紫外诱变选育秦巴山区黑木耳耐高温菌株的研究

以秦巴山区黑木耳主要栽培菌株为材料,采用不同紫外照射时间和温度梯度筛选高温黑木耳品种。结果表明,黑木耳原生质体紫外诱变的最佳时间为120¹80 s,再生菌落的致死率为64%180 s,再生菌落的致死率为64%⁸2%,通过40℃高温初步筛选得到适合秦巴山区栽培的黑木耳菌株YBZ4。     

xyl-d二型乙醇脱氢酶(SADH)G198R突变体的研究

本研究通过设计简并引物及文献参考,PCR 克隆得到嗜热厌氧菌菌株 xyl-d 的SADH,并利用定点诱变技术将该酶的198位苷氨酸(Gly)突变为精氨酸(Arg)。将野生型和突变基因连接到原核表达载体pET28a,IPTG诱导表达,再经镍柱纯化,然后比较了两个蛋白分别以 NAD/NADH,NADP/NADPH 为辅因子、异丙醇或异丁醛为底物、55℃条件下的酶活。发现突变后蛋白的总体催化活性相对于野生型有所下降,其中突变蛋白对 NAD/NADH 的亲和性分别下降了约8倍和6倍,而且对NADPH 的亲和性下降也非常明显,但对NADP的亲和性变化却不大。     

柠檬酸高产黑曲霉菌株选育与发酵新底物研究

本研究利用Aspergillus niger AS3.3928作为对照菌株以废弃苹果榨汁为底物,设计了四因素四水平的正交实验。在pH6,温度31℃,甲醇含量2%,发酵时间4d的最适条件下,黑曲霉的发酵最好。将土壤中分离培养获得的20株黑曲霉,选出菌株透明圈相对较大的A1、A2、A5、A11这4株。在最适条件下,根据4株菌发酵废弃苹果榨汁生产柠檬酸的情况来进行复筛,得到菌株A2;进而对A2进行鉴定,初步确定所得菌株为黑曲霉。在最适条件下,A2发酵废弃苹果榨汁其柠檬酸的产量为1.539 mg/mL,经对A2进行T21紫外诱变,筛选得到A2-1。结果表明A2-1菌株柠檬酸产量比其父本增加了36.6%,比对照菌株AS3.3928增加了38.53%。     

水稻白叶枯病菌致病相关基因筛选系统的建立

水稻白叶枯病菌Xanthomonas oryzae pv.oryzae是水稻生产中最严重的细菌性病害之一。本研究采用Tn5转座子随机插入突变的方法构建水稻白叶枯病菌广西菌株K74的突变体库,Southern杂交显示Tn5随机单拷贝插入基因组。通过水稻致病性检测试验,目前获得15个致病力降低50%以上的突变体,为定位Tn5插入突变基因,经质粒拯救、测序、序列分析后发现:4个突变基因为gum基因簇基因,5个为LPS基因簇基因,2个为metB基因,1个为hrpB1基因,2个是与糖合成转移酶相关基因,1个为编码假定蛋白的基因。其中14个突变基因是已知的与致病相关的基因。实验结果表明本研究成功建立了一个筛选水稻白叶枯病菌致病相关基因的系统,为进一步研究水稻白叶枯病致病相关基因,阐明该病的致病机理奠定基础。     

DNA transposons in vertebrate functional genomics

Genome sequences of many model organisms of developmental or agricultural importance are becoming available. The tremendous amount of sequence data is fuelling the next phases of challenging research: annotating all genes with functional information, and devising new ways for the experimental manipulation of vertebrate genomes. Transposable elements are known to be efficient carriers of foreign DNA into cells. Notably, members of the Tc1/mariner and the hAT transposon families retain their high transpositional activities in species other than their hosts. Indeed, several of these elements have been successfully used for transgenesis and insertional mutagenesis, expanding our abilities in genome manipulations in vertebrate model organisms. Transposon-based genetic tools can help scientists to understand mechanisms of embryonic development and pathogenesis, and will likely contribute to successful human gene therapy. We discuss the possibilities of transposon-based techniques in functional genomics, and review the latest results achieved by the most active DNA transposons in vertebrates. We put emphasis on the evolution and regulation of members of the best-characterized and most widely used Tc1/mariner family.Received 8 June 2004; received after revision 26 October 2004; accepted 18 November 2004     

人胰岛素原类似物（B-Arg-Arg-A）基因的构建和表达

用聚合酶链反应（PCR）法将C肽为6个氨基酸残基的胰岛素原类似物基因删除突变成C肽仅为2个精氨酸残基的胰岛素原类似物（BArg-Arg-A）基因,即把pUC18BC'A改建为pUC18BR₂A。再将pUC18BR₂A与pJG105重组为表达质粒pJG107,使B-Arg-Arg-A与pJG105编码的一种多肽构成融合蛋白在大肠杆菌体系中表达。融合蛋白占细菌蛋白总量的58%。表达产物具有人胰岛素放射免疫活性。     

Molecular dynamics simulation of site-directed mutagenesis of HIV-1 Tattrans-activator

The Cys-rich domain, core region and basic domain are highly conserved and very important to thetrans-activation activity of HIV-1 Tattrans-activator. The three-dimensional structures of 6 mutants of HIV-1 Tat protein were constructed with the methods of molecular dynamics simulation. The variations of the structures of the mutants have been analyzed and the factors that led to abolishment oftrans-activation activity have been discussed.     

链霉素抗性突变理性筛选链霉素高产菌株

以灰色链霉菌(Streptomyces griseus) CICC11002为出发菌株,采用紫外诱变并结合链霉素抗性筛选法育种,以期获得高产菌株.灰色链霉菌孢子悬液经90％紫外致死剂量诱变后,在含有链霉素最小抑制浓度(0.8μg/mL)的培养基平板上,分离得到105株链霉素抗性突变菌.其中链霉素产量高于出发菌株的有19株,正突变率高达18.1％,同时获得了产链霉素能力约为出发菌株2倍的突变株SM-55.     

利用原生质体融合技术筛选红霉素优良菌株

以红霉素高产菌HE-08-6及低产优组分菌HA-08-20为出发菌株,其原生质体经紫外诱变后在适宜条件下进行融合,再生培养后挑取融合子89株,经筛选得到3株具有二亲本优良性状的高产菌株HEA-08-01,HEA-08-02,HEA-08-03.其化学效价较出发菌株HA-08-20提高约25%,产品的红霉素A组分含量较出发菌株HE-08-6提高约26%.传代培养证明该菌株具有一定的遗传稳定性,在50L-Fus智能发酵罐上连续3批发酵结果表明,该优良菌种在提高红霉素产品质量及效价上取得较大进展,具有推广应用价值.