庆大霉素生物合成基因genA的功能

【目的】在绛红色小单孢菌G1008(Micromonospora purpurea G1008)上构建genA基因缺失工程菌,通过分析其次级代谢产物的变化,推测genA基因功能。【方法】构建用于gen A基因框内敲除的质粒pAB103,经接合转移导入绛红色小单孢菌G1008,安普抗性及PCR扩增筛选获得genA缺失工程菌GA1048。【结果】与出发菌G1008相比,工程菌GA1048不再合成庆大霉素C族组分,主要积累中间代谢产物庆大霉素A2。【结论】genA基因失活导致庆大霉素生物合成代谢流中断,暗示gen A基因参与加洛糖胺C-3″位的氨甲基化。     

甲基营养菌MP688甲醇脱氢酶基因mpq1818的敲除及功能研究

目的:研究甲醇脱氢酶基因mpq1818在甲基营养菌MP688生长代谢中的作用。方法:利用同源重组原理构建中间为庆大霉素抗性基因Gmr、两侧mpq1818基因上下游序列同源的敲除载体pAK0-up-Gmr-down,接合转移导入MP688,通过庆大霉素抗性和组合PCR方法筛选基因敲除菌,并检测其生长、甲醇脱氢酶活性、甲醇利用及吡咯喹啉醌(PQQ)生物合成能力等方面的差异。结果:抗性和PCR验证显示mpq1818缺失株构建成功;与野生菌相比,缺失株的甲醇脱氢酶活力及利用甲醇的能力降低,而且菌株的生长和PQQ产量也有显著下降。结论:基因mpq1818的缺失影响菌株前期生长与PQQ合成。     

一株产G418单组分工程菌的构建

【目的】定向改造庆大霉素产生菌绛红小单孢菌G1008,获得产G418单组分工程菌。【方法】以温敏型穿梭质粒p KC1139为载体,构建敲除基因genQ*重组质粒p QB303,通过接合转移,导入绛红小单孢菌G1008中,影印筛选和PCR鉴定genQ*框内缺失突变菌,利用TLC和MS分析其代谢产物组分。【结果】获得一株genQ*缺失工程菌Micromonospora purpurea GQ175,其代谢产物为G418单组分,生物效价达828 mg/L,与出发菌G1008的产抗能力相当。【结论】工程菌GQ175产G418单组分,具有很好的工业开发价值。同时,证明绛红小单孢菌G1008中,庆大霉素C-6′脱氢酶基因为genQ*,且无其他同功酶基因。     

弗氏志贺菌 M90T 株毒力相关膜蛋白复合物组成基因缺失突变体的构建

目的：构建弗氏志贺菌M90T株毒力相关膜蛋白复合物组成蛋白基因的缺失突变株。方法：分别扩增目的基因的上、下游同源臂和卡那霉素抗性基因片段,利用重叠延伸PCR技术将这3个片段融合为打靶片段,电击转化M90T／pKD46感受态细胞,在bRed重组系统的作用下,通过同源重组将目的基因置换为卡那霉素抗性基因,之后在辅助质粒pCP20的作用下切除FRT位点之间的卡那霉素抗性基因,得到基因缺失突变体。结果与结论：分别构建了M90T株毒力相关膜蛋白复合物4个组成蛋白Apy、DnaK、CIpB、YdgA的基因缺失突变体,为进一步研究各基因的功能提供了突变体。     

肠出血性大肠杆菌Ⅲ型分泌系统ATP水解酶escN基因突变株的构建

目的:利用λ噬菌体的Red重组系统敲除肠出血性大肠杆菌O157∶H7的Ⅲ型分泌系统ATP水解酶Esc N,构建大肠杆菌esc N基因缺失突变株。方法:以O157∶H7为模板,PCR扩增目的基因两侧的同源臂序列,分别酶切连接于p UC19-kan质粒上,PCR获得中间嵌合卡那霉素抗性基因(带有FRT位点)的同源线性片段,利用质粒p KD46和p CP20介导的重组技术敲除esc N基因,并去除抗性标记;PCR及测序验证目的基因缺失后,测定缺失株及野生菌株的生长曲线。结果:敲除了肠出血性大肠杆菌O157∶H7的esc N基因,突变株与野生株的生长曲线相近。结论:构建了Ⅲ型分泌系统缺陷菌株,为进一步研究Ⅲ型分泌系统因子在肠出血性大肠杆菌致病过程中的作用奠定了基础。     

醛脱氢酶基因敲除的K.pneumoniae重组菌的构建

在利用KlebsiellapneumoniaeM5aL厌氧发酵甘油生产1,3丙二醇的过程中,一部分甘油通过氧化代谢途径经醛脱氢酶(ALDH)催化合成大量副产物乙醇,降低了1,3丙二醇的产量和得率。首次以醛脱氢酶ALDH为改造目标,利用同源重组技术获得了ALDH基因敲除的K.pneumoniae重组菌。首先,采用PCR的方法分别从K.pneumoniaeM5aL基因组DNA及质粒pBR322上扩增得到ALDH基因和四环素抗性基因(Tcr);然后将ALDH基因定向插入到质粒pUC18的多克隆位点得到中间载体pUC18ALDH,该载体与Tcr基因分别用AvaI和BsaAI双酶切后进行连接,得到ALDH基因敲除的重组载体pUCAT;经DNA测序及限制性酶切电泳分析,构建的载体由5′ALDHTcr3′ALDHpUC18组成,与设计结果相符;最后,利用该载体通过同源重组技术得到两株K.pneumoniae重组菌06231hb及06231hc,经菌落PCR及酶活鉴定,两株菌的ALDH基因均已缺失。与出发菌株K.pneumonaieM5aL相比,重组菌的乙醇合成浓度降低了43%～53%,1,3丙二醇合成浓度提高了27%～42%。     

利用λ-Red重组系统和平衡致死系统改造抗药性致腹泻工程疫苗

质粒pMM085是含有猪毒素源性大肠杆菌(ETEC)的黏附素K88与无毒肠毒素LTA-B 基因的重组质粒,含氯霉素抗性基因,由此构建的菌苗株带有抗药性。利用平衡致死系统改建此疫苗株,即将质粒上的氯霉素抗性基因cat替换成asd基因,并把新构建的质粒转移到缺失asd基因的大肠杆菌X6097中。但由于质粒pMM085是一个23kD的大质粒,传统的基因工程操作不易进行,利用λ-Red重组系统,将表达Red重组蛋白的质粒pKD46转化含pMM085的大肠杆菌X6097,并用两端各带有39ntcat基因同源区、含全长asd基因的PCR产物电击转化此感受态细胞,在λ-Red重组系统的帮助下,成功实现了asd基因对cat基因的置换。     

猪繁殖与呼吸综合征病毒M+N基因重组腺病毒载体的构建

本研究构建了M+N基因的重组腺病毒载体。首先用反转录聚合酶链反应(RT-PCR)的方法分别扩增出猪繁殖与呼吸综合征病毒M基因和N基因,将两者用口蹄疫病毒2A序列串联起来,将连接好的M+N基因插入腺病毒穿梭载体pAdTrack-CMV,经筛选获得了重组质粒pAdTrack-CMV/M+N;然后在大肠杆菌BJ5183内将此重组质粒和腺病毒骨架载体pAdEasy-1进行同源重组,获得了插有外源基因的重组腺病毒质粒pAd/M+N;最后,经PacⅠ酶切线性化后转染HEK-293细胞,成功获得含有M+N基因的重组腺病毒,为重组腺病毒活载体疫苗的研究奠定基础。     

肠炎沙门菌lpfC基因缺失株的构建及基本生物学特性研究

[目的]为研究与Lpf菌毛合成相关的lpfC基因对肠炎沙门菌致病性的影响。[方法]利用自杀质粒介导的同源重组技术构建肠炎沙门菌C50041株的lpfC基因缺失株C50041ΔlpfC,并进行PCR和测序鉴定。同时,将lpfC基因克隆至质粒pBR322并电转入缺失株中,构建回复株C50041ΔlpfCR。比较野生株C50041、缺失株C50041ΔlpfC及回复株C50041ΔlpfCR的基本生物学特性。[结果]成功构建了缺失株C50041ΔlpfC及回复株C50041ΔlpfCR,且lpfC基因的缺失不影响C50041的生长特性和生化特性。该缺失株具有良好的遗传稳定性,但缺失株对BALB/c小鼠的LD50是野生株的2倍。[结论]lpfC基因的缺失使肠炎沙门菌对BALB/c小鼠的毒力降低,为进一步研究肠炎沙门菌Lpf菌毛的功能奠定了基础。     

λRed重组系统用于沙门菌质粒毒力基因spvC敲除株的构建

[目的]利用λRed重组系统敲除沙门菌质粒毒力基因spvC。[方法]首先以质粒p KD4为模板,扩增得到两侧含spvC同源臂、中间为卡那霉素抗性基因的线性DNA片段。再将此线性片段电转入具重组功能的感受态沙门菌菌株,发生重组后,卡那霉素平板筛选阳性转化子。最后利用表达FLP重组酶的质粒p CP20,将FRT位点之间的卡那霉素抗性基因消除,用PCR鉴定。Western Blot检测野生沙门菌和spvC敲除株感染的He La细胞ERK磷酸化水平。[结果]沙门菌质粒毒力基因spvC敲除株构建成功,spvC敲除株感染的He La细胞内ERK磷酸化水平升高。[结论]成功构建沙门菌质粒毒力基因spvC敲除株,验证了spvC基因的功能。     

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     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV