簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.

超声与磁粉强化含单宁废水生物处理研究

单宁是一种典型的有毒难降解污染物，在制革、造纸、制药、印染等行业废水中广泛存在，对水环境造成污染并且影响废水生物处理效果。本研究针对含单宁废水生物处理效率低、较高浓度时微生物受抑制且污泥容易膨胀等问题，采用超声和磁粉来强化含单宁废水生物处理，研究超声和磁粉对微生物活性、污染物去除及污泥沉降性能的影响，并对其作用机理进行了分析和探讨。 研究结果表明，活性污泥系统中单宁酸容积负荷可以达到1.8kgCOD/(m3·d)，单宁酸和COD去除率分别达到85.2%和79.6%，但如果负荷进一步增大则微生物活性迅速降低。系统在pH 5~8、温度20~35℃、DO>1 mg/L的条件下具有较好的单宁酸降解效果和处理稳定性。单宁降解动力学参数为：μmax =0.208h-1；Ks=226mg/L；Ki=522mg/L；kd=0.0092h-1；Y =0.594。 磁粉对系统处理效果和污泥沉降性能有一定的促进作用，且效果要优于外磁场。适宜的磁粉粒径和投加量分别为0.05~0.15mm和1.0g/L，COD去除率比对照系统提高6.4%，SVI降低28.6%，污泥絮体结构紧密。磁粉强化主要是通过其对污泥菌胶团的凝聚、吸附作用以及对微生物活性的强化作用实现。 在适当强度（0.4W/cm2）和辐照时间（20min）的超声作用下污泥絮体和细胞膜通透性增大，酶分泌也增多，系统的COD去除率比对照提高了8.8%，单宁酶酶活提高了11%。但超声也使污泥絮体结构松散，沉降性能下降，SVI比对照系统升高9.3%。 由于污泥流失加剧导致污泥浓度相对较低，声磁联合强化系统相对于磁粉强化系统其处理效果并没有提高。但相对于单纯活性污泥系统，声磁联合作用下系统处理效果、污泥沉降性能以及系统运行稳定性都得到明显改善。本研究为难降解废水的生物处理提供了一个新的思路。 Tannins are typical refractory and toxic pollutants that commonly exist in wastewater from dye, medicine, paper and leather industries and cause many problems associated with environmental pollution and biological treatment of wastewater. Biological treatment efficiency of tannin-containing wastewater is usually low owing to its biological toxicity and low biodegradability, microbes are usually inhibited under high tannin concentration and sludge bulking frequently occurs. In this study, ultrasound and magnetic powder were used to improve the biological treatment performance of simulated tannic acid-containing wastewater. The effects of ultrasonic irradiation and magnetic powder on microbial activity, tannic acid degradation rate and sludge sedimentation were investigated. The augmentation mechanisms were analyzed and discussed. The experimental results showed that the microbes were prominently inhibited under high tannic acid concentration, but moderate degradation efficiency can be maintained under a tannic acid load of up to 1.8kgCOD/(m3·d), with the tannic acid degradation and COD removal percentage of 85.2% and 79.6% respectively. The highest degradation rates and treatment stability were achieved at pH range of 5~8, temperature range of 20~35℃ and DO concentration of above 1mg/L. The kinetic parameters were estimated, including: μmax =0.208h-1；Ks=226mg/L；Ki=522mg/L；kd=0.0092h-1；Y =0.594. The microbial activity, tannic acid degradation rate and sludge sedimentation were improved by adding Fe3O4 magnetic powder, and the augmentation performance was better than external magnetic field. The appropriate particle size and dosage of magnetic powder were found to be 0.05~0.15mm and 1.0g/L, respectively, under which the COD removal percentage was improved by 6.4% and SVI value decreased by 28.6%, and compact floc structure was observed. This was mainly caused by the flocculation and adsorption effects of magnetic powder against sludge floc and the stimulation of microbial activity under appropriate magnetic field. Under appropriate ultrasonic irradiation (ultrasonic intensity 0.4W/cm2, ultrasonic irradiation time 20min), the permeability of floc and cell membrane are improved, transfer of substrate and oxygen were reinforced; meanwhile, more enzyme were produced by microbes under the slight damage caused by ultrasound. However, the floc structure became loose under ultrasonic irradiation, leading to relatively poor sedimentation, with the SVI value 9.3% higher than the control system. Although the magnetic powder-ultrasonic irradiation combined augmentation system showed no improvement in treatment performance compared with sole magnetic augmentation system owing to its relatively low sludge concentration, it guaranteed the stable operation of system, meanwhile the tannic acid degradation and sludge sedimentation were significantly improved compared with sole activated sludge system. This study gives a new idea for biological treatment of refractory wastewater.

Simultaneous determination of both the calibration constant in an electrochemical quartz crystal microbalance and the active surface area of a polycrystalline gold electrode

A novel method is employed for the simultaneous determination of both the calibration constant of an electrochemical quartz crystal microbalance (EQCM) and the active surface area of a polycrystalline gold electrode. A gold electrode: is immersed into a 1 mM KI/1 M H2SO4 solution and on which forms a neutral monolayer. The adsorbed iodine can then be completely oxidized into IO3-. The active surface area of a gold electrode can be obtained from the net electrolytic charge of the oxidation process, and the calibration constant in the EQCM can be calculated from the corresponding frequency shift. The result shows that this method is simple, convenient and valid. (C) 2000 Elsevier Science S.A. All rights reserved.

A preliminary study on chemistry of phenoxynaphthacenequinones

A preliminary study on the chemistry of 6-phenoxy-5,12-naphthacenesuinone derivatives was reported, It was found that the main product was 6-(N,N-dimethylamino)-5,12-naphthacenequinone when a reaction of 6-chloro-5,12-naphthacenequinone with 4-hydroxyazobenzenes in DMF was carried out under certain reaction conditions. When the reaction of 6-[4-(4-hydroxyphenyl)isopropyl)phenoxy]-5, 12-naphthacenequinone and epichlorohydrin was carried out in an acetone/DMF(V/V=2/1) mixed solvent and in the presence of K2CO3 and KI, a new phenoxynaphthacenequinone derivative,6,6'-[1-methylethylidenebis(4,1-phenyleneoxy)]bis(5,12-naphthacenequinone), was obtained.

萃取-非水介质氢化物发生-ICP-AES中的干扰及Ni-Fe基合金的分析

本文结合溶剂萃取研究了非水介质中氢化物发生-ICP-AES的分析条件、干扰等因素的影响。利用KI~+H_2SO_4/MIBK萃取体系将As,Sb,Bi萃取到MIBK中而与基体元素分离,然后将该有机相与甲酸按等体积混合后,即可直接进行氢化物发生-ICP-AES分析。还对影响萃取和氢化物发生的一些因素及共存元素的干扰进行了讨论。该方法应用于Ni-Fe基合金中As和Sb的分析,取得了较满意的结果。

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

Community structure and seasonal dynamics of planktonic ciliates along salinity gradients

The ciliate community structure and seasonal dynamics in a solar saltern of the Yellow Sea were studied based oil 4 sampling dates and 8 stations with salinities from 27.7 parts per thousand to 311.0 parts per thousand. The effects of the type and concentration of the fixative used (Lugol's and Bouin's) were tested at the first sampling date. Fixative type and fixative concentration had significant effects on ciliate abundance and blovolume, with 1% Lugol's giving the best results. A detailed investigation using live observations and protargol staining techniques revealed a total of 98 morphospecies from 8 sampling stations. There was obvious seasonal variation in species composition at most of the stations, but this tended to be less distinct with increasing salinity, as the dominant ciliate group shifted from oligotrichs to heterotrichs. Ciliate abundance varied from 4.40 x 10(1) to 2.11 x 10(5) cells l(-1) and biomass ranged between 2.39 and 9.87 x 10(3) mu g Cl-1 (at a salinity of 147.6 parts per thousand). Both abundance and biomass decreased abruptly when salinity exceeded 100-150 parts per thousand. Statistical analyses Suggested that the dynamics of ciliate abundance and biomass were regulated by both salinity and by season, but those of diversity and species richness were mainly controlled by salinity and both significantly decreased with increasing salinity. (C) 2009 Elsevier GmbH. All rights reserved.

Morphology and Molecular Phylogeny of Trimyema koreanum n. sp., a Ciliate from the Hypersaline Water of a Solar Saltern

A new ciliate, Trimyema koreanum n. sp., isolated from hypersaline water (salinity of 293 parts per thousand) from a solar saltern in Korea, was investigated using live observation, protargol impregnation, and gene sequencing. Trimyema koreanum is about 30 x 13 mu m in vivo, has usually 23 longitudinal ciliary rows forming two distinct ciliary girdles visible both in vivo and in protargol impregnation. A third indistinct ciliary girdle as well as a girdle of mucocysts is distinguishable only in impregnated cells. We suggest T. koreanum as a new species, differing from the most similar species, T. marinum, by the presence of two distinct ciliary girdles (T. marinum usually has six ciliary girdles clearly visible in living cells and three anterior spirals that encircle the cell completely). Although the number of known 18S rRNA sequences in the genus Trimyema was limited, the Trimyema group including T. koreanum forms a strong clade. The phylogenetic position confirms that the isolate belongs to the genus Trimyema and is different from previously sequenced species. Trimyema koreanum is able to consume both prokaryotes and small eukaryotes (specifically, the alga Dunaliella sp.).

Kiitricha minuta n. Sp., a peculiar hypotrichous ciliate (Ciliophora, Spirotrichea) from the Yellow Sea

The morphology and infraciliature of a new ciliate, Kiitricha minuta n. sp., isolated from the Yellow Sea, were investigated using live observation and protargol impregnation. Kiitricha minuta represents a third member of the rarely known order Kiitrichida. It is unique in the subclass Hypotrichia in having many rows of small uniform cirri along the right side of the body and the dorsal kineties composed of dikinetids, most of which bear two cilia. Kiitricha minuta n. sp. is ovoid and measures about 60 x 45 mu m in vivo. It has a huge buccal cavity occupying about 80% of the body length, numerous body extrusomes, one macronucleus and two micronuclei, 27-27 adoral membranelles, 9-12 frontoventral cirral rows, a submarginal row of 7-9 cirri, 6 or 7 transverse cirri, and roughly 7-9 dorsal kineties. This new species differs distinctly from its only congener Kiitricha marina by its smaller size (60 mu m vs. 80-150 mu m), the presence of body extrusomes (vs. absent), the different macronuclear pattern (one vs. two nodules), and the lower number of frontoventral cirral rows (9-12 vs. 21-26), which terminate at the anterior two-thirds of body (vs. extend to the posterior). The new term "submarginal cirral row" is introduced to distinguish from the marginal cirral row in typical hypotrichs sensu lato. Based on our new observations and the literature, an improved diagnosis for the genus Kiitricha is provided and its phylogenetic importance is discussed.