裸燕麦EMS突变体库筛选与分析

燕麦是重要的粮饲兼用作物,构建燕麦EMS突变体库对燕麦功能基因组学研究和遗传改良有重要意义。本试验利用化学诱变剂甲基磺酸乙酯(EMS,ethyl methane sulfonate)处理燕麦品种花早2号,获得了4083株M1材料;对其中2000个单株种植了M2株行,进行全生育期调查,鉴定其表型变化;对2份黄化苗突变材料种植了M3家系,进行相关突变性状的稳定性验证。结果表明,燕麦经EMS处理后代变异巨大,在M2发现表型突变材料196份,变异率为9.8%,变异类型非常丰富,包括幼苗习性、叶片性状、分蘖、株高、穗部形态及成熟期等突变株系。M3证实突变的黄化苗特性可以稳定遗传。本研究建立了燕麦EMS诱变体系,获得的燕麦变异类型丰富,为燕麦功能基因组学研究和燕麦遗传改良奠定了材料基础。     

低双乙酰啤酒酵母菌株BEZ112的选育

以啤酒酿造生产菌株啤酒酵母(Saccharomyces cerevisiae)FB作为出发菌株,用甲基磺酸乙酯(EMS)诱变,经分离筛选得到一株优良的啤酒酵母菌株BEZ112。该菌株的絮凝性、发酵度、酒精度、发酵液的总酯和总高级醇的含量等特性保持了亲株的优良性状。但以12°Bx麦芽汁为培养基用500mL三角瓶在12℃下发酵,该菌株发酵至第4d,发酵液中的双乙酰含量达到峰值(0.291mg/L),比出发菌株FB发酵4d的峰值降低了30％,发酵至第8d,BEZ112发酵液中的双乙酰含量比出发菌株FB的降低了23％。以12°Bx麦芽汁为培养基用500L罐在12℃下发酵8d,BEZ112发酵液中的双乙酰含量(0.091mg/L)比出发菌株FB的(0.124mg/L)降低了27％。发酵得到的啤酒口感纯正清爽。     

EMS诱变技术在植物育种中的研究进展

甲基磺酸乙酯（Ethyl methane sulfonate,EMS）是一种常用的化学诱变剂,能诱发产生高密度的系列等位基因点突变。在当前种质资源极为匮乏,基因资源日益枯竭的状况下,采用EMS诱发突变技术创造有用基因资源具有极其重要的意义。本文通过对EMS的诱变原理和技术要领、应用实例、以及该技术在现代分子生物学中的应用前景加以阐述,对EMS诱变技术在农业生产中的应用具有重要作用。     

A new approach to random mutagenesis in vitro

Random mutagenesis is a powerful tool for studying the effects of a large number of permutations of a particular DNA sequence and its encoded products. Here we describe a new strategy of conducting in vitro random mutagenesis using ethyl methane sulfonate (EMS). The Bacillus aprN18 gene, coding for a serine protease with fibrinolytic activity, was used as a target gene. To study the mutations of the coding region, rather than the whole plasmid, the 1.4 kb gene fragment was cut out from an expression plasmid and treated with 10 mM EMS at 37 degrees C for 1 h. The treated fragment was then ligated back into the original expression vector and a library of random mutants was constructed in a protease-deficient Bacillus subtilis strain. A plate assay-based screening method was used to select for mutant clones with altered enzyme activity, and the change of activity was then confirmed by a semi-quantitative enzyme assay using liquid culture supernatant. The inserts of five clones with altered enzyme activity were randomly chosen for sequencing analysis. Among the point mutations detected, GC --> AT transition accounts for 42.1%, AT --> GC transition 34.2% and GC/CG transversion 23.7%, respectively. To our knowledge this is the first application of EMS for in vitro mutagenesis of a defined DNA sequence.     

鱼腥草组培体系优化及耐低温突变体的筛选

为解决豫北地区没有鱼腥草栽培,缺乏耐低温能越冬材料的需求,本实验以鱼腥草茎段为外植体进行不同植物生长调节剂及其浓度的组织培养诱导侧芽发生,优化了鱼腥草侧芽组织培养体系;使用不同浓度的甲基磺酸乙酯（EMs）诱变侧芽,诱导耐低温突变体,通过测定诱变材料可溶性糖含量、过氧化氢酶（CAT）和超氧化物歧化酶（SOD）活性变化鉴定材料耐寒性。结果表明茎段侧芽诱导的最佳植物生长调节剂组合培养基MS＋6-BA2．0mg·L-1＋NAA0．2mg·L-1;诱变处理的最佳组合为0．4％EMS处理6h,获得8株耐低温突变体;侧芽生根的最佳浓度组合为1／2MS＋NAA0．8mg·L-1。     

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.     

芦苇耐盐变异植株及其细胞学鉴定

用甲基磺酸乙酯（ＥＭＳ）处理芦苇（Ｐｈｒａｇｍ ｉｔｅｓｃｏｍ ｍ ｕｎｉｓ Ｔｒｉｎ．）胚性愈伤组织。从处理后的愈伤组织诱导获得芦苇耐盐变异植株Ｒ５００２－１２。变异植株能在含有１％ ＮａＣｌ的ＭＳ培养基上生长。细胞学检查变异植株是混倍体,染色体数目变异范围在１００至３３ 之间。分蘖植株具有相似的形态学及染色体变异特性     

A Salt Tolerant Variant of Phragmites communis and Its Cytological Characterization

A salt tolerant varaint, R5002-12 of Phragmites communis Trin. has been obtained by treating the embryogenic callis with ethyl methane sulfonate. It could grow in MS medium containing 1% W/V sodium chloride. The plant regenerated was mixoploid with chromosome number ranging from 100 to 33 and its tillers showed similar variation in morphology and chromosome.     

Influence of high-pressure homogenization,ultrasonication, and supercritical fluid on free astaxanthin extraction from β-glucanase-treated Phaffia rhodozyma cells

In this study astaxanthin production by Phaffia rhodozyma was enhanced by chemical mutation using ethyl methane sulfonate. The mutant produces a higher amount of astaxanthin than the wild yeast strain. In comparison to supercritical fluid technique, high-pressure homogenization is better for extracting astaxanthin from yeast cells. Ultrasonication of dimethyl sulfoxide, hexane, and acetone-treated cells yielded less astaxanthin than β-glucanase enzyme-treated cells. The combination of ultrasonication with β-glucanase enzyme is found to be the most efficient method of extraction among all the tested physical and chemical extraction methods. It gives a maximum yield of 435.71 ± 6.55 µg free astaxanthin per gram of yeast cell mass.     

The subunit interface of the Escherichia coli ribosome: Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane

The 70 S ribosomes of Escherichia coli were treated with 2-iminothiolane with the resultant addition of 110 sulfhydryl groups per ribosome. The modified ribosomes were oxidized to promote disulfide bond formation, some of which formed intermolecular crosslinks. About 50% of the crosslinked 70 S ribosomes did not dissociate when exposed to low concentrations of magnesium in the absence of reducting agent. Dissociation took place in the presence of reducing agents, which indicated that the subunits had become covalently linked by disulfide linkages. Proteins extracted from purified crosslinked 70 S ribosomes were first fractionated by polyacrylamide/urea gel electrophoresis. The proteins from sequential slices of these gels were analyzed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Monomeric proteins derived from crosslinked dimers appeared below the diagonal containing non-crosslinked proteins, since the second electrophoresis, but not the first, is run under reducing conditions to cleave the crosslinked species. Final identification of the proteins in each dimer was made by radioiodination of the crosslinked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of non-radioactive total 70 S proteins as markers. This paper describes the identification of 23 protein dimers that contained one protein from each of the two different ribosomal subunits. The proteins implicated must have some part of their structure in proximity to the other ribosomal subunit and are therefore defined as “interface proteins”. The group of interface proteins thus defined includes 50 S proteins that are part of the 5 S RNA: protein complex and 30 S proteins at the initiation site. Correlations between the crosslinked interface proteins and other functional data are discussed.     

Phosphorylation in isolated chloroplasts coupled to dichlorophenyldimethylurea-insensitive silicomolybdate reduction

1. The electron transport in isolated chloroplasts with silicomolybdate as electron acceptor has been reinvestigated. The silicomolybdate reduction has been directly measured as ΔA₇₅₀ or indirectly as O₂ evolution (in the presence or absence of ferricyanide).2. Silicomolybdate-dependent O₂ evolution is inhibited to a similar extent by 3-(3,4-dichlorophenyl) 1, 1-dimethylurea (DCMU) or dibromothymoquinone (DBMIB), indicating the existence of two different sites of silicomolybdate reduction: one before the DCMU block (i.e. at Photosystem II) and one after the DBMIB block (i.e. at Photosystem I).3. Silicomolybdate-dependent O₂ evolution is coupled to ATP synthesis with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 1.0 to 1.1. The presence of ferricyanide inhibits this ATP synthesis ($\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio then is about 0.3).4. Silicomolybdate-dependent O₂ evolution is also coupled to ATP-synthesis in the presence of DCMU with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 0.6–0.8 characteristic of Site II; in this case the electron transport itself is not affected by uncouplers or energy-transfer inbihitors.5. The data are interpreted as a further demonstration that the water-splitting reaction is responsible for the conservation of energy at Photosystem II.     

E-cadherin和KLF 4表达对胃癌侵袭转移的作用

观察E-cadherin,Krüppel-like factor 4(KLF4)蛋白在胃癌和正常胃黏膜组织中的表达,分析其与胃癌浸润、转移的关系.应用免疫组织化学SP法检测84例手术切除的胃癌标本及对应正常胃黏膜组织中E-cadherin,KLF4蛋白的表达.各指标之间相关因素的差异性比较采用χ2检验,E-cadherin,KLF4相关性研究采用Spearman相关分析.结果显示,与正常胃组织相比,E-cadherin、KLF4蛋白在胃癌组织中均呈低表达或者缺失(分别42.9%vs.95.24%,8.3%vs 81%,P<0.05).E-cadherin、KLF4蛋白的阳性表达率与组织分级(P<0.05)、肿瘤浸润深度(P<0.05)、淋巴转移(P<0.05)明确相关.Spearman相关分析显示KLF4蛋白与E-cadherin蛋白的表达呈正相关(P<0.05).因此,E-cadherin,KLF4蛋白水平低表达可能与胃癌浸润和转移有关,而联合检测更能有效判断胃癌这一生物学行为.     

An expeditious synthesis of 4-hydroxy-2E-nonenal (4-HNE), its dimethyl acetal and of related compounds

The facile one step synthesis of 4-hydroxy-2(E)-nonenal and its dimethyl acetal via a cross-metathesis reaction between commercially available octen-3-ol and acrolein or its dimethyl acetal is reported. The method was extended to the synthesis of C₆ and C₁₂ 4-hydroxy-2(E)-enals, their dimethyl acetal and of the 4-hydroxy-2(E)-nonenoic acid (4-HNA).     

Mutagenicity of hycanthone in drosophila: Additional results and a comparison with some analogs

The data reported in this paper extend earlier results on the effects of hycanthone in Drosophila. The main findings are the following. (1) A refined brood-pattern analysis of hycanthone-induced sex-linked recessive lethals confirmed the specific sensitivity of mid- and late spermatids. Injection of young males (0–20 h old) did not cause a shift in the brood pattern, but tended to produce higher rates of recessive lethals than injection of 4-day-old males, although the difference was not significant. (2) An autosomal recessive lethal test (chromosome 2) similarly showed a low sensitivity of premeiotic stages. (3) Feeding of hycanthone was much less effective than injection. This difference was not observed for the methyl analog lucanthone. From the observation that hycanthone- and lucanthone-induced mutations exhibited different germ-cell-stage sensitivity patterns, it was concluded that lucanthone does not (at least not exclusively) act via metabolic activation to hycanthone. (4) After injection, the hycanthone analogs IA-4-N-oxide and IA-4-N-oxide were marginally mutagenic. (5) It was shown previously that hycanthone was ineffective in producing breakage events, in Drosophila. In this report, hycanthone is shown to be weakly active in inducing ring-X chromosome loss. This emphasizes the relat ive sensitivity of the ring-X-loss test, in comaprison with the tests that etect translocations or dominant lethals.     

蛋白质翻译中的反转运

在蛋白质的翻译过程中,氨酰-tRNA进入核糖体,解密mRNA上的一个密码子,并带着mRNA向其5'的方向运动,直到空载的tRNA离开核糖体,整个过程tRNA在核糖体内始终沿着一个方向运动.但随着LepA(EF4)蛋白的发现和其功能的明确,tRNA在核糖体内的新运动形式--"反转运"被揭示,即tRNA带着mRNA倒退一步,向其3'的方向运动.通过对tRNA反向运动生理意义的研究,引发了对蛋白质翻译调控的深入思考.     

适合高产铁载体细菌筛选、检测体系的改进与探析

采用pH6．8的磷酸缓冲液代替通用CAS固体检测培养基中的MM9缓冲体系,不加哌嗪二乙醇磺酸,得到颜色稳定、检测效果明显、配制方便,适合高产铁载体细菌筛选与检测的新型检测平板。根据CAS检测液连续吸收光谱比较分析结果,将CAS液体定量检测体系中的检测波长由630nm改为680nm,可以克服OD630读数偏高、易受干扰的问题,而且OD680与铁载体浓度线性关系不变,但采用OD680检测不同细菌产铁载体能力的差异更为明显,因此提高了CAS定量检测的灵敏度,更适合高产铁载体细菌筛选与检测。     

Carbon balance of a European mountain bog at contrasting stages of regeneration

Carbon dioxide and methane (CH4) fluxes were measured in a cutover bog of the Jura Mountains (France) together with biotic and abiotic variables for two entire vegetation periods in order to compare the carbon balance of the bog at three stages of regeneration. Among all factors, air temperature and vegetation index (including leaf area of vascular plants, bryophyte density and bryophyte desiccation) were the two main determinants of ecosystem respiration and gross photosynthesis at light saturation. During 2004 and 2005, the vegetated plots acted as carbon sinks. Net carbon exchange ranged between 67 and 166 g C m(-2) yr(-1) for the Eriophorum-dominated plots and between 93 and 183 g C m(-2) yr(-1) for the Sphagnum-dominated plots. The bare peat plots represented a net carbon source (between -19 and -32 g C m(-2) yr(-1)). Methane fluxes accounted for a very small part of the total carbon efflux (< 2%). The recovery of vegetation in our naturally regenerating bog was beneficial for the carbon sequestration after the relatively short period of 20 yr.     

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5'', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.     

Specific labelling of the active site of the phosphate translocator in spinach chloroplasts by 2,4,6-trinitrobenzene sulfonate

Phosphate transport across the chloroplast envelope is rapidly inactivated by the amino-group reagent 2,4,6-trinitrobenzene sulfonate. Subsequent exposure to [³H]NaBH₄ leads to an incorporation of the trinitrophenyl moiety into envelope membrane preparations. From the membrane proteins only a polypeptide with 29000 dalton molecular weight is labelled. The inactivation of phosphate transport and the incorporation of radioactivity are both specifically reduced by the presence of substrates.The results lead to the conclusion that a polypeptide with a molecular weight of 29000 dalton and containing a lysyl residue at the substrate binding site is involved in the phosphate translocator function.