拟南芥海藻糖酶基因克隆及其在大肠杆菌中高效表达与功能研究

从拟南芥幼苗中提取RNA,通过RT-PCR克隆得到海藻糖酶基因后,将其构建到原核高效表达载体pET30a( )上并在大肠杆菌BL21菌株中进行高效诱导表达,继而对纯化得到的海藻糖酶蛋白进行活性检测和酶学特性研究.实验结果表明,植物源的海藻糖酶基因在异体大肠杆菌中能够高效表达,纯化获得的海藻糖酶蛋白在试管条件下具有较高的海藻糖水解活性,其活性最适温度为45℃.通过GC-MS分离检测,可以明显地看到酶反应过程中底物海藻糖和产物葡萄糖的含量随反应时间变化的消长关系,这充分证明克隆基因在大肠杆菌中的表达产物具有海藻糖酶的功能.     

拟南芥海藻糖酶基因克隆及其在大肠杆菌中高效表达与功能研究

从拟南芥幼苗中提取RNA, 通过RT-PCR克隆得到海藻糖酶基因后, 将其构建到原核高效表达载体pET30a(+)上并在大肠杆菌BL21菌株中进行高效诱导表达, 继而对纯化得到的海藻糖酶蛋白进行活性检测和酶学特性研究。实验结果表明, 植物源的海藻糖酶基因在异体大肠杆菌中能够高效表达, 纯化获得的海藻糖酶蛋白在试管条件下具有较高的海藻糖水解活性, 其活性最适温度为45℃。通过GC-MS分离检测, 可以明显地看到酶反应过程中底物海藻糖和产物葡萄糖的含量随反应时间变化的消长关系, 这充分证明克隆基因在大肠杆菌中的表达产物具有海藻糖酶的功能。     

CMO与BADH双基因表达载体构建及在烟草中的表达

本研究的目的是将甜菜碱合成关键酶CMO与BADH基因构建到同一表达载体中,利用转基因方法将该表达载体导入植物体内,完善植物体内的甜菜碱合成途径,提高植物的抗旱性和耐盐性。以pC1303质粒为基础,构建了均由35S启动子驱动的CMO基因和BADH基因的植物双基因表达载体pC35SC35SB1303。利用冻融法将其导入农杆菌LBA4404中,通过农杆菌介导法分别将CMO基因、BADH基因以及该双基因表达载体导入烟草中,PCR检测和Northern杂交分析表明,外源基因已整合到受体植物基因组中并正常表达。对转基因植株及对照植株甜菜碱含量的检测结果表明,转双基因植株的甜菜碱含量明显高于转BADH基因植株、转CMO基因植株及对照植株。     

白背飞虱海藻糖合成酶基因的表达特性及其在糖代谢调控中的作用

【目的】昆虫中海藻糖主要通过海藻糖合成酶(trehalose-6-phosphate synthase, TPS)在脂肪体中合成,当昆虫受极端环境胁迫时TPS能够诱导海藻糖累积从而起到保护作用。本研究旨在分析白背飞虱Sogatella furcifera两个TPS基因的发育和组织表达模式及其对糖类物质代谢调控功能,探究TPS基因在白背飞虱生长发育中的具体作用。【方法】基于实验室前期获得的两个海藻糖合成酶基因SfTPS1和SfTPS2片段序列,在本实验中也进行了基因克隆和测序筛选,比对两者确定了白背飞虱两个TPS基因序列。并通过MEGA 7.0软件构建基于氨基酸序列的白背飞虱与其他昆虫TPS的系统发育树。利用qRT-PCR技术检测这两个基因在白背飞虱不同发育阶段(4龄第1天若虫至3日龄成虫)和成虫不同组织(头、足、翅、中肠、脂肪体、表皮和马氏管)中的表达情况。合成这两个基因的dsRNA,并注射到白背飞虱5龄第1天若虫中进行RNAi。在RNAi 48和72 h后检测白背飞虱海藻糖酶基因TRE1-1,TRE1-2和TRE2的表达变化,海藻糖、葡萄糖和总糖原含量以及海藻糖酶活性。【结果】克隆获得白背飞虱SfTPS1和SfTPS2,ORF分别为2 424和2 115 bp,编码氨基酸数目分别为807个和704个,预测蛋白质分子量分别为90.37和80.56 kD,等电点分别为6.08和6.10。而且白背飞虱2个TPS氨基酸序列与褐飞虱Nilaparvata lugens TPS1和TPS2的一致性最高。发育阶段表达模式表明,白背飞虱TPS基因SfTPS1和SfTPS2在4龄若虫到成虫阶段都有表达;组织表达模式表明,SfTPS1和SfTPS2在成虫马氏管、中肠和表皮中的表达较为显著。当SfTPS1被RNAi后,TRE1-1和TRE2的表达水平与对照组(dsGFP注射组)相比分别为略有上升和显著升高,TRE1-2的相对表达水平在SfTPS1被RNAi 48 h后显著上升而在72 h后显著下降;可溶性海藻糖酶活性无显著变化,膜结合型海藻糖酶活性显著增加;白背飞虱5龄若虫体内海藻糖、葡萄糖和总糖原含量显著上升。TRE1-2和TRE2基因的表达水平在SfTPS2被RNAi 48 h后显著升高,而在72 h后两基因的表达水平却显著下降;TRE1-1基因的表达水平在注射dsSfTPS2 48和72 h后均显著上升。可溶性海藻糖酶活性在SfTPS2被RNAi 48 h后显著下降,72 h后显著上升;膜结合型海藻糖酶活性在SfTPS2被RNAi 72 h后显著增加。白背飞虱5龄若虫体内葡萄糖含量在SfTPS2基因RNAi 48 h后显著减少,但在72 h后海藻糖、葡萄糖和总糖原含量显著上升。【结论】通过调节白背飞虱体内TPS基因的表达影响TRE1-1,TRE1-2及TRE2基因的表达水平,进而调控体内海藻糖的含量,该结果为后期采用TPS为靶标基因用于害虫防治提供理论依据。     

柑橘大实蝇滞育和非滞育蛹体内海藻糖含量的调控

【目的】通过比较柑橘大实蝇Bactrocera minax蛹滞育期与滞育前和滞育后以及滞育蛹与非滞育蛹体内海藻糖和葡萄糖含量的变化、海藻糖合成代谢途径中关键酶的活力变化以及关键酶基因的表达量变化,明确蛹滞育期间海藻糖合成代谢途径中关键酶对海藻糖含量的调控。【方法】利用分光光度法检测柑橘大实蝇滞育前(1日龄蛹)、滞育期(30,60和90日龄蛹)以及滞育后(120和150日龄蛹)蛹体内海藻糖与葡萄糖含量的变化,以及海藻糖合成代谢途径中的海藻糖-6-磷酸合成酶(TPS)、海藻糖-6-磷酸磷酸酯酶(TPP)和海藻糖酶(Tre)活力的变化;利用实时定量荧光PCR(qPCR)检测TPS,TPPB,TPPC-1,TPPC-2和Tre-1基因表达量的变化。向1日龄蛹体内注射20-羟基蜕皮酮(20E)作为处理(以注射10%乙醇为对照),分别于注射后1和30 d比较处理组与对照组蛹体内海藻糖与葡萄糖含量、关键酶活力以及上述基因表达量的差异。【结果】柑橘大实蝇蛹进入滞育后,海藻糖含量显著升高,葡萄糖含量无显著变化; TPS和TPP的酶活力以及TPS,TPPC-1和TPPC-2表达量在化蛹后逐渐升高,于滞育期达到最高水平,维持至羽化前显著下降;TPPB表达量在整个蛹期无显著差异; Tre酶活力以及Tre-1表达量在化蛹后逐渐升高,于滞育早期达到最高水平,随后显著下降,羽化前再次显著上升。注射20E后1 d,与对照组相比,处理组蛹体内海藻糖与葡萄糖含量、关键酶(TPS,TPP和Tre)活力以及TPS,TPPC-2和Tre-1表达量无显著变化,TPPB表达量显著下降,TPPC-1表达量显著上升;注射后30 d,与对照组滞育蛹相比,处理组非滞育蛹海藻糖含量显著上升,葡萄糖含量、TPS和Tre酶活力、TPS和Tre-1表达量显著下降,TPP酶活力以及TPPB和TPPC-2表达量无显著差异。【结论】柑橘大实蝇蛹体内海藻糖的含量在合成代谢途径中关键酶的调控下,随着滞育状态发生变化,表明海藻糖与滞育之间存在密切的关系,但其作用机理仍待进一步研究。     

红色亚栖热菌TPS/TPP海藻糖合成途径中相关基因的克隆、表达及功能鉴定

通过构建红色亚栖热菌(Meiothermus ruberCBS-01)的基因组DNA文库,克隆得到该嗜热菌海藻糖合成途径中的磷酸海藻糖合成酶(TPS)和磷酸海藻糖磷酸酯酶(TPP)基因。以pET21a为表达载体,将磷酸海藻糖合成酶和磷酸海藻糖磷酸酯酶在大肠杆菌中进行表达并纯化,利用薄层层析的方法验证了这两个酶的活性。同时,本研究检测了红色亚栖热菌在各种环境压力下细胞内含物成分的变化情况,发现在高渗环境压力的诱导下,该菌会在胞内积累大量的6-磷酸海藻糖,而并非海藻糖,这为进一步研究TPS/TPP和TreS途径在细胞体内的作用奠定了基础。     

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。     

甜瓜海藻糖-6-磷酸合成酶基因鉴定及表达

为了明确甜瓜海藻糖-6-磷酸合成酶基因(CmTPS)家族信息及对逆境信号的响应,该研究采用生物信息学方法,通过拟南芥TPS家族基因与甜瓜基因组数据库比对,从甜瓜基因组中共鉴定出7个海藻糖-6-磷酸合成酶基因,按照其在染色体上的位置分别命名为CmTPS1~7。系统进化分析结果显示,CmTPS4和CmTPS7为第1类,二者均含有16个内含子,推测其编码产物均具有海藻糖-6-磷酸合成酶(TPS)活性;其余5个CmTPS基因归为第2类,分别含有2~4个内含子;在这7个甜瓜TPS中,除CmTPS3只有TPS结构域外,其余CmTPS都含有TPS、TPP及UDP-forming结构域;蛋白序列比对结果显示,甜瓜TPS家族各成员间相似性较低(15.90%~57.31%);亚细胞定位预测表明,CmTPS1、CmTPS2和CmTPS6定位在细胞核内,其余4个CmTPS定位在细胞质内。qRTPCR表达分析表明,低温胁迫下甜瓜叶片可能以CmTPS4为主要的TPS编码基因;CmTPS基因家族对盐胁迫较为敏感,同时在ABA信号传递中起调控作用。这为进一步研究甜瓜TPS基因家族奠定了基础。     

人干扰素α-2b基因植物表达载体的构建及转化

目的:通过构建人干扰素α-2b(hIFNα-2b)基因的植物表达载体,为后期将其导入胡萝卜愈伤组织中作准备。方法:采用PCR技术从人基因组DNA扩增hIFNα-2b编码基因及全长基因,将其克隆于pMD19-T载体中,双酶切hIFNα-2b基因及植物表达载体pBI121,回收目的片段,T4DNA连接酶连接得到植物表达载体pBI121-hIFN。采用三亲交配法将后者导入根瘤农杆菌。结果:经PCR检测,双酶切及DNA序列测定表明hIFNα-2b编码基因及全长基因已分别插入植物表达载体pBI121中,重组表达载体pBI121-IFN已成功转化根瘤农杆菌LBA 4404。结论:成功构建了植物表达载体pBI121-hIFN并转入根瘤农杆菌。     

干旱诱导性启动子驱动的海藻糖－6－磷酸合酶基因载体的构建及转基因烟草的耐旱性

The trehalose-6-phosphate synthase gene (TPS) from Saccharomyces cerevisiae Hansen and the drought-responsive promoter from Arabidopsis thaliana (L.) Heynh. have been cloned by PCR procedure. A plant expression vector with TPS under control of Prd29A has been constructed and used for the genetic transformation of tobacco. The transgenic tobacco with Prd29A/TSP demonstrated TPS expression with increased drought tolerance under drought stress. Some obvious morphological changes including dwarf and fine shoot, lancet-shaped leaves and vigorous auxiliary buds have been observed in a few transformed plants.     

Genetic Transformation of Tobacco with the Trehalose Synthase Gene from Grifola frondosa Fr. Enhances the Resistance to Drought and Salt in Tobacco

Trehalose is a non-reducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In the present study, we report on the expression of the Grifolafrondosa Fr. trehalose synthase (TSase) gene for manipulating abiotic stress tolerance in tobacco (Nicotiana tabaccum L.). The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and was transferred into tobacco by Agrobacterium tumefaciens EHA105. Compared with non-transgenic plants, transgenic plants were able to accumulate high levels of products of trehalose, which were increased up to 2.126-2.556 mg/g FW, although levels were undetectable in non-transgenic plants. This level of trehalose in transgenic plants was 400-fold higher than that of transgenic tobacco plants cotransformed with Escherichia coli TPS and TPP on independent expression cassettes, twofold higher than that of transgenic rice plants transformed with a bifunctional fusion gene (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of E. coli, and 12-fold higher than that of transgenic tobacco plants transformed the yeast TPS1 gene.It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and had morphological alterations of their roots. Interestingly, our transgenic plants have obvious morphological changes, including thick and deep-coloured leaves, but show no growth inhibition; moreover, these morphological changes can restore to normal type in T2 progenies. Trehalose accumulation in 35S-35S:TSase plants resulted in increased tolerance to drought and salt, as shown by the results of tests on drought, salt tolerance, and drought physiological indices, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, and the activity of superoxide dismutase and peroxidase in excised leaves. These results suggest that transgenic plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought and salt.     

TREHALOSE PHOSPHATE SYNTHASE11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae

Agricultural productivity is limited by the removal of sap, alterations in source-sink patterns, and viral diseases vectored by aphids, which are phloem-feeding pests. Here we show that TREHALOSE PHOSPHATE SYNTHASE11 (TPS11) gene-dependent trehalose metabolism regulates Arabidopsis thaliana defense against Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA). GPA infestation of Arabidopsis resulted in a transient increase in trehalose and expression of the TPS11 gene, which encodes a trehalose-6-phosphate synthase/phosphatase. Knockout of TPS11 function abolished trehalose increases in GPA-infested leaves of the tps11 mutant plant and attenuated defense against GPA. Trehalose application restored resistance in the tps11 mutant, confirming that the lack of trehalose accumulation is associated with the inability of the tps11 mutant to control GPA infestation. Resistance against GPA was also higher in the trehalose hyper-accumulating tre1 mutant and in bacterial otsB gene-expressing plants, further supporting the conclusion that trehalose plays a role in Arabidopsis defense against GPA. Evidence presented here indicates that TPS11-dependent trehalose regulates expression of the PHYTOALEXIN DEFICIENT4 gene, which is a key modulator of defenses against GPA. TPS11 also promotes the re-allocation of carbon into starch at the expense of sucrose, the primary plant-derived carbon and energy source for the insect. Our results provide a framework for the signaling function of TPS11-dependent trehalose in plant stress responses, and also reveal an important contribution of starch in controlling the severity of aphid infestation.     

拟南芥冷诱导型启动子CBF 3的克隆及活性检测

目的：构建冷诱导型启动子CBF3基因的植物表达载体,并将其转入烟草。方法：以拟南芥基因组DNA为模板,通过特异PCR扩增,克隆冷诱导表达启动子CBF3（C-repeat binding factor）。用CBF3启动子替换pBI121载体上的35S启动子构建新的载体pBC-GUS,通过农杆菌介导的叶盘法转化烟草。结果：获得了转基因烟草,转基因烟草的GUS组织化学染色及PCR分析结果表明,在低温诱导下,CBF3启动子可增强GUS基因表达。结论：CBF3启动子可应用于植物抗冷基因工程研究。     

地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。     

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.     

Improved drought tolerance without undesired side effects in transgenic plants producing trehalose

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.     

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana

Summary. Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana. Correspondence and reprints: Laboratório de Biotecnologia de Células Vegetais, ITQB, Apartado 127, Avenida da República (E.A.N.), 2781-901 Oeiras, Portugal.