Pi-ta+基因和几丁质酶基因双价基因导入水稻的研究

为了提高‘云资粳41’和‘云资粳43’的抗稻瘟病能力,利用农杆菌介导法将二价表达载体pCAMBIA1300-Pi-ta⁺-Bchi转化到水稻愈伤组织中。经组织培养获得再生苗,再通过氯酚红显色法、PCR检测和抗稻瘟病鉴定法获得抗稻瘟病的转基因植株,为进一步创建持久、广谱抗稻瘟病水稻新材料奠定基础。结果显示:(1)抗性愈伤组织经分化和生根培养后,共获得T⁰代水稻再生苗137株,其中‘云资粳41’14株,‘中花11’82株,‘云资粳43’41株。(2)经氯酚红显色法和PCR对再生苗检测,‘中花11’、‘云资粳41’、‘云资粳43’的转化苗阳性率分别为66%、43%和55%。证明2个外源基因已经整合到水稻基因组中。(3)对转化阳性植株温室接种稻瘟病病菌66b鉴定结果显示,转基因植株较非转基因植株对稻瘟病的抗性明显增强,而且转Pi-ta⁺基因和几丁质酶基因双价的水稻植株比转单价Pi-ta⁺基因或几丁质酶基因的水稻植株抗稻瘟病能力强。（4）氯酚红检测结果存在一定的假阳性,PCR检测结果更真实可靠,但氯酚红显色法方便、快速,结果观察直观,可对大量的转基因植株进行初步筛选。研究表明,转Pi-ta⁺基因和几丁质酶基因双价基因的水稻植株具有更高的抗稻瘟病能力。     

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.     

核糖体bS22、bL37基因的敲除增强分枝杆菌对抗生素的敏感性

近年来,利用冷冻电子显微镜在分枝杆菌(mycobacteria)的核糖体中新发现了两个蛋白：位于30S亚基解码中心附近的bS22和位于50S亚基肽酰转移酶中心附近的bL37。由于这两个蛋白均邻近抗生素与核糖体结合区,推测它们可能会影响相关药物与靶点的结合。因此我们利用同源重组的方法,在野生型耻垢分枝杆菌(Mycolicibacterium smegmatis) mc2155中分别敲除这两个蛋白的编码基因,并测定菌株的生长曲线和对相关抗生素的敏感性。结果表明,与野生型相比,2株敲除株的生长速率均没有显著变化,但菌株ΔbS22相比于野生型对卷曲霉素、卡那霉素、阿米卡星、链霉素、庆大霉素、巴龙霉素和潮霉素B的敏感性增加,菌株ΔbL37相比于野生型对利奈唑胺的敏感性增加,并且这种敏感性的变化通过基因回补均得到恢复。研究结果暗示了核糖体蛋白bS22、bL37作为药物设计靶点的可能性。     

Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: Implications for cdc2+ protein structure and function

Summary The cdc2 ⁺ gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis. Previously the cdc2 ⁺ gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity. Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time. We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughtout the cdc2 protein. Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2 ⁺ activity and regulation. These include regions which may be involved in the interaction of the cdc2 ⁺ gene product with the proteins encoded by the wee1 ⁺, cdc13 ⁺ and suc1 ⁺ genes.     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

The dose effect ofma-l+ andry+ on xanthine dehydrogenase activity inDrosophila melanogaster

Summary Two loci,ma-l ⁺ andry ⁺, necessary for xanthine dehydrogenase activity inDrosophila melanogaster have been studied for dosage effects utilizing deficiencies and duplications induced for this purpose. Comparisons of one, two and three doses ofma-l ⁺ in the female or one and two doses in the male indicate that there is no increase in specific enzyme activity with dose. On the other hand, comparisons of one, two and three doses ofry ⁺ in the male and female reveal an increase in enzyme activity that is roughly proportional to dose. Since dosage ofry ⁺ is limiting, whereas that ofma-l ⁺ is not, the final concentration of xanthine dehydrogenase is shown to depend on the number of doses ofry ⁺.The implications of these findings with respect to the hypothesis of dosage compensation and to the mechanism of control of enzyme and protein concentration are discussed.Operated by Union Carbide Corporation for the U.S. Atomic Energy Commission.     

Identification of the DNA damage-responsive elements of therhp51+ gene, arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51 ⁺ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51 ⁺ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51 ⁺ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51 ⁺ expression.     

Na+、K+、Mg2+、Ca2+和葡萄糖溶液作为授精-激活介质对中华鲟精子受精率的影响

以不同浓度的NaCl、KCl、MgCl2、CaCl2溶液和葡萄糖溶液作为授精介质,研究了中华鲟(Acipenser sinensis)的受精效果.结果显示,适量的阳离子和葡萄糖作为激活授精介质时中华鲟卵受精率都有所提高.在实验设置浓度范围内25 mmol/L NaCI溶液、0.1 mmol/L KCl溶液、1 mmol/L MgCl2溶液、1 mmol/LCaCh溶液和50 mmol/L葡萄糖溶液浓度下,受精率分别可达到最高值,依次为87.72%、86.82%、82.24%、89.76%、80.92%.随着实验浓度继续增加,受精率反而呈下降趋势.结果显示,作为人工配制的中华鲟精子授精一激活介质,最适NaCI溶液浓度在25 mmol/L附近,最适葡萄糖溶液浓度在25 mmol/L附近,最适KCI溶液浓度≤0.1 mmol/L,最适MgCl2溶液浓度≤1 mol/L,最适CaCh溶液浓度≤1 mmol/L.     

不同藻密度下Zn2+ 浓度对萼花臂尾轮虫实验种群增长参数的影响

为了比较不同食物密度下污染物浓度对受试生物的慢性毒性,筛选出以轮虫为受试生物对水环境中Zn2+污染进行监测的敏感指标,在不同斜生栅藻(Scenedesmus obliquus)密度(1.0×106、2.0×106和4.0×106个/m L)下不同浓度(0、0.1、0.3、0.5、0.7、0.9 mg/L)的Zn2+对萼花臂尾轮虫(Brachionus calyciflorus)实验种群增长参数的影响。结果表明,25℃以及1.0×106、2.0×106和4.0×106个/m L藻密度下Zn2+对萼花臂尾轮虫的24 h LC50值分别是6.647、8.102和5.873 mg/L。与各食物密度下的对照组相比,当食物密度为1.0×106个/m L时,各浓度的Zn2+对萼花臂尾轮虫的各种群增长参数均无显著性影响(P0.05)。当食物密度为2.0×106个/m L时,各浓度的Zn2+均显著延长了轮虫的生命期望、世代时间和平均寿命,提高了轮虫的净生殖率;除0.3 mg/L外,其他浓度的Zn2+显著提高了轮虫的种群内禀增长率。当食物密度为4.0×106个/m L时,0.1、0.3和0.7mg/L的Zn2+显著提高了轮虫的种群内禀增长率,0.7和0.9 mg/L的Zn2+显著提高了轮虫的后代混交率。藻密度对轮虫的生命期望、世代时间、净生殖率、种群内禀增长率、平均寿命和后代混交率均有极显著性影响(P0.01),Zn2+浓度对轮虫的生命期望、世代时间、净生殖率、种群内禀增长率和后代混交率均有极显著性影响(P0.01),藻密度和Zn2+浓度之间的交互作用对轮虫的生命期望、种群内禀增长率和后代混交率均有显著性影响(P0.05)。2.0×106个/m L食物密度下,Zn2+浓度与轮虫的生命期望、世代时间、净生殖率和平均寿命之间具有显著的剂量-效应关系;4.0×106个/m L食物密度下,Zn2+浓度与轮虫的后代混交率间也具有显著的剂量-效应关系。     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

A molecular analysis of terminase cuts in headful packaging of Salmonella phage P22

Summary Fragments of DNA molecules of Salmonella phage 22 which represent the molecular termini created by the terminase reaction have been cloned and sequenced. The terminase cleavage separates a headful-sized piece of DNA from the concatemeric precursor; by successful cloning strategy it was shown that the terminase produces blunt ends. The termini of 20 different phage DNA molecules fall into a region located between about 600 and 4000 bp from the pac signal and show a Gaussian distribution. The average terminal redundancy was calculated to be about 2230 by (=5.3%) and is therefore higher than was previously reported. A comparison of the nucleotides flanking the terminal bases of 20 different end clones does not support the suggestion that the terminase recognizes some specific sequence and/or structural information in determining the actual cleavage site.     

Correlation of activity with phenotypes of Escherichia coli partial function mutants of rnh, the gene encoding RNase H

Summary The rnh gene of Escherichia coli encodes RNase H. rnh mutants display at least two phenotypes: (1) they require functional RecBCD enzyme for growth; thus rnh-339::cat recB270 (Ts) and rnh-339::cat recC271 (Ts) strains are temperature sensitive for growth; (2) rnh mutants permit replication that is independent of the chromosomal origin, presumably by failing to remove RNA-DNA hybrids from which extra-original replication can be primed. We report here that manifestation of these two phenotypes occurs at different levels of RNase H function; we have examined partially functional rnh mutants for their in vitro RNase H activity, their ability to rescue viability in recB or recC cells and their ability to permit growth of mutants incapable of using oriC [dnaA (Ts)].     

Cd2+和Pb2+对花翅摇蚊(Chironomus kiiensis)幼虫生长发育的影响及富集效应

河流、湖泊等水生环境中普遍存在的重金属污染破坏水生生态系统并间接威胁人类健康。为探究重金属胁迫下水生昆虫花翅摇蚊（Chironomus kiiensis）生态毒理,测定了重金属Cd²⁺和Pb²⁺胁迫对花翅摇蚊化蛹率和羽化率的影响,检测了摇蚊的口器致畸与富集效应。研究结果表明,Cd²⁺和Pb²⁺影响摇蚊幼虫化蛹和羽化过程,单一重金属离子处理14 d Pb²⁺处理组的化蛹率和羽化率分别为22.22%和8.89%,低于Cd²⁺的化蛹率（25.56%）和羽化率（11.11%）,表现出更强的抑制效应。混合离子1：2和2：1配比处理组化蛹率和羽化率均为11.11%和4.44%,显著低于单一重金属离子胁迫下的化蛹率和羽化率。单一重金属离子及混合离子处理均能导致花翅摇蚊幼虫口器致畸,表现为上颚前齿断裂,中齿和基齿磨损、缺失,下唇板齿部不规则,下唇板边缘齿与中央齿磨损、断裂、增生、缺失。不同重金属离子处理下幼虫口器致畸率不同,并与暴露时间呈正相关,其中1：2配比处理14 d致畸率达到40.61%。重金属离子在摇蚊幼虫体内产生生物富集效应,单一重金属离子处理下的Pb²⁺富集含量7 d至14 d由11.46 mg/kg上升至31.32 mg/kg,不同配比混合离子处理下Pb²⁺富集含量均呈增加趋势,其中1：2配比处理组由15.48 mg/kg上升至42.50 mg/kg,而Cd²⁺在单一重金属与1：1混合离子处理组7 d至14 d的富集含量无显著性变化,2：1配比处理组由14.20 mg/kg下降至9.52 mg/kg,1：2配比由5.85 mg/kg上升至20.99 mg/kg。这些研究结果表明Cd²⁺和Pb²⁺胁迫影响花翅摇蚊幼虫生长发育且口器出现畸型,与重金属在幼虫体内的富集密切相关,为研究重金属对水生生态系统多重效应提供了理论依据。     

长叶红砂液泡膜Na~+/H~+逆向转运蛋白基因的克隆及表达特性

为研究长叶红砂(Reaumuria trigyna)离子转运分子机制,利用RT-PCR和RACE技术,克隆到其液泡膜Na+/H+逆向转运蛋白基因(NHX1)的全长cDNA片段,命名为RtNHX1(NCBI序列号为KR919802)。结果表明:RtNHX1的cDNA片段全长2 622bp,开放阅读框1 662bp,5′非编码区509bp,3′非编码区451bp,编码553个氨基酸,推测分子量为60.91kD。该蛋白含有12个跨膜结构域,为疏水蛋白,与其他植物液泡膜Na+/H+逆向转运蛋白NHX1的亲缘关系较近。实时荧光定量PCR对其在NaCl胁迫下的表达检测显示,不同时间和不同浓度NaCl胁迫下,RtNHX1表达量变化均呈先升高后降低趋势,在100mmol/L NaCl胁迫6h和200mmol/L NaCl胁迫后达到最高,表达量分别超过或约是对照的3倍,一定程度反应出RtNHX1参与长叶红砂的盐胁迫应答,是该植物离子转运体系的重要元件。     

西伯利亚白刺质膜Na+/H+逆向转运蛋白基因NsSOS1的分离及表达分析

采用同源克隆技术分离了西伯利亚白刺(Nitraria sibirica)质膜Na~+/H~+逆向转运蛋白基因NsSOS1,并对其在不同胁迫条件下的表达特性进行了分析。NsSOS1包含3 516bp开放阅读框(ORF),编码1 171个氨基酸,蛋白分子量为128.34kD。生物信息学分析显示,NsSOS1包含12个跨膜结构域,具有植物SOS1蛋白的保守结构域。系统发育分析表明,NsSOS1与其他植物质膜Na~+/H~+逆向转运蛋白处于同一个次级分化群,与锦葵科海滨锦葵KvSOS1亲缘关系较近。实时荧光定量RT-PCR分析显示,NsSOS1基因在西伯利亚白刺的根和叶中表达量较高;其表达受到非生物胁迫(NaCl、低温、干旱)和外源激素(MeJA和GA)的诱导,表明NsSOS1基因在西伯利亚白刺抵御逆境胁迫过程中发挥重要作用。     

Effects of heavy metals Cd2+, Pb2+ and Zn2+ on DNA damage of loach Misgurnus anguillicaudatus

The effects of heavy metals Cd²⁺, Pb²⁺ and Zn²⁺ at 0.05, 0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1–35 days exposure were examined by single cell gel electrophoresis (SCGE). For each test group, 20 loaches with similar body size (5.17–7.99 g; 11.79–13.21 cm) were selected and kept in aquaria with dechlorinated water at (22±1)°C and fed a commercial diet every 48 h. According to the percentage of damaged DNA with tail and its TL/D (tail length to diameter of nucleus) value, the relationship between DNA damage degree and heavy metal dose and exposure time was determined. Results showed that the percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time. The highest percentage (84.85%) of damaged DNA was shown in 5.0 mg/L Zn²⁺ group after 28 days exposure and the biggest TL/D value (2.50) in all treated groups after 35 days exposure. During the first treated week, the damnification of DNA was mainly recognized as the first level, after that time, the third damaged level was mostly observed and the percentage of damaged DNA was beyond 80%. The joint toxic effects among Cd²⁺, Pb²⁺ or Zn²⁺ revealed much complexity, but it generally displayed that the presence of Cd²⁺ could enhance the genotoxicity of Pb²⁺ or Zn²⁺. In conclusion, the results suggested that there was a significant time-and dose-depended relationship between the heavy metal and DNA damage in hepatopancreas of loach, and SCGE could represent a useful means to evaluate the genotoxicity of environmental contamination on aquatic organisms. __________ Translated from Acta Hydrobiologica Sinica, 2006, 30(4): 399–403 [译自: 水生生物学报]