Marker gene expression driven by the maize ubiquitin promoter in transgenic wheat

Selecting a promoter for driving transgene expression is one of the most important factors to consider in a transformation project. Information about the native regulation of the promoter activity is important, but it is also necessary to consider how that activity will be affected when integrated into the genome of the transformed plants. Study of a promoter performance in individually transformed lines provides useful information in this area. The maize ubiquitin 1 (Ubi‐1) promoter has been widely used to drive constitutive transgene expression in monocotyledonous plants. However, lack of data on its activity in individual transformed wheat lines constitutes a gap in the understanding and predictability of this promoter's performance. In this paper, we began addressing this problem by examining the expression of the marker gene uidA, coding for β‐glucuronidase (GUS), under the control of the maize Ubi‐1 promoter in individual transgenic wheat (Triticum aestivum L.) lines from different wheat varieties. The expression of uidA driven by this promoter depended to a great extent on the specific transformation event. Whilst expression was strong and constitutive in all tissues in some of the lines analysed, there were also transgenic lines in which GUS activity was restricted to only a few tissues. In general the maize Ubi‐1 promoter had strong activity in young, metabolically active tissues and in pollen grains.     

Transient gene expression in aleurone protoplasts isolated from developing caryopses of barley and wheat

Methods have been developed for the isolation of aleurone protoplasts from developing caryopses of Hordeum vulgare and Triticum aestivum in order to study transient expression of introduced genes. Chimaeric gene constructs were introduced into aleurone protoplasts by polyethylene glycol (PEG). Transient expression directed by the 35S promoter from cauliflower mosaic virus (CaMV) of the reporter gene encoding chloramphenicol acetyl transferase (CAT) was detected in aleurone protoplasts from developing barley and wheat grains. Using a similar construct, CAT activity increased when the alcohol dehydrogenase intron 1 fragment from maize was ligated between the 35S promoter and the CAT coding region. The demonstration of transient expression in protoplasts from developing aleurone layers indicates that they may be useful for investigating tissue and developmental control of genes coding for cereal seed proteins.     

小麦HMW-G12亚基基因启动子克隆及序列分析

为了研究高分子量谷蛋白基因启动子在种子中的特异性表达,以小麦品种“东农7742”的基因组DNA为模板,根据已发表序列设计并合成引物,用PCR的方法克隆了小麦贮藏蛋白中高分子量谷蛋白12亚基基因的上游调控序列。序列测定结果表明：所克隆的启动子片段大小为424bp与Thomspon报道的序列比较,同源性为97.9%,有9个核苷酸发生了改变。推测的TATA box位于-27— -30bp,Prolamin-box位于-175— -181bp,认为该元件可能与转录速率的调控有关。     

β-N-acetylglucosaminidase and β-galactosidase from aleurone layers of resting wheat grains

We have examined the characteristics of binding to wheat germ agglutinin-Sepharose of β-N-acetylglucosaminidase and β-galactosidase from aleurone layers of resting wheat grains. Although the enzymes interacting with wheat germ agglutinin-Sepharose could be extracted by a procedure which did not involve any solubilizing treatments, the highest activity of these enzymes was obtained by extracting and sonicating the tissues in the presence of 0.5% Triton X-100. The pH optimum and time-course of binding as well as the effect of some divalent ions on the binding were studied. The largest part of the bound enzymes was eluted at low concentration of N-acetyl-D-glucosamine (0.05 M), although smaller amounts were still eluted at higher molarities (0.1 and 0.2 M). D-Mannose, D-glucose and L-fucose failed to replace N-acetyl-D-glucosamine in eluting the enzymes bound to wheat germ agglutinin-Sepharose, whereas N-acetyl-D-galactosamine was much less effective than N-acetyl-D-glucosamine. The catalytic properties of the enzymes remained unchanged after the binding to wheat germ agglutinin-Sepharose, although the K_m values of the free and lectin-bound enzymes were slightly different. A rapid and easy three-step procedure of purification, mainly based on affinity chromatography on wheat germ agglutinin-Sepharose, is described. It allows purification of β-galactosidase and β-N-acetylglucosaminidase over 200-fold. β-N-Acetylglucosaminidase has been further purified to electrophoretic homogeneity and also characterized.     

A cathepsin B-like enzyme from mackerel white muscle is a precursor of cathepsin B

A cathepsin B-like enzyme from the white muscle of common mackerel Scomber japonicus was a cysteine protease that hydrolyzed Z-Arg-Arg-MCA, the substrate for cathepsin B. In a partial purified cathepsin B-like enzyme preparation at 4 degrees C left over time, a converted enzyme that hydrolyzes Z-Arg-Arg-MCA and Z-Phe-Arg-MCA appeared in the preparation. The converted enzyme was purified from the cathepsin B-like enzyme, characterized and was identified as mackerel cathepsin B. These results suggested that the mackerel cathepsin B-like enzyme was a precursor of cathepsin B. Mackerel cathepsin B formed in the purified cathepsin B-like enzyme preparation by adding of a small amount of the purified cathepsin B to the preparation. Therefore, mackerel cathepsin B-like enzyme was converted to the mature form of cathepsin B by autoactivation. The conversion of the cathepsin B-like enzyme (molecular mass 60 kDa) to cathepsin B (molecular mass 23 kDa) was detected by immunoblotting by using human anti-(cathepsin B) antibody. The intermediate forms of 40 kDa and 38 kDa were also detected during the conversion.     

Molecular cloning, sequence analysis and expression studies of a novel GAmyb homologous gene, hvmyb

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An Escherichia coli gene divergently transcribed from a promoter overlapping the metA promoter

The upstream region of the metA gene in Escherichia coli contains two promoters. We have identified by lacZ fusion an additional promoter in this region, and showed that it is transcribed in the opposite orientation from the metA gene. The putative translation product corresponds to a peptide of 147 amino acids – ORF19 by molecular mass. This peptide is probably not essential for growth, as an insertion mutant is viable.     

两种HMW-GS基因启动子片段的克隆及分析

利用聚合酶链反应(PCR)技术从小偃6号中获得400bp左右的扩增产物,将其与pGEM-T Easy载体连接后转入大肠杆菌,经过筛选获得HMW-8-P和HMW-38-P两种类型克隆。序列分析表明：HMW-38-P包括了HMW-GS14基因上游启动子及信号肽对应编码区,而另一段(HMW-8-P)为一未知HMW-GS基因启动子区及信号肽对应的编码区。将两序列和GenBank中已知的35种HWM-GS基因启动子区序列进行多序列比对,最后获得HMW-GS启动子的系统发生树。通过系统发生树可以清晰地看出位于不同染色体上的不同亚基类型的HMW-GS基因的进化关系,并可确定HMW-8-P为Glu-D-1类型HMW-GS的启动子区,小偃6号中Glu-D-1类型的亚基为2亚基,所以HMW-6-P为2亚基启动子区序列。     

小鼠T-bet启动子荧光素酶报告基因载体的构建及活性鉴定

为了研究T-bet在T细胞中的转录调控机制,并研究其在多发性硬化症中的信号通路,本研究构建小鼠TBX21(编码T-bet)基因启动子区和增强子区萤火虫荧光素酶报告基因载体。在对小鼠TBX21基因5?侧翼区进行详尽生物信息学特征分析后,设计相应引物,用PCR的方法从小鼠基因组中扩增出TBX21基因5?侧翼区–1 000 bp-28 bp片段长为1 028 bp的启动子区(以翻译起始点ATG为+1)和–3 308 bp-–2 000 bp片段长为1308 bp的非编码区保守序列(No-coding conserved sequence,CNS),再用定向克隆的方法将这两个片段定向重组入专门用于启动子活性研究的萤火虫荧光素酶报告基因载体(p GL4.10)中,构建出包含小鼠TBX21基因启动子区和CNS区的萤火虫荧光素酶报告基因载体(p GL4.10-TBX21pr-CNS),电泳与测序鉴定,最后再将p GL4.10-TBX21pr-CNS与内参p RL-TK用lipofectamine 2000共转染293T细胞和Jurkat细胞中,通过双荧光素酶报告基因检测系统鉴定p GL4.10-TBX21pr-CNS的启动子和增强子活性,并用独立样本t检验方法进行统计分析。对照组共转染p GL4.10与内参p RL-TK。结果表明,成功构建出荧光素酶报告基因重组质粒p GL4.10-TBX21pr-CNS。与转染空质粒p RL-TK组相比,293T细胞(P=0.012 2)和Jurkat细胞(P=0.002 2)中转染p GL4.10-TBX21pr-CNS组荧光素酶活性升高。研究结果表明在293T细胞和Jurkat细胞中p GL4.10-TBX21pr-CNS可以表现出启动子活性,为后续小鼠T-bet转录调控研究提供了基本材料。     

拟南芥ats1A基因启动子的克隆和功能分析

通过PCR扩增,从拟南芥中克隆出ats1A基因启动子(包括叶绿体转运肽),将此启动子与GUS基因相连构建植物瞬时表达载体,用基因枪法将之导入烟草进行瞬时表达。GUS基因检测分析表明,ats1A基因启动子能特异的启动GUS基因在烟草叶片中高效表达。     

Expression and promoter activity of the desiccation-specific Solanum tuberosum gene, StDS2

Environmental stresses induce the expression of several plant genes via multiple and cross‐talking signalling pathways. Previously it was shown that ScDS2, a gene of the wild potato species, Solanum chacoense, is highly inducible by dehydration but not by abscisic acid (ABA), the mediator of many plant stress responses. Herein it is shown that ScDS2‐related genes are present in the cultivated potato, Solanum tuberosum (StDS2) and also in the non‐tuberizing Solanum species, Solanum brevidens (SbDS2). We show that expression of StDS2 is dehydration‐specific, is not inducible by cold, heat, salt, hypoxia or oxidative stresses, and is independent of ABA. Signalling of StDS2 induction, however, is dependent on the synthesis of novel proteins because cycloheximide can block StDS2 expression. To analyse the promoter region of StDS2 a genomic library of Solanum tuberosum was established and 1140 and 498 bp regions of the StDS2 promoter were isolated. The promoter fragments were fused to the β‐glucuronidase (GUS) reporter gene and tested in transgenic potato plants. Both promoter fragments were able to induce GUS activity in response to dehydration. This result suggests that drought‐specific cis‐elements are located within 498 bp upstream to the StDS2 coding sequence.     

sgna基因小麦高效表达载体的构建

利用在禾谷类作物中表达效率较高的启动子Ubi对含目的基因sgna的现有载体进行了改造,并引入筛选标记基因bar;为提高目的基因的表达水平,在目的基因5′端引入了Ω和kozak序列,3′端引入了poly(A)序列,成功构建了适用于小麦的抗虫基因植物表达载体pGU4AGBar和pGBIU4AGBar,基因pGU4AGBar含有顺向连接的Ubi-sgna及Ubi-bar基因表达盒,pGBIU4AGBar含有T-DNA边界序列,并在其左右边界中间插入了含有顺向连接的CaNV35S-nptⅡ,Ubi-sgna和Ubi-bar基因表达盒,人工合成的雪花莲外源凝集素基因sgna可以编码对同翅目昆虫具有毒杀作用的蛋白。