小蓬NeMT2基因的克隆及其植物表达载体的构建

该研究利用RACE ( Rapid amplification of cDNA ends)技术从小蓬中成功分离编码金属硫蛋白( Metal-lothionein,MT)的cDNA序列,命名为NeMT2,在GenBank中登录号为KT835290。该基因全长590 bp,开放阅读框为237 bp,编码78个氨基酸,编码的氨基酸序列中含有14个半胱氨酸残基( Cys,C),呈C-C,C-X-C,C-X-X-C排列,集中分布在肽链的N端和C端,基因编码蛋白的分子量为7.6036 kD,等电点为4.71。系统发育分析表明,小蓬金属硫蛋白NeMT2与藜科的海蓬子( AEF01492)和盐穗木( AHI62953)同源性最高,其次是甜菜( XP 010667708.1)。生物信息学分析表明,金属硫蛋白NeMT2无信号肽结构,属于非跨膜亲水性蛋白;疏水性分析表明,NeMT2蛋白的35~45个氨基酸之间有较强的疏水性,其中第41位Asp具最强的疏水性(1.444);结构预测分析该蛋白质二级结构的主要元件是无规则卷曲。通过RT-PCR对NeMT2基因的表达分析发现, NeMT2基因在铜矿区和非铜矿区的小蓬叶片中均有表达,但该基因在铜矿区小蓬叶片的表达量明显高于非铜矿区。将小蓬NeMT2基因定向克隆到植物表达载体pCAMBIA1300的35S 启动子下游,构建该基因的植物超表达载体pCAMBIA1300＋NeMT2。该研究结果为进一步研究该基因的功能和小蓬响应重金属胁迫的分子机制提供了一定基础。     

The influence of drought on the relationship between leaf and conducting sapwood area in Eucalyptus globulus and Eucalyptus nitens

 Development of the relationship between leaf area (A _l ) and sapwood area (A _s ) was investigated in two important hardwoods, Eucalyptus globulus (Labill) and E. nitens (Deane and Maiden) Maiden, growing in an experimental plantation established in a low rainfall zone (approx. 515 mm year^–1) of Tasmania. The experiment compared irrigated controls and a rainfed treatment which was subjected to cyclical summer droughts from age 1 to 6 years old. Leaf area and sapwood area were determined by destructive sampling at ages 2, 3 and 6 years old. There was no effect of stand age on A _l :A _s when sapwood area was measured at crown break. At age 3 years old A _l :A _s was significantly greater in the rainfed than the irrigated trees. It was concluded that this difference was due to earlier canopy closure in the irrigated trees. When the plantation was 6 years old A _l :A _s was significantly greater in the irrigated than the rainfed treatment. An analysis based on an equation which links A _l :A _s with transpiration and volumetric flow rate (Whitehead et al. 1984) was used to infer a positive correlation between stem hydraulic conductivity (k _h ) and water availability. Independent of water availability E. globulus maintained a higher A _l :A _s than E. nitens at all ages. Received: 20 March 1997 / Accepted: 30 December 1997     

BcMAF2 activates BcTEM1 and represses flowering in Pak-choi (Brassica rapa ssp. chinensis)

Key message

BcMAF2 plays a key role in flowering regulation by controlling BcTEM1, BcSOC1 and BCSPL15 in Pak-choi.

Abstract

Flowering is a key event in the life cycle of plants. Flowering time shows an extensive variation from different Pak-choi (Brassica rapa ssp. chinensis) cultivars. However, the regulation mechanism of flowering in Pak-choi remains rarely known. In this study, a systematic identification and functional analysis of a Pak-choi MADS Affecting Flowering (MAF) gene, BcMAF2, was carried out. BcMAF2 encoded a protein containing a conserved MADS-box domain, which was localized in the nucleus. QPCR analysis indicated that the expression of BcMAF2 was higher in the leaves and flowers. Overexpression of BcMAF2 in Arabidopsis showed that BcMAF2 repressed flowering, which was further confirmed by silencing endogenous BcMAF2 in Pak-choi. In addition, Tempranillo 1 (TEM1) expression was up-regulated and MAF2 expression was down-regulated in the BcMAF2-overexpressing Arabidopsis. The expression of BcMAF2 and BcTEM1 was down-regulated in BcMAF2-silencing Pak-choi plants. The yeast one-hybrid, dual luciferase and qPCR results revealed that BcMAF2 protein could directly bind to BcTEM1 promoter and activate its expression, which was not reported in Arabidopsis. Meanwhile, a self-inhibition was found in BcMAF2. Taken together, this work suggested that BcMAF2 could repress flowering by directly activating BcTEM1.

     

Determination of redox potential levels critical for cell respiration and suitable for L-leucine production

The effect of oxygen supply on L -leucine fermentation was investigated employing a leucine-producing mutant of Brevibacterium lactofermentum. Since it was not possible to measure oxygen tension below 0.01 atm by a Teflon-coated oxygen electrode, the degree of satisfaction of the cells' oxygen demand (cells' respiration rate/maximum oxygen demand of cells, r_ab/KrM) and the redox potential of the culture medium (E. mV) were used as indices to oxygen supply in cultures under low oxygen tension. When the oxygen demand of the cells was satisfied (r_ab/KrM = 1.0) and the E value was between ?90 and ?110 mV, L -leucine formation was 26.5 mg-ml. When the oxygen demand of the cells was not satisfied (r_ab/KrM = 0.85) and the E value was between ?200 and ?220mV, L -leucine accumulation was 29.7 mg/ml. When the oxygen supply was extremely limited (r_ab/KrM = 0.27) and the E value was ?280 mV. L -leucine formation was 12.9 mg/ml. A new method which simultaneously measures the redox potential and dissolved oxygen was applied to the determination of the critical dissolved oxygen level for cell respiration (P_{L crit}), which was too small to be detected by conventional oxygen electrodes. The value of P_{L crit} of the leucine producer was estimated as 0.0002 atm.     

Characterisation of Triticum vavilovii-derived stripe rust resistance using genetic,cytogenetic and molecular analyses and its marker-assisted selection

Stripe rust resistance was identified in Triticum vavilovii (T. vavilovii Aus22498)-derived Russian wheat aphid (RWA)-resistant germplasm. Inheritance studies indicated monogenic control of resistance. The resistance gene was tentatively designated as Yrvav and was located on chromosome 1B by monosomic analysis. A close association (1.5±0.9% recombination) of Yrvav with a T. vavilovii-derived gliadin allele (Gli-B1vav) placed it in chromosome arm 1BS. Yrvav was allelic with Yr10. Tests with Yr10 avirulent and virulent pathotypes showed that Yrvav and Yr10 possess identical pathogenic specificity. Yrvav and Yr10 showed close genetic associations with alternate alleles at the Xpsp3000 (microsatellite marker), Gli-B1 and Rg1 loci. Based on these observations Yrvav was named as Yr10vav. The close association between Xpsp3000 and Gli-B1 was also confirmed. The Yr10vav-linked Xpsp3000 allele (285 bp) was not present in 65 Australian cultivars, whereas seven Australian wheats lacking Yr10 carried the same Xpsp3000 allele (260 bp) as Yr10 carrying wheat cultivar Moro. Xpsp3000 and/or Gli-B1 could be used in marker-assisted selection for pyramiding Yr10vav or Yr10 with other stripe rust resistance genes. Yr10vav was inherited independently of the T. vavilovii-derived RWA resistance. Received: 5 December 2000 / Accepted: 3 April 2001     

Food attraction and population growth of fungivorous nematodes with different fungi

Food attraction of the fungivorous nematodes Aphelenchus avenae and Aphelenchoides spp. to seven fungal species (Pyrenochaeta lycopersici, Botrytis cinerea, Rhizoctonia solani strains AG 3 and AG 2‐1, Verticillium dahliae, Pochonia bulbillosa, Mortierella hyalina and Trichoderma harzianum) was determined on agar plates by counting the number of test nematodes present on the mycelium of each fungus 24 h after inoculation. Population growth of A. avenae and Aphelenchoides spp. on five of the seven fungi included in the attraction test (P. lycopersici, R. solani strain AG 3, V. dahliae, P. bulbillosa and T. harzianum) was also determined on agar plates by counting nematode numbers every week during a 6‐week period. A. avenae and Aphelenchoides spp. were attracted to all the fungi tested. A. avenae was preferentially attracted to V. dahliae (P < 0.0001), and Aphelenchoides spp. did not show any preference except for low attraction to R. solani. A. avenae and Aphelenchoides spp. reproduced on all fungal species tested. After 6 weeks of incubation, the highest number of nematodes was found on P. lycopersici and P. bulbillosa, while the lowest number occurred on R. solani for A. avenae and on T. harzianum for Aphelenchoides spp. The suitability of a fungus as a host was not clearly related to the attraction to that fungus.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Asexual reproductive organ-specific expression of the glyceraldehyde-3-phosphate dehydrogenase 2 gene of Pilobolus crystallinus

Pilobolus crystallinus has three putative glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes (pcgapdh1, pcgapdh2 and pcgapdh3). The results of this study demonstrate that expression of pcgapdh2 was increased by irradiation and that this increased expression was correlated with the formation of asexual reproductive organs (trophocysts). Interestingly, expression of pcgapdh2 was restricted to trophocysts. The formation of trophocysts was likely promoted by light, and the expression of pcgapdh2 was increased as a result of trophocyst formation. This is the first report that shows the regulation of a gapdh gene in an organ-specific manner in fungi.     

The disruption of JEN1 from Candida albicans impairs the transport of lactate

A lactate permease was biochemically identified in Candida albicans RM1000 presenting the following kinetic parameters at pH 5.0: K_m 0.33±0.09 mM and V_max 0.85±0.06 nmol s^?1 mg dry wt^?1. Lactate uptake was competitively inhibited by pyruvic and propionic acids; acetic acid behaved as a non-competitive substrate. An open reading frame (ORF) homologous to Saccharomyces cerevisiae gene JEN1 was identified (CaJEN1). Deletions of both CaJEN1 alleles of C. albicans (resulting strain CPK2) resulted in the loss of all measurable lactate permease activity. No CaJEN1 mRNA was detectable in glucose-grown cells neither activity for the lactate transporter. In a medium containing lactic acid, CaJEN1 mRNA was detected in the RM1000 strain, and no expression was found in cells of CPK2 strain. In a strain deleted in the CaCAT8 genes the expression of CaJEN1 was significantly reduced, suggesting the role of this gene as an activator for CaJEN1 expression. Both in C. albicans and in S. cerevisiae cells CaJEN1-GFP fusion was expressed and targeted to the plasma membrane. The native CaJEN1 was not functional in a S. cerevisiae jen1Δ strain. Changing ser²¹⁷-CTG codon (encoding leucine in S. cerevisiae) to a TCC codon restored the permease activity in S. cerevisiae, proving that the CaJEN1 gene codes for a monocarboxylate transporter.     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.     

拉雅松遗传多样性及同域分布种间亲缘关系分析

拉雅松是广西西北部稀有的乡土用材、用脂树种,有较高的经济价值,但其遗传多样性状况及种间进化关系未知。利用SSR分子标记检测拉雅松群体遗传多样性,希望对该物种保护策略的制定提供参考依据。此外,鉴于该地区自然分布的松属树种仅有拉雅松、细叶云南松与马尾松三个种,试图利用SSR分子标记信息分析拉雅松与细叶云南松、马尾松的种间亲缘关系。结果表明:7对SSR引物在拉雅松群体共检测到14个等位基因。有效等位基因数为1.653,观测杂合度为0.577,期望杂合度为0.374,Shannon信息指数为0.540,Nei多样性指数为0.367,表明拉雅松具有较高的遗传多样性。拉雅松与马尾松遗传距离最近为0.0175,与3个细叶云南松群体距离较远,平均为0.0525。拉雅松与马尾松、细叶云南松平均共祖系数(Θ)分别为0.094、0.066,据此推测拉雅松可能与马尾松存在较近的亲缘关系。讨论了拉雅松的遗传多样性保护策略。     

Construction of Uracil and Tryptophan Auxotrophic Mutants from Sake Yeasts by Disruption of URA3 and TRP1 Genes

Uracil auxotrophic mutants were constructed from the sake yeasts K-701 and K-901 by successive URA3 gene disruption. First, as sake yeast is diploid, one URA3 gene was disrupted with pURA38 (AURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (Δ URA3) and homozygous URA3 disruptants were isolated on FOA (5-fluoro-orotic acid) plate on which only ura3 mutants can grow. Direct URA3 gene disruption with pURA36 (Δ URA3) was also done and the uracil auxotrophic mutant was isolated. Four types of URA3 disruptants were isolated, two of which had no bacterial DNA.

A tryptophan auxotrophic mutant was constructed from one of the URA3 disruptant using pTRP14 (Δ TRP1 URA3) by gene disruption. This TRP1 disruptant was also lacking bacterial DNA.

Laboratory scale sake brewing using the auxotrophic mutants showed that these strains are very useful as recipient strains for molecular breeding of sake yeasts.     

cDNA cloning and expression analysis of the juvenile hormone acid methyltransferase from seabuckthorn carpenterworm,Holcocerus hippophaecolus (Lepidoptera: Cossidae)

In this study, we report the cDNA cloning and sequence determination of Hh‐JHAMT from the seabuckthorn carpenterworm, Holcocerus hippophaecolus, by using rapid amplification of cDNA ends. The full‐length cDNA of putative Hh‐JHAMT was 1659 bp and contained a highly conserved Motif I, SAM motif I, which showed that Hh‐JHAMT like enzyme was a member of SAM‐dependent MTases. Moreover, putative Hh‐JHAMT had high homology to the other members of the JHAMT peptide family: 59% with Spodoptera litura, 54% with Bombyx mori and 54% with Helicoverpa armigera. Multiple alignments and phylogenetic analysis revealed that Hh‐JHAMT was closely related to JHAMT from Lepidoptera. Real‐time quantitative PCR experiments showed that Hh‐JHAMT mRNA expression was highest in the corpora allata (CA) complex, and was also detected at high levels during earlier larval and adult stages. The JHAMT mRNA level gradually declined during larval development, and the lowest amount of expression was observed in the pupal stage, while it increased to a higher level during adult stages. The pattern of Hh‐JHAMT expression was similar to the mode of JH biosynthesis. These results provided information concerning molecular characteristics of Hh‐JHAMT, whose expression profile suggests that the Hh‐JHAMT gene might be changed with larval development, metamorphosis and adult reproduction of the H. hippophaecolus.     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

Mapping of Rym16 Hb , the second soil-borne virus-resistance gene introgressed from Hordeum bulbosum

Rym16 ^Hb , a gene conferring resistance to soil-borne viruses, was introgressed from Hordeum bulbosum to barley chromosome 2HL. Mechanical inoculation with BaMMV and field tests on a plot contaminated with different viruses demonstrated that Rym16 ^Hb is effective against all European viruses of the soil-borne virus complex (BaMMV, BaYMV-1, -2). Genetic analysis revealed a dominant inheritance of the resistance controlled by Rym16 ^Hb . Using 2HL anchor markers, the size of the introgression was estimated to be about 30 M. In its proximal part, the introgression was characterized by a rearrangement of markers Xbcd266, ABC153 and ABC252, accompanied with pronounced linkage drag by factor 4 in segregating mapping populations. The introgression was found to be associated with a recessive lethality factor, l ^Hb , which was closely linked to the markers mentioned above. Recombination occurring within the introgressed H. bulbosum segment allowed us to separate l ^Hb from Rym16 ^Hb and to reduce the size of the introgression to 23 cM or less.     

Engineering the lycopene synthetic pathway in <Emphasis Type="Italic">E. coli</Emphasis> by comparison of the carotenoid genes of <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> and <Emphasis Type="Italic">Pantoea ananatis</Emphasis>

The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-d-thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli. Sang-Hwal Yoon and Ju-Eun Kim: These authors contributed equally to this work.     

Display of <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus GP64 on the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Spore Coat

To investigate whether Bombyx mori immunized with Bacillus subtilis spore displaying GP64 escape from the B. mori nucleopolyhedrovirus (BmNPV) attack, a recombinant integrative plasmid named pJS700-GP64 was constructed, which carries a recombinant cotC-Gp64 gene under the control of the cotC promoter. In this study, pJS700-GP64 was transformed into B. subtilis 168 (trp⁻) competent cells, an amylase (amyE) inactivated mutant was selected, and was confirmed to be a double cross-over integrant, cotC-Gp64 fragment of which was integrated into B. subtilis chromosome. Gp64 was expressed on the spore surface and recognized by Gp64-specific antibody. Results of B. mori when challenged with BmNPV indicated that B. mori vaccinated with the recombinant spores possessed resistance to the invasion of BmNPV at some degree.     

Occurrence of Ochratoxin A-Producing Fungi in Commercial Corn Kernels in Argentina

Potentially ochratoxigenic Aspergillus and Penicillium species were identified and the natural occurrence of ochratoxin A (OTA) in corn kernels was evaluated. Likewise, the capacity to produce OTA by Aspergillus section Nigri and Circumdati was investigated. A total of 50 corn samples for human consumption was collected in the south of Córdoba Province. The surface-disinfected method for mycobiota determination was used. The OTA detection was performed by HPLC. OTA production was tested in strains belonging to section Nigri and Circumdati. Statistical analysis demonstrated that the specie A. flavus was isolated in higher frequency (p<0.01) from corn kernels in DRBC and DG18 media. The percentage of corn kernels contaminated by A. niger var. niger was similar in DRBC and DG18 media. The frequency of grains contaminated by A. flavus and A. niger var. awamori was higher than A. niger var. niger and A. japonicus var. japonicus (p<0.01) in DG18 media. The other potentially ochratoxigenic species, A. ochraceus, was isolated between 5% and 10% of the corn kernels in DG18 and DRBC media, respectively. The OTA producing species P. verrucosum was not isolated. All samples of corn were OTA negative (<1 ng g⁻¹). Thirty strains (25%) of the black Aspergillus were OTA producers. From four strains of A. ochraceus isolated, only one produced OTA. Due to the storage variable conditions could not be adequate in this substrate, the presence of ochratoxigenic strains of section Nigri and OTA needs to be evaluated for a longer time to establish the toxicological risk for human beings. The contamination of stored corn kernels with A. flavus and Aspergillus section Nigri was significant.     

Degradative pathway of p-aminoazobenzene by Bacillus subtilis

Summary p-Aminoazobenzene was degraded by Bacillus subtilis to aniline and p-phenylenediamine by reductive fission of an azo bond. The aniline was then acetylated to acetanilide while the p-phenylenediamine underwent 2 successive acetylations to yield p-aminoacetanilide and p-phenylenediacetanilide. In addition, another pathway was found in Bacillus subtilis in which p-aminoazobenzene was metabolised to p-acetamidoazobenzene.