Cloning and analysis of the antagonistic related genes of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> B8

To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.     

厦门港浮游动物体内色氨酸的荧光法测定

建立了海洋浮游动物体内色氨酸的荧光分析方法.将1 mg左右样品置于安瓿瓶中,加1 mL 5.5 mol/L NaOH为水解液,在110℃水解20 h.水解产物用HCl中和,然后用KH2PO4-NaOH缓冲液调节pH为10.5,在激发波长225 nm,发射波长350 nm处测定荧光强度.方法线性范围0~0.07 mg/L,检测限为2.5×10-3mg/L,回收率99.5%~109.3%.应用该方法测量了7种从厦门港采集的浮游动物的色氨酸含量.     

花特异表达启动子的克隆及花色调节基因Lc表达载体的构建

克隆了矮牵牛花特异表达基因CHSA启动子，并定向插入到已含有花色调节基因Lc的质粒pBI121中，取代原有的CaMV 35S启动子．所构建的新表达载体可用于花色改良研究．     

酵母合成的15NL-色氨酸中N元素代谢通量的分析

以葡萄糖为碳源，硫酸铵为氮源，¹⁵N邻氨基苯甲酸为前体物，利用酵母发酵合成¹⁵NL-色氨酸，是合成¹⁵NL-色氨酸的一种新方法。为了有效的利用含氮的化合物，实验过程中，采用¹⁵N标记的邻氨基苯甲酸为示踪剂，分析了N元素在¹⁵NL-色氨酸合成过程中的代谢通量。结果表明，93％外加的邻氨基苯甲酸，被转化为¹⁵NL-色氨酸。硫酸铵在促进菌体生长的同时，0.05％硫酸铵被用于合成邻氨基苯甲酸，并进一步被转化为L-色氨酸，5.95％硫酸铵被直接用于合成¹⁵NL-色氨酸。     

Cloning and enzymology analysis of farnesyl pyrophosphate synthase gene from a superior strain of<Emphasis Type="Italic">Artemisia annua</Emphasis> L.

A cDNA(af1) encoding farnesyl pyrophosphate synthase AaFPS1(FPS,EC2.5.1.1/EC2.5.1.10) from a high yield Artemisia annua strain 025 has heen cloned from its cDNA library .Sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with molecular weight of 39 kD.Deduced aa sequence of the cDNA was similar to FPS from other plants,yeast and mammals,containing 5 conserved domains found in both prenyl trans-ferase and polyprenyl synthase,The expression of the cDNA in Escherichia coli showed measurable specific activity of FPS in vitro.The enzyme was purified by ion exchange chromatography and its kinetics was measured ,These results would further promote the molecular regulation of artemisnin biosynthesis.     

地方芽孢杆菌A.4041耐高温α—淀粉酶色氨酸残基和巯基的化？…

用Ｎ－溴代琥泊酰亚胺和Ｎ－乙基丁二酰亚胺分别为Ａ．４０４１α－淀娄酶的主要组分α－Ⅲ的色氨酸残基和巯基进行修饰。结果表明，５０％的Ｔｒｐ残基ＮＢＳ修饰，可使酶活力完全丧失，Ｃａ＾２＋和底物可减少修饰酶的失活；加Ｃａ＾２＋或底物后修饰酶的荧光强度较对照修饰酶有显著提高。     

固氮鱼腥藻HB686固氮_放氢与谷氨酰胺合成酶的关系

固氮鱼腥藻HB686(Anabaena azotica HB686)的固氮和放氢被氨抑制与蛋氨酸砜亚胺(MSX)解除氨抑制的作用呈平行关系.谷氨酰胺能抑制固氮和放氢,而对谷氨酰胺合成酶(GS)和吸氢酶却有激活作用.固氮血腥藻在Mg~(2+)介质中的乙炔还原活性和放氢量皆比在Mn~(2+)介质中的高,而GS的活性则相反.不同波长的光照射对固氮、放氢和GS活性有不同的影响.     

吲哚胺2，3-双加氧酶下调HLA-I类分子的表达

目的：探讨人类吲哚胺2,3-双加氧酶（IDO）对细胞表面HLA-I类分子的影响。方法：用脂质体法将含IDO基因的重组质粒pEGFP-IDO转染293T细胞,用Western blot检测IDOGFP在转染细胞中的表达,荧光抗体染色结合流式细胞术检测细胞表面的HLA-I类分子的表达。结果：Western blot证实转染重组质粒的细胞表达IDO。转染空载体的细胞与转染重组质粒的细胞HLA-A,B,C-PE染色的平均荧光强度分别为299.46±62.65及42.51±8.19,两者相比有显著性差异（P〈0.01）;转染重组质粒的细胞,未加色氨酸组与色氨酸组HLA-A,B,C-PE染色的平均荧光强度分别为309.17±34.89及692.12±33.42,两者相比亦有显著性差异（P〈0.01）。结论：IDO下调细胞表面HLA-I类分子的表达,色氨酸能够逆转IDO所致的HLA-I类分子的下调。     

流动注射-化学发光法测定色氨酸

在酸性条件下,色氨酸对Ru(phen)32+-Ce(Ⅳ)反应体系化学发光有显著的增强作用,据此建立了流动注射-化学发光法检测色氨酸的新体系.对影响流动注射化学发光的各因素进行了实验研究,优化了反应条件和各项测定参数.结果表明,在优化条件下,色氨酸在7.5×10-8～8 0×10-6g/mL的浓度范围内发光强度与其浓度呈良好的线性关系,检测限(3σ)为5 16×10-8g/mL,对浓度为2 5×10-7g/mL的色氨酸进行11次平行测定,相对标准偏差(RSD)为2 2%.对合成样品中色氨酸测定的回收率在99 1%～100.8%之间,采样频率为90次/h.