木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

克隆木糖还原酶XYL1基因的工程菌YT发酵玉米芯水解液的研究

克隆毕赤氏酵母(Pichia stipitis)木糖还原酶基因XYL1,将其连接到适用于酿酒酵母工业菌株的多拷贝载体pYMIKP中,构建得到表达质粒pYMIKY-XYL1,转化酿酒酵母工业菌株Saccharomyces cerevisiae 6508.利用G418筛选转化子,得到含高拷贝木糖还原酶基因的酿酒酵母重组菌YT,以YT发酵玉米芯工业水解液生产木糖醇,研究其发酵特性和规律,为工业上生物转化法生产木糖醇提供参考.     

酿酒酵母中双链RNA介导基因沉默技术研究GRE3基因表达抑制

RNA沉默技术作为探索基因功能的实验手段应用于多种生物.以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象,检测酿酒酵母双链RNA介导的基因沉默效应.以pESC-LEU为骨架,构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499.用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调.结果表明,双链RNA介导的基因沉默技术,能够用作降低酿酒酵母某一特定基因表达水平的工具.并有助于理解芽殖酵母的RNA干扰现象.     

重组菌Ralstonia eutropha W50-EAB D-木糖代谢相关的限速靶点

【目的】通过代谢工程改造真养罗氏菌(Ralstonia eutropha)W50-EAB木糖代谢的相关限速靶点,进一步提高R.eutropha W50-EAB的D-木糖利用效率,为获得高效利用纤维素水解液的菌株奠定基础。【方法】利用PCR技术扩增R.eutropha转酮酶基因tkt A,cbb T2和转醛酶基因tal,将扩增的tkt A,cbb T2和tal基因分别构建到表达载体p BBR1MCS-3上,获得重组质粒p WL1-TKT,p WL1-CBBT2,p WL1-TAL。通过电转的方式将质粒分别转化W50-EAB获得重组菌W50-KAB,W50-CAB和W50-TAB。利用基因敲除的方法,获得醛还原酶基因h16_A3186敲除株W50’-EAB。通过电转的方式将重组质粒p WL1-TAL导入敲除株W50’-EAB获得重组菌株W50’-TAB。通过摇瓶发酵研究重组菌株W50-KAB,W50-CAB,W50-TAB,W50’-EAB以及W50’-TAB的发酵特性。【结果】酶活分析结果表明,转酮酶和转醛酶基因实现表达。摇瓶发酵结果表明,转酮酶基因过表达菌株W50-KAB和W50-CAB相比于对照菌株W50-EAB/p3,表现出降低的木糖利用能力;而转醛酶基因过表达重组菌株W50-TAB以及敲除菌株W50’-EAB对木糖的利用得到一定的提高。在0.1 mol/L木糖的发酵培养基中,W50-EAB的最大比生长速率为0.035 h-1,PHB干重比为16.2±1.01%;而W50-TAB的最大比生长速率提高到0.039 h-1,PHB干重比达到20.5±0.76%;醛还原酶基因敲除菌株W50’-EAB最大比生长速率提高到0.040 h-1,PHB含量提高到19.8±1.05%。结果显示转醛酶基因的过表达与醛还原酶基因的敲除对木糖利用均表现出一定的优势,将这两种优势组合获得菌株W50’-TAB,摇瓶发酵分析结果为最大比生长速率达到0.042 h-1,PHB积累达到27.9±0.47%,相比于对照菌株提高了72.2%。另外,在含有葡萄糖(0.01 mol/L)和木糖(0.09 mol/L)的混合糖培养下,重组菌株W50-TAB,W50’-EAB和W50’-TAB相比于在纯木糖培养下都表现出更高的生物量和胞内PHB积累量。【结论】磷酸戊糖途径关键酶转醛酶基因的过表达加速了木糖代谢流,从而可以高效利用木糖积累一定量的PHB。醛还原酶对木糖代谢有阻碍作用,敲除该酶基因后木糖代谢能力有了一定的提高,而两者协同作用可以进一步提高重组菌株的木糖利用效率和PHB积累能力。     

spt15突变基因对酿酒酵母木糖利用的影响

利用基因工程手段得到重组菌YPH499-3中的spt15有效突变基因,通过表达载体pYX212转化入酿酒酵母原始菌株YPH499中,重新获得酿酒酵母重组菌株。对其性状进行研究,结果表明该菌株能有效利用木糖并共发酵木糖和葡萄糖。在30oC、200r/min,发酵72h时,50g/L木糖的利用率为82.0%,乙醇产率为28.4%;当木糖和葡萄糖以质量比1:1混合发酵时,木糖和葡萄糖的利用率分别为80.4%和100%,乙醇产率为31.4%;同时发现木糖醇的含量极低。从而验证了有效突变基因spt15-10对酿酒酵母共发酵木糖和葡萄糖产酒精的影响。     

酵母整合型载体的构建及其功能分析

选取酿酒酵母基因组rDNA序列中25S和5S rDNA之间的一段约2.2 kb序列作为整合载体同源重组到酿酒酵母基因组中的靶位点,PCR扩增得到该段序列左右2段大小分别为865 bp和1 350 bp的片段,将其与 URA3、磷酸丙糖异构酶启动子和终止子序列依次克隆入pUC18中,构建成整合型载体pNK.以绿色荧光蛋白基因为报告基因,荧光显微镜下观察到酿酒酵母菌株INVSc1转化子发出较强荧光,Southern 杂交产生目的条带,这些结果表明载体 pNK 具有整合并表达外源基因功能.通过对转化子连续培养证明构建的整合型载体的稳定性较高.     

带有木糖还原酶基因和木糖醇脱氢酶基因的重组酿酒酵母的构建

采用双载体系统,将携带有瑞氏木霉木糖醇脱氢酶基因的表达质粒pAJ401－Xdh1转化已带有树干毕赤氏酵母木糖还原酶基因的重组酿酒酵母H475,构建了同时带有毕赤氏酵母木糖还原酶基因和瑞氏木霉木糖醇脱氢酶基因的重组酿酒酵母HX1。研究了重组酿酒酵母HX1对木糖的转化利用情况。     

稳定遗传的染色体组合整合酿酒酵母重组菌株的构建

染色体整合表达可获得稳定遗传的基因工程菌,是工业酿酒酵母育种的重要手段。pAUR135整合载体是抗生素标记基因可循环使用的载体,添加特定的同源臂后可构建在酵母染色体上稳定遗传的菌株。目前对酵母菌的分子育种常需要对多个基因进行过表达,利用pAUR135载体可将不同基因分别整合在不同染色体或相同染色体的不同位点,这种组合整合表达方法可对不同基因的表达强度比例进行调节,构建表型优化的工业酿酒酵母菌株。本研究以木糖代谢途径基因为例,构建了3个pAUR135整合载体,将3个木糖代谢基因依次整合到工业酿酒酵母染色体的不同位点,获得了染色体组合整合表达的代谢工程菌株。与将这3个基因整合在同一个位点的对照重组菌株相比,染色体组合整合的重组菌株木糖利用率提高了24.4%–35.5%。多基因染色体组合整合方法从新的角度对工业酵母进行代谢工程改造,所获得的工程菌株不带有任何外来基因和选择标记,可以保持性状的稳定,是工业酿酒酵母分子育种的新方法。     

代谢木糖和葡萄糖的重组酿酒酵母的构建

为使酿酒酵母（Saccharomyces cerevisiae）YS58代谢木糖产乙醇,采用PCR方法克隆得到树干毕赤酵母（Pichia stipitis）木糖醇脱氢酶基因xy12,并将该基因和克隆得到的休哈塔假丝酵母（Candida shehatae）缺终止子的木糖还原酶基因xyl1一起连接到酵母表达载体pYES2的强启动子GAL下,得到融合表达载体pYES2-P12。通过醋酸锂转化的方法将pY-ES2-P12转入S.cerevisiae YS58中,得到S.cerevisiae YS58-12。利用所构建的重组酿酒酵母进行术糖发酵实验,结果表明该重组酵母能发酵木糖,使木糖利用率得到进一步提高,最高达到81．3％,而且能代谢木糖产生乙醇。     

轮状病毒VP7基因的克隆与表达

应用聚合酶链式反应技术(PCR)扩增轮状病毒VP7基因,并将其克隆到pMD18-T simple载体上,对重组子进行PCR检测和限制性内切酶分析,并测定DNA全序列。结果显示,克隆片段全长为981 bp。将轮状病毒VP7基因定向的克隆到原核表达载体pET-32a启动子下游,构建原核表达载体pET-32aVP7。将质粒pET-32aVP7转化Transetta表达菌株进行诱导表达,裂解菌体细胞抽提蛋白质进行SDS-PAGE。结果表明,轮状病毒壳蛋白VP7基因在Transetta表达菌株内得到成功表达。     

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.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.