猪bmp15基因报告载体的构建及其特异性

为了研究骨形态发生蛋白15(bmp15)基因的表达和调控特性,通过克隆猪bmp15基因2.2 kb启动子片段,构建pBMP15-EGFP报告载体,实现监测干细胞向类卵母细胞分化的过程。以猪卵巢组织和中国仓鼠卵巢细胞(CHO)、成肌细胞(C2C12)、猪羊水干细胞(pAFSC)为材料,通过RT-PCR、免疫荧光、细胞转染、显微注射检测bmp15组织特异性表达,并且通过单层细胞诱导检测该基因体外示踪类卵母细胞获得过程的能力。RT-PCR结果显示bmp15在猪的卵巢组织中特异表达,在CHO中表达,而在C2C12和pAFSC中不表达。卵巢组织切片免疫荧光检测结果显示bmp15表达于卵泡发育的各个阶段。瞬时转染不同细胞发现启动子只在CHO中有活性,而在C2C12和pAFSC中均无活性。显微注射重组质粒片段结果显示增强绿色荧光蛋白(Enhanced Green Fluorecence Protein,EGFP)在卵母细胞体外成熟18 h启动表达,并能够持续至4-细胞期胚胎。单层细胞诱导结果显示诱导12 d的pAFSC出现携带EGFP的圆形细胞团。说明bmp15具有表达特异性和示踪干细胞诱导分化为类卵母细胞的潜能。     

Development of a shuttle vector for screening of strong promoter sequences

A new promoter probe plasmid, pMOL618, has been specifically designed to be selective for strong promoter sequences. The plasmid contains two origins of replication which allow it to replicate both in Bacillus subtilis and Escherichia coli, as well as an indicator gene which also functions in both backgrounds. The plasmid is therefore useful in the screening of promoter sequences in both organisms. The stringency of the promoter selection is demonstrated using a known strong promoter.     

Tissue-specific activation of the osmotin gene by ABA,C2H4 and NaCl involves the same promoter region

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene -glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between –248 to –108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C₂H₄ and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C₂H₄ and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C₂H₄ in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C₂H₄ was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C₂H₄. However, significant C₂H₄-induced activity was obtained with a promoter fragment containing the AT and PR elements together.     

Hybrid genes in the analysis of transformation conditions: II. Transient expression vs stable transformation—analysis of parameters influencing gene expression levels and transformation efficiency

Reports on direct gene transfer have dealt with either the obtention of stable transformants and transgenic plants, or described the use of reporter genes to analyse different aspects of gene expression in plant protoplasts and conditions for their use in transient gene expression assays.
In this paper we present comparisons between several transformation techniques, show species-specific differences in efficiencies of stable transformants and in the levels of transient gene expression, and report on the identification of major parameters responsible for DNA uptake as judged from transient chloramphenicol acetyl transferase (CAT) expression levels and from efficiencies of transformation based on kanamycin-resistance. The described procedures have been simplified, optimized and standardized and should allow routine use with a great variety of plant species.     

褪黑素合成酶基因转化烟草及转化植株抗氧化系统变化

通过根癌农杆菌(含植物表达载体YXu55)介导的转化技术,将褪黑素生物合成酶-芳烷基胺N-乙酰转移酶(Arylalkylamine N-acetyltransferase,AANAT)与羟基吲哚O-甲基转移酶(Hydroxyindole O-methyltransferase,HIOMT)基因导入到烟草(秦烟95)中。对所获得的庆大霉素抗性烟草株系进行Southern blotting和RT-PCR分子生物学检测,结果表明,AANAT-HIOMT基因已成功地整合到烟草基因组中,并且可以在mRNA水平上进行转录。用反相高效液相色谱法(RP-HPLC)测定转化株系的褪黑素含量表明,转AANAT-HIOMT基因烟草株系的褪黑素含量均明显高于pZP122(不含AANAT和HIOMT基因的空白质粒)转基因株系和未转基因的对照植株,证明AANAT-HIOMT基因在转基因植株中的表达增强了褪黑素的合成能力。对不同株系抗氧化系统的部分指标进行了测定,并与其亲本对照植株比较,发现AANAT-HIOMT基因在转基因植物中的表达引起超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性增加,谷胱甘肽(GSH)浓度...     

普通烟草LBD基因家族的全基因组序列鉴定与表达分析

LBD是一类具有LOB(lateral organ boundaries)结构域的基因家族,在植物发育过程中起到非常重要的作用。采用生物信息学方法,根据拟南芥LBD基因序列鉴定了普通烟草基因组中的LBD基因,并对家族成员进行了序列特征、系统发育和表达谱分析。结果表明:普通烟草基因组中共有98个LBD基因成员,其基因结构相对简单,一般含有1~3个外显子。LBD基因家族可分成I和II两大类,两类均含有CX_2CX_6CX_3C保守结构域,但II类不含有LX_6LX_3LX_6L形成的"卷曲螺旋"二级结构,根据与拟南芥LBD蛋白构建的系统发育树则可细分成5个亚家族(Ia、Ib、Ic、Id和II)。将LBD基因与表达序列标签(EST)比对,发现36个基因有EST证据;EST、芯片数据和转录组数据分析表明:LBD基因具有不同的组织表达模式,部分基因表现出组织特异性。这些研究结果为普通烟草LBD基因家族功能的深入研究奠定了基础。     

gfP基因甘蓝质体表达载体构建及原核表达分析

以甘蓝自交系‘03097’为试材,PCR扩增并克隆了甘蓝质体的trnI-trnA基因区段,利用该基因区段作为定点整合外源基因的同源重组片段,构建了甘蓝质体定点转化载体,并进行了原核表达分析。结果表明,所扩增的基因区段大小为2.7 kb。进行序列分析后,经酶切、连接和转化,构建成含Prrn-gfp-aadA-TpsbA表达盒的质体表达载体,酶切鉴定表明,所构建载体符合预期设计;gfp基因能够在质体特异性启动子Prrn及终止子TpsbA的调控下高水平表达,表达量约占总可溶性蛋白的41.0%,包涵体占菌体蛋白的38.0%。该载体的构建为后期甘蓝质体转化体系的建立和其它功能基因导入甘蓝质体进行性状改良奠定了基础。     

Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter

A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.     

Mutational analysis of a baculovirus major late promoter

We have used a linker-scan mutation strategy to analyze P_cap99, the proximal promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) gene encoding the major capsid protein. A series of recombinant viruses expressing the cat reporter gene under the control of selected mutants of this promoter was constructed. Only mutations that altered bases within a region extending from 8 bp upstream to 6 bp downstream from a TAAG sequence had a significant effect on expression from the late gene promoter. A synthetic promoter consisting of only these 18 bp (P_capmin) was sufficient to direct late expression. Aside from this small region surrounding the TAAG, no evidence for distinct late activating or repressing sequence elements was obtained. Experiments comparing and combining late and very late gene promoter sequences suggest that late expression is intrinsically determined by the presence and immediate context of a TAAG sequence and that very late expression [as previously shown in Ooi et al. J. Mol. Biol. 210 (1989) 721–736] results from additional modulation of TAAG-dependent expression by downstream promoter elements placed in an appropriate context. A compact combination promoter (95 bp), constructed by fusing P_capmin to linker-modified very late polyhedrin promoter, directs strong expression at late and very late times post-infection.     

烟草乙烯转录因子ERF基因NtTOE3的克隆、载体构建及表达分析

ERF（Ethylene-responsive factor）是一类具有AP2特征结构域的乙烯应答转录因子,在响应植物的逆境胁迫应答中起关键作用。为明确ERF家族成员TOE3在烟草中的生物学功能和调控机制,同源克隆普通烟草K326中NtTOE3基因cDNA全长,利用实时荧光定量PCR（qRT-PCR）表征基因表达模式,结合生物信息学分析,对基因功能和调控机制进行初步预测。结果表明,该基因全长1 356 bp,编码451个氨基酸残基,预测分子量为49.84 ku。生物信息学分析表明,该蛋白是一个亲水蛋白,可能定位在细胞核,且与美花烟草（Nicotiana sylvestris）NsTOE3有96%的同源性,故命名为NtTOE3。组织表达分析发现,该基因在烟草根、茎、叶、花中均有表达,其中茎中表达量最高。逆境胁迫试验表明,该基因能快速响应低钾、高盐、干旱、H₂O₂、ABA和4℃等逆境处理。表明NtTOE3转录因子在烟草非生物胁迫中起着重要调节作用。     

Isolation and functional analysis of a strong specific promoter in photosynthetic tissues

Constitutive promoters such as CaMV (cauliflower mosaic virus) 35S and nos (nopaline syn-thase) have been used extremely as useful tools in many plant transgenic researches. Because of lacking temporal and spatial regulation, constitutive promoters have a number of potential drawbacks in genetically improved crops[1]. For example, constitutive expression of viral capsid proteins in plants may increase the risk of transvapsidation or viral recombination to generate new strains of phytopathogen…     

食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达

为构建乳酸乳球菌食品级分泌表达载体,通过PCR扩增质粒pMG36e的p32启动子片段及乳酸乳球菌MG1363未知分泌蛋白(Usp45)基因的核糖体结合位点、分泌信号肽和成熟肽前11个氨基酸的编码序列(SPusp45),克隆到食品级载体pSH91中,构建食品级分泌性表达载体pSQ;克隆报告基因金黄色葡萄球菌核酸酶(NucA)成熟肽的编码序列nucA到pSQ中分泌信号后,转化乳酸乳球菌MBP71,构建了乳酸乳球菌食品级分泌性表达系统L lactis/pSQ-nucA;通过TB-D法和酶谱法检测L lactis/pSQ-nucA的表达形式、表达量并与以前构建的L lactis/pSQZ-nucA系统表达能力进行比较,结果发现L lactis/pSQ-nucA能够分泌性表达NucA,分泌性表达的NucA量大约是胞内NucA的10倍;L lactis/pSQ-nucA的表达量高于lactis/pSQZ-nucA.为进一步目的蛋白的的分泌性表达及食品级疫苗的研制奠定了基础.     

Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter

Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5 deletions of the osmotin promoter cloned into a promoter-less GUS-NOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from –108 to –248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to –1052) of enhancer-like sequence and then a sequence upstream of –1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the –248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.     

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.     

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。