Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1

A magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved three putative ORFs; the mini-Tn5 lacZ2 was inserted into ORF1. Functional complementary test indicated that the 3073-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The majority of proteins, which had homology with the protein encoded by ORF1, were the cation transporter. Transmembrane domain analysis showed that the protein encoded by ORF1 contained four transmembrane domains. It may be a transmembrane protein. The protein encoded by ORF1 contained two putative conserved domains: COG0053 and PRK09509. The MMT1 and FieF, containing conserved domains COG0053 and PRK09509 too, were Fe²⁺ transporter (cation diffusion facilitator superfamily). It was suggested that the protein encoded by ORF1 might take part in the magnetosomes biosynthesis as Fe²⁺ transporter. Supported by National Natural Science Foundation of China (Grant No. 30570023) and Scientific Research Project of Huaibei City, Anhui Province (Grant No. 070114)     

Functional analysis of hydrogenases and their effects on cell growth and magnetosome synthesis in <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis>

This study addressed the effect of hydrogen metabolism on cell growth and magnetosome synthesis in Magnetospirillum gryphiswaldense strain MSR-1. Two deletion mutants were generated: L206, with single deletion of the hupL gene encoding H2-uptake [NiFe] hydrogenase; and B206, with double deletion of the hyaB gene encoding H2-producing [NiFe] hydrogenase and the hupL gene. The wild-type and mutant strains were compared in terms of hydrogen uptake capability, hydrogen yield, growth rate, and iron uptake, and o...     

Submerged culture of Magne-tospirillum gryphiswaldense under N2-fixing condition and regulation of activity of nitrogen fixation

A submerged culture technique for Magnetospirillum gryphiswaldense under the nitrogen-fixing condition (microaerobic and N-limited) was set up. In N-limited medium with Na-lactate as a sole carbon source, the optical density (A600 nm) and activity of nitrogen fixation of cells were 1.3 and 217 nmol of ethylene produced per hour per A600nm respectively within 21 h by three times of feeds. The pH and temperature were controlled at 7.2 and 30℃ respectively, and the oxygen concentration was controlled by sparging with N2 containing 0.4%-0.8% of O2. The activity of nitrogen fixation of cells was obviously inhibited by oxygen and ammonium. It indicated that the posttranslational regulation of nitrogenase existed in M. gryphiswaldense.     

一株黄河三角洲盐碱地耐盐菌的分离、鉴定及耐盐能力分析

从黄河三角洲盐碱地土壤分离到一株耐高盐菌株NM-1.对该菌株生理生化性质、耐盐性以及系统分类位置进行了分析.结果表明:菌株NM-1为长杆状,革兰氏阴性,菌落黄色,呈圆形,湿润、光滑.能利用葡萄糖、乳糖和蔗糖,不能利用淀粉和明胶.生长pH范围为8.0~10.0,最高耐盐浓度(NaCl)为30%,为中度嗜盐微生物,最适生长的Na2CO3浓度为0.25 M.16SrDNA序列分析表明:该菌株与Halomonas variabilis(JN645866)以及Halomonas variabilis(AY204638)序列的相似性均为98%,结合其形态、生理生化性质,认定该菌株属于耐盐单胞菌属,暂命名为Halomonas sp.NM-1(AB917466).     

Cloning and expression pattern of a dehydrin-likeBDN1 gene from drought-tolerantBoea crassifolia Hemsl.

A 500-bp cDNA fragment was amplified via RT-PCR from drought-induced total RNA of the drought-tolerant B. crassifolia Hemsl. using primers based on the sequence of published dehydrin conserved region. By using 5′RACE, full-length coding region (1 148 bp) of BDN1 gene was produced. It is a new member of the dehydrin gene family. Southern analysis indicated that BDN1 is present in the B. crassifolia genome as a single-copy gene. Northern analysis revealed that its expression is inducible by drought and cold stresses as well as ABA application.     

VMP1基因的克隆及其抗体的制备与鉴定

为了得到效价高、特异性强、能用于检测内源性VMP1蛋白的抗体，采用RT-PCR法，从L929细胞株中克隆鼠VMP1基因，重组入pCMVS载体，用PCR扩增与其氨基酸132-239区域对应的DNA片断，将该片段连接到原核表达载体pGST parallel中，在大肠杆菌BL21菌株中大量表达，经谷胱甘肽-琼脂糖树脂纯化后，得到高纯度的VMP1抗原．免疫新西兰兔，获得抗VMP1蛋白的兔抗血清，将血清纯化后即得到抗VMP1的多克隆抗体．用Western blot分析手段检测了该抗体的特异性及效价．在用该抗体检测外源性和内源性VMP1蛋白的试验中，证实该抗体效价高，特异性强，效果很理想．此方法可用于获得大量廉价的抗VMP1蛋白的高滴度和高特异性的抗体，为进一步研究VMP1在细胞内，尤其是在信号转导通路中的功能和作用奠定了基础．     

Knob-associated tandem repeats on mitotic chromosomes in maize,Zea diploperennis and their hybrids

Knob-associated tandem repeats, 180-bp repeats and TR-1 elements, together with 45S rDNA were located on mitotic chromosomes of Zea diploperennis (DP),maize inbred line F102 and their hybrid. In DP, knobs on the short arm of chromosomes 1 and 4 and on the long arm ofthe chromosomes 4 and 5 are composed predominantly of the 180-bp repeats. In addition, 180-bp repeats existed together with TR-1 elements were also detected on the short arm ofchromosomes 2 and 5 and on the long arm of the chromosomes 2, 6, 7, 8 and 9. In maize inbred line F102, 180-bp repeats were present in chromosomes 7S and one homologue of chromosomes 8L. TR-1 elements appeared on satellite of chromosome 6 and no detectable hybridization site co-located with 180-bp repeats was observed in maize F102.Polymorphism of size, number, and distribution of 180-bp and TR-1 signals were revealed among different chromosomes in these two species and heteromorphism existed between some homologous chromosomes in the same species.Using these excellent landmarks, the interspecific hybrid of maize and DP were identified. The results suggest that comparative analysis of 180-bp repeats and TR-1 elements may help understand the genome organization and the evolution in Zea.     

NM/FS1/FS2/NM结的隧穿电导和隧穿磁电阻

利用平均场近似和转移矩阵方法,对NM/FS1/FS2/NM结(NM为非磁金属,FS1和FS2为铁磁半导体层)的隧穿磁电阻(TMR)与FS层厚度及Rashba自旋轨道耦合的关系进行了研究.结果表明NM/FS1/FS2/NM结中TMR值随半导体层厚度的改变发生周期性变化,选择适当的半导体层的厚度和Rashba自旋轨道耦合系数可以得到大的TMR值.     

Analysis of the Cotton E6 Promoter

An E6 gene from sea island cotton （Gossypium barbadense） was expressed specifically in cotton fiber cells to transfer functions to cultivated species for better transgenic engineering. The regulatory activity of the E6 promoter region was then studied by isolating a 614-bp fragment of the 5＇-flanking region from upland cotton （Gossypium hirsutum CR1-12） to produce a green fluorescent protein （GFP） reporter construct for analysis of tissue-specific expression in transgenic tobacco seedlings. Fluorescent analyses indicate that the relatively short E6 promoter is sufficient to direct green fluorescent protein expression specifically in the leaf trichomes （hair cells） of the transgenic tobacco plants. As cotton fibers are also unicellular trichomes that differentiate from epidermal cells of developing cotton ovules, the result suggests that the relatively short E6 promoter can serve as a fiber-specific expression promoter for genetic engineering to improve cotton fiber quality.     

纳豆激酶基因的克隆及其在大肠杆菌中的表达

纳豆激酶是一种良好的天然蛋白酶类溶栓物质。国内外许多学者对该酶进行了基因工程研究,但在克隆表达过程中出现了许多长短不同的基因片段。本研究通过原产日本的优质纳豆中分离鉴定出高产纳豆杆菌N07并提取该菌株的全基因组;通过PCR手段扩增出能编码纳豆激酶信号肽,前导肽和成熟肽的前纳豆激酶酶原基因NK1,以及能编码纳豆激酶成熟肽的纳豆激酶基因NK2,构建了纳豆激酶基因的表达载体pET30a-NK1和pET30a-NK2,转化E.coli BL21后在大肠杆菌中表达,并进行了活性分析。结果发现,纳豆激酶酶原基因片段NK1能成功表达出有活性的分泌型纳豆激酶;而纳豆激酶基因片段NK2的表达产物为无活性的包涵体。在对NK1和NK2的比较研究后可知,纳豆激酶酶原基因片段NK1能在大肠杆菌中很好的分泌表达,这将为纳豆激酶基因工程的深入研究奠定基础。     

含磁性非磁性金属插入层磁隧道结的隧穿特性研究

讨论了一种新型FM1/NM/FM/I/FM2磁性隧道结,该隧道结结构可获得高质量的I层,从而具有重要的应用价值.利用Slonezewski的自由电子模型和转移矩阵方法,对这种隧道结中的隧穿电导(TC)和隧穿磁电阻(TMR)与NM、FM层的厚度以及和势垒高度的关系进行了研究.同时还通过和FM1/NM/I/FM2型隧道结的相应结果的比较讨论了FM层在FM1/NM/FM/I/FM2磁性隧道结中的作用.     

复合诱变和原生质体融合选育S-腺苷甲硫氨酸酿酒酵母高产菌株

对生产S-腺苷甲硫氨酸（SAM）的酿酒酵母（S.cerevisiae）菌株R1进行复合诱变和原生质体融合。采用酶解法获得出发菌株S.cerevisiae R1的单倍体菌株hR10,对其进行UV和DES复合诱变,筛选出了4个单倍体正突变株DR1-3、NM14、NM39和NM45。制备了单倍体正突变株的原生质体,采用热灭活和UV灭活法灭活双亲原生质体,在聚乙二醇（PEG）的诱导下进行原生质体融合,筛选得到融合子F35,其生产 SAM的产量为22.5 mg/L,与出发菌株R1（11.1 mg/L）相比,提高了103%,并且能够稳定遗传。采用正交试验优化了F35发酵生产SAM的培养基和发酵时间,优化了的培养基配方为麦芽糖100 g/L,蛋白胨10 g/L,NH₄H₂PO₄4g/L,NH₄Cl₅g/L,MgSO₄0.1g/L,KH₂PO₄6 g/L,发酵时间为72h。     

以精子载体法制备转基因小鼠的初步研究

将外源融合基因BaLA-HI与DOSPER脂质体等比较混合,加入获能精子悬液中,37度,5％CO2共培养0．5h,以这种处理精子作为转基因载体乾鼠的体外受精及胚胎移植,在获得的40只移植后代后,经PCR特异片段扩增和Southern杂交,共检测出2只呈相性的转基因小鼠,证明人胰 基因已在小鼠染色体上实现了整合,基因整合率为5％。     

Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1-25 amino acid sequence is a predicted signal peptide and the other 26-216 amino acid sequence is a mature peptide. The 26-45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+- dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

视黄酸对胃癌细胞NM—IF系统变化的影响

选择性抽提整装扫描与透射电镜观察显示,人胃腺癌ＭＧｃ８０－３细胞核骨架纤维和中间纤维数量较少、分布不均匀,核纤层为厚薄不一结构,与两类纤维联系不密切．经１０－６ｍｏｌ／ＬＲＡ处理后,细胞核骨架纤维和中间纤维数量增多、结构层次丰富,分布均匀并相互交织成规则网络,两类纤维通过薄层均一的核纤层发生密切联系,形成贯穿整个细胞核质区域的完整体系．表明经ＲＡ诱导处理后ＭＧｃ８０－３细胞的核骨架－中间纤维系统产生了与正常细胞相似的恢复性改变．这种变化是癌细胞恶性表型逆转的重要形态特征和功能表现．