海洋放线菌Streptomyces sp.大片段DNA基因组文库的构建

目的:构建稀有海洋放线菌Streptomyces sp.基因组文库.方法:以稀有海洋放线菌Streptomyces sp.为实验材料,随机剪切提取的总DNA,5'-磷酸末端补平回收40kb左右的DNA片段,与pWEBTM载体连接,经包装蛋白包装成噬菌体后侵染宿主细胞E.coli EPI100,构建该菌株的基因组文库,并对该文库进行质量鉴定.结果:成功构建了稀有海洋放线菌Streptomyces sp.的基因组文库,效价达9.0×104CFU/mL,得到4000个阳性克隆子,远远大于按覆盖率为99%计算至少所需的837个阳性克降子数,且平均插入片段长度为36kb,重组率100%.阳性克隆子保存于96孔板中,-80℃保存.结论:所构建文库的各项指标均达到要求,为了进一步评估Streptomyces sp.所能合成的所有潜在天然产物,还需要进一步检测该文库中包含有生物合成基因簇的大肠杆菌的表达情况.     

对虾养殖水环境宏基因组Fosmid文库的构建

采用包埋法提取大片段基因组DNA,通过低熔点琼脂糖酶法回收40 kb左右的DNA,经补平磷酸化、与pCC2FOS载体连接、体外包装和转染EP1300-T1R,构建对虾养殖水环境Fosmid文库.对文库进行鉴定,该文库平均插入片段大小约35 kb,共保存8 000个克隆,包括了大概8×108个微生物细胞.用几丁质酶筛选平板对文库进行初步筛选,筛选到4个阳性克隆子.本试验构建Fosmid文库,初步获取了对虾养殖生态系统的微生物遗传信息,对于从虾池原位环境鉴定并筛选具有潜在益生作用的功能微生物意义重大.     

莱茵衣藻基因组文库的构建

以莱菌衣藻CC-849为材料,提取基因组DNA,利用BamHⅠ和BglⅡ对基因组DNA进行酶解,获得了可用于构建基因组文库的6-12 kb的基因组片段,并浓缩至200 ng/μL。该片段与λDNA载体连接,经噬菌体蛋白包装、侵染大肠杆菌XL1-blue后,获得了莱菌衣藻基因组文库。该文库的滴度为2.12×10~5 pfu/mL,共有转化子4.26×10~4个,插入片段的平均长度约为9kb,扩增后基因组文库滴度为9.5×10~6 pfu/mL。     

一种用于构建红色红曲霉基因组Cosmid文库的大片段DNA制备方法

摘要：【目的】制备用于构建红色红曲霉cosmid文库的大片段基因组DNA。【方法】采用优化的酚氯仿抽提法制备DNA,并利用Sau3AI切割至平均大小为40 kb,然后使用Stratagene包装蛋白构建cosmid文库。基于PCR法使用同源探针从该文库中进行了目的基因的筛选。【结果】制备了浓度为5 μg/μL,平均片段大小大于48 kb的红色红曲霉大片段基因组DNA。利用该DNA构建的cosmid文库基因组覆盖倍数为10,并筛选到了含有目的片段的cosmid。【结论】通过该方法制备红色红曲霉大片段基因组D     

稀有海洋放线菌Salinispora arenicola非核糖体肽合成酶和卤代酶生物合成基因簇核心区的克隆及序列分析

克隆稀有海洋放线菌Salinispora arenicola的非核糖体肽合成酶(NRPS)和卤代酶生物合成基因簇核心区基因片段。根据已发表的放线菌NRPS和卤代酶生物合成基因簇核心区的核苷酸序列保守区设计两对简并性引物,采用PCR的方法扩增NRPS和卤代酶生物合成基因簇核心区基因片段,使用分子生物学软件进行序列分析。获得两段大小分别为662bp和557bp的基因片段,编码220个和185个氨基酸。这两段序列与海洋放线菌Salinispora arenicola CNS-205的NRPS和卤代酶生物合成基因簇核心区基因核苷酸序列的同源性分别为99%和98%。成功地获得了稀有海洋放线菌Salinispora arenicola的NRPS和卤代酶生物合成基因簇核心区基因片段,该基因片段的获取将为分离全长基因簇以及研究该基因簇在生物合成中的功能奠定基础。     

毛足棒角蝗基因组DNA文库的构建

纯化毛足棒角蝗 (Dasyhippusbarbipes)虫体组织的高分子量基因组DNA ,经限制性内切酶Sau3AI部分消化后 ,10 %～ 4 0 %蔗糖密度梯度离心分离 10～ 2 0kb的DNA片段。以λEMBL3为克隆载体 ,与 10～ 2 0kb的DNA片段进行连接和包装 ,构建了毛足棒角蝗的基因组DNA文库。经测定该文库的滴度是 2× 10 5pfu/mL ,根据计算 ,99%的毛足棒角蝗的基因包含在该基因组文库中。     

武夷菌素产生菌Fosmid文库的构建及文库探针的获得

以不吸水链霉菌武夷变种CK-15为材料,提取基因组DNA,经Sau 3A Ⅰ部分酶切后回收35-40 kb之间的片段,连接到pCC1FOS载体上,经过包装转染涂布后构建得到了CK-15基因组的Fosmid文库,文库的滴度为8.8×105CFU/mL.随机挑取16个阳性克隆,经EcoR Ⅰ和Hind Ⅲ双酶切电泳分析,样品插入片段平均长度大于35 kb,插入率为100%,符合构建文库的要求.根据多氧霉素、尼克霉素生物合成基因及大环内酯类聚酮合成酶基因(PKS)设计特异性引物,以基因组为模板进行PCR扩增,筛选特异性引物探针.结果用大环内酯类聚酮合成酶基因设计引物扩增出1 693 bp的片段经Blast比对其与大环内酯类抗生素生物合成基因相似性为95%以上.武夷菌素产生菌CK-15 Fosmid文库的构建及文库探针的获得,为武夷菌素生物合成基因的克隆奠定了基础.     

一种快速简便构建DNA病毒测序文库的方法

为了对1株中国棉铃虫核型多角体缺失病毒HZ-9进行基因组测序,采用了一种新的方法,通过超声波振断HaBacHZ9细菌人工染色体质粒（bacterial artificial chromosome plasmid,Bacmid）基因组DNA,用Taq酶在DNA片段两端加腺噤呤A,胶回收后得到预期的1—2kb的DNA片段,然后与pGEM-Teasy载体连接,构建了中国棉铃虫缺失病毒HaBacHZ9的亚克隆文库。结果随机挑选10个克隆子酶切分析,显示9个克隆子有1500bp左右的插入片断,并对HaBaeHZ9进行了全基因组测序。结论成功构建了HaBaeHZ9的DNA测序文库,为HZ-9功能基因组学研究奠定了基础,这是一种简单快速的构建DNA病毒测序文库的方法。     

以可转化人工染色体(TAC)载体为基础的百脉根基因组文库的构建及筛选

可转化人工染色体(transformation-competentartificial chromosome,TAC)载体是具有克隆和转移大片段DNA特征的新型载体,是植物基因克隆和转化的有效工具.该研究把它用于豆科植物百脉根(Lotus japonicus)基因组文库的构建.此文库由1.8×105个克隆组成,平均插入片段大小为15kb左右,约覆盖百脉根基因组6倍.文库保存在12块96孔板中,每个孔中约含150个不同的重组克隆.用与花发育相关的同源基因Ljcen1片段为探针,筛选得到6个阳性克隆,酶切后验证这些阳性克隆,结果表明这些克隆含有同一个基因片段.此基因组文库可直接用于植物转化,为百脉根功能基因组的研究打下基础.     

新疆军垦型细毛羊基因组BAC文库的构建

选用新疆军垦型细毛羊为材料,.构建了含有190 464个克隆的BAC文库,文库平均插入片段大小为133 kb,同时文库92.5%的克隆插入片段大于100 kb,而且有部分克隆甚至大于300 kb,这将满足大多数基因筛选的要求.假定绵羊的基因组含有3x106 kb,根据文库的平均插入片段大小,计算的文库基因组覆盖率为8倍.因此,从文库筛选到目的片段的概率为98.2%.由于该文库中插入的外源片段来自新疆军垦型细毛羊的基因组,这对于研究新疆军垦型细毛羊的特殊性状的基因与其他绵羊品种和物种之间的差异,及构建其全基因组物理图谱和基因图谱地完善是非常有利的.     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various