枸杞蔗糖磷酸合成酶基因的克隆及组织表达分析

利用RT-PCR及RACE技术,从药用植物枸杞中克隆了1个编码蔗糖磷酸合成酶(SPS)基因的全长cDNA,命名为LbSPS(GenBank登录号KC834608)。序列分析表明:LbSPS基因长3 677bp,开放阅读框为3 165bp,编码1 033个氨基酸,分子量为118.457 5kD,理论等电点6.05。系统进化分析显示,LbSPS编码的氨基酸序列与甜瓜、马铃薯、番茄等蔗糖磷酸合成酶基因编码氨基酸序列一致性为66%~98%。qRT-PCR分析显示,LbSPS基因在枸杞花中表达量最高,叶中表达水平较低。该研究为进一步了解LbSPS在枸杞生长发育、逆境胁迫等过程中的生物学功能奠定了基础。     

铁皮石斛蔗糖磷酸合成酶基因的克隆与原核表达

应用同源序列克隆法克隆了铁皮石斛蔗糖磷酸合成酶(SPS)基因cDNA全长,并进行了原核表达分析,为进一步研究该基因的时空表达、功能分析及多糖合成机理提供理论依据。结果表明:(1)铁皮石斛SPS基因cDNA全长3 502bp,编码区3 186bp,GenBank登录号JF423929。该基因编码1 061个氨基酸,与文心兰的SPS基因氨基酸序列的一致性最高为93%,与其他科植物SPS基因的氨基酸序列的一致性均高于60%。(2)原核诱导表达结果显示,SPS基因在大肠杆菌中的重组蛋白分子质量约为118.7kD,其表达与序列分析推测的结论一致。(3)生物信息学分析表明,铁皮石斛SPS基因的二级结构包括了螺旋、β-折叠和无规则卷曲,是非跨膜结构的亲水性不稳定蛋白,有2个功能结构域,分别是蔗糖合成功能域及糖基转移功能域。     

酿酒酵母海藻糖—６—磷酸合成酶基因克隆及植物表达载体的构建

从耐热性极强的酿酒酵母菌株ＡＳ２．１４１６中分离纯化出总ＲＮＡ和mRNA,以ＡＭＶ逆录酶合成cDNA,采用保守引物,从该cDNA中扩增克隆出tps1基因,对该基因的全序列分析表明,该基因含有１５０７个核苷酸,与国外报道相关基因的同源性达９９．６５。利用ＢamＨⅠSacⅠ切点将tps1基因插入植物表达载体pBin438多克隆位点上,得到tps1基因植物表达载体重组质粒。     

蔗糖磷酸合成酶(SPS)与果实品质及成熟衰老的研究进展

从蔗糖磷酸合成酶（sucrose phosphate synthase,SPS）基本性质、SPS与果实糖的积累、相关酶类和外源刺激、乙烯和呼吸、果实软化的火系及SPS基因的克隆与表达调控等几方面综述了国内外近年来对SPS的研究进展。     

酿酒酵母海藻糖-6-磷酸合成酶基因克隆及植物表达载体的构建

从耐热性极强的酿酒酵母菌株AS21416中分离纯化出总RNA和mRNA,以AMV逆转录酶合成cDNA,采用保守引物,从该cDNA中扩增克隆出tps1基因,对该基因的全序列分析表明,该基因含有1507个核苷酸,与国外报道相关基因的同源性达99.6%。利用BamHⅠ和SacⅠ切点将tps1基因插入植物表达载体pBin438多克隆位点上,得到tps1基因植物表达载体重组质粒。     

植物蔗糖磷酸合成酶研究进展

蔗糖磷酸合成酶(Sucrose Phosphate Synthase,以下简称SPS)是植物体内控制蔗糖合成的关键酶。植物体内蔗糖的积累与SPS活性正相关,SPS还参与植物的生长和产量形成,并在植物的抗逆过程中起重要作用。高等植物中至少存在A、B、C三个家族的SPS,而禾本科植物至少存在A、B、C、DIII和DIV五个家族的SPS。不同植物体内不同家族的SPS基因的表达特性不同,它们所发挥的功能也存在差异。SPS的活性在基因表达调控和SPS蛋白磷酸化共价修饰作用两个层面受到植物生长发育、光照、代谢产物、外源物质如激素和糖类等多种因素的复杂调控。转基因研究表明,转SPS基因是提高作物产量和品质、增强作物抗逆性的有效途径,值得深入研究。全面总结了国内外在植物蔗糖磷酸合成酶方面的研究进展,并提出问题与研究展望,期望为进一步研究并利用植物SPS基因改良作物品种提供参考。     

阔叶薰衣草芳樟醇合成酶基因的克隆与表达载体构建北大核心CSCD

以阔叶薰衣草(Lavandula latifolia)的鲜叶为试材,通过RT-PCR技术,获得阔叶薰衣草芳樟醇合成酶基因的cDNA序列。该序列长度为1809bp,编码602个氨基酸,包含一个1809 bp的开放阅读框,与NCBI上已公布的阔叶薰衣草的芳樟醇合成酶蛋白序列相似度为98%,有21个核苷酸差异,11个氨基酸差异,将其命名为Lslis。序列分析研究表明:Lslis具有多数单萜烯类合成酶基因典型的保守结构域,即DDXXD,RRX8W和(N,D)D(L,I,V)X(S,T)XXXE;具有芳樟醇合酶活性必需的DDXXD功能基团。Lslis推导氨基酸序列的预测分子质量和等电点分别为70.36和5.66 kD;疏水性分析结果表明其大部分氨基酸区域为亲水结构,该基因为亲水多肽。Lslis和已公布的阔叶薰衣草芳樟醇合酶Lllis的蛋白二级结构预测结果表明,两者在整体结构上基本保持一致,但Lslis相对于Lllis要少一个α螺旋结构。将其连接到pMD18-T的载体上,通过中间载体pCAMBIA1303,构建了Lslis基因的植物表达载体,并将其导入农杆菌,经菌落PCR鉴定和测序结果表明该片段已插入表达载体。     

毛白杨纤维素合成酶基因(PtoCesA1)克隆、序列分析及其植物表达载体的构建

以毛白杨形成层为材料克隆了毛白杨纤维素合成酶基因（PtoCesA1）,序列分析表明该基因序列为3215bp,与欧洲颤杨的PtCesA1基因同源性为97％具有开放的阅读框,编码区在52～2985碱基之间,编码区为2925bp。通过同义突变引入BamHI酶切位点,将全长基因克隆到植物表达栽体pBll21中,经酶切和PCR鉴定确认载体构建正确,为下一步ptoCesA1转基因功能研究打下了基础。     

番茄蔗糖代谢关键基因的分子生物学研究

蔗糖代谢相关的酶类主要有转化酶、蔗糖合成酶和蔗糖磷酸合成酶。该文着重对番茄中这些酶的编码基因表达与转基因方面的近期研究进行分析评述。     

Over-expression of an arabidopsis family A sucrose phosphate synthase (SPS) gene alters plant growth and fibre development

The objective of this study was to manipulate the intracellular pools of sucrose by differentially expressing exogenous sucrose phosphate synthase (SPS) and investigating its role in regulating plant growth and fibre development. Tobacco (Nicotiana tabacum cv. Xanthi) plants were transformed with an arabidopsis SPS gene under the regulation of the ubiquitously expressed tandem repeat of the 35S cauliflower mosaic virus promoter, and subject to growth trials and fibre characterization. It was apparent that over-expression of SPS resulted in substantially elevated concentrations of sink sucrose pools compared to wild-type plants, while source tissue sucrose pools remained the same. All transformed plants had significantly increased stem height, which was ascribed to internode elongation, and greater stem diameters, longer fibers and increased total dry biomass relative to the control plants. Difference in the chemical composition of either the storage or structural carbohydrates of the wild-type and SPS transgenic lines were only minor. The correlation between increased stem sucrose content and plant phenotypes with elevated SPS gene expression confirm a role for sucrose availability in controlling plant growth and fibre elongation.     

Gene cloning and expression in lactic streptococci

Abstract Recent developments have made the mesophilic lactic streptococci, widely used in dairy fermentations, accessible to genetic manipulation. Several host-vector systems have been described which currently are used in the cloning and expression of homologous and heterologous genes. The essential elements of these systems, the various cloning strategies and the first successful cloning experiments are described with emphasis on the molecular organization of proteinase genes. In addition, the organization and nucleotide sequence of signals which are involved in gene expression in lactic streptococci are summarized.     

大豆球蛋白G1启动子的克隆及植物表达载体构建

从大豆冀nf37和冀豆15中克隆了大豆球蛋白G1基因的启动子片段。序列分析表明,两种启动子片段均为688bp,与GenBank现有的3种启动子序列(四川大豆(DQ250808)、南农87-c38(AY649096)和Dare(X15121))间的同源性在96.4%～99.6%之间。其中来自冀nf37的启动子片段除Legumin盒上有一个碱基差异外,其它元件与DQ250808完全相同,据此推测该启动子片段具有种子特异性启动子活性。将其与已有γ-生育酚甲基转移酶基因连接,构建了种子特异性表达载体pBG1TMT,为通过代谢工程手段调控油料作物种子维生素E组成、提高其营养品质奠定了基础。     

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.     

植物肉桂酰辅酶A还原酶基因克隆研究进展

木质素单体合成的过程中涉及了许多酶的参与,而肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase,CCR)是该过程中的一个关键酶。综述了CCR基因在植物体内的克隆、基因功能及在植物组织中的表达情况,并介绍了该基因在植物的抗病虫害和抗逆性研究、饲草和能源上的应用潜力,为进一步研究CCR基因生物学功能和利用奠定了基础。     

构建定向T载体用于基因克隆和表达

传统的T载体克隆方法需要烦琐的后续步骤来筛选和鉴定重组子,并且无法实现目的基因的定向克隆。为了克服这些问题,本研究在pET-23a(+)的基础上构建了定向T载体pETG,首先通过定点诱变消除pET-23a(+)上的两个BfuⅠ位点得到PET-23aM;设计一对引物在5端各引入一个BfuⅠ位点,下游引物紧邻BfuⅠ位点引入13 bp的部分LacO序列,用该引物从pHBM2002上扩增Prrn-gfp表达盒,插入PET-23aM的NdeⅠ和XhoⅠ位点,得到定向T载体pETG。PCR扩增的目的基因通过下游引物引入7 bp剩余的LacO序列,该基因片段与BfuⅠ酶切制备的定向T载体连接、转化大肠杆菌DH10β感受态细胞,通过补加了X-gal的平板筛选蓝色重组子。质粒酶切和PCR鉴定表明蓝色菌落全部为定向插入的重组子,重组效率100%,利用本方法成功地定向克隆了103个人类肝蛋白编码基因cDNA,克隆过程无需复杂的步骤筛选鉴定重组子。随机选择了其中的8个基因的克隆进行表达,结果显示8个克隆均在大肠杆菌中获得成功表达。该结果表明定向T载体构建成功,并且该载体非常适合基因的克隆和表达。     

肺炎支原体双蛋白多抗原表位表达载体的构建

目的构建肺炎支原体(Mp)双蛋白多特异抗原表位表达载体,提高重组蛋白抗原的敏感性。方法应用生物信息学方法筛选Mp P116粘附蛋白抗原表位序列,PCR点突变技术获取P116蛋白基因片段,与pMD-T载体重组,转入大肠埃希菌JM109,通过限制性酶切图谱和基因序列分析鉴定重组质粒。酶切回收P116基因片段与pGEX 6P-1-P1 DNA重组,转入大肠埃希菌JM109菌株。用Glutathione Sepharose 4B纯化重组蛋白,SDS-PAGE分析表达产物的相对分子量,用Mp免疫血清进行免疫印迹试验,鉴定重组蛋白的免疫原性。结果 PCR点突变扩增Mp黏附蛋白P116的基因片段为597 bp,该基因片段与已知的基因库序列分析比较,除两个突变位点由UAG突变为UGG外,其余核苷酸序列同源性为100%。SDS-PAGE分析多表位重组蛋白相对分子质量(Mr)为77.8 kDa。免疫印迹结果显示,Mp兔多价血清能与纯化的78KDa的重组蛋白发生免疫反应。结论本研究成功构建了Mp双蛋白多表位的表达载体。该表达载体表达的重组蛋白具有Mp特异的免疫反应性。重组蛋白的敏感性有待进一步鉴定。     

Gene expression in nematode-infected plant roots

Summary A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.     

Electrophoretic analysis of sucrose synthetase proteins in the complementing heterozygotes at the Shrunken locus in maize

Summary Electrophoretic comparisons of sucrose synthetase (SS) proteins in complementing heterozygotes and the corresponding in vitro mixtures of extracts from the homozygotes are described. The latter revealed two protein bands in the expected fashion. The SS protein pattern in the hybrid was different from that of the mixtures. The possibility of heteromeric SS molecules, formed by random polymerization of subunits of the tetramer coded by each allele in the heterozygote, was considered. Such an interaction was expected to form a multiple of five SS proteins that could be visualized after gel electrophoresis. However, only two SS bands were seen in the hybrids. The basis of this marked deviation remains to be explained.Cooperative Investigation, United States Department of Agriculture and Institute of Food and Agricultural Sciences, University of Florida, Florida Agricultural Experiment Station Journal Series No. 2470.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable