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σs因子的调节。细胞的周质海藻糖酶(treA)将外源海藻糖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 。     

一种快速筛选海藻糖合成酶突变体体系的建立

利用λRed重组系统对大肠杆茵JM109的6-磷酸海藻糖合成酶基因OtsA和6-磷酸海藻糖酯酶基因otsB进行敲除,获得otsA和otsB基因失活的大肠杆菌突变体,该突变体由于对渗透压变得非常敏感,在舍5%NaCl和0.5%麦芽糖的培养基中生长缓慢,然而当导入有活力的外源海藻糖合成酶基因后,由于能在胞内合成海藻糖,增强了细胞抗高渗透压培养基的能力,因而可让otsA和otsB基因缺失后的JM109得以恢复其生长能力.通过比较它们在高渗透压的培养基中的生长速度,可以筛选出含有不同活力的海藻糖合成酶突变体.     

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。     

改造大肠杆菌合成海藻糖途径以高效合成海藻糖

海藻糖是相容性溶质的一种,因其具有多种生物学功能,在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法,虽然这种方法的转化效率高,但是却存在着副产物的问题,难以得到高纯度的海藻糖产品,严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径,通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因(tpsp),采用E.coli pTac-HisA高效表达载体,实现海藻糖双功能合成酶基因(tpsp)高效表达,利用高效表达菌株进行全细胞催化,将葡萄糖高效转化为海藻糖。结果表明C.hutchinsonii海藻糖合成酶基因(tpsp)在E.coli中成功实现表达,该酶能够在胞内将葡萄糖高效转化为海藻糖,并将其转运到胞外,实现海藻糖的高效率合成,海藻糖的产量提高到1.2 g/L,相对转化率为21%。当将此高产菌株在发酵罐中进行转化时,海藻糖的产量达到13.3 g/L,葡萄糖的相对转化率达到48.6%。采用C.hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道,海藻糖的转化率及产率都已达到文献报道最高水平,本研究为开拓海藻糖生产新技术奠定了基础。     

垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析

海藻糖-6-磷酸合成酶(Trehalose-6-phosphate synthse, TPS)是植物海藻糖合成途径的关键酶, 在旱生卷柏等复苏植物对逆境胁迫应答中起重要作用。文章以我国特有旱生植物垫状卷柏(Selaginella pulvinata)为材料, 采用同源扩增与RACE技术相结合的方法克隆了海藻糖-6-磷酸合成酶基因SpTPS1, cDNA全长3 223 bp, 包括一个2 790 bp的开放阅读框, 推导的氨基酸序列与模式物种的海藻糖-6-磷酸合成酶具有较高的序列相似性, 催化活性中心保守位点基本一致。酵母功能互补实验证明, 用SpTPS1基因开放阅读框转化的海藻糖合成酶基因突变(tps1△)酵母菌株, 可恢复在以葡萄糖作为唯一碳源培养基上的生长, 说明垫状卷柏海藻糖-6-磷酸合成酶基因SpTPS1的编码蛋白具有生物活性, 可应用于植物抗逆性的转基因改良。     

昆虫几丁质合成及其调控研究前沿

几丁质合成与降解是昆虫最重要的生理过程之一。本文根据国外和作者自己的研究,综述了昆虫几丁质合成及其调控研究进展。昆虫几丁质的生物合成通路始于海藻糖,终止于几丁质,其中共有8个酶参与。目前研究最多的为海藻糖酶和几丁质合成酶。昆虫存在2个海藻糖酶基因和2个几丁质合成酶基因。可溶性海藻糖酶基因对昆虫表皮的几丁质合成影响更大,而膜结合海藻糖酶基因则主要影响中肠的几丁质合成。几丁质合成酶A主要负责表皮和气管几丁质的合成,而几丁质合成酶B则负责中肠围食膜的几丁质合成。目前,昆虫几丁质合成的调控途径主要有两种:利用RNAi技术和几丁质合成抑制剂。     

海藻糖酶法合成途径及其酶基因的重组表达研究

在生物抗逆研究中,海藻糖合酶基因是继甘露醇、脯氨酸、甜菜碱合成酶基因之后又一个与抗逆相关的基因。海藻糖具有独特的生物学功能,能提高生物体对干旱、高温、冷冻和渗透压的抗性,发现以来就受到人们的普遍关注。随着对海藻糖化学性质、生理功能、作用机理及代谢途径等方面研究的深入,其在生物制品、食品、医药、作物育种及精细化工等领域广阔的应用前景日益显现。就海藻糖在生物体中的合成途径,以及海藻糖合成酶的基因工程研究进展进行了综述。     

转基因烟草中的海藻糖测定

The \%E.coli\% trehase synthalose gene(\%otsA\%) was transferred into \%Nicotiana tabacum\% mediated by \%Agrobacterium\%, but the method for detecting low concentration of trehalose in transgenic plant was not available.The high performance liquid chromatograph(HPLC) with evaporative light\|scatting detector (ELSD) using water:methyl cyanide(1∶2\^6 v/v) as mobile phase was established in this work. An ODS column Zorbax RX\|SIL was employed. The trehalose detection limits of ELSD was 5mg/L. From the linear…     

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。     

Overexpression of <Emphasis Type="Italic">Citrus junos</Emphasis> mitochondrial citrate synthase gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> confers aluminum tolerance

Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.     

Expression of insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco

To elucidate the function of antifreeze protein from Microdera puntipennis dzhungarica for freezing stress tolerance in plant, the construct of MpAFP149 gene with the signal peptide sequence responsible for secreting the native MpAFP149 into the apoplast space under control of a cauliflower mosaic virus 35S promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. The observation of immunogold localization by TEM (transmission electron microscope) showed that the heterologous MpAFP149 protein was mainly distributed on the cell wall in apoplast of the transgenic tobacco plant. T1 generation transgenic tobacco plants displayed a more frost resistant phenotype and kept the lower ion leakage ratio and MDA (malondialdehyde) content in the leaves compared with wild-type ones at -1 degrees C for 3 days. The results showed that MpAFP149 provided protection and conferred cold tolerance to transgenic tobacco plant during freezing stress.     

杨树HDA902基因在低温胁迫应答反应中的功能

组蛋白去乙酰化酶在植物非生物胁迫应答反应中具有重要的调控作用。利用RT-PCR的方法从毛果杨中克隆了组蛋白去乙酰化酶基因HDA902。利用农杆菌介导法将其遗传转化到烟草中,并对转基因植株进行低温耐受性分析。研究结果表明,HDA902在烟草中的表达显著提高了转基因株系对低温的耐受性。叶片NBT和DAB染色结果表明,在低温处理后转基因烟草比野生型烟草产生较少的活性氧。丙二醛和脯氨酸含量测定结果表明,在低温条件下,转基因烟草叶片的脯氨酸含量显著高于野生型烟草,而丙二醛含量显著低于野生型烟草。这些研究结果表明,HDA902参与低温胁迫应答反应,其过量表达提高了植株耐低温的能力。     

Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance

Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4DeltaC) from which its C-terminal, with the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full-length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T-DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.     

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice

We engineered a salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar-type Na+/H+ antiporter in the transgenic rice plants was eight-fold higher than that in wild-type rice plants. Salt tolerance assays followed by non-stress treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 300 mM NaCl for 3 days while the wild-type rice plants could not. These results indicate that overexpression of the Na+/H+ antiporter gene in rice plants significantly improves their salt tolerance.     

Over-expression of a cytosolic isoform of the HbCuZnSOD gene in Hevea brasiliensis changes its response to a water deficit

Hevea brasiliensis is the main commercial source of natural rubber. Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering was undertaken to study the strengthening of plant defences by antioxidants. To that end, Hevea transgenic plant lines over-expressing a Hevea brasiliensis cytosolic HbCuZnSOD gene were successfully established and regenerated. Over-expression of the HbCuZnSOD gene was not clearly related to an increase in SOD activity in plant leaves. The impact of HbCuZnSOD gene over-expression in somatic embryogenesis and in plant development are presented and discussed. The water deficit tolerance of two HbCuZnSOD over-expressing lines was evaluated. The physiological parameters of transgenic plantlets subjected to a water deficit suggested that plants from line TS4T8An displayed lower stomatal conductance and a higher proline content. Over-expression of the HbCuZnSOD gene and activation of all ROS-scavenging enzymes also suggested that protection against ROS was more efficient in the TS4T8An transgenic line.     

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。     

Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene PvSR2

Heavy metal pollution such as Cd, Hg, Pb, As and Se is an increasing environment problem worldwide. These metals and metalloids have toxic effect on both plants and animals, which are strongly poisonous to metal-sensitive enzymes, resulting in growth inhibition and death of the organism[1]. Contamination of soils with heavy metals, either by natural causes or due to pollution, often has pronounced effects on the vegetation, resulting in the appearance of metallophytes, and heavy-metal tolera…