链霉菌负调控因子突变筛选系统的构建

摘要:【目的】构建用于阻遏链霉菌隐性次级代谢基因簇表达的负调控因子筛选的报告系统。【方法】通过“REDIRECT (Rapid Efficient Directed Recombination Time Saving)”技术结合链霉菌温和噬菌体BT1整合酶的体内位点特异性重组技术,对链霉菌中多基因进行无痕敲除。以链霉菌隐性次级代谢基因簇中受阻遏的启动子驱动链霉菌中保守的inoA 构建报告质粒,针对阻遏次级代谢基因簇表达的负调控基因的突变进行检测,以验证报告系统的可行性。【结果】本研究首先通过对天蓝色链霉菌的肌醇从头合成途径关键酶基因inoA,及合成黄色聚酮类隐性抗生素(yellow cryptic polyketide,yCPK)的途径特异性负调控基因scbR2依次进行了无痕敲除,以构建进一步筛选所用的受体菌,再以scbR2阻遏的cpkO启动子控制inoA 的表达构建了报告质粒pIJ8660::PcpkO::inoA。结果显示沉默的cpkO 启动子在突变的受体菌中被激活并使inoA得到了表达,可以使inoA的光秃型突变表型在不添加肌醇的培养基上恢复到产孢的野生型表型。【结论】inoA可以作为新的链霉菌普遍适用的报告基因,可方便地通过表型变化的观察进行筛选,同时可针对性对负调控基因的突变进行检测,可应用于链霉菌隐性抗生素激活的研究。     

茶树脯氨酸转运蛋白基因鉴定及表达分析

脯氨酸转运蛋白在植物体内脯氨酸的分配及响应多种非生物逆境胁迫过程中发挥着重要作用。为明确茶树体内脯氨酸转运蛋白家族情况,该研究从全基因组水平鉴定获得茶树脯氨酸转运蛋白家族成员,进行了系统进化关系、蛋白结构、基因表达特异性等分析。结果表明:(1)茶树中有6个脯氨酸转运蛋白基因,长度为1 326～1 725 bp之间,编码氨基酸数目在441～574 aa之间,蛋白质分子质量在48.5～63.0 kD之间,等电点为8.51～9.41,大部分为碱性蛋白,其结构中含有大量的α-螺旋和自由卷曲,少量的延长链和β-转角结构。(2)亚细胞定位分析结果显示,茶树CsProT1、CsProT2、CsProT4、CsProT5和CsProT6蛋白定位于细胞膜,CsProT3蛋白则定位于高尔基体。(3)CsProTs蛋白中含9～11个典型的跨膜结构域,其三级结构与保守基序特征均与拟南芥高度相似,具有高度的保守性,不同成员间氨基酸序列相似性达40.14%。(4)基因表达特异性分析显示,CsProT1,CsProT2和CsProT3基因在各个组织部位的表达量均较高,CsProT4、CsProT5和CsProT6表达量均较低,且CsProT1基因的表达量最高;除CsProT5基因外,CsProTs蛋白家族的基因均受到NaCl、干旱及冷胁迫的诱导表达。(5)蛋白相互作用分析结果显示,CsProTs蛋白可与脯氨酸氧化酶ERD5,脯氨酸生物合成限速酶P5CS1、P5CS2和δ-吡咯啉-5-羧酸脱氢酶ALDH12A1等脯氨酸合成,转运及降解有关的蛋白相互作用,共同调控茶树体内脯氨酸的含量。研究认为,茶树6个CsProTs蛋白可共同参与茶树体内脯氨酸的转运平衡及对多种非生物逆境胁迫响应的过程。     

流域景观格局与河流水质的多变量相关分析

流域内的景观格局改变是人类活动的宏观表现,会对河流水质产生显著影响,因此明确影响水质变化的关键景观因子,对于深入了解景观对水质的影响机制具有重要的研究价值。选择广东省淡水河流域为研究对象,以2007年ALOS卫星影像以及水质监测数据为基础,运用空间分析和多变量分析方法,分析淡水河流域景观格局与河流水质的相关关系。用包括流域和河岸带尺度的景观组成和空间结构信息的景观指数表征景观格局,用Spearman秩相关分析、多元线性逐步回归模型和典型相关分析(CCA)研究景观指数和水质指标的相关关系。研究结果表明:林地、城镇用地和农业用地占淡水河流域总面积超过90%,其中城镇用地超过20%。多元线性逐步回归分析和CCA结果说明水质指标受到多个景观指数的综合影响,反映了景观格局对水质的复杂影响机制。流域景观格局对河流水质有显著影响,流域尺度的景观指数比河岸带尺度的景观指数对水质影响更大。城镇用地比例是影响耗氧污染物和营养盐等污染物浓度最重要的景观指数,林地和农业用地对水质的影响较小。另外,景观破碎化对pH值、溶解氧和重金属等水质指标有显著影响。CCA的第一排序轴解释了景观指数与水质指标相关性的54.0%,前两排序轴累积能解释景观指数与水质指标相关性的87.6%,前两轴分别主要表达了城市化水平和景观破碎化水平的变化梯度。淡水河流域的景观格局特征从上游到下游呈现出城市—城乡交错—农村的景观梯度,水质变化也对应了这个梯度的变化,说明人类活动引起的流域土地覆盖及土地管理措施变化会对水质变化产生显著影响。     

Establishment of a Genetic Transformation System and Its Application in Thermoanaerobacter tengcongensis

The whole-genome sequence of Thermoanaerobacter tengcongensis,an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China,was completed in 2002.However,in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system.In order to establish such a system,the plasmid pBOLOl containing the replication origin of the T.tengcongensis chromosome and a kanamycin resistance cassette,in which kanamycin resistance gene expression was controlled by the ttel482 promoter from T.tengcongensis,was constructed and introduced into T.tengcongensis via electroporation.Subsequently,the high transformation efficiency occurred when using freshly cultured T.tengcongensis cells without electroporation treatment,suggesting that T.tengcongensis is naturally competent under appropriate growth stage.A genetic transformation system for this strain was then established based on these important components,and this system was proved to be available for studying physiological characters of T.tengcongensis in vivo by means of hisG gene disruption and complementation.     

Purification and biological characterization of halocin C8, a novel peptide antibiotic from<Emphasis Type="Italic"> Halobacterium</Emphasis> strain AS7092

Halocins are bacteriocin-like proteins or peptides produced by many species of the family Halobacteriaceae. Halocin C8, excreted by the Halobacterium strain AS7092, is a single 6.3-kDa polypeptide with an isoelectric point of 4.4, which is sensitive to proteinase K but not to trypsin. Halocin C8 is quite stable, as it can be desalted, boiled, frozen, subjected to organic solvents, and stored in culture supernatant at 4°C or in dH₂O at –20°C for more than 1 year without losing activity. The purification of this halocin was achieved by combination of tangential flow filtration (TFF), Sephadex G50 and DEAE-sepharose chromatography. The N-terminal amino acid sequence was also determined by Edman degradation. Halocin C8 appeared to have a very wide activity spectrum, including most haloarchaea and even some haloalkaliphilic rods. When a sensitive strain of Halorubrum saccharovorum was exposed to halocin C8, the treated cells swelled at the initial stage, the cell wall appeared to be nicked and the cytoplasm was then extruded out, and the whole cell was eventually completely lysed. These results indicate that halocin C8 is a novel microhalocin and its primary target might be located in the cell wall of the sensitive cells.Communicated by W.D. Grant     

Structure and Function of sanV: a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes

A 6.3-kb BamHI- BglII DNA fragment was cloned from cos20 by using chromosome walking strategy. It was partially sequenced with the result that there is a possible ORF of 1272 nucleotides. The ORF designated sanV was deposited in GenBank under accession no. AF469955. Database search indicated that the deduced protein of sanV shows 28% identity and 44% similarity over 405 amino acid residues to the large component (E) of glutamate mutases from Clostridium cochlearium. Gene disruption was performed to study the function of sanV. It was found that sanV disruptants exhibited much poorer inhibition to the test strain than the wild-type S. ansochromogenes 7100. Furthermore, HPLC analysis indicated that the sanV disruptants almost did not produce nikkomycins X and Z, whereas they accumulated new nikkomycins O(x) and O(z), which revealed that sanV is an important gene involved in the biosynthesis of the peptidyl moiety of nikkomycins.     

Susceptibility of dipel-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae) to individual Bacillus thuringiensis protoxins

Dipel-resistant and -susceptible strains of Ostrinia nubilalis (Hübner) were evaluated for larval mortality and growth inhibition when fed diets containing individual Bacillus thuringiensis protoxins. Resistance ratios for four of the protoxins in Dipel (Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa) were 170-, 205-, 524-, and > 640-fold, respectively, considerably higher than the 47-fold resistance to Dipel. The Dipel-resistant strain was 36-fold resistant to Cry1Ba, a protoxin not present in Dipel. Another non-Dipel protoxin, Cry1Ca, did not cause significant mortality for either resistant or susceptible larvae with doses as high as 1.0 mg/ml. In an evaluation of larval growth inhibition, resistance to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ba was significant at concentrations of 0.054 and 0.162 microg/ml. However, growth inhibition with Cry2Aa was not significant at either dose. These data provide information on the spectrum of resistance and cross-resistance to individual Cry protoxins in this strain.     

Cloning, Sequencing, and Function of sanF: A Gene Involved in Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 111-bp DNA fragment related to nikkomycin biosynthesis of Streptomyces ansochromogenes 7100 was obtained with the method of reverse genetics. Then, a 2.2-kb DNA fragment was cloned from the DNA library of S. ansochromogenes 7100 by using the 111-bp fragment as a probe. Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 219-amino acid (aa) protein, and this gene was designated sanF (GenBank Accession No. AF223971). The function of the sanF gene was studied by a strategy of gene disruption, and the resulting sanF mutants lost the ability to synthesize biologically active nikkomycin, indicating that sanF is essential for nikkomycin biosynthesis. Received: 17 April 2000 / Accepted: 23 May 2000