变构菌素产生菌 S.griseochromogenes 接合转移体系建立与优化

以整合型质粒pSET152为出发质粒,建立并优化了变构菌素产生菌Streptomyces griseochromogenes(S.griseochromogenes)与Escherichia coli(E.coli)的属间接合转移体系,确定了适用于变构菌素产生菌灰产色链霉菌的最佳接合转移条件.研究发现Ca2+和甘氨酸都能够提高接合转移的效率,尤其是甘氨酸的效果非常显著.这一工作对于S.griseochromogenes的基因操作、小分子药物生物合成机制的研究以及具有自主知识产权的药物衍生物的开发具有普遍的应用价值.     

曲张链菌素产生菌壮观链霉菌NRRL2494菌株遗传操作体系的建立

曲张链菌素属于安莎类抗生素,具有显著的生物活性.在从壮观链霉菌NRRL2494菌株的发酵产物中分离和鉴定了多组分曲张链菌素的基础上,探索和优化了外源DNA通过接合转移进入壮观链霉菌NRRL2494菌株的操作方法和培养条件.以游离型质粒pJTU1278为载体,在体外构建了一个曲张链菌素酰胺合酶基因svaF敲除的质粒,通过接合转移转入到壮观链霉菌NRRL2494野生型菌株中,所获得的svaF基因缺失突变株失去了产生曲张链菌素的能力.该遗传操作体系的成功建立和优化,使得在体内分析和鉴定曲张链菌素生物合成基因的功能成为可能,同时也为建立其他类似放线菌的遗传操作体系提供了参考.     

一个诱动接合性柯斯载体的构建及其应用

用ｐＨＺ１３２在大肠杆菌中构建基因文库,并用特异的探针进行菌落杂交获得了有意义的ＤＮＡ片段,可将其衍生质粒转化到携带接合性大肠杆菌质粒如ＲＰ４的细胞中。通过与萌发后的链霉菌孢子进行两亲本杂交,或将携带ｐＨＺ１３２的大肠杆菌细胞和携带ＲＰ４衍生质粒ｐＰＫ２０７３的大肠杆菌细胞与萌发后的链霉菌孢子混合起来进行三亲本杂交,在接合性质粒ＲＰ４的转移功能的诱导下,携带插入片段的ｐＨＺ１３２衍生质粒即可转移到     

井冈霉亚基胺A高产突变株的构建

通过接合转移将一个带有阿泊拉抗性基因和双交换臂的穿梭质粒pJTU609导入井冈霉素高产菌株吸水链霉菌井冈变种(Streptomyces hygroscopicus var.jinganggensis)DX546中,将负责井冈霉素A生物合成最后一步糖基转移中的糖基转移酶基因valG置换突变,多聚酶链式反应结果显示valG被阿泊拉霉素抗性基因成功置换,通过高压液相色谱检测证实突变株的发酵产物中井冈霉亚基胺A的产量得到大幅提高.     

厦门霉素生物合成基因簇在白色链霉菌及吸水链霉菌FR-008中的异源表达

将携带有厦门霉素生物合成的整合质粒p LMO09404,通过供体菌大肠杆菌ET12567(p UZ8002)与受体菌白色链霉菌及吸水链霉菌FR-008进行接合转移,将得到的接合转移菌株经PCR验证后进行发酵,发酵液经HPLC和质谱检测.结果显示,厦门链霉菌来源厦门霉素基因簇可以在白色链霉菌及吸水链霉菌FR-008中异源表达.     

质粒在大肠杆菌和吸水链霉菌井冈5008之间的属间接合转移

利用一个链霉菌－－大肠杆菌双功能柯斯载体ｐＨ１３２建立了吸水链霉菌井冈变种５００８（以下简称井冈５００８）的属间接合转移系统,将携带ｐＨ１３２的大肠杆菌细胞ＬＥ３９２和携带ＲＰ４衍生质粒ｐＲＫ２０７３的大肠杆菌细胞ＥＤ８７６８与萌发后的井冈５００８孢子混合起来进行三亲本杂交,在接合性质粒ＲＰ４转移功能的诱导下,ｐＨＺ１３２即可从大肠肝菌细胞ＬＥ３９２转移到井冈５００８孢子中去,所用培养基是利用２ＣＭ：ＬＡ＝４：１（体积比）的混合培养基,井冈５００８最佳孢子萌发时间为３．５～４小时。     

具有抗稻瘟病菌活性的ⅡW1菌株的分离鉴定及其接合转移方法的建立

从湖北省神农架地区土壤中筛选出一株具有抗稻瘟病菌活性的ⅡW1菌株,采用菌块法测定了ⅡW1菌株对稻瘟病菌的抑菌活性,通过形态结构、生理生化和16S rDNA基因序列同源性比对对ⅡW1菌株进行鉴定,同时对链霉菌ⅡW1菌株attB序列进行克隆,并建立接合转移的方法.结果表明:ⅡW1菌株属假灰色链霉菌,链霉菌ⅡW1菌株对稻瘟病NO-1菌株和168菌株的抑菌圈直径分别为29.7 mm和27.4 mm;链霉菌ⅡW1菌株attB序列具有位点特异性重组所必须的核心位点5′-TT-3′.含ΦC31attP位点的整合型质粒pSET152通过两亲接合转移导入链霉菌ⅡW1菌株.     

一株产环己酰亚胺的链霉菌遗传操作研究

 对一株高产环己酰亚胺的链霉菌菌株系统进行遗传操作体系的建立研究,并初步探索了相关生物合成的基因.采用了接合转移,电击转化菌丝体和PEG介导的原生质体转化3种方法进行外源基因的导入实验.使用了7种配方差异较大的培养基进行发酵,并使用HPLC对发酵产物进行分析,摸索了合适的发酵及检测条件.最后通过依赖于λ-RED介导的PCR-targeting方法进行了可能参与环己酰亚胺生物合成的PKS基因片段的敲除.通过实验,最终确定了使用优化的接合转移方法并采用新鲜孢子能够有效地导入外源基因,获得了较高的转化效率(10^-6),成功确立了遗传操作体系,获得了合适的发酵条件和HPLC检测方法.在建立遗传操作体系的基础上,成功找到了负责该类化合物生物合成的PKS基因片段,为深入研究其生物合成机理并进一步通过遗传工程改良以获得新化合物及提高化合物产量打下基础,并为链霉菌遗传操作研究提供了新的参考.     

高温链霉菌与细菌的相互作用及其自溶和分枝机制

链霉菌（Streptomyces sp.2)与金黄色葡萄球苏，枯草杆菌之间有相互抑制作用，链霉菌对大肠杆菌有抑制作用，但大肠杆菌则对链霉菌无抑制作用，细菌的存在能诱导链霉菌提前产生可溶性紫色色素，Streptomyces sp.2在5度下，YM，YG，YA培养基上产生细胞自溶的最短培养时间为2天，全部细胞自溶需要5天，在37度下及培养基上全部自溶的时间为8天，Streptomyces SP.1在55度或37度，培养基上细胞全部自溶的时间超过14天，Streptomyces SP.2孢子萌发的时间为4小时左右，产生的菌丝有分节现象，其细小分枝产生在菌丝及孢子的任何部位，低浓度（4mg/L)氨苄青霉素对链霉菌的孢子萌发及生长无影响，链霉菌菌丝在生长延伸时表现出相互靠扰和吸引，形成网络结构，各细胞间相互直辖市，形成一定形状，大小的菌落，在菌落的特定部位产生孢子，表现出一定的社会性。     

不同荧光蛋白基因标记利迪链霉菌

 利迪链霉菌（Streptomyces lydicus）A01 是一株分离自北京郊区对植物病原真菌具有广谱抑制作用的放线菌,在植物真菌病害防治中具有良好的应用前景。为了检测红霉素启动子在利迪链霉菌A01 中的活性,为后期对菌株A01 进行遗传改造提供技术支持,同时对生防菌A01 进行遗传标记以研究和阐明其在生态环境中的生物学行为规律,本实验采用两亲本接合的方法,将增强绿色荧光蛋白（EGFP）和红色荧光蛋白（RFP）基因片段克隆到携带红霉素启动子（ermE^*）的链霉菌表达载体pIB139 中,成功构建了以egfp 和rfp 为报告基因的重组载体pIB139-EGFP 和pIB139-RFP,并转化利迪链霉菌A01,突变株在荧光显微镜下观察到较强的绿色荧光和红色荧光,同时PCR 鉴定结果正确。这表明重组载体pIB139-EGFP 和pIB139-RFP 成功转入菌株A01,并且ermE^*启动子成功启动egfp 和rfp 基因表达。     

Markedness and its Interpretation in Language Use

Language markedness is a common phenomenon in languages, and is reflected from hearing, vision and sense, i.e. the variation in the three aspects such as phonology, morphology and semantics. This paper focuses on the interpretation of markedness in language use following the three perspectives, i.e. pragmatic interpretation, psychological interpretation and cognitive interpretation, with an aim to define the function of markedness.     

Lower Paleozoic oil relationships within Williston Basin, Canada

The Williston Basin is a significant petroleum province, containing oil production zones that include the Middle Cambrian to Lower Ordovician, Upper Ordovician, Middle Devonian, Upper Devonian and Mississippian and within the Jurassic and Cretaceous. The oils of the Williston Basin exhibit a wide range of geochemical characteristics defined as "oil families", although the geochemical signature of the Cambrian Deadwood Formation and Lower Ordovician Winnipeg reservoired oils does not match any "oil family". Despite their close stratigraphic proximity, it is evident that the oils of the Lower Palaeozoic within the Williston Basin are distinct. This suggests the presence of a new "oil family" within the Williston Basin. Diagnostic geochemical signatures occur in the gasoline range chromatograms, within saturate fraction gas chromatograms and biomarker fingerprints. However, some of the established criteria and cross-plots that are currently used to segregate oils into distinct genetic families within the basin do not always meet with success, particularly when applied to the Lower Palaeozoic oils of the Deadwood and Winnipeg Formation.     

An Analysis of Catherine＇s Character——Wuthering Heights

Wuthering Heights, Emily Bronte＇s only novel, was published in December of 1847 under the pseudonym Ellis Bell. The book did not gain immediate success, but it is now thought one of the finest novels in the English language. Catherine is the key character of this masterpiece, because everybody and everything center on her though she had a short life. We can understand this masterpiece better if we know Catherine well.     

Characterization of Ordovician carbonate reservoirs, southeastern Saskatchewan, Canada

The discovery of the prolific Ordovician Red River reservoirs in 1995 in southeastern Saskatchewan was the catalyst for extensive exploration activity which resulted in the discovery of more than 15 new Red River pools. The best yields of Red River production to date have been from dolomite reservoirs. Understanding the processes of dolomitization is, therefore, crucial for the prediction of the connectivity, spatial distribution and heterogeneity of dolomite reservoirs.The Red River reservoirs in the Midale area consist of 3～4 thin dolomitized zones, with a total thickness of about 20 m, which occur at the top of the Yeoman Formation. Two types of replacement dolomite were recognized in the Red River reservoir: dolomitized burrow infills and dolomitized host matrix. The spatial distribution of dolomite suggests that burrowing organisms played an important role in facilitating the fluid flow in the backfilled sediments. This resulted in penecontemporaneous dolomitization of burrow infills by normal seawater. The dolomite in the host matrix is interpreted as having occurred at shallow burial by evaporitic seawater during precipitation of Lake Almar anhydrite that immediately overlies the Yeoman Formation. However, the low δ18O values of dolomited burrow infills (-5.9‰～ -7.8‰, PDB) and matrix dolomites (-6.6‰～ -8.1‰, avg. -7.4‰ PDB) compared to the estimated values for the late Ordovician marine dolomite could be attributed to modification and alteration of dolomite at higher temperatures during deeper burial, which could also be responsible for its 87Sr/86Sr ratios (0.7084～0.7088) that are higher than suggested for the late Ordovician seawaters (0.7078～0.7080). The trace amounts of saddle dolomite cement in the Red River carbonates are probably related to "cannibalization" of earlier replacement dolomite during the chemical compaction.     

Features of Women's language

Language is a means of verbal communication. People use language to communicate with each other. In the society, no two speakers are exactly alike in the way of speaking. Some differences are due to age, gender, statue and personality. Above all, gender is one of the obvious reasons. The writer of this paper tries to describe the features of women＇s language from these perspectives： pronunciation, intonation, diction, subjects, grammar and discourse. From the discussion of the features of women＇s language, more attention should be paid to language use in social context. What＇s more, the linguistic phenomena in a speaking community can be understood more thoroughly.     

Design and analysis of generic LBS application developing platform

Location based services is promising due to its novel working style and contents.A software platform is proposed to provide application programs of typical location based services and support new applications developing efficiently. The analysis shows that this scheme is easy implemented, low cost and adapt to all kinds of mobile nework system.     

橡胶颗粒沥青混合料低温抗裂性试验研究

以AC-13级配为基础,将橡胶颗粒代替部分集料掺入混合料中,以低温弯曲试验为评价方法对不同橡胶颗粒掺量下沥青混合料的低温抗裂性进行研究,并引入应变能密度值对混合料的低温抗裂性进行综合评价.试验结果表明:橡胶颗粒沥青混合料试件的破坏微应变均超过2 300,满足冬寒区的技术指标;无论是否掺加橡胶颗粒,随着温度的下降,沥青混合料破坏时的最大弯拉强度增大,弯拉应变降低,劲度模量增大;弯曲应变能密度在胶粒掺量为1%左右时具有较大的弯曲应变能密度值,此时橡胶颗粒沥青混合料具有较好的低温抗裂性.     

A computer generator for randomly layered structures

AcomputergeneratorforrandomlylayeredstructuresYUJia shun1,2,HEZhen hua2(1.TheInstituteofGeologicalandNuclearSciences,NewZealand;2.StateKeyLaboratoryofOilandGasReservoirGeologyandExploitation,ChengduUniversityofTechnology,China)Abstract:Analgorithmisintrod…     

低渗透非均质砂岩油藏启动压力梯度研究

理论推导与室内实验相结合,建立了低渗透非均质砂岩油藏启动压力梯度确定方法。首先借助油藏流场与电场相似的原理,推导了非均质砂岩油藏启动压力梯度计算公式。其次基于稳定流实验方法,建立了非均质砂岩油藏启动压力梯度测试方法。结果表明:低渗透非均质砂岩油藏的启动压力梯度确定遵循两个等效原则。平面非均质油藏的启动压力梯度等于各级渗透率段的启动压力梯度关于长度的加权平均;纵向非均质油藏的启动压力梯度等于各渗透率层的启动压力梯度关于渗透率与渗流面积乘积的加权平均。研究成果可用于有效指导低渗透非均质砂岩油藏的合理井距确定,促进该类油藏的高效开发。     

Quantitative trait loci （QTLs） for quality traits related to protein and starch in wheat

Quality traits in wheat （Triticum aestirum L.） were studied by quantitative trait locus （QTL） analysis in a recombinant inbred line （RIL） population, a set of 131 lines derived from Chuan 35050 × Shannong 483 cross （ChSh）. Grains from RILs were assayed for 21 quality traits related to protein and starch. A total of 35 putative QTLs for 19 traits with a single QTL explaining 7.99-40.52% of phenotypic variations were detected on 10 chromosomes, 1D, 2A, 2D, 3B, 3D, 5A, 6A, 6B, 6D, and 7B. The additive effects of 30 QTLs were positive, contributed by Chuan 35050, the remaining 5 QTLs were negative with the additive effect contributed by Shannong 483. For protein traits, 15 QTLs were obtained and most of them were located on chromosomes 1 D, 3B and 6D, while 20 QTLs for starch traits were detected and most of them were located on chromosomes 3D, 6B and 7B. Only 7 QTLs for protein and starch traits were co-located in three regions on chromosomes 1D, 2A and 2D. These protein and starch trait QTLs showed a distinct distribution pattern in certain regions and chromosomes. Twenty-two QTLs were clustered in 6 regions of 5 chromosomes. Two QTL clusters for protein traits were located on chromosomes 1D and 3B, respectively, three clusters for starch traits on chromosomes 3D, 6B and 7B, and one cluster including protein and starch traits on chromosome 1D.