水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用in vivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

甲基营养菌MB200中mutS的缺失及高浓度甲醇和甲醛诱变

【背景】甲基营养菌(Methylobacterium)是一类能够以单碳或非C-C键低碳化合物(如甲烷、甲醇、甲醛等)为底物生长,并可生产多种代谢产物如氨基酸、工业酶和辅助因子、多羟基烷酸酯(polyhydroxyalkanoates,PHA)、多糖和类胡萝卜素等的革兰氏阴性细菌。【目的】通过突变甲基营养菌MB200的mutS基因,在胁迫条件下定向诱导,以获得可以耐受高浓度甲醇和甲醛的生产菌株。【方法】利用三亲本结合构建mutS基因缺失的高突变菌株MB200sTB,逐步提升培养液中甲醇、甲醛的浓度进行定向诱导突变,对获得的高耐受性突变株进行回补,分析菌株的生长情况。【结果】构建了mutS基因的缺失突变体MB200sTB,并且得到了高耐受甲醇和甲醛的菌株MB200sHBc和MB200sHBq。MB200sHBc与野生株MB200相比其甲醇耐受性得到了极显著的提高,甲醇耐受浓度从8g/L提升到44g/L,但生长量不受影响。MB200sHBq在以甲醛为0.45g/L的碳源条件下,生长量相较于野生型MB200提高了1.69倍。【结论】通过定向诱导缺失mutS基因的突变体,可获得具有生产应用潜力的...     

水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用invivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

硝磺草酮抗性菌株的筛选及抗性基因的克隆表达

【目的】从采集的土壤中筛选出硝磺草酮的抗性菌株,并从中克隆对羟苯基丙酮酸双加氧酶抗性基因。【方法】以酪氨酸为唯一碳源,采用富集培养法筛选分离硝磺草酮抗性菌株,利用16S rRNA基因序列分析对菌株进行初步鉴定。通过PCR扩增获得其HHPD基因序列,构建pETH4表达载体并在大肠杆菌Escherichia coli BL21(DE3)中进行异源表达。通过检测色素在440 nm处的吸收值分析菌株E. coli BL21(DE3)-pETH4对硝磺草酮的抗性特性。【结果】在含10 mmol/L硝磺草酮和1 g/L酪氨酸的选择培养基上,分离得到7株硝磺草酮抗性细菌,1株为不动杆菌属,2株为无色杆菌属,4株为假单胞菌属。从抗性最佳的Pseudomonas sp. AM-H4中扩增得到HPPD的基因片段为1 056 bp,其序列与Acinetobacter baumannii基因组中HPPD的基因序列相似性达到99%,341位点由天冬氨酸突变为丙氨酸。HPPD基因在大肠杆菌中实现异源表达,蛋白分子量大小约40 kD。菌株E. coli BL21(DE3)-pETH4在40 μmol/L硝磺草酮酪氨酸LB培养基中的色素吸收值显著降低,能够耐受高于200 μmol/L的硝磺草酮。【结论】克隆获得的HPPD具有良好的硝磺草酮抗性,将在新除草剂抗性作物选育中有一定的应用潜力。     

甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达

八氢番茄红素脱氢酶( CrtI)催化八氢番茄红素经过4次脱氢合成番茄红素,或者经过3次脱氢合成链孢红素,在类胡萝卜素的生物合成中发挥重要的作用.以甲基营养菌Methylobacterium sp MB200为原始菌株,首先采用转座子突变技术构建部分突变体库共11552株,筛选得到33株颜色发生变化的目的突变体,随后利用分子克隆技术从目的突变体中获得crtI基因的完整ORF,长为1539 bp,编码512个氨基酸.与来自M.populi BJ001、M.chloromethanicum CM4和M.extorquens AM1的crtI一致性均为93％.将crtI与载体pCM80连接得到重组质粒pCM80-crtI,导入原始菌株中得到重组菌MB200/pCM80-crtI.测定原始菌株与重组菌株的CrtI酶活,结果发现,重组菌株CrtI的酶活与原始菌株相比约提高了40％.实验结果为完善甲基营养菌中类胡萝卜素的生物合成代谢途径提供了理论参考.     

ht-Pam基因在山羊β-酪蛋白基因座定位整合的研究

利用体细胞基因打靶与核移植技术制备动物乳腺生物反应器是当今转基因定位整合表达的一种新技术。分别克隆山羊的β-酪蛋白基因5′调控区的6.3kb片段,外显子7、外显子8和9三个基因片段,并与克隆的人tPA突变体cDNA一起构建了含有neo和tk正负筛选标记基因的β-酪蛋白基因打靶载体P^GBC4tPA,并验证了neo基因、tk基因以及Cre-LoxP系统的有效性。将线性化的P^GBC4tPA通过电转染整合到山羊胎儿成纤维细胞基因组中,利用G418和GANC进行抗性细胞克隆的药物筛选,初步获得抗性细胞克隆244个,PCR检测后获得阳性细胞克隆31个,其中初步验证2个细胞克隆转植基因整合位点重组后的基因序列正确,并且该细胞克隆能够有效扩增。这为下一步基因打靶体细胞核移植制备山羊乳腺生物反应器奠定了基础。     

兽疫链球菌中与透明质酸合成相关新基因的高通量筛选及克隆

【目的】兽疫链球菌(Streptococcus equi subsp. zooepidemicus)中透明质酸主要的生物合成途径和相关基因已经被研究得比较透彻,探究一种挖掘与透明质酸合成相关新基因的策略。【方法】利用自杀质粒pSET4s::sacB在宿主基因组中的随机整合作用,筛选具有表型差异突变菌株构建突变体库,进一步利用连接介导PCR (Ligation mediated PCR,LM-PCR)方法和全基因组重测序,检测质粒整合位点,通过基因无痕敲除和回补实验验证插入位点。【结果】构建了包含150株具有表型差异突变株的突变体库;以荚膜合成能力缺失的1号突变株(M1)作为基础研究对象,检测到自杀质粒整合到基因组458 960位点上,破坏了编码塔格糖-6-磷酸激酶的lacC基因;无痕敲除lacC基因得到ΔlacC,表型分析发现ΔlacC表现为粘性荚膜特性;进一步全基因组重测序发现,除了lacC基因位点存在插入突变,206 613位点存在碱基G缺失,导致编码透明质酸合成酶的hasA基因发生移码突变,且回补hasA基因后,M1恢复粘性荚膜合成能力。【结论】M1突变株粘性荚膜合成能力的缺失由hasA基因功能缺失引起,与lacC基因功能缺失无关。初步建立了兽疫链球菌中高通量筛选与透明质酸合成相关新基因的策略,为今后挖掘新基因奠定了基础。     

阴沟肠杆菌B8拮抗活性基因‘admA’及上游调控序列的克隆与功能鉴定

为了阐明水稻白叶枯病拮抗菌阴沟肠杆菌B8的作用机理,文章采用转座子标签法和染色体步移技术克隆到突变株B8B中Tn5插入位点周边拮抗活性相关片段,并通过基因敲除验证了获得的拮抗相关片段admA’上游调控序列的功能。以转座子中Kan抗性基因为标签,克隆了B8B菌株中Tn5插入位点左侧2 608 bp序列,经两次染色体步移得到Tn5插入位点右侧的2 354 bp序列。序列拼接后获得B8菌株拮抗相关序列4 611 bp的Bcontig。生物信息学分析显示该序列含有7个ORF,分别对应于3-磷酸甘油醛脱氢酶(GADPH)基因的部分编码区、2个LysR家族转录调控因子、弧菌假设蛋白VSWAT3-20465及成团泛菌(Pantoea agglomerans)andrimid生物合成基因簇的admA、admB和部分admC基因序列。B8B菌株Tn5插入分别位于同源于弧菌假设蛋白的anrPORF及‘admA’基因上游200 bp和894 bp处。通过同源重组技术,借助敲除质粒pMB-BG,获得拮抗活性消失的突变株B-1和B-3。结果表明B8B突变株中Tn5的插入可能影响了anrP蛋白的转录和表达,进而调控拮抗物质编码基因簇的生物合成。B8菌株中拮抗物质相关基因是类似于andrimid生物合成基因簇的基因家族,其上游调控区对该抗生素的生物合成具有重要的作用。     

解淀粉芽胞杆菌启动基因的克隆及其对地衣杆菌α-淀粉酶基因的调控

用大肠杆菌启动基因探针质粒pHE5克隆了解淀粉芽胞杆菌的启动基因。供体菌DNA的HindⅢ片段与pHE5重组后转化大肠杆菌,获得一批带启动基因的抗四环素转化子,其中四株的抗性达到200μg/mL,从这四株高抗性转化子中提取质粒,并对其中的一个重组质粒pAE23的插入片段进行限制性图谱分析和缺失研究,获得了一个缺失了部份片段,四环素抗性仍达到200μg/mL的衍生质粒pAED23,经酶切分析证明其上的启动基因位于0.8kb的EcoR Ⅰ—HindⅢ片段上。点杂交分析证实该片段来自解淀粉芽胞杆菌的DNA。将地衣杆菌的α-淀粉酶基因亚克隆至pAED23启动基因下游的HindⅢ位点上能增强该基因在大肠杆菌中的表达。     

来自西尔斯山羊草的抗小麦白粉病基因Pm57抗性丧失突变体的筛选与鉴定

小麦近缘种属来源的抗白粉病基因是培育小麦抗病品种,防治白粉病危害的最重要基因来源。Pm57是位于西尔斯山羊草2S^s#l染色体长臂上的一个外源基因,对小麦白粉病具有苗期和成株期广谱抗性。为了创制Pm57白粉病抗性丧失突变体,利用基于基因突变体的植物抗病基因克隆新兴技术分离Pm57基因,选用0.625%的甲基磺酸乙酯(EMS)对1万粒小麦-西尔斯山羊草Pm57易位系89(5)69种子进行了诱变处理,M1大田密播种植,收获了1598个M2可育株系。初步对其中300个M2株系进行苗期白粉病抗性接种鉴定,并利用2个Pm57基因特异分子标记X2L4g9P4/HaeⅢ和X284274及小麦全国区试品系DUS测试所用的42对SSR核心引物对Pm57抗性丧失突变体进行鉴定,筛选出来自27个M2株系的真实抗性丧失突变体70个,Pm57基因抗性丧失突变体频率达到9.0%。本研究所获得的白粉病抗性丧失突变体为Pm57基因的后续克隆与抗白粉病分子机理研究提供了重要的材料基础。     

Inactivation of Metabolic Genes Causes Short- and Long-Range dys-Regulation in Escherichia coli Metabolic Network

The metabolic network in E. coli can be severely affected by the inactivation of metabolic genes that are required to catabolize a nutrient (D-galactose). We hypothesized that the resulting accumulation of small molecules can yield local as well as systemic effects on the metabolic network. Analysis of metabolomics data in wild-type and D-galactose non-utilizing mutants, galT, galU and galE, reveal the large metabolic differences between the wild-type and the mutants when the strains were grown in D-galactose. Network mapping suggested that the enzymatic defects affected the metabolic modules located both at short- and long-ranges from the D-galactose metabolic module. These modules suggested alterations in glutathione, energy, nucleotide and lipid metabolism and disturbed carbon to nitrogen ratio in mutant strains. The altered modules are required for normal cell growth for the wild-type strain, explaining why the cell growth is inhibited in the mutants in the presence of D-galactose. Identification of these distance-based dys-regulations would enhance the systems level understanding of metabolic networks of microorganisms having importance in biomedical and biotechnological research.     

Isolation and enzyme determination of Candida tropicalis mutants for DCA production

Techniques, named two-step enrichment and double-time replica-plating method (TEDR), are described that allow a mutated population of Candida tropicalis to be enriched efficiently for mutants deficient in the alkane degradation pathway (Alk(-)) and to be selected easily for mutants increasing in the DCA (dicarboxylic acids) excretion pathway. After C. tropicalis was mutated with ethyl methane sulphonate and ultraviolet, the Alk(-) mutants were enriched (the first step enrichment, up to eightfold in one round of enrichment) by treatment with nystatin in medium SEL1-1. The mutagen-treated cells were then cultured in medium YPD containing chlorpromazine for further enriching (the second-step enrichment, up to threefold in one round) the mutants with an increasing capacity of alpha- and omega-oxidation. On the other hand, the Alk(-) mutants were readily isolated by the SEL1 replica-plating method by using alkane or glucose as the sole carbon source. A total of 43 Alk(-) mutants were isolated from 2x10(8) mutagen-treated cells. In the following steps, by using SEL2 replica plating, the screening studies showed that of the 43 Alk(-) mutants, 11 strains could accumulate DCA greatly from alkane, and strains 1-12 and 1-3, especially, could produce nearly three times as much DCA as the wild-type organism could. The results showed that the strains had more cytochrome P450 activity and a higher converting capacity of alkane.     

Assessment of the Toxicity of CuO Nanoparticles by Using Saccharomyces cerevisiae Mutants with Multiple Genes Deleted

To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P < 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles.     

Mutants of yeast with altered oxidative energy metabolism: selection and genetic characterization

Isolation of a series of mutants, characterized by decreased ability to utilize nonfermentable carbon sources for growth and presence of all cytochromes, is reported. A total of 161 mutants, showing deficient growth on glycerol but able to reduce 2,3,5-triphenyltetrazolium chloride, were isolated, purified, and characterized by ability to grow on various carbon sources. Mutants showing decreased growth were examined by low-temperature spectroscopy, and the 35 strains shown to possess all cytochromes were retained for further studies. These strains were characterized by growth on various nonfermentable carbon sources, relative yield on glucose medium, and respiration (Q(O2)) of glucose and ethyl alcohol. Genetic studies revealed that at least 19 of the 35 mutants are the result of mutation in single nuclear genes. Furthermore, at least 11 complementation groups are represented among these 19 mutants. Mutants within two complementation groups were shown to be very similar in various properties. These studies demonstrate that a large number of nuclear genes control oxidative energy metabolism and that the characteristics of mutants of the general class are extremely diverse.     

Carbohydrate Metabolism Mutants of a Bacillus Species

Mutants which could not utilize d-gluconate, l-arabinose, sorbitol, pyruvate or l-glutamate as a sole carbon source and which required shikimic acid for their growth were isolated. Characterization of these mutants by the patterns of carbohydrate utilization revealed that various kinds of carbohydrate metabolism mutants including those of the non-oxidative limb of the pentose phosphate pathway were isolated.

Ability of inosine formation of these mutants and transformants from them was investigated. Consequently, slightly improved strains were found among transformants in comparison with the parent strain.     

Correlation between the serine sensitivity and the derepressibility of the ilv genes in Escherichia coli relA- mutants.

Summary Upon addition of excess one carbon metabolites (including serine) bacteria stop growing because of isoleucine starvation. After such treatment stringent bacteria rapidly resume normal growth whereas relaxed mutants remain unable for some time to grow. We show here that this is due to a lack of derepressibility of ilv genes after the starvation period. Results are also presented which show that RNA polymerase structural mutants may be selected among the clones resistant to a mixture of serine, methionine and glycine, in relA ^-strains. Finally circumstancial evidence suggests that the one carbon metabolism may be involved in a process controlling isoleucine metabolism.Abbreviations: Throughout this work we have represented the mixture of amino acids serine, methionine and glycine (1 mM each) by the letters SMG. Para amino benzoic acid represented by the letters PABA     

Utilization of γ-Aminobutyric Acid as the Sole Carbon and Nitrogen Source by Escherichia coli K-12 Mutants

Wild-type strains of Escherichia coli K-12 cannot grow in media with gamma-aminobutyrate (GABA) as the sole source of carbon or nitrogen. Mutants were isolated which could utilize GABA as the sole source of nitrogen. These mutants were found to have six- to ninefold higher activities of gamma-aminobutyrate-alpha-ketoglutarate transaminase (EC 2.6.1.19) and succinate semialdehyde dehydrogenase (EC 1.2.1.16) than those of the wild-type parent strains. Secondary mutants derived from these GABA-nitrogen-utilizing strains were able to grow on GABA as the sole source of carbon and nitrogen. They also grew faster on a variety of other carbon and nitrogen sources, and their growth was more strongly inhibited by different metabolic inhibitors than was that of the parent strains. The nature of the two mutations and the possible genes involved are discussed. A scheme of the pathway for GABA breakdown in E. coli K-12 is presented.     

Genes aroA and serC of Salmonella typhimurium constitute an operon.

Genetic analysis of aroA554::Tn10 derivatives of two mouse-virulent Salmonella typhimurium strains, "FIRN" and "WRAY," and of a nonreverting derivative of each constructed for use as a live vaccine, showed the site of the insertion among mapped aroA point mutants. The WRAY live-vaccine strain gave no aro+ recombinants in crosses with aroA point mutations to one side of the insertion, indicating a deletion from Tn10 through the sites of these point mutations. The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants; it probably has a deletion or inversion extending from Tn10 into aroA but not as far as the nearest point mutation. Some tetracycline-sensitive mutants of aroA554::Tn10 strains required serine and pyridoxine, indicating loss of serC function, and some that were found to be SerC- did not produce gas from glucose, indicating a loss of pfl function. These results show the gene order pfl-serC-aroA, as in Escherichia coli. Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with Tn10 insertions near aroA (i.e., zbj::Tn10 strains) yielded Aro- SerC- Pfl-, Aro- SerC+ Pfl+, and Aro- SerC- Pfl+ mutants but none which were Aro+ SerC-. All of the mutants are explicable by deletions or inversions extending clockwise from zbj::Tn10 into or through an operon comprising serC (promoter-proximal) and aroA. Such an operon was also shown by the identification of two Tn10 insertions causing phenotype Aro- SerC-, each able to revert to Aro+ SerC+ by precise excision. serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base sequencing of a cloned aroA segment of S. typhimurium (Comai et al., Science 221:370-371, 1983). Both serine and chorismate are precursors of enterochelin; this may be why serC and aroA are in a single operon.     

Method for isolating mutants overproducing nicotinamide adenine dinucleotide and its precursors

A procedure has been developed for isolating mutants which are defective with respect to nicotinamide adenine dinucleotide (NAD) metabolism. It is based on the well known V-factor requirement of Haemophilus parainfluenzae. This procedure was used to isolate a series of mutants from Escherichia coli. The pyridine metabolism of wild-type and mutant E. coli cells falls in one of four distinct classes. Class A includes wild-type E. coli and represents strains that are normal with respect to pyridine metabolism. Class B mutants have altered internal pools of NAD. The intracellular NAD concentration of different class B mutants varies over a 10-fold range. Class C mutants excrete pyridine mononucleotides, and class D mutants excrete NAD. The production of pyridine nucleotides by class C and D mutants exceeds that of wild-type E. coli by a factor of at least ten. The mutant strains generally have normal generation times and achieve normal cell densities in minimal medium.