Escherichia coli FF4169 和FF4050,对转化株的质粒DNA酶切分析表明均含有1．5kb PCR克隆片段,生长曲线实验证明,带有克隆片段的转化株在含0．5mol/L NaCl的高渗透压基础培养基中生长良好;用高效液相色谱（HPLC）结合蒸发散射（ELSD）技术测定细胞内海藻糖实验证明转化株能够合成海藻糖。     

海藻糖合酶产生菌筛选、鉴定及目的基因克隆

目的:为筛选出一株产海藻糖合酶的菌株,并以此菌的全DNA为模板,克隆出产海藻糖合酶的目的基因片段。方法:实验过程中采用了常规筛选菌种、快速提取细菌全基因、显微镜观察菌种、热启动PCR技术、电泳纯化回收基因片段、EcoRⅠ和HindⅢ双酶切鉴定目的基因片段等方法。结果:在电镜下可观察到有芽孢、杆菌;菌株16S rRNA基因扩增产物共计1490个碱基;PCR方法扩增出阳性克隆大约1700bp的基因片段。结论:通过生理、形态、结构特征分析及16S rRNA基因全序列比较得出结论:筛选到一株短小芽孢杆菌;PCR扩增出阳性克隆片段,全长1722bp,为实验所要的编码海藻糖合酶的基因片段。     

小麦—簇毛表6VS／6AL易位系可转化人工染色体（TAC）文库的构建

可转化人工染色体（Ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｃｏｍｐｅｔｅｎｔ Ａｒｔｉｆｉｃｉａｌ Ｃｈｒｏｍｏｓｏｍｅ,ＴＡＣ）是具有克隆和转移大片段基因能力的新型载体,是植被基因克隆和转化的有效工具。为了克隆泪科抗白粉病基因和其它基因,本研究用ＴＣＡ载体ｐＹＬＴＡＣ１７构建了带有抗白粉病基因Ｐｍ２１的小麦＝簇毛麦６ＶＳ／６ＡＬ易位系的基因组ＤＮＡ文库。该文库包含２１０万个克隆平均插入征段３５ｌｂ,相当于     

稻瘟净（Blasticidin S）脱氨酶基因的克隆

从一株抗稻瘟净（ＢＳ）的Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ菌中克隆到一个ｂｌａｓｔｉｅｉｄｉｎＳ脱氨酶基因,命名为ｂｓｒＡＳ。ＤＮＡ序列分析表明ｂｓｒＡＳ不含内含子。编码区长３９０ｂｐ,编码１３０个氨基酸。将ｂｓｒＡＳ转化到稻瘟菌中,能使受体菌表达出ＢＳ脱氨酶的活性,从而产生抗药性。该基因可作为抗药标记基因使用,建立稻瘟菌的基因转化系统。     

α-乙酰乳酸脱羧酶的基因克隆及在大肠杆菌和酿酒酵母中的表达

以ｐＵＣ１９质粒为载体,以Ｅ．ｃｏｌｉ ＪＭ１０９为受体,构建了含α－乙酰乳酸脱羧酶（α－ＡＬＤＣ）基因的地衣芽孢杆菌Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ ＡＳ１０１０６的基因文库,得到４８００个重组转化子中均含有４～１０ｋｂ的外源插入ＤＮＡ片段,从基因文库中筛选到６个阳性克隆,对其中１个克隆的α－乙酰乳酸脱羧酶基因片段进行亚克隆分析表明,该α－乙酰乳酸脱羧酶基因位于１．６ｋｂ的ＢａｍＨⅠ     

海藻糖合酶基因的克隆及其植物表达载体的构建

以担子菌灰树花的菌丝体中提取总ＲＮＡ,并纯化出ｍＲＮＡ,ｍＲＮＡ经反转录合成ｃＤＮＡ第一链,以ｃＤＮＡ第一链为模板经ＰＣＲ扩增海藻糖合酶（Ｔｓａｓｅ）基因,获得一长约２．２ｋｂ的片段,把该片段连接一ｐＧＥＭ－Ｔ－ｅａｓｙ ｖｅｃｔｏｒ上进行测序,其全长共２１９９ｂｐ。随后将此片段以正向插入植物表达载体ｐＢＩ１２１的ＨｉｎｄⅢ＋Ｘｂａｌ位点构建ｐＵＢ,再把海藻糖合酶基因以正向插入载体ｐＵＢ的ＢａｍＨ     

汉滩病毒A9株强毒和弱毒克隆M基因片段G1糖蛋白编码区核苷酸序列…

采用逆转录－ＰＣＲ方法,用３对引物分３个片段扩增强毒克隆（Ａ９ｖ）及弱毒克隆（Ａ３９ｓ）的Ｍ基因片段ｃＤＮＡ并克隆入ｐＧＥＭ－Ｔ载体中,采用Ｓａｎｇｅｒ双脱氧法测定了Ａ３９ｓ,Ａ９ｖ病毒Ｇ１糖蛋白编码区的全序列,发现二者均由１９７８个核苷酸组成,比７６／１１８株少６个核苷酸,并发现Ａ３９ｓ在Ｇ１编码区有３３个碱基及８个氨基酸与Ａ９ｖ不同,进一步与７６／１１８,ＨＶ１１４的相关序列比较,发现Ｇ１编码     

Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.     

Directed evolution of operon of trehalose-6-phosphate synthase/phosphatase from Escherichia coli

Trehalose is a nonspecific protective agent for biomacromolecules. Trehalose-6-phosphate synthase (OtsA)/phosphatase (OtsB), which is encoded by the gene operon otsBA located at -42 of the Escherichia coli genome, is the main enzyme system that catalyzes the synthesis of trehalose in E. coli. We cloned the operon and modified it by directed evolution. Unlike in the previously reported work, we modified the whole operon and screened the positive mutant simultaneously. Thus we believe that the gene complex solves the negative effects between two enzymes if one of them diversifies its structure or functions and finds the form most suitable for trehalose synthesis. It thus mimics the natural process, in which the functional improvement of organisms is related to alterations in coordinated enzymes. The evolution procedure was carried out in a sequence of error-prone PCR, shuffling PCR, and then strict screening of the mutants. After screening of a library of more than 4000 colonies, about 15 positive colonies were analyzed, resulting in a higher concentration of trehalose than control. One of them, E. coli TS7, shows 12.3-fold higher trehalose synthesis ability than E. coli DH5alpha. In contrast, we introduced the cDNA sequence of the tps1 gene from Saccharomyces cerevisiae, which has 54% identity with the gene otsA, as one of the templates in shuffling PCR. By hybrid evolution and screening, we obtained 10 positive colonies with higher concentrations of trehalose than control. E. coli TS22 appears to have 5.3-fold higher trehalose synthesis ability than E. coli DH5alpha and 1.6-fold more than E. coli DEF3(pOTS11). This result demonstrated that coevolution and hybrid evolution, as powerful protocols in protein engineering, are effective in modifying enzyme. It indicates that repeating the process of genomic evolution in nature is feasible.     

Identification at the species and symbiovar levels of strains nodulating Phaseolus vulgaris in saline soils of the Marrakech region (Morocco) and analysis of the otsA gene putatively involved in osmotolerance

Salinity is an increasing problem in Africa affecting rhizobia-legume symbioses. In Morocco, Phaseolus vulgaris is cultivated in saline soils and its symbiosis with rhizobia depends on the presence of osmotolerant strains in these soils. In this study, 32 osmotolerant rhizobial strains nodulating P. vulgaris were identified at the species and symbiovar levels by analysing core and symbiotic genes, respectively. The most abundant strains were closely related to Rhizobium etli and R. phaseoli and belonged to symbiovar phaseoli. A second group of strains was identified as R. gallicum sv gallicum. The remaining strains, identified as R. tropici, belonged to the CIAT 899(T) nodC group, which has not yet been described as a symbiovar. In representative strains, the otsA gene involved in the accumulation of trehalose and putatively in osmotolerance was analysed. The results showed that the phylogeny of this gene was not completely congruent with those of other core genes, since the genus Ensifer was more closely related to some Rhizobium species than others. Although the role of the otsA gene in osmotolerance is not well established, it can be a useful protein-coding gene for phylogenetic studies in the genus Rhizobium, since the phylogenies of otsA and other core genes are coincident at the species level.     

Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose

Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.     

Distribution of genes for synthesis of trehalose and Mannosylglycerate in Thermus spp. and direct correlation of these genes with halotolerance

In this study we correlate the presence of genes leading to the synthesis of trehalose and mannosylglycerate (MG) in 17 strains of the genus Thermus with the ability of the strains to grow and accumulate these compatible solutes in a defined medium containing NaCl. The two sets of genes, namely, otsA/otsB for the synthesis of trehalose and mpgS/mpgP for the synthesis of MG, were necessary for the growth of Thermus thermophilus in a defined medium containing up to 6% NaCl. Strains lacking a complete otsA gene did not grow in defined medium containing >2% NaCl. One strain of T. thermophilus lacking the genes for the synthesis of MG did not grow in a medium with >1% NaCl. We did not identify any of these genes in the type strains of the other seven species of Thermus, and none of those strains grew in defined medium with 1% NaCl. The results strongly indicate that the combined accumulation of trehalose and MG is required for optimal osmotic adjustment.     

Role of trehalose in growth at high temperature of Salmonella enterica serovar Typhimurium

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of Salmonella enterica serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45 degrees C. One of these mutations proved to be in the seqA gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with pgm (which encodes phosphoglucomutase), it is likely to be polar on the expression of the pgm gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the seqA::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an otsA mutation, which inactivates the first step of the trehalose biosynthetic pathway. The otsA mutation caused a growth defect at 45 degrees C in minimal medium containing 0.2 M NaCl that was similar to that caused by the pgm mutation, but otsA did not affect growth rate in this medium at 37 degrees C. These results suggest that the growth defect of the seqA-pgm mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45 degrees C had a fivefold higher trehalose pool size than cells grown at 30 degrees C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.