巴斯德毕赤酵母甲醇诱导表达磷脂酶A2的转录组学分析

【目的】基于转录组学技术研究表达磷脂酶A_2的毕赤酵母重组菌在甲醇诱导表达外源蛋白时的基因表达差异,从而解析外源蛋白高效诱导表达机制,为进一步工程菌株的改造提供理论支撑。【方法】以一株产磷脂酶(PLA_2)的毕赤酵母为出发菌株,采用RNA-Seq二代测序方法,研究在甘油培养和甲醇诱导两种条件下,重组毕赤酵母转录组基因表达差异情况。【结果】重组毕赤酵母中共鉴定到5225个转录本。甘油培养与甲醇诱导相比,共有857个基因发生显著变化。依据代谢途径分类,差异基因集中在核糖体成分、甲醇代谢、磷酸戊糖途径、糖酵解途径、柠檬酸循环、乙醛酸循环以及蛋白质加工过程。【结论】通过分析甲醇诱导前后的差异表达基因,结果表明碳源改变对胞内代谢会产生全局影响。本研究结果为进一步研究毕赤酵母表达外源蛋白的机制提供了基础。     

酿酒酵母对数生长后期代谢重构的全基因组表达谱芯片分析

为了探讨酵母进入对数生长后期以后酒精生产速度降低的原因,我们利用酵母表达谱芯片技术对酿酒酵母细胞从对数生长中期进入对数生长后期时的全基因组表达谱进行了分析,发现酵母在对数生长中期的表达谱非常稳定,而一旦进入对数生长后期.则出现明显的代谢重构现象.许多氨基酸合成和代谢相关的基因、离子转移以及与能量的生成和储存等功能相关的基因出现了不同程度的上调;而许多涉及酵母转座和DNA重组的基因则表达下调;一些中心代谢途径也发生了代谢重构.包括:琥珀酸和α-酮戊二酸生成途径基因的一致上调,都与氨基酸合成和代谢相关基因表达的结果相吻合.结果表明:由于氨基酸合成的需求量增加,进入对数生长后期酵母的代谢转向TCA循环和乙醛酸循环,导致酒精的生产速率降低.     

酿酒酵母对数生长后期代谢重构的全基因组表达谱芯片分析

为了探讨酵母进入对数生长后期以后酒精生产速度降低的原因, 我们利用酵母表达谱芯片技术对酿酒酵母细胞从对数生长中期进入对数生长后期时的全基因组表达谱进行了分析, 发现酵母在对数生长中期的表达谱非常稳定, 而一旦进入对数生长后期, 则出现明显的代谢重构现象。许多氨基酸合成和代谢相关的基因、离子转移以及与能量的生成和储存等功能相关的基因出现了不同程度的上调; 而许多涉及酵母转座和DNA重组的基因则表达下调; 一些中心代谢途径也发生了代谢重构, 包括: 琥珀酸和a-酮戊二酸生成途径基因的一致上调, 都与氨基酸合成和代谢相关基因表达的结果相吻合。结果表明: 由于氨基酸合成的需求量增加, 进入对数生长后期酵母的代谢转向TCA循环和乙醛酸循环, 导致酒精的生产速率降低。     

鲁氏接合酵母海藻糖耦合发酵过程的转录组差异分析

[目的]为探究鲁氏接合酵母(Zygosaccharomyces rouxii)在海藻糖耦合发酵过程中适应高温胁迫的基因表达差异及内在代谢调节机制。[方法]采用GC色谱仪和Illumina HiSeq^TM高通量测序技术对海藻糖耦合发酵过程(无调控阶段、补料响应阶段、补料与中温刺激耦合响应阶段、补料与高温刺激耦合响应阶段)的生物量、胞内海藻糖含量、基因转录序列等指标进行检测。[结果]转录组测序结果显示，与无调控阶段对比，三个调控响应阶段分别筛选得到1717、522、2038个差异表达基因。海藻糖耦合发酵过程中的鲁氏接合酵母细胞差异表达基因主要富集到次级代谢产物的生物合成、核糖体、氨基酸的生物合成等代谢途径上，高温胁迫条件下(海藻糖高速合成过程)分别涉及158、97、58个差异表达基因，次级代谢主要涉及赖氨酸等氨基酸和嘌呤/嘧啶核苷酸，氨基酸的生物合成主要涉及赖氨酸代谢、半胱氨酸和谷氨酸代谢。[结论]通过转录组分析揭示了鲁氏接合酵母主要通过调控次级代谢产物的生物合成与氨基酸代谢来适应高温热激胁迫。     

MXR1基因对毕赤酵母工程茵代谢调控的影响

为了研究毕赤酵母中转录因子Mxrlp在毕赤酵母代谢调控中所起的作用,构建一株以南极假丝酵母脂肪酶B基因(CALB)作为报告基因,MXR1基因完全缺失的毕赤酵母基因工程菌株.将重组质粒pPIC9K-CALB转化毕赤酵母GS115,利用三丁酸甘油酯平板筛选得到分泌表达CALB的重组茵GS115/pPIC9K-CALB.通过重叠延伸PCR方法获得一段中间含有博来霉素抗性基因sh ble,两翼大约各有1 200 bp与毕赤酵母MXR1基因上下游同源的基因片段,将此片段用氯化锂法转化毕赤酵母细胞GS1 15/pPIC9 K-CALB后,利用博来霉素抗性及CALB酶活力丧失双重筛选的方法得到一株MXR1基因完全缺失的毕赤酵母基因工程菌株.该菌株在以甲醇为唯一碳源的培养基中不生长,在以乙醇、葡萄糖或者甘油为唯一碳源的培养基中生长缓慢.结果表明转录Mxrlp因子在毕赤酵母中的多条代谢途径中起着关键性的作用,主要涉及甲醇、乙醇、甘油和葡萄糖等代谢途径.     

降低诱导温度提高毕赤酵母甲醇代谢关键酶基因转录水平和猪-α干扰素生产性能

以一株表达猪-α干扰素(pIFN-α)重组毕赤酵母为研究对象,考察了诱导温度对pIFN-α表达、细胞代谢活性及甲醇代谢关键酶的影响.在5 L发酵罐开展毕赤酵母高密度流加培养生产表达pIFN-α研究,利用实时PCR技术对不同诱导温度、诱导稳定期的胞内甲醇代谢关键酶(包括解毒酶)基因转录水平进行定量分析.低温(20℃)可以...     

毕赤酵母基因工程菌胞内AOX酶的检测方法

巴斯德毕赤酵母(Pichia pastoris)作为外源基因的表达宿主,已成功表达出一系列胞内和胞外蛋白[1～6],并已建立起了一套较成熟的发酵工艺.巴斯德毕赤酵母基因工程菌的外源基因,由胞内AOX酶(乙醇氧化酶)基因启动子调控.在非甲醇碳源条件下(如甘油或葡萄糖),AOX酶基因表达被抑制,外源基因也处于不表达状态.而以甲醇为唯一碳源时,AOX酶在胞内大量合成,同时外源基因被调控表达.在一般情况下,AOX酶的变化直接反映了外源基因的表达状况,因此通过分析检测胞内AOX酶的含量和变化速率,就可以确定外源基因所处的状态.     

利用毕赤酵母系统表达重组人生长激素的研究

为了获得重组人生长激素在毕赤酵母中高表达的菌株,按毕赤酵母基因密码子偏爱性,人工合成hGH的全基因序列.该基因被克隆到穿梭质粒pPIC9K中,PEG1000介导转入毕赤酵母GS115细胞,通过G418筛选获得高拷贝转化子.在甲醇的诱导下.实现了hGH在毕赤酵母中的成功表达.通过发酵条件的优化.发酵上清中的表达量达1537 mg/L经过超滤和两步层析,重组蛋白的得率这35%,纯度为97%,相对分子质量测定表明重组蛋白的相对分子质量与理论值相近.N-端氨基酸测序证实hGH基因在毕赤酵母中获得正确的表达.     

酵母SOD1基因缺失突变体应答刚果红胁迫的转录组学分析（英文）

SOD1基因编码的铜锌超氧化物歧化酶是酵母细胞中最重要的抗氧化酶.前期研究发现,SOD1基因缺失(sod1Δ)导致酵母细胞对真菌细胞壁抑制剂刚果红(Congo red,CR)的敏感性增加,提示细胞抗氧化能力与细胞壁稳定性相关.本研究采用酵母全基因组表达谱芯片,比较了CR胁迫条件下,野生型酵母细胞和sod1Δ酵母细胞的转录表达谱.结果表明,与野生型酵母细胞相比,sod1Δ酵母细胞中260个基因发生了显著差异表达(140个基因表达上调、120个基因表达下调).随机选取12个差异表达基因采用定量PCR验证,结果与芯片分析结果一致.差异表达基因功能主要涉及细胞壁(几丁质合成)、细胞代谢、细胞防御(抗氧化和热冲击蛋白)、蛋白质合成以及大量功能未知基因.进一步研究发现,CR处理后,细胞壁几丁质含量和细胞内氧化应激指标丙二醛(MDA)含量在sod1Δ酵母细胞中显著升高,而在野生型酵母细胞中无明显变化,与芯片筛选差异表达基因的生物学功能分析结果一致.本研究提供了在全基因组水平上对SOD1基因与细胞壁应激反应之间关联的新认识.     

基于转录组分析的金针菇冷诱导原基形成的调控网络

金针菇是低温结实型菌类,原基形成需要低温诱导,子实体发育也需要较低的温度,因此在工厂化生产过程中能源消耗大,生产成本高。本研究利用转录组测序对金针菇菌丝体和原基进行RNA-Seq分析,筛选得到7 935个差异表达基因,在原基形成后,有4 025个基因上调表达以及3 910个基因下调表达。通过GO注释和KEGG通路注释等生物信息学手段对代谢途径进行分析,可推测冷诱导形成原基的代谢调控为：当金针菇菌丝细胞接受到冷信号后,糖分转运相关的基因表达量下降,导致碳源摄取效率下降,因而糖酵解途径大部分相关基因表达量下调,进而导致三羧酸循环的底物乙酰辅酶A（乙酰CoA）合成量减少,整个细胞能量产出下降。此负反馈信号使细胞内储存的脂质进行氧化代谢的基因表达上调,产出乙酰CoA以供三羧酸循环产能。此负反馈导致不饱和脂肪酸的合成基因表达上调,以调节细胞流动性;同时磷脂和鞘脂代谢通路的相关基因表达大多上调,合成增多,细胞膜的组分因此改变,因此细胞进行重构进入另一种状态。与DNA复制、RNA转录和蛋白质合成的相关基因表达均大部分上调,表明了原基形成时细胞正处于增殖旺盛时期。本研究结果从分子水平上揭示了金针菇原基的形成伴随着能量来源的转变,各个代谢途径的相互调控以及相关基因的表达影响,为有目的培育高温型金针菇新品种,减少栽培能耗提供理论依据。     

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