Relationship between dose of hCREG and proliferation of vascular smooth muscle cells in vitro

AIM: To elucidate the relationship between the dosage of human cellular repressor of E1A-stimulated genes (hCREG) and the proliferation of vascular smooth muscle cells (VSMCs). METHODS: hCREG cDNA was cloned into a retroviral response vector (pRevTRE) controlled by tetracycline responsive element. The recombinant vector (pRevTRE-hCREG) was constructed and confirmed by sequence analysis. The pRevTet-On was transferred into human VSMCs-HITASY in vitro and selected by G418. The pRevTRE-hCREG was transferred into pRevTet-On-HITASY clones and selected by hygromycin. The expression of G418 and TRE was detected by RT-PCR analysis. The hCREG expressions in cell lysate and in supernant were evaluated by Western blotting and IP-Western respectively after the HITASY-hCREG cells cultured in medium at different concentrations of doxycycline. The relationship between dosage of hCREG and the proliferation of HITASY cells was assessed by flow cytometry analysis. RESULTS: The recombinant pRevTRE-hCREG was constructed successfully. The fragment inserted was confirmed by sequencing. The pRevTet-On and pRevTRE-hCREG were transferred into human HITASY cells and the clones (HITASY-hCREG) expressed double-resistant genes stably were obtained. RT-PCR identified that HITASY-hCREG cells expressed a 500bp band for G418 and a 899bp band for TRE, respectively. The expressions of hCREG both in lysate and in supernant were upregulated in parallel with the doxcycline increase in a dose dependent manner. Flow cytometry analysis showed that the distribution of G1 stage in HITASY-hCREG cells was also enhanced in accordance with the increase in hCREG expression. CONCLUSION: The expression of hCREG inhibits the proliferation of human VSMCs in a dose dependent manner.     

甘蓝Ogura 细胞质雄性不育相关基因BoMF1启动子的克隆及功能分析

 通过TAIL-PCR 染色体步移技术,从甘蓝（Brassica oleracea L. var. capitata L.）基因组中克 隆到胞质雄性不育（OguCMS）相关基因BoMF1 翻译起始位点上游521 bp 的启动子序列。软件分析预测 表明,该启动子序列中存在多个顺式作用元件,包括TATA-box、CAAT-box、MYB 结合位点、植物激 素响应单元等。为了研究该启动子的表达特性,亚克隆了BoMF1 转录起始位点上游521 bp 序列,将其置 换pBI121 中的CaMV35S 启动子,驱动其下游的GUS 基因,构建植物表达载体pBI121-BoMF1P,以pBI121 空载体作为阳性对照,通过农杆菌（LBA4404）介导法转入拟南芥。结果表明,甘蓝BoMF1 启动子序列 能驱动GUS 基因在拟南芥花药发育晚期的花药和花粉中特异表达,表达具有组织特异性。     

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Expression of GFP and Luc reporter genes driven by human myocardial myosin light chain gene promoter in human cardiomyocytes

AIM:To construct the lentiviral vectors with green fluorescent protein(GFP) and luciferase(Luc) reporter genes driven by human myosine light chain 2v gene promoter(pMLC2v) and to investigate their expression in human cardiomyocyte(HCM) cell line and human lung cancer cell line A549. METHODS:Human pMLC2v-specific lentiviral vectors with GFP(pMLC2v-GFP) or Luc(pMLC2v-Luc) were constructed and transfected into HCM and A549 cell lines. The expression characteristics of the reporter genes were observed by confocal fluorescent microscopy and bioluminescence detection. Common(nonspecific) promoter-driven GFP(GFP_C) or red fluorescent protein(RFP_C) lentiviral vectors were used as controls. RESULTS:Both cell lines expressed GFP and RFP 3 days after transfected with the nonspecific vectors. HCM specifically expressed GFP and Luc 3 weeks after transfected with the pMLC2v-GFP or pMLC2v-Luc vectors. However, A549 cells didn't show the similar expression pattern. CONCLUSION:The pMLC2v-GFP and pMLC2v-Luc lentiviral vectors are specific for newly proliferative cardiomyocytes, indicating that they can be used as reliable tools for tracking the differentiation of stem cells into cardiomyocytes in vivo.     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

Construction and identification of recombinant adenovirus vector carrying a N-terminal phosphorylation sites-deleted human IκBα mutant gene

AIM:To optimize the IκBα mutant (IκBαM) gene derived from human placenta tissue by deleting N-terminal phosphorylation sites of serine 32/36,and to construct and identify its replication-deficient recombinant adenovirus (AdIκBαM).METHODS:The IκBαM gene (203-1 003 bp) was acquired by positional cloning,followed by subcloning it into pShuttle and pGEM-T vectors for further PCR,double digestion,DNA sequencing and homology analysis.Subsequently,the expression unit of pShuttle-IκBαM containing CMV promoter,IκBαM cDNA and poly A signals was inserted into Ad5 vector,after which the resultant recombinant adenovirus AdIκBαM was packaged in 293 cells by cotransfection with lipofectamine.Western blotting analysis and electrophoretic mobility shift assay were utilized to detect the AdIκBαM-mediated expression of IκBαM gene in 293 cells and its suppressive effect on phorbol myristate acetate (PMA)-induced nuclear factor κB (NF-κB) activation in ECV304 cells,respectively.RESULTS:The relevant nucleotide and amino acid sequence of IκBαM gene was consistent with that of GenBank (accession number M69043).The titer of the prepared AdIκBαM was 4.0×1012 pfu/L.Moreover,the IκBαM gene was expressed in 293 cells,and potently inhibited the PMA-induced NF-κB activation in ECV304 cells in a dose-dependent manner.CONCLUSION:AdIκBαM is a nonvel vector for both efficient transfer and expression of IκBαM gene as well as specific inhibition of NF-κB activity,providing a promising future for gene therapy of asthma.     

庆祝《园艺学报》创刊50 周年

今年是《园艺学报》创刊发行50周年。50年来,《园艺学报》坚持为学术交流服务,为促进学科发展作贡献的办刊原则,以"科学性;创新性;对生产和科研有参考启迪作用"的标准,收录和发表了大量高水平的论文,记载了几代科技工作者呕心沥血创新之作,反映了中国园艺科学技术和园艺产业的发展历程。     

砂梨果实ACC氧化酶cDNA克隆及其反义表达载体构建

利用RT-PCR和RACE技术从'早生新水'砂梨成熟果实cDNA中克隆出ACC氧化酶基因保守片段及其5'和3'端,拼接后获得砂梨果实ACC氧化酶cDNA全长。该cDNA全长1 225 bp,将其命名为Pyp-ACO,GenBank登录号为EF451060。Pyp-ACO核苷酸序列有一个945 bp的开放读码框,5'端非翻译区为63 bp,3'端非翻译区为217 bp,与西洋梨和苹果的编码区核苷酸序列有较高的相似性,分别为98.3%和98.1%。Pyp-ACO推导编码314个氨基酸,该序列具备非血红素二价铁离子依赖型的氧化/加氧酶类的12个保守氨基酸和催化活性所需的3个氨基酸。将Pyp-ACO编码区序列反向插入pYPX145载体,构建了由双35S启动子所控制的双元表达载体,同时已成功将表达载体导入根癌农杆菌菌株LBA4404,为耐贮藏转基因梨选育奠定了基础。     

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.     

香石竹ACC氧化酶基因的克隆及其正义反义表达载体的构建

利用在Genebank上已报道的香石竹ACC氧化酶(ACO)基因的CDNA序列设计特异引物,以香石竹品种'MASTER'的eDNA为模板进行PCR扩增,克隆香石竹ACC氧化酶(AC0)基因.测序结果显示,克隆基因的序列与报道序列完全一致.将克隆的ACC氧化酶(AC0)基因分别连接到植物表达载体PBI121启动子CaMV 35S的上游及下游,构建香石竹ACC氧化酶(Aco)基因的正义表达栽体PBI121-ACO及反义表达载体PBI121-anti ACO.经PCR鉴定,基因已成功构建到表达裁体上.为香石竹抗衰老基因工程育种奠定了基础.     

甘菊BADH基因启动子的克隆与瞬时表达分析

为了给菊花 [Dendronthema ×grandiflora (Ramat.) Kitam.] 的转基因育种提供诱导型启动子,借鉴5′RACE策略,利用锚定PCR步移的方法克隆了甘菊 [Dendranthema lavandulifolium (Fisch. ex Trautv.) Makino] 甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase,BADH）基因的4个启动子序列,分别命名为DBP11、DBP12、DBP21和DBP22（GenBank登录号：DQ497620~DQ497623）,序列长度分别为1 230、1 249 、1 273 和574 bp。4个启动子序列对应区域的同源性在89%以上。其中DBP12和DBP21分別是甘菊BADH基因DlBADH1和DlBADH2（GenBank登录号：DQ011151和DQ011152）的启动子序列,DBP11和DBP22为甘菊BADH基因家族中其它成员的启动子序列。序列分析表明,上述启动子序列中均含有多处与水分胁迫和脱落酸诱导相关的顺式作用元件。在表达载体pCAMBIA1305.2的基础上,用所克隆的4个甘菊BADH基因启动子序列分别置换驱动其报告基因表达的35S启动子,建立了新的植物表达载体。用其转化农杆菌,并用叶盘法侵染甘菊,瞬时表达的结果表明,这些启动子序列均具备驱动报告基因表达的功能。     

银耳芽孢内源gpd启动子的克隆与功能鉴定

利用反向长距离PCR技术从银耳(Tremellafuciformis)芽孢gpd基因出发克隆上游gpd启动子序列,将预测的gpd启动子片段与多功能纤维素基因(mfc)连接构建表达载体,与潮霉素抗性质粒pBgGl-hph共转化银耳芽孢,对拟共转化子进行酶活发酵试验。结果表明:4轮反向长距离PCR克隆得到了1761bp的上游序列,经预测启动子落在上游1000bp左右区域内,包含两个高分值的起始转录位点,将gpd启动子分成gpd-Tre1(885bp)、gpd-Tre2(708bp)、gpd-Tre3(466bp)3段区域,分别与多功能纤维素基因(mfc)构建表达载体pgTre1-mfc、pgTre2-mfc和pgTre3-mfc。拟共转化子酶活发酵试验结果发现3个表达载体的转化子均能检测到多功能纤维素酶活,转化子T1-2包含gpd-Tre1启动子大片段,整体酶活最高,CMC酶活为14.12U/mL,比出发菌株Tr01提高34.3%,比工程菌株yLes3提高25.7%,木聚糖酶活为34.8U/mL,比Tr01酶活提高26.3%,略低于yLes3。3个启动子片段均具有表达活性,相比之下885bp的大片段gpd启动子表达活性更高。