基于cDNA微阵列技术对激活蛋白处理水稻后信号传导及防卫反应相关基因的研究

为探讨激活蛋白对植物抗病防虫作用的机理,应用水稻10368条非冗余序列标签(ESTs)所制备的cDNA微阵列及荧光信号Cy3、Cy5杂交体系,对激活蛋白处理水稻后信号传导及防卫反应相关基因进行了研究,并利用半定量RT-PCR方法验证了相关基因的表达.结果表明:激活蛋白处理水稻后1～5d内,诱导了NPR1和bZIP等信号传导相关基因的上调表达,同时对APX、GST、CHS及PR1a等防卫基因的表达也有促进作用.激活蛋白诱导水稻启动了水杨酸介导的系统获得抗性(systemic acquired resistance, SAR)反应.     

2009大流行H1N1流感病毒与经典H1N1猪流感病毒核酸扩增检测标准物质研究

根据已发表的A/California/04/2009(H1N1)基因序列,通过基因合成获得该毒株的完整M、HA和NA的基因序列,并克隆于pET32a.从经典猪流感病毒A/swine/2003(H1N1)提取的RNA中扩增了完整M、HA和NA的基因序列,并克隆于pGEM-T.测序后采用体外转录方法制备6种RNA纯品.初步定量稀释后,将2个病毒的3个基因分别混合分装,采用荧光定量RT-PCR方法进行均匀性和稳定性检验,并委托外部实验室采用外标实时定量RT-PCR方法对所有RNA片段进行定值.均一性结果显示瓶间差异小于5%,稳定性实验表明室温20～25℃(相对湿度20%～50%)14天,2～8℃6个月以及-20℃保存1年的含量均无明显变化.     

抑制消减杂交法分离血管内皮细胞中表达的TNFα相关基因

介绍使用快速而高效地分离差别表达基因的新方法———抑制消减杂交法 ,克隆了在TNFα作用后血管内皮细胞中的差异表达cDNA。经反向Northern杂交试验证实。为进一步研究血管内皮细胞中表达的TNFα作用相关基因打下基础。     

牙鲆抗菌肽hepcidin基因在大肠杆菌中的融合表达

根据从实验室克隆的牙鲆抗菌肽hepcidin基因(GenBank登陆号DQ129693)的序列,设计了特异性引物,PCR扩增其成熟肽片段.重组至融合表达载体pGEX-4T-1中,构建成牙鲆抗菌肽hepcidin基因融合表达载体pGEX-fhep,转化至大肠杆菌BL21(DE3)plyS中,筛选得到的阳性克隆用IPTG进行诱导.SDS-PAGE电泳显示在29kD处有特异性的蛋白条带出现,Western-blotting 检测表明已经成功表达了融合蛋白,表达的融合蛋白最高占总蛋白的21%.纯化得到的GST-fhep用凝血酶切割后,用亲合层析得到牙鲆的抗菌肽hepcidin.抑菌实验结果表明抗菌肽对大肠杆菌(ATCC25922)有一定程度的抑制作用.     

苏云金芽孢杆菌营养期杀虫蛋白基因vip3A的研究

根据已知vip3A基因序列设计一对特异性引物VIP3／VIP5,对218株Bt菌株进行PCR鉴定,有51株含有vip3A类基因,其中12株为不同亚种的标准菌株,有4株标准菌株不含有该基因。从Bt C9菌株中分离克隆了vip—C9基因,并插入表达载体pET—21b,转化大肠杆菌BL21,诱导表达出分子量88．6kDa的可溶性蛋白,表达产物对甜菜夜蛾和棉铃虫具有较高的杀虫活性,LC50分别为0．42ng／mg和25．95ng／mg,对小菜蛾活性较低。构建了缺失蛋白Vip—C9—N(N端去除39个氨基酸组成的信号肽序列),杀虫活性测定结果表明其对甜菜夜蛾的活性显著降低,说明N端39个氨基酸对甜菜夜蛾的活性是必需的。     

猪、牛成纤维细胞α-1,3-半乳糖基转移酶基因的敲除以及人白细胞抗原-G1基因的敲入

构建了敲除猪α-1,3-GT并敲入人白细胞抗原HLA-G1基因的打靶载体pZJ,其中以新霉素抗性基因(Neo)为正筛选基因,绿色荧光蛋白基因(GFP)为负筛选基因.采用优化的脂质体转染法将打靶载体pZJ转染于一个长势良好的胎猪成纤维细胞系中,经7天G418(600 μg/ml)药物筛选,共获得30个Neo抗性细胞克隆,其中12个为非绿色荧光细胞克隆,7个扩大培养良好,提取阳性克隆细胞的基因组DNA并进行PCR检测.经PCR结果检测,打靶载体转入到胎猪成纤维细胞中,其中3个非荧光阳性单克隆细胞在细胞基因组中进行了定点整合.以同样的脂质体转染条件将pZJ转染牛胎儿成纤维细胞,经筛选并对其进行PCR检测,其中2个均为定点整合阳性.该研究为今后克隆出表达HLA-G1而不表达α-1,3-GT基因的个体猪,从而提供可以真正用于临床的异种器官打下了基础,也为猪牛之间的跨物种敲除提供了一个佐证.     

转"全鱼"GH基因鲤鱼胸腺发育差异表达基因的筛选与鉴定

采用显微注射方法,获得了转"全鱼"生长激素(GH)基因鲤鱼(转基因鱼),应用抑制性差减杂交(SSH)技术,构建了4月龄F3转基因鱼胸腺发育差减cDNA文库,筛选并鉴定了与胸腺发育相关的81个与已知基因同源的表达序列标签(EST).这些EST至少代表69个基因.根据基因的作用将其分为5类:18个与免疫和细胞防御有关;23个参与细胞生长、发育和分化等代谢过程;3个参与细胞信号传导和周期调控;8个在转录和表达调节中发挥作用;17个为核糖体蛋白基因,表明转基因鱼胸腺细胞处于活跃的蛋白合成状态.应用RT-PCR和虚拟Northern杂交技术进一步证实其中的若干基因在转基因鱼胸腺组织中的表达量增加.实验结果为阐明转植GH基因促进鲤鱼胸腺发育的相关分子机制提供了依据.     

灭活病毒诱导牙鲆传代细胞差减cDNA文库的构建及分析

以灭活大鳞鲆弹状病毒诱导的牙鲆(Paralichthys olivaceus)传代细胞作为检测子(tester),正常对照细胞作为驱赶子(driver),分别提取mRNA,逆转录合成双链cDNA。经两轮差减杂交,两轮抑制性PCR扩增后,取正向差减的PCR产物与T载体连接,构建抑制差减cDNA文库,随机挑取7400余个克隆,对其中4200多个克隆进行PCR扩增鉴定,发现近4000个克隆中均含有插入片段,大小在250～1000bps之间,进一步对含有插入片段的近4000个克隆进行了斑点杂交筛选,得到近668个可能含有抗病毒或与免疫相关的阳性克隆。初步的结果表明所建立的差减cDNA文库适合进一步克隆鱼类抗病毒相关新基因。     

用差减抑制杂交方法鉴定太谷核不育小麦花药败育过程中的特异表达基因

以太谷核不育小麦败育花药为对照群体，可育花药为目标群体，进行抑制差减杂交。用经过败育花药差减的可育花药cDNA群体构建了一个差减抑制杂交文库。结果显示，插入片段的大小主要集中在300－500bp之间。随机选取8个克隆进行Northern杂交分析，结果其中7个克隆在可育花药和不育花药之间的表达存在差异。对其中16个插入cDNA片段测序后与GenBank同源性比较表明，共获得未重复序列13条，占81％，其中与已知功能基因同源的3条，与EST库中的序列同源的7条，新的cDNA片段3条。     

番茄乙烯信号转录因子LeERF2在大肠杆菌中的表达和纯化

从破色期番茄果实中提取了总RNA,用RT-PCR方法从中克隆到了627bp全长LeERF2基因.测序结果分析表明,该LeERF2基因序列与已发表序列的同源性为100%.将LeERF2基因亚克隆至载体pET-30a上,构建了原核表达载体pE-LeERF2,将其转化到大肠杆菌BL21中.蛋白质诱导表达的温度为37℃,诱导剂IPTG浓度为1mmol/L,诱导时间为3 h,LeERF2表达蛋白在细菌沉淀中约占菌体总蛋白的58%.应用Ni-NTA亲和层纯化回收的LeERF2表达蛋白的纯度为99%.银染凝胶阻滞实验表明,LeERF2蛋白能与含有GCC盒的逆境相关的几丁质酶启动子基因TLBP1结合.     

抗稻瘟病新基因pi-hit-1的克隆与功能研究

采用差异显示法(DD-PCR)寻找易感稻瘟病的突变品系与野生型对照品系中的差异表达基因。结果发现与野生型对照相比,Rubisco小亚基、pi-hit．1(AF450251)和pi-hit-2(AF448491)基因在易感稻瘟病的突变品系中高表达,pi-hit-1和pi-hit-2是功能未知的新基因。进一步采用电子克隆和RT-PCR方法克隆了pi-hit-1基因的全长eDNA(AF514859),该基因编码的蛋白质属于ATP依赖的Clp蛋白酶家族。分析pi-hit-1基因的组织特异性表达发现,此基因在叶片组织特异表达。进一步设计接种诱导实验研究pi-hit-1基因表达与水稻稻瘟病易感性的关系。在易感稻瘟病的突变品系中pi-hit-1受稻瘟病菌诱导,接种后其表达量明显升高,而野生型对照品系pi-hit-1基因表达在接种前后无明显变化。比较易感稻瘟病的突变品系与对照品系pi-hit-1基因序列差异发现,稻瘟病敏感品系中pi-hit-1基因第一外显子发生缺失突变使pi-hit-1蛋白功能缺失,导致突变品系易感稻瘟病。这些实验结果提示野生型pi-hit-1是稻瘟病抗性基因。     

Effects of Boron on the Microstructure,Ductility-dip-cracking,and Tensile Properties for NiCrFe-7 Weld Metal

The distribution of boron and the microstructure of grain boundary(GB) precipitates(M_(23)(C,B)_6 and M_2B)have been analyzed with their effects on the susceptibility of ductility-dip-cracking(DDC) and tensile properties for NiCrFe-7 weld metal,using optical microscopy(OM),secondary ion mass spectroscopy(SIMS),scanning electron microscopy(SEM),and transmission electron microscopy(TEM).The results show that boron segregates at GBs in NiCrFe-7 weld metal during the welding process.The segregation of boron at GBs promotes the formation of continuous M_(23)(C,B)_6 carbide chains and M_2B borides along GBs.The addition of boron aggravates GB embrittlement and causes more DDC in the weld metal,by its segregation at GBs presenting as an impurity,and promoting the formation of larger and continuous M_(23)(C,B)_6 carbides,and M_2B borides along GBs.DDC in the weld metal deteriorates the ductility and tensile strength of the weld metal simultaneously.     

Profile of differentially expressed genes in blood vessels and blood cells of hyperlipidemia rats using suppression subtractive hybridization

Platelets, macrophages, endothelial cells, and smooth muscle cells can all contribute to atherosclerosis. To investigate the molecular events leading to atherosclerosis involving platelets, macrophages, endothelial cells, and smooth muscle cells, we carried out suppression subtractive hybridization (SSH) to generate a profile of genes overexpressed in the aorta and blood cells in high fat diet rats. From 85 random SSH-cDNA clones, we have screened 23 clones by Northern blotting, which were scored as overexpressed in the aorta and blood cells in high fat diet rats compared to normal diet rats. Sequencing showed that the gene corresponded to the known gene in the public databases, previously shown to be overexpressed in atherosclerosis, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and local production was seen in vascular smooth muscle and endothelial cells by RT-PCR and Western blotting. These results show that SSH provides a very efficient means to produce a profile of differentially expressed genes in atherosclerosis. The identified gene may provide novel points of therapeutic intervention and pathophysiological mechanisms in atherosclerosis.     

水稻小GTP蛋白基因Osrab5B基因的克隆和鉴定

用已知遗传图位的BAC克隆片段,对水稻（Oryza sativa)小穗cDNA文库进行筛选,获得一个水稻小GTP结合蛋白（small GTP-binding protein,SGP)的相关序列,序列测定后以该cDNA序列为基础将４个表达序列标记（EST）进行了电子克隆（electronic cloning),根据电子克隆结果设计引物,利用RT－PCR方法获得了一个水稻（Oryza stiva)中尚未鉴定的cDNA克隆Osrab5B,长1016bp。它与龙船海棠属（Mesembryanthemum crystallinum)和百脉根属（Lotus japonicus)命名为rab5B基因（序列登录号分别是AJ006225和Z73939）有着高度同源性（cDNA核苷酸序列ID值分别大于74％和76％）,同属rab家族的一个新的亚族。进而根据Osrab5B的cDNA序列设计引物,扩增得到Osrab5B的基因组克隆,序列分析表明它至少有7个内含子和8个外显子。     

条斑紫菜丝状孢子体表达序列标签分析

为获得条斑紫菜丝状孢子体特异表达基因和抗逆相关基因，进行了条斑紫菜丝状孢子体表达序列标签分析，共获得170条表达序列标签。与数据库中条斑紫菜叶状配子体表达序列标签比较，发现73条新标签，占42．94％。这些新标签可能是条斑紫菜丝状孢子体特异表达的基因。获得的170条表达序列标签可分成106组，其中的43组，占46．6％，在蛋白质数据库中存在同源蛋白。已知功能的43组标签中，大部分与能量代谢和蛋白质合成相关，只有3组与生长发育，6组与抗逆与防御相关。上述研究结果是构建条斑紫菜分子标记连锁图谱和探讨条斑紫菜养殖性状遗传机理的重要基础。     

奥氏体中Fe-C-Me晶胞原子杂化状态的确定

本文以一元合金奥氏体为研究对象,应用原子状态判定因子w来确定Fe-C-Me晶胞中各原子的杂化状态.分析结果表明,Fe-C-Me晶胞中FeC、Fef原子的杂化状态因Me原子的作用而与Fe-C晶胞中的FeC、Fef原子的杂化状态相比发生了改变,不同的Me原子使FeC、Fef原子的杂化状态发生改变的程度不同.在FeC-Cr晶胞中,Cr、FeC、Fef原子的杂化状态分别为A8(或A7)、B14和B15;在Fe-C-W晶胞中,W、FeC、Fef原子的杂化状态分别为C1(或C2)、B13和B14;在Fe-C-Si晶胞中,Si、FeC、Fef原子的杂化状态分别为A3(A1或C2)、B15和B16(或B17);而在Fe-C-Mn晶胞中,Mn、FeC、Fef原子的杂化状态则分别为C11、B14(或B13)和B15.     

芦荟耐盐差异表达基因文库的构建

为了弄清景天酸植物芦荟的耐盐、耐旱机理，应用抑制性消减杂交技术，构建了库拉索芦荟的盐诱导组织与正常组织差异表达的cDNA消减文库，并做了初步鉴定。分别从库拉索芦荟的正常叶片组织及高盐诱导叶片组织中提取poly(A)^ RNA，依次合成单链及双链cDNA，酶切成平均大小为400～600bp的片段；将高盐诱导组织eDNA分为两组，分别与两种不同的接头连接，再与正常组织cDNA进行两次消减杂交及两次抑制性PCR；获得的产物与T／A载体连接构建cDNA消减文库，并转染大肠杆菌进行文库扩增。经检验证明，构建的cDNA消减文库具有很强的消减效率，非特异性cDNA片段被有效地消减，而特异表达的cDNA得到富集。所构建的库拉索芦荟高盐诱导组织cDNA消减文库为进一步大批量筛选、克隆高盐特异性表达的未知新基因奠定了基础。     

ESTMAP: a system for expressed sequence tags mapping on genomic sequences

The completion of a number of large genome sequencing projects emphasizes the importance of protein-coding gene predictions. Most of the problems associated with gene prediction are caused by the complex exon-intron structures commonly found in eukaryotic genomes. However, information from homologous sequences can significantly improve the accuracy of the prediction. In particular, expressed sequence tags (ESTs) are very useful for this purpose, since currently existing EST collections are very large. We developed an ESTMAP system, which utilizes homology searches against a database of repetitive elements using the RepeatView program and the EST Division of GenBank using the BLASTN program. ESTMAP extracts "exact" matches with EST sequences (>95% of homology) from BLASTN output file and predicts introns in DNA comparing ESTs and a query sequence. ESTMAP is implemented as a part of the WebGene system (http://www.cnr.it/webgene).     

Cone pigment gene expression in individual photoreceptors and the chromatic topography of the retina

Human trichromatic vision is based on three classes of cones: L, M, and S (long-, middle-, and short-wavelength sensitive, respectively). Individuals can have more than one M and/or more than one L pigment gene on the X chromosome along with an S pigment gene on chromosome 7. In some people the X-linked pigment gene array can include polymorphic variants that encode multiple, spectrally distinct cone photopigment subtypes. A single-cell, polymerase chain reaction approach was used to examine visual pigment gene expression in individual human cone cells and identify them as L or M. The ratio of L:M pigment gene expression was assayed in homogenized retinal tissues taken from the same eyes. Results indicate that there is a close correspondence between the cone ratio determined from counting single cells and the L:M pigment mRNA ratio estimated from homogenized pieces of retina. The results also show that the different pigment genes in one array are often expressed at very different levels, giving rise to unequal numbers of L and M cones. Expression of only one photopigment gene was detected in each cone cell. However, individual males can have more than the classically described three spectrally distinct cone types in their retinas.