工业酿酒酵母PGK启动子的克隆与功能分析

为了获得适用于构建工业酿酒酵母整合型表达载体的组成型启动子,以工业酿酒酵母南阳K基因组DNA为模板,采用PCR方法扩增得到了磷酸甘油酸激酶基因起始密码前的启动子片段2个,其中长片段781bp,命名为PGK1(GenBank Acession No.FJ415226)。NCBIBlast软件分析结果表明,PGK1核苷酸序列与酿酒酵母染色体Ⅲ上PGK启动子(GenBank Acession No.X59720)相似性为99%,序列中含有基因表达所需的基本调控元件TATA-box和CAAT-box等。功能分析表明PGK1能驱动整合在工业酿酒酵母基因组上的外源基因葡萄糖淀粉酶基因的表达。综上表明成功克隆得到PGK1启动子,为工业酿酒酵母表达载体的构建奠定了基础。

PGK1基因变异致磷酸甘油酸激酶1缺乏症1例临床与遗传学分析

目的探讨PGK 1基因变异致Ⅸ型糖原累积病(磷酸甘油酸激酶1缺乏症)的临床特点及诊疗方法。方法回顾分析1例磷酸甘油酸激酶1缺乏患儿临床资料,分析其临床特点及实验室检查结果,采用二代测序分析基因变异筛查结果。结果患儿男性,3岁11个月首次就诊。患儿每次均以抽搐起病,起病后病情迅速加重,出现反复抽搐发作,每次病程中均有溶血性贫血,第三次住院期间出现严重的横纹肌溶解症,且发病后有明显的智力、运动发育落后,基因检测显示患儿PGK 1基因错义变异(c.150C>G),该变异导致第50号氨基酸由Cys变为Trp,来自于母亲,其母为杂合子,符合X连锁隐性遗传方式,既往文献及数据库未见报道。根据ACMG指南,此变异判定为可能致病性(基因检测时,尚未出现横纹肌溶解症),后期结合临床表现及基因检测结果,确诊为PGK 1缺乏。结论磷酸甘油酸激酶缺乏症是一类罕见的X连锁隐性遗传病,由PGK 1基因变异所致,该变异未见报道,扩充了Ⅸ型糖原累积病基因变异数据库。

日本血吸虫pcDNA3.1(+)-SjPGK重组质粒的构建及鉴定

目的 构建日本血吸虫中国大陆株磷酸甘油酸激酶基因片段 (SjPGK )的真核表达重组质粒 ,寻找新的预防日本血吸虫病的候选疫苗分子。 方法 采用逆转录 -聚合酶链反应 (RT -PCR)技术 ,从日本血吸虫总RNA中体外扩增SjPGK基因片段 ;克隆入pMD18-T载体中 ,然后挑取阳性克隆经双酶切分析、PCR鉴定 ;亚克隆入真核表达载体pcDNA3 .1(+ )中 ,阳性克隆经鉴定后进行脱氧核糖核酸序列测定 ;运用Blast程序 ,将测序结果及其推导的编码氨基酸序列与NCBI数据库在核苷酸水平和氨基酸水平进行同源性比较。 结果 RT -PCR特异性扩增出一条长约 83 0bp大小的条带 ;重组质粒pMD18-T -SjPGK和 pcDNA3 .1(+ ) -SjPGK经双酶切和以质粒为模板进行PCR扩增 ,均可获得一条与RT -PCR产物一致的DNA片段 ;脱氧核糖核酸序列测定和分析结果表明 :SjPGK基因片段长为 83 0bp ,与SmPGK的核苷酸同源性为 85 % ,分值为 672 ;氨基酸同源性为 94% ,分值为 473。 结论 成功地构建 pcDNA3 .1(+ ) -SjPGK重组质粒 ,为构建其核酸疫苗提供了条件。

Differential gene expression of phosphoglyceric kinase (PGK) and hypoxic adaptation in chicken

Four single-nucleotide polymorphisms (SNP) of the Phosphoglyceric Kinase (PGK) gene were discov- ered based on comparison of the sequences from an altiplano chicken breed (Tibetan chicken) and two lowland breeds (White Leghorn and Shouguang chicken). Gel-shift results indicate that one of these SNPs, an A→G mutation at position 59 in exon10, is able to bind hypoxia-induced factor-l (HIF-1), functioning as a hypoxia response element (HRE). The mutant gene results in M→T mutation at position 379 amino acid. The combined activity of this HRE and HIF-1 could increase correspondingly under a hypoxic stimulus. Hypoxia leads to increased death rates of chicken embryos; while the M→T mutation described herein is prevalent in healthy embryos grown under hypoxic conditions, thus it may repre- sent an adaptation to hypoxia. Fluorescence quantitative reverse transcription PCR results revealed that HIF-1 upregulates the transcript level of the glycolytic enzyme PGK in the brain and skeletal mus- cle of animals subjected to hypoxia. Thus, a large amount of ATP is produced by increased glycolysis, allowing the organism to meet energy metabolism demands. As such, we believe this SNP to be an adaptation to the external anoxic environment.

PGK1-coupled HSP90 stabilizes GSK3βexpression to regulate the stemness of breast cancer stem cells

Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.

水杨酸对蛋白核小球藻(Chlorella pyrenoidosa)生长及抗逆相关基因的影响

以蛋白核小球藻(Chlorella pyrenoidosa)为研究对象,首先克隆了热激蛋白70(HSP70)和磷酸甘油酸激酶(PGK)基因全长序列,然后研究了0—20μg/m L水杨酸对高温胁迫下HSP70和PGK基因表达,以及蛋白核小球藻生长、可溶性糖和蛋白含量的影响。结果获得了蛋白核小球藻HSP70基因的c DNA序列2405 bp,包括60 bp的5’-非编码区(UTR)、1959 bp的开放阅读框(ORF)和386 bp的3’-UTR和PGK基因的c DNA序列1664 bp,包括35 bp的5’-UTR,1398 bp ORF和231 bp 3’-UTR。序列比较和分析表明该蛋白核小球藻HSP70序列与其它绿藻的同源性高达83%—91%,PGK为70%—75%。荧光定量PCR结果显示随着水杨酸浓度的增加,HSP70和PGK的表达量在12h和24h均有所增加,而在10μg/m L水杨酸浓度下表达量最大,12h时分别为对照组的2.57倍和1.56倍,24h则为1.71倍和1.79倍。1—20μg/m L水杨酸可促进该藻的比生长速率、可溶性糖和蛋白含量的升高。本文结果表明一定浓度水杨酸对高温胁迫蛋白核小球藻有缓解作用,且5和10μg/m L水杨酸的效果最显著。

热带假丝酵母磷酸甘油酸激酶基因启动子功能鉴定

热带假丝酵母是重要的工业生产菌株,但表达系统还不完善导致代谢工程育种手段受到限制。探索新的表达元件对热带假丝酵母的基因工程育种十分重要。本文通过多重序列比对从热带假丝酵母ATCC 20336基因组中扩增得到一个具有较高活性的磷酸甘油酸激酶基因(PGK1)启动子,以酵母增强型绿色荧光蛋白(yeGFP3)作为报告基因,整合到热带假丝酵母基因组上并对其表达活性进行研究。通过分离300、550、750 bp和846 bp4个长度的启动子片段分别表达yeGFP3,转化XZX宿主菌获得P-1、P-2、P-3和P-4共4个转化子。荧光显微镜结果表明,随着启动子长度的截短,荧光逐渐减弱,前3个转化子的相对荧光强度分别为P-4的4.98%、9.84%和48.4%。同时分析了4个转化子的yeGFP3转录水平,转录水平分别为P-4的5.6%、13.8%和60%。初步判断PGK1启动子至少存在2个上游激活序列(UAS)。

载有磷酸甘油激酶基因启动子的新表达载体的构建以及在发酵性丝孢酵母中启动外源基因的表达研究(英文)

利用简并PCR技术从一株丝孢酵母(Trichosporon sp.)中克隆到磷酸甘油激酶基因的部分序列,然后利用染色体步移的方法克隆到了已知片段的上游序列约950bp。通过启动子序列分析软件分析,发现序列中含有启动子所需的必须元件如TATA BOX和CAAT BOX等,因此确定克隆到的基因片段含有启动子序列。将潮霉素基因置于该启动子下构建了丝孢酵母整合型表达载体pTFPH,并转化发酵性丝孢酵母(Trichosporon fermentans),转化后的酵母能够在含有潮霉素的抗性选择性平板长出,而未进行转化的对照菌株则不能生长。以上试验证明:丝孢酵母的磷酸甘油激酶基因启动子具有启动异源基因在发酵性丝孢酵母中表达的功能,这个结果为油脂酵母工程菌的构建和开发新的酵母表达宿主奠定了基础。