从小麦-簇毛麦易位系TAC文库中筛选Hv-S/TPK基因

本实验室已经通过基因芯片技术筛选到一个白粉菌诱导后上调表达的抗病相关基因Hv-S/TPK, 并获得了它的全长cDNA序列。利用Hv-S/TPK的特异引物筛选小麦-簇毛麦6VS/6AL易位系基因组可转化人工染色体(Transformation-competent artificial chromsome, TAC)文库, 获得了阳性TAC单克隆, 并进一步获得了含有Hv-S/TPK cDNA序列的5160 bp(GenBank Accession No. EU153366)的亚克隆。对亚克隆的序列分析结果表明, Hv-S/TPK基因在起始密码子和终止密码子之间有3个内含子和4个外显子, 4个外显子序列与簇毛麦上已得到的Hv-S/TPK的cDNA序列100%同源。对起始密码子上游序列分析结果表明, 该基因的调控序列中, 含有W-Box、OCS-element等与抗病相关的元件。以TAC克隆为探针与小麦-簇毛麦6VS/6AL易位系有丝分裂中期染色体进行荧光原位杂交(Fluorescence in situ hybridization, FISH), 结果表明含有Hv-S/TPK基因的TAC克隆来自于簇毛麦。     

以可转化人工染色体(TAC)载体为基础的百脉根基因组文库的构建及筛选

可转化人工染色体(transformation-competentartificial chromosome,TAC)载体是具有克隆和转移大片段DNA特征的新型载体,是植物基因克隆和转化的有效工具.该研究把它用于豆科植物百脉根(Lotus japonicus)基因组文库的构建.此文库由1.8×105个克隆组成,平均插入片段大小为15kb左右,约覆盖百脉根基因组6倍.文库保存在12块96孔板中,每个孔中约含150个不同的重组克隆.用与花发育相关的同源基因Ljcen1片段为探针,筛选得到6个阳性克隆,酶切后验证这些阳性克隆,结果表明这些克隆含有同一个基因片段.此基因组文库可直接用于植物转化,为百脉根功能基因组的研究打下基础.     

药用野生稻TAC克隆转化籼稻的体系初探

采用已构建的药用野生稻TAC文库,比较研究4个籼稻品种在不同培养基的诱导率及愈伤组织对潮霉素抗性。通过2种不同诱导方法产生的愈伤组织,将携带药用野生稻大片段DNA的TAC克隆转化到籼稻品种中的结果表明,除了‘粤香占’以外,其它3个品种在心诱导培养基中添加微量B,后,其愈伤诱导率最高。不同品种的潮霉素筛选适合浓度存在差异,其中‘华粳籼74’的适合筛选浓度为50mg．L-1,‘粤香占’为40mg．L-1。用预培养5d的愈伤组织进行遗传转化,在潮霉素筛选之前先以头孢拉定抑菌处理的,容易获得转基因植株。4个品种中以‘粤香占’的抗性愈伤组织筛选率和分化率最高,分别为22．58％和14．86％。分子检测11株转基因水稻的结果表明,其中8株含有抗性标记基因。据此认为,药用野生稻TAC文库在水稻创新育种中可能有一定的利用前景。     

A subnanosecond resolving spectrofluorimeter for the analysis of protein fluorescence kinetics

A spectrofluorometer is described consisting of an excitation source, optics, detector and time resolving electronics. The excitation source consists of a mode-locked Ar ion laser, synchronously pumps a dye laser, followed by a frequency doubling device. The repetition frequency of the U.V. pulses (FWHM some ps) has been reduced by an extra-cavity electro-optical modulator. Provisions have been made in the optical configuration to determine both time-resolved fluorescence spectra and fluorescence anisotropy decay curves. The commercially avialable electronics have been optimized for maximum time resolution. The spectral output of the excitation source is confined between 280 and 310 nm, which encompasses the region for eliciting protein fluorescence. The performance of the complete system has been tested with single lifetime standards line p-terphenyl in cyclohexane or with $\text{N-}\text{acetyl}\text{-}\text{L}\text{-}\text{tryptophanamide}$ in pH 7.5 buffer. Serum albumins from human and bovine sources have been employed as examples for time resolved fluorescence spectra and for the demonstration of anisotropy decay curves. Using these methods protein dynamics in the (sub)nanosecond time region can be directly explored.     

Structural analysis of a Lotus japonicus genome. V. Sequence features and mapping of sixty-four TAC clones which cover the 6.4 mb regions of the genome.

We determined the nucleotide sequences of 64 TAC (transformation-competent artificial chromosome) clones selected from genomic libraries of Lotus japonicus accession Miyakojima MG-20 based on the sequence information of expressed sequence tags (ESTs), cDNAs, genes and DNA markers from L. japonicus and other legumes. The length of the DNA regions sequenced in this study was 6,370,255 bp, and the total length of the L. japonicus genome sequenced so far is 32,537,698 bp together with the nucleotide sequences of 256 TAC clones previously reported. Five hundred forty-eight potential protein-encoding genes with known or predicted functions, 127 gene segments and 224 pseudogenes were assigned to the newly sequenced regions by computer prediction and similarity searches against the sequences in protein and EST databases. Based on the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was genetically localized onto the linkage map of two accessions of L. japonicus, MG-20 and Gifu B-129. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Structural analysis of a Lotus japonicus genome. IV. Sequence features and mapping of seventy-three TAC clones which cover the 7.5 mb regions of the genome.

Using the sequence information of expressed sequences tags (ESTs), cDNAs and genes from Lotus japonicus and other legumes, 73 TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of L. japonicus accession MG-20, and their nucleotide sequences were determined. The length of the DNA sequenced in this study was 7,455,959 bp, and the total length of the DNA regions sequenced so far is 26,167,443 bp together with the nucleotide sequences of 183 TAC clones previously reported. By similarity searches against the sequences in protein and EST databases and prediction by computer programs, a total of 699 potential protein-encoding genes with known or predicted functions, 163 gene segments and 267 pseudogenes were assigned to the newly sequenced regions. Based oil the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was located onto the linkage map of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Structural analysis of a Lotus japonicus genome. I. Sequence features and mapping of fifty-six TAC clones which cover the 5.4 mb regions of the genome.

A total of 56 TAC clones with an average insert size of 100 kb were isolated from a TAC library of the Lotus japonicus genome based on the expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined according to the shot-gun based strategy. The total length of the sequenced regions is 5,473,195 bp. By comparison with the sequences in protein and EST databases and analysis with computer programs for gene modeling, a total of 605 potential protein-encoding genes with known or predicted functions, 69 gene segments, and 172 pseudogenes were identified. The average density of the genes assigned so far is 1 gene/8120 bp. Introns were identified in approximately 78% of the potential genes. There was an average of 3.8 introns per gene and the average length of the introns was 375 bp. DNA markers were generated based on the nucleotide sequences obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of L. japonicus Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

新辅助化疗后保乳手术治疗三阴性乳腺癌的临床疗效分析

目的:探讨不同方案新辅助化疗后行保乳手术治疗三阴性乳腺癌的临床疗效及安全性。方法:收集2006年1月至2015年6月于我院确诊的可手术三阴性乳腺癌患者90例作为研究对象,按照随机数字表法分为观察组及对照组各45例。观察组患者在保乳手术前给予AC-T方案进行新辅助化疗,对照组患者在保乳手术前给予TAC方案进行新辅助化疗。观察并比较两组患者治疗效果、化疗相关不良反应发生情况、术后并发症发生情况以及生活质量的改善情况。结果:观察组总有效率为82.22%,对照组为73.33%;观察组控制率为95.56%,对照组为91.11%,两组总有效率及控制率比较差异无统计学意义(P0.05);观察组患者中性粒细胞减少、胃肠道不良反应及口腔溃疡发生率均低于对照组(P0.05),而两组脱发情况比较差异无统计学意义(P0.05)。两组术后并发症发生率分别为6.67%及11.11%,组间比较差异无统计学意义(P0.05)。观察组患者术后生活质量改善总有效率为86.67%,对照组总有效率为82.22%,两组相比差异无统计学意义(P0.05)。结论:在保乳手术前采用AC-T序贯化疗方案进行新辅助化疗治疗三阴性乳腺癌与TAC方案疗效相当,但不良反应明显减少,值得临床推广应用。     

Engineered marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125: a promising micro-organism for the bioremediation of aromatic compounds

Aims:  The recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 ( P. haloplanktis TAC/ tou ) expressing toluene- o- xylene monooxygenase (ToMO) can efficiently convert several aromatic compounds into their corresponding catechols in a broad range of temperature. When the genome of P. haloplanktis TAC125 was analysed in silico , the presence of a DNA sequence coding for a putative laccase-like protein was revealed. It is well known that bacterial laccases are able to oxidize dioxygenated aromatic compounds such as catechols.
Methods and Results:  We analysed the catabolic features, conferred by recombinant ToMO activity and the endogenous laccase enzymatic activity, of P. haloplanktis TAC/ tou engineered strain and its ability to grow on aromatic compounds as sole carbon and energy sources.
Conclusions:  Results presented highlight the broad potentiality of P. haloplanktis TAC/ tou cells expressing recombinant ToMO in bioremediation and suggest the use of this engineered Antarctic bacterium in the bioremediation of chemically contaminated marine environments and/or cold effluents.
Significance and Impact of the Study:  This paper demonstrates the possibility to confer new and specific degradative capabilities to a bacterium isolated from an unpolluted environment (Antarctic seawater) transforming it into a bacterium able to grow on phenol as sole carbon and energy source.     

The role of a 2-on-2 haemoglobin in oxidative and nitrosative stress resistance of Antarctic Pseudoalteromonas haloplanktis TAC125

The 2-on-2 haemoglobins, previously named truncated, are monomeric, low-molecular weight oxygen-binding proteins that share the overall topology with vertebrate haemoglobins. Although several studies on 2-on-2 haemoglobins have been reported, their physiological and biochemical functions are not yet well defined, and various roles have been suggested. The genome of the psychrophilic Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is endowed with three genes encoding 2-on-2 haemoglobins. To investigate the function played by one of the three trHbs, PhHbO, a PhTAC125 genomic mutant strain was constructed, in which the encoding gene was knocked-out. The mutant strain was grown under controlled conditions and several aspects of bacterium physiology were compared with those of wild-type cells when dissolved oxygen pressure in solution and growth temperature were changed. Interestingly, inactivation of the PhHbO encoding gene makes the mutant bacterial strain sensitive to high solution oxygen pressure, to H₂O₂, and to a nitrosating agent, suggesting the involvement of PhHbO in oxidative and nitrosative stress resistance.