苹果ABP2基因克隆及其表达载体构建

以红富士苹果为材料,从苹果枝条形成层组织中提取总RNA,以随机引物及oligodT反转录成cD-NA。根据已知序列设计PCR引物并对其进行修饰,运用PCR技术扩增出苹果ABP基因片段和全长,经测序分析得知从红富士苹果中克隆到的基因ABP2(GenBank:HQ610832)为ABP(生长素结合蛋白)的突变体,同时完成了ABP2基因表达载体的构建。文章在苹果RNA提取、全长基因克隆、ABP2基因表达载体的构建方面积累了经验,并为进一步的转基因研究奠定了基础。     

人C6orf210基因原核表达载体的构建及蛋白的初步表达

为了构建人C6orf210基因原核表达载体,并在E．coliBL21中表达并纯化。采用RT—PCR扩增人C6orf210基因片段,并将其克隆到原核表达载体Pet21a中,构建重组质粒pET21a—C6ort210。经限制性内切酶EcoRI与XhoI双酶切鉴定及序列测定后,转化E．coliBL21,经IPTG诱导表达融合蛋白。结果显示获得全长为1188bp的人的CAiorf210基因片段。以构建的重组质粒pET21a—C6orf210转化E．coliBL21后,经IPTG诱导,表达出相对分子质量（Mr）约66000的融合蛋白。经SINS—PAGE、Western blot分析显示,诱导表达的蛋白为C6orf210。     

新基因SNC6在大肠癌中的表达研究

应用非同位素标记的原位杂交技术,配对检测了SNC 6基因在37例大肠癌病人的正常粘膜、癌旁粘膜和癌组织中的表达情况,并结合临床病理资料,探讨SNC 6表达与大肠癌发生、发展的相关性。结果表明,SNC 6基因只表达在大肠粘膜的上皮结构中,在大肠癌中发生了明显的表达降低或表达缺乏。该基因的表达与癌症病人的性别、年龄、Dukes分期、分化程度及浸润深度无关,但与有无淋巴结转移有关。提示SNC 6可能参与大肠癌发生发展后期的次级分子事件。     

假单胞茵产弹性蛋白酶基因的克隆与表达

从假单胞菌（Pseudomonassp．）XZG36中克隆弹性蛋白酶基因,构建原核表达载体,实现其在大肠杆菌（Escherichiacoli）中的高效表达,并对表达产物进行酶学性质分析,为微生物发酵生产弹性蛋白酶奠定基础．以假单胞菌基因组DNA为模板,PCR扩增弹性蛋白酶基因,并将其开放阅读框（0RF）克隆至融合表达载体pET30a（＋）进一步IPTG诱导表达;表达产物经His·Bind亲和层析纯化后对弹性蛋白酶进行酶学性质分析．实验成功克隆了弹性蛋白酶基因,DNA基因片段为1672bp、编码497个氨基酸残基的多肽,与预计长度相符合;实现了其在E．coli中的高效表达,表达量约占菌体总蛋白的20％;经SDS-PAGE分析,相对分子质量为48000,与预期的一致;提纯后的表达蛋白SDS-PAGE分析可见单一条带,纯度可达92％以上．表达蛋白具有良好活性．     

大肠癌的差异蛋白质组学研究

文章通过初步观察大肠癌患者癌组织与正常成人肠黏膜蛋白质的差异表达,为进一步探讨大肠的分子机制奠定基础。应用蛋白质组学的双向电泳技术,对大肠癌患者与正常成人肠黏膜蛋白质表达进行比较,观察其差异点。得到分辨率和重复性均好的大肠癌患者与正常成人直肠黏膜蛋白的双向凝胶电泳图谱,发现二者之间在表达上有明显差异,差异表达蛋白质点数共26个差异点,其中在大肠癌组织中表达上调点11个,下调的点15个。共分析了10个差异蛋白点,在10个蛋白质点中有5个蛋白质点得到鉴定。结论是大肠癌原发灶和正常肠黏膜蛋白质组表达有显著差异,HSP27蛋白、S100A9蛋白和GST-π蛋白表达上调,GRP75蛋白表达下调有可能与大肠癌发生相关。     

3～6岁儿童情绪表达规则认知研究

目前我国关于情绪表达规则认知的研究,还主要集中在语言表达能力已经有了一定发展的学龄儿童或成人,对3～6岁儿童情绪表达规则认知的研究还比较少。本研究借鉴国外研究范式,探讨在中国文化背景下年龄、性别、情境这三个因素对学前儿童情绪表达规则认知的影响。研究结果表明,年龄和情境对儿童情绪表达规则认知存在影响,但性别不是影响儿童情绪表达规则认知的因素。     

猪干扰素基因的克隆与原核表达载体的构建

干扰素(Interferon)是一种广谱抗病毒剂,具有抗肿瘤、抗病毒和免疫调节等多种作用而被广泛应用于病毒性疾病的预防和治疗。本实验以猪肝脏组织为材料,提取猪肝脏组织DNA,根据Gen Bank收录的猪干扰素基因序列设计特异性引物,以猪肝脏DNA为模板进行高保真扩增得到IFN基因的ORF片段,然后进行了T载体克隆。再利用引入Eco RⅠ、SalⅠ酶切位点的特异性引物对IFN成熟肽段的基因进行亚克隆,经双酶切、连接、转化后,成功构建了p ET28a-ifn原核表达重组质粒,为干扰素基因的原核表达奠定了基础。     

“思辨性阅读与表达”的内涵及其实现

“思辨性阅读与表达”任务群回应核心素养“思维能力”的目标指向,旨在培育学生理性思维和理性精神。落实“思辨性阅读与表达”任务群,需要深刻理解其内涵和意义,把握结构化的课程内容安排,以素养为导向重建教学形态和教学评价,同时要处理好与其他任务群之间的关系,实现形象思维与理性思维的平衡发展。     

Cult3D技术在网络虚拟实验开发中的应用研究

Cu lt3D作为一种新的网络3D技术,具有高压缩性、强交互力、跨平台和易于掌握等优点,应用于网络虚拟实验的开发可极大提高实验的仿真效果和交互能力。简要介绍了Cu lt3D技术及其项目开发步骤,以超导磁悬浮列车模型虚拟实验为例详细阐述了Cu lt3D的应用开发过程。     

甲壳动物卵黄蛋白原基因的分子克隆和表达研究进展

对近年来甲壳动物卵黄蛋白原基因的分子克隆和该基因在体内表达研究等方面进行分析,显示甲壳动物卵黄蛋白原的cDNA及其推断的氨基酸序列有很高的相似性,且有类似的分裂位点.卵巢和肝胰腺是甲壳动物卵黄蛋白原基因表达的主要位点.     

链霉菌查尔酮合成酶基因克隆及原核表达载体构建

根据Genbank数据库已知的链霉菌的查尔酮合成酶基因的保守区设计chs基因特异简并引物,土壤总DNA为模板,利用PCR技术扩增得到该1条chs基因编码区,通过TA克隆、测序和同源比对及进化分析表明：该基因为放线菌来源chs基因．分别在该基因5＇末端和3’末端分别引入的限制酶NcoI和EcoRI酶切位点,利用上述2种限制酶分别酶切导入到psimple—T／chs载体和原核表达pET32a,凝胶回收目的片段后,将二者连接并转化大肠杆菌感受态细胞,转化子经菌液PCR筛选、双酶切鉴定后,3730测序结果表明,该基因全长编码区为1089bp,推测该基因编码全长为362个氨基酸残基,等电点（PI）为5．41、分子量为3965道尔顿含有cHs保守功能区的酸性蛋白质．分析表明,该基因与Streptomyceslividans来源的查尔酮合成酶RppA基因核苷酸相似性高达93％,氨基酸序列相似性高达87．70％．测序结果表明,该基因已经成功插入到pET32a载体中．     

食道癌和胃癌组织中MDM2基因蛋白表达的研究

为探讨MDM2基因蛋白在胃、食道癌中表达的特点。我们利用SABC免疫组织化学方法,检测了69例食道癌和胃癌标本。结果表明:MDM2总阳性率为56.52％,在食道癌中阳性率为63.47％,胃癌中阳性率为46.42％;在高分化癌组织中阳性率为77.78％;低分化癌阳性率为29.41％;两者差异有显著性(P<0.05)。文中对MDM2基因在以上两种癌组织中表达的意义进行了讨论。     

大肠水疗用于大肠癌术前肠道准备的临床观察

[目的]探讨大肠癌术前肠道准备的有效方法.[方法]选择180例大肠癌患者随意分成治疗组与对照组,分别行大肠水疗与传统的肠道灌洗,进行临床观察,统计学分析.[结果]大肠水疗组并发症如急性肠梗阻及离子紊乱、缺水少(P<0.05),术后吻合口漏的发生率相等(P>0.05).[结论]大肠水疗法是安全、有效、痛苦小的术前肠道准备方法,尤其适用于年老体弱代偿能力差的患者.     

马疯树硬酯酰-酰基载体蛋白脱饱和酶基因的克隆及特征

Using degenerate primers and RT-PCR,RACE techniques,a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase(SAD)is cloned from developing seeds of Jatropha curcas L.The segment contains a 1191 bp of complete open reading frame(ORF).Analysis in the BLAST on NCB! shows that Jatropha curcas SAD(JSAD)gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids.The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs.The nucleotide and peptide identity of JSAD to Ricinus communis SAD(RSAD)is up to 89% and 96.2% respectively.Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.     

Quantitative analysis of a panel of gene expression in prostate cancer——with emphasis on NPY expression analysis

Objective: To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods: A set of laser captured microdissected (LCM) specimens from 300 prostate cancer (PCa) patients undergoing radical prostatectomy (RP) were defined. Ten patients representing "aggressive" PCa, and 10 representing "non-aggressive" PCa were selected based on prostate-specific antigen (PSA) recurrence,Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups.Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens.The expressions of a panel of genes, including NPY, PTEN, AR,AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR (TaqMan), and correlation was analyzed with clinicopathological features. Results: The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas (P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test). The lower expression of NPY showed association with "aggressive" PCas based on a larger PCa patient cohort analysis (P=0.0037,univariate generalized linear model (GLM) analysis). Conclusion: Despite widely noted heterogeneous nature of PCa, gene expression alterations of AMACR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mechanisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.     

Effect of siRNA-mediated knockdown of eIF3c gene on survival of colon cancer cells

Eukaryotic initiation factor subunit c(eIF3c) has been identified as an oncogene that is over-expressed in tumor cells and,therefore,is a potential therapeutic target for gene-based cancer treatment.This study was focused on investigating the effect of small interfering RNA(siRNA)-mediated eIF3c gene knockdown on colon cancer cell survival.The eIF3c gene was observed to be highly expressed in colon cancer cell models.The expression levels of the gene in eIF3c siRNA infected and control siRNA infected cells were compared via real-time polymerase chain reaction(PCR) and western blotting analysis.Cell proliferation levels were analyzed employing 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide(MTT) and colony formation assays.Furthermore,the effects of eIF3c gene knockdown on the cell cycle and apoptosis were analyzed using flow cytometry.The results showed that suppression of eIF3c expression significantly(P<0.001) reduced cell proliferation and colony formation of RKO colon cancer cells.The cell cycle was arrested by decreasing the number of cells entering S phase.Further,apoptosis was induced as a result of eIF3c knockdown.Collectively,eIF3c deletion effectively reduced the survival of colon cancer cells and could be used as a therapeutic tool for colon cancer therapy.     

Spatio-temporal expression of the pathway-specific regulatory gene redD in S. coelicolor

INTRODUCTION Streptomycetes are gram-positive soil bacteriarenowned for their ability to produce a large numberof different secondary metabolites. Their life cycleinvolves complex morphological differentiation,which is believed to be closely co-ordinated with geneexpression that promotes a variety of physiologicaland structural changes including the onset of antibi-otic biosynthesis. S. coelicolor has been extensivelystudied as a model system with the aid of genetic andmolecular technique…     

烟草中NAC转录因子NtNAC8的克隆与序列分析

利用数据库资源和RT-PCR技术从烟草中克隆出一个NAC转录因子新成员--NtNAC8,并进一步对NtNAC8转录因子的序列进行分析;同时对该基因编码蛋白质的理化性质、蛋白质结构及其生物学功能等进行预测.结果表明,NtNAC8编码一个由301个氨基酸残基组成的相对分子质量为34.98 kD的NAC转录因子,并推测其可能...