鹅源腺病毒全基因组DNA酶切片段的克隆

利用1株鹅源腺病Y81G4株,经鹅胚增殖后收获尿囊液,用差速离心法纯化病毒子并提取病毒基因组DNA,病毒DNA经Hind Ⅲ酶切后共产生10个片段,分别回收各酶切片段,与经Hind Ⅲ单酶切的pUC18连续,获得8个不同的重组质粒。对于未克隆到的两末端片段,经碱处理去除末端蛋白后,以平端和HindⅢ粘端与经SmaⅠ和 HindⅢ双酶切的pUC18连接,获得了重组质粒pGAHC和pGAHI。克隆的各酶切片段,通过酶切电泳和Southern blotting结果鉴定,证明已分别克隆到该病毒基因组DNA HindⅢ酶切片段,且各酶切片段大小之和约为32．9kb.     

氧化亚铁硫杆菌启动子片段的克隆及酶切分析

本文利用DNA体外重组技术,将氧化亚铁硫杆菌染色体DNA的Hind Ⅲ酶切片段插入启动子探测质粒pSDSI(Ap~r,Tc~s,5.65kb)的Hind Ⅲ位点上,获得了一批具有启动子活性的染色体DNA片段.其中抗性最强的一株可在240μg/ml的四环素平板上生长,对此抗性最强的质粒pSDRF12进行了限制性酶切图谱分析,并利用其上的PstI和EcoRI位点分别酶切,得到了两个亚克隆pSDRF121和pSDRF122.新构建的两个亚克隆在其氧化亚铁硫杆菌启动子后只有一个Hind Ⅲ位点,可通过该位点插入外源目的基因,进一步观察其表达情况。     

B7-1和新型隐球菌DHA1基因嵌合重组质粒的构建

目的构建包含新型隐球菌细胞免疫主要相关基因-迟发超敏反应抗原基因(delayed-type hypersentivity antigen 1，DHA1)与小鼠共刺激分子B7-1的嵌合重组质粒。方法设计合成两对寡核苷酸引物。用PCR法分别从pUCmB7-1TM和新型隐球菌重组质粒pcDNA3-DHA1中特异扩增出编码B7-1和DHA1的基因片段(921和984bp)。分别用HindⅢ、EeoRⅠ、XbaⅠ酶切后。逐个定向连接到质粒pcDNA3中，转化宿主菌DH-5α．分别用上述内切酶酶切及DNA测序鉴定重组质粒。结果酶切鉴定示所切下的片段大小均与预计相符，测序结果与献报道序列及预计结果一致。证实符合表达框架。结论本实验成功构建了新型隐球菌嵌合重组质粒pcDNA3-B7-1-DHA1。为进一步研究新型隐球菌DNA免疫奠定了基础。     

A组乙型溶血性链球菌DNaseB基因亚克隆及pET-28a(+)-DNaseB载体构建

运用PCR扩增技术,以质粒pMD18-T-DNaseB为模板,扩增出0.5 kb截短的DNaseB基因,先将该基因片段与pMD19-T载体连接,确定该序列正确并进行大量繁殖后,再将DNaseB基因定向克隆入pET-28a( )表达载体中,构建新的原核表达载体pET-28a( )-DNaseB,转化该重组质粒至受体菌E.coliDH5a中,采用SDS碱裂解法提取该质粒DNA,经EcoRⅠ和HindⅢ双酶切鉴定和核苷酸序列分析,证实插入的基因片段具有正确的DNaseB基因核苷酸序列,将pET-28a( )-DNaseB转化至大肠杆菌BL21(DE3)中,经过IPTG诱导其目的蛋白得到了表达.     

h TNF-α cDNA的酶切鉴定

本文对Sepharose 2B层析分离后的重组质粒pGEM-1用E CORI及HindⅢ限制性内切酶进行切割,电泳分析结果表明用Sepharose 2B层析分离的质粒纯度可满足于克隆片段的酶切图谱分析。     

黑麦Rubisco大亚基基因(rbc L)的克隆及其限制性内切酶图谱的构建

以小麦的rbc L为探针,分别与黑麦ctDNA EcoR I,BamH I,Hind III的酶切谱带进行Southern杂交。结果发现,E13(2.15 kb),B2(10.7 kb),H2(10.4 kb)均含有rbc L基因。用pUC9为载体克隆黑麦ctDNA的B2片段,Southern杂交后表明,得到的重组质粒含有rbc L基因,将其命名为pRCB2。设计了一种快速准确的分析方法,构建出pRCB2质粒DNA的限制性内切酶图谱,rbc L被定位在一个2.8 kb的区域内。     

用α—珠蛋白基因探针对两例Hb—Bart''''s胎儿双亲的α—基因的探测

采用限制性内切酶印迹杂交放射性自显影技术和α—珠蛋白基因探针分析了二例Hb—Bart's胎儿水肿综合征及其双亲的α—珠蛋白基因,其分析结果表明:Hb—Bart's胎儿水肿综合征没有α—基因;其双亲则以内切酶HindⅢ消化其DNA得到17、4.5、3.8kb α—基因特异片段,用内切酶Bgl Ⅱ消化则得12.3、7.2kb二个α—基因特异片段。其基因图谱与正常人相似,为正常人的一半。     

禽类多瘤病毒APV-1的cDNA病毒突变体构建

为进一步研究禽类多瘤病毒晚期基因多顺反子翻译调控的分子机制,设计和构建了与野生型病毒APV-1相同的在AUG位点有agno-1a突变区和7个插入氨基酸的新型重组病毒.用碱裂解法提取了APV-1 cDNA克隆pHL1003,全长线性目的片段由Acc I部分酶切后回收,再用T4连接酶将合成的agno插入片段磷酸化后与目的片段连接得到了重组质粒,最后转化至E.coli DH10b感受态细胞中筛选阳性克隆.经PCR,PAGE和DNA测序验证获得了APV-1突变cDNA克隆.     

Mn~(++)及二甲基亚砜(DMSO)对限制酶HindⅢ识别顺序的影响

本文以质粒pBR322及噬菌体λcI857S7DNA 为底物研究了Mn~(++)及DMSO 对限制酶Hind Ⅲ对底物DNA 识别顺序的影响.在10mMTris-HClpH8.4,10mM MgCl_2,60mMNaCl 及7mM 巯基乙醇的正常反应条件下,HindⅢ在pBR322DNA 上只有一个切点,酶切后在凝胶电泳上可看到一条带.用10mM Mn~(++)代替Mg~(++)或在反应液中加入DMSO 进行酶切,在电泳上出现许多新的带.以λcI857S7DNA 为底物也得到类似的结果.新产生的片段可以用DNA 连接酶重新连接成线状分子.Mn~(++)及DMSO还能使Hind Ⅲ切割细菌本身的DNA.这些数据表明:Mn~(++)及DMSO 确实能降低Hind Ⅲ对底物的专一性,放松对识别顺序的要求.讨论了专一性降低的机制及其在核酸研究及基因工程中的意义。     

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.     

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.     

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.     

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.     

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.     

On Tragic Consciousness In The Works By Ernest Hemingway

As an American modern novelist who were famous in the literary world, Hemingway was not a person who always followed the trend but a sharp observer. At the same time, he was a tragedy maestro, he paid great attention on existence, fate and end-result. The dramatis personae's tragedy of his works was an extreme limit by all means tragedy on the meaning of fearless challenge that failed. The beauty of tragedy was not produced on the destruction of life, but now this kind of value was in the impact activity. They performed for the reader about the tragedy on challenging for the limit and the death.