高低转移卵巢癌细胞株差异表达基因在染色体定位及其功能

用基因芯片技术研究高（H）、低（L）转移卵巢癌细胞株（HO-8910PM和HO-8910）和正常卵巢上皮（C）基因表达谱差异,筛选与卵巢癌转移相关的基因,并利用生物信息学方法对检测结果进行差异基因在染色体定位和功能分析。结果：高、低转移卵巢癌细胞株比较表达差异2倍以上共有409个基因,其中表达上调（信号比的对数值[SLR]≥1）有271个,表达下调（SLR≤-1）有138个。从表达差异的基因在染色体定位分析,发现除1个基因未知其定位外,其余所有差异表达基因散在分布于各条染色体上,但以1号染色体最多有43个（占10．7％）。其次是6号染色体有39个（占9．6％）,第三是2号染色体有29个（占7．1％）。第四是17号染色体有28个（占6．9％）。第五是3号染色体有25个（占6．2％）。第6是5号和11号染色体各有24个（各占5．9％）。而差异表达的基因发生在染色体短臂（q）的有264个（占64．7％）,在13,14,15,21和22号仅发现在q都有异常表达。从表达差异基因的分子功能分类看,属于酶和酶调控子基因为最多（104个,占25．4％）,其次是信号传导基因（43个,占10．5％）。第3类是核酸结合基因（42个,占10．3％）。第4类是蛋白结合基因（34个,占8．3％）。以上4大类共占基因总数54．5％。还有功能未知的基因有76个,占18．6％。高、低转移卵巢癌细胞株差异表达基因散在分布在各条染色体上,但以1、6、2、17、3、5和11号染色体差异表达基因居多。肿瘤的转移是多基因共同作用的结果。4大类（酶和酶调控子、信号传导、核酸结合和蛋白结合）相关基因异常是我们今后研究卵巢癌转移的重要基因。     

用基因芯片筛选高转移卵巢癌细胞系转移相关基因

用标准化的Affymetrix公司生产U133A基因芯片技术研究高(H)转移卵巢癌细胞株(HO-8910PM)和正常卵巢上皮(C)基因表达谱差异,筛选与卵巢癌转移相关的基因及其在染色体的定位和功能。结果发现高转移卵巢癌细胞株和正常卵巢上皮比较表达差异8倍以上共有1,237个基因,其中表达上调(信号比的对数值SLR≥3)有597个,表达下调(SLR≤-3)有640个。从表达差异的基因在染色体定位分析,发现除1个基因未知其定位外,其余所有差异表达基因散在分布在各条染色体上,但以1号染色体最多,有115个(9.3%)。其次是2号染色体有94个(7.6%),第三是12号染色体有88个(7.1%)。第四是11号染色体有76个(6.1%)。第五是X染色体有71个(5.7%)。第6是17号染色体有69个(5.6%)。而差异表达的基因发生在染色体短臂(q)上有805个(占65.1%),在13,14,15,21和22号仅发现在q上有差异表达基因。从表达差异的基因分子功能分类看,属于酶和酶调控子基因最多(306个,占24.7%),其次是核酸结合基因(144个,占11.6%)。第三类是信号传导基因(137个,占11.1%)。第四类是蛋白结合基因(116个,占9.4%)。以上4大类共占基因总数56.8%。还有功能未知的基因有207个,占16.7%。结论:高转移卵巢癌细胞株差异表达基因散在分布在各条染色体上,但以1、2、12、11、17和X染色体差异表达基因居多,肿瘤的转移是多基因共同作用的结果。4大类(酶和酶调控子活性、核酸结合活性、信号传导活性、蛋白结合活性)差异表达基因是我们今后研究卵巢癌转移相关的重要基因。     

RASSF1A和Runx3基因启动子区甲基化在胃癌组织中的表达及意义

目的：检测胃癌组织中RASSFlA和Runx3基因启动子区甲基化状态,探讨二者与胃癌发生发展的关系。方法：采用甲基化特异性PCR（MSP）技术检测57例胃癌组织和相应癌旁组织及30例正常胃黏膜组织中RASSFlA和Runx3基因启动子区甲基化状态。结果：RASSFlA和Runx3甲基化在正常组未见表达。胃癌组RASSFlA基因甲基化率为64．9％（37／57）,明显高于癌旁组的7．0％（4／57）,差异有统计学意义（P〈0．05）,胃癌组Runx3基因甲基化率为49．1％（28／57）,明显高于癌旁组5．3％（3／57）,差异有统计学意义（P〈O．05）。胃癌组RASSFlA和Runx3基因甲基化率为68．4％（39／57）,明显高于癌旁组的8．8％（5／57）,差异有统计学意义（P〈0．05）。结论：RASSFlA和Runx3基因启动子区高甲基化与胃癌的发生密切相关,有望为胃癌的早期诊治提供理论依据。     

一步法RT-PCR克隆水稻线粒体磷转运蛋白基因及其序列特征分析

以水稻广亲和品种Cpslo17幼穗为材料,用一步法RT—PCR（逆转录聚合酶链式反应）克隆了一个长度为1118bp的编码线粒体磷转运蛋白的OsMPT基因。序列分析表明其包含了基因完整的编码序列,编码由368个氨基酸组成的线粒体磷转运蛋白,它与玉米、大豆、Lotus japonicus、Betula pendula、拟南芥的线粒体磷转运蛋白氨基酸序列相似率分别为93．5％,85．6％,83．8％,83．7％,81．1％。氨基酸疏水谱分析显示它有线粒体磷转运蛋白家族高度保守的6个跨膜结构域。水稻线粒体磷转运蛋白N端富含精氨酸（Arginine）、丙氨酸（Alanine）和丝氨酸（Serine）。iPSORT预测其蛋白N端具有定位于线粒体的信号肽序列,进一步分析表明此编码区段有6个外显子和5个内含子。RT—PCR结果表明,OsMPT基因在水稻两个亚种粳稻和籼稻的叶片中均有表达,在Cpslo17营养器官和生殖器官中都有高水平表达。水稻线粒体磷转运蛋白的克隆和表达分析将为研究其结构和生物学功能奠定基础。     

胃癌组织中Bmi-1基因表达的研究

目的：多梳基因家族的Bmi．1被认为是一种癌基因,在多种肿瘤组织中均有表达。本研究主要是检测Bmi-1基因在胃癌组织中的表达及探讨其临床意义。方法：应用RT—PCR和Westernblotting方法检测45例胃癌中Bmi-1基因的表达情况,并结合患者的临床病理资料分析与其相关性。结果：Bmi-1基因阳性表达率在胃癌中为88．9％（40／45）,癌旁组织为17．7％（8／45）,差异有显著性（P〈0．05）;Bmi-1基因阳性表达率与患者年龄、性别、肿瘤大小及有无淋巴结转移无关,与肿瘤分化程度及TNM分期有关（P〈0．05）。结论：Bmi-1基因在胃癌中高表达,与胃癌的疾病进展密切相关,检测Bmi-1的表达可作为胃癌生物学行为的一项评估指标。     

玉米△12脂肪酸脱氢酶基因（FAD2）在酿酒酵母中的表达

玉米△12脂肪酸脱氢酶是催化油酸形成亚油酸的关键酶。将其编码基因FAD2（GenBank登陆号：DQ496227）克隆到酿酒酵母表达载体pYES2．0中,构建成重组质粒pYE／FAD2,转化到酿酒酵母进行诱导表达,同时以pYES2．0转化子为对照。气相色谱（Gc）分析表明,重组转化子亚油酸的含量占酵母总脂肪酸的1．54％,而对照未检测到亚油酸。表明FAD2基因具有编码△12脂肪酸脱氢酶的功能。为探索转译起始密码子周边序列的改变对FAD2基因表达产生的影响,将该基因的起始密码子上游序列进行修改,构建重组表达载体pYE／FAD2—1,转化酿酒酵母进行表达。GC分析表明,pYE／FAD2—1转化子的亚油酸含量占总脂肪酸含量的8．81％,是对照pYE／FAD2转化子的近5倍。     

低磷胁迫水稻根部基因表达谱研究

磷是植物体内重要的营养元素．土壤中含磷总量丰富,但能被植物直接吸收利用的可溶性磷含量却很低,这成为制约农作物产量的重要因素．本研究利用水稻寡核苷酸芯片分析了水稻根部在正常营养条件和低磷胁迫6,24,72h3个时间点的全基因组表达谱．和正常营养条件下相比,低磷胁迫水稻根部共发现795个差异表达基因．差异表达基因功能分析发现：（1）磷酸盐转运蛋白、酸性磷酸酶、RNA酶等基因上升表达;（2）糖酵解等与能量代谢相关基因先上升后下降表达;（3）氮吸收和脂代谢相关基因改变其表达;（4）蛋白质降解、细胞衰老相关基因上升表达;（5）部分跨膜转运蛋白基因表达上调．研究结果为进一步揭示植物低磷胁迫反应机制,改善作物对磷吸收利用效率提供了有用的信息．     

启动子诱捕在棉花基因组中的功能分析

利用根癌农杆菌介导的遗传转化,将启动子诱捕（Promoter trapping）元件插入到棉花基因组,获得141个独立的转化子,其中97％的转化子经PCR扩增为阳性。不同组织中GUS基因的表达频率为：根部48％,茎的微管组织9．2％,叶5．2％,花51％;同时检测了不同植株中GUS基因的表达模式,发现GUS基因在不同株系的植株间的表达模式呈现较大的差异,有些植株中GUS基因是组织特异表达,有些则是器官特异表达,有些则在多个器官中均有表达。所建立的启动子诱捕系统中的GUS基因高频率、多模式和时空特异性表达为分离基因及其调节序列、开展功能基因组研究奠定了坚实的基础。     

拟南芥CAS基因的生物信息学分析及超表达载体构建

对拟南芥氰丙氨酸合酶(Cyanoalanine synthase,CAS)基因进行了生物信息学分析,并构建了CAS合酶基因的超表达载体,以期为后续功能研究奠定基础。生物信息学分析结果表明,编码拟南芥CAS合酶的CYS-C1和CYS-D1基因含有10个外显子和9个内舍子,定位于3号染色体,而CYS-D2基因有9个外显子和8个内含子,定位于5号染色体。三个基因编码的蛋白氨基酸序列相似度较高,CYS-C1蛋白偏碱性且主要在线粒体中起作用,而CYS-D1和CYSD2蛋白偏酸性,主要在细胞质中起作用;通过RT-PCR扩增了拟南芥CYS-C1,CYS-D1和CYS D2基因片段,并构建了超表达载体pBI121-35S-CYS-C1,pBI121-35S-CYS-D1和pBI121-35SCYS-D2,经检测重组质粒已转化到农杆菌GV3101中。     

应用基因表达数量性状基因座定位（eQTL）研究PINK1表达调控

Pink 1基因编码定位于线粒体上的丝/苏氨酸激酶（即PARK6）,为常染色体隐性遗传性帕金森病（Parkinsons disease, PD）连锁的基因．该基因在遗传性和散发性PD的发病中起重要作用,但其发病机理尚未明确．本研究以近交系C57BL/6J (B6) 和DBA/2J (D2)小鼠制作MPTP诱导的PD鼠为模型,借助基因表达数量性状基因座(eQTL),结合分子生物学方法,分析Pink1的表达调控．结果显示,Pink 1基因在PD模型组中表达显著升高．区间连锁分析检测显示,引起Pink 1基因表达水平差异的染色体区域,定位于4号染色体上,距Pink 1基因自身5 Mb范围之类,属于顺式调节eQTL．Pearson相关分析表明,在BXD 基因重组近交系（recombinant inbred,RI）小鼠脑中,Camk2n等30个基因的表达与Pink 1基因高度相关,相互间可能存在一定的协同作用．Pink 1基因在行使特定生物学功能时,很可能协同这些基因一起发挥相应的作用,这部分基因是深入研究Pink 1基因在PD发病中分子机制的重要靶点．     

Function and Chromosome Location of Differentially Expressed Genes in Gastric Cancer

Using Affymetrix U133A oligonucleotide microarrays, screening was done for genes that were differentially expressed in gastric cancer (T) and normal gastric mucosa (C), and their chromosome location was characterized by bioinformatics. A total of 270 genes were found to have a difference in expression levels of more than eight times. Of them 157 were up-regulated (Signal Log Ratio [SLR]≥3), and 113 were down-regulated (SLR≤-3). Except for, four genes with unknown localization, a vast majority of the genes were sporadically distributed over every chromosome. However, chromosome 1 contained the most differentially expressed genes (26 genes, or 9.8%), followed by chromosomes 11 and 19 (both 24 genes, or 9.1%). These genes were also more likely to be on the short-arm of the chromosome (q), which had 173 (65%). When these genes were classified according to their functions, it was found that most (67 genes, 24.8%) belonged to the enzymes and their regulators groups. The next group was the signal transduction genes group (43 genes, 15.9%). The rest of the top three groups were nucleic acid binding genes (17, 6.3%), transporter genes (15, 5.5%), and protein binding genes (12, 4.4%). These made up 56.9% of all the differentially expressed genes. There were also 50 genes of unknown function (18.5%). Therefore it was concluded that differentially expressed genes in gastric cancer seemed to be sporadically distributed across the genome, but most were found on chromosomes 1, 11 and 19. The five groups associated genes abnormality were important genes for further study on gastric cancer.     

Identification of Differentially Expressed Genes in the High and Low Metastatic Human Ovarian Cancer Cell Lines and Analyses of Their Chromosomal Localizations and Functions

Oligonucleotide microarrays were used to study the differences of gene expressions in high (H) and low (L) metastatic ovarian cancer cell lines and in normal ovarian tissues (C). Bioinformatics was used to identify novel genes and their functions as well as chromosomal localizations. A total of 409 genes were differentially expressed between the high and low metastatic ovarian cancer cell lines. Of them, 271 genes were up regulated (Signal Log Ratio[SLR] ≥1), and 138 genes were down regulated (SLR≤-1). Except one gene whose location was unknown, all these genes were localized randomly on all the chromosomes, with a majority of them localized to Chromosomes 1, 6, 2, 17, 3, 5 and 11. Chromosome 1 contained, 43 of them (10.7%), the most for a single chromosome. A total of 264 genes (64.7%) were localized on the short arm of the chromosome (q). Functional classification showed that the 104 (25.4%) genes coding for enzymes and enzyme regulators made up the largest functional group, followed by signal transduction activity genes (43, 10.5%), nucleic acid binding activity genes (42, 10.3%), and proteins binding activity genes (34, 8.3%). These four groups accounted for 54.5% of all the differentially expressed genes. In addition, the functions of 76 genes (18.6%) were unknown. Tumor metastasis is the result of a number of genes acting in concert. The four functional groups of genes classified among these genes and their abnormalities would be the focus of further studies on ovarian cancer metastasis.     

杨盘二孢菌诱导的杨树差异表达基因的功能分析和染色体定位

利用cDNA芯片技术从含有2,952个克隆的杨树芯片中筛选出1,160个受杨盘二孢菌诱导的基因。功能分析表明,该1,160个基因分别属于11个功能类别,除了功能未知基因外,参与新陈代谢、防御反应、信号传导及转录调控的基因最多,这4大类基因约占基因总数的42%。1,160个差异表达基因中有926个基因被定位于19条染色体上,其中被定位于第Ⅱ条染色体上的差异基因最多,共102个(11.0%),其次是第Ⅰ条染色体,共93个(10%),被定位到第ⅩⅦ条染色体上的差异基因最少,仅有11个,基因在染色体上的分布则表现为在部分染色体的末端区域存在大量的聚集,在中间区段则相对较少和排列稀疏,基因的这种分布情况与植物抗病的关系有待进一步研究。     

Identification of differentially expressed genes in normal and tumor human gastric tissue

The search for differentially expressed genes in gastric cancer may help define molecular alterations and molecular diagnosis of gastric cancer. Using the differential display PCR technique, we identified 18 genes that are differentially expressed between normal and tumor human gastric tissues. Their expressions were verified with reverse Northern blot analysis and Northern blot analysis. Oxidative phosphorylation-related genes, antizyme inhibitor of ornithine decarboxylase, protein phosphatase-1beta, 35-kDa peroxisomal membrane protein, and cystic fibrosis transmembrane conductance receptor were highly expressed in tumor tissue, whereas pepsinogen A, Na-K ATPase alpha subunit, nerve growth factor receptor, and alpha-tropomyosin were highly expressed in normal tissue. In addition, 3 unknown genes were found to be differentially expressed in paired gastric tissues. These differentially expressed genes may provide significant opportunities for further understanding of gastric carcinogenesis and the molecular diagnosis of gastric cancer.     

胃癌组织中肿瘤相关成纤维细胞与正常成纤维细胞之间转录组学研究及验证

胃癌组织中肿瘤相关成纤维细胞(carcinoma associated fibroblasts, CAFs)是胃癌微环境的重要成分,主要来源于正常成纤维细胞(normal fibroblasts, NFs)的活化,对胃癌的发生发展有重要作用,但是两者之间的基因表达差异并不完全清楚。本研究选取从人胃癌组织中分离获得的CAFs及NFs 各3组,进行转录组学研究,筛选出3组细胞中交集且差异倍数较大的基因12个,用Omicsbean在线工具对差异基因进行Gene Ontology (GO)功能及KEGG通路富集,构建蛋白质相互作用调控网络;最后用RT-qPCR验证CAFs和NFs中差异基因的表达。结果显示,筛选出的12个差异表达基因主要参与NF-κB信号、炎症、细胞黏附、细胞表面受体和细胞因子等功能,上述功能均与肿瘤的发生发展密切相关。RT-qPCR检测发现,与NFs相比,CAFs中BCL2A1、NKX3-2、CXCL12、TNFAIP3、FOS、CDH4及CLDN1表达上调;ATF3、CYFIP2、CCL11、KLF2及GDF15基因表达下调,差异均具有统计学意义(P＜0.05)。结果提示,胃癌CAFs与NFs中存在肿瘤相关的差异表达基因,这些差异基因可能在胃癌微环境中发挥重要作用。     

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.     

Buffering of Segmental and Chromosomal Aneuploidies in Drosophila melanogaster

Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4^th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4^th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF).