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。     

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.     

条斑紫菜锰超氧化物歧化酶基因克隆与序列分析

为研究紫菜抗逆抗病的分子机制,以本实验室建立的条斑紫菜表达序列标签(EST)和全长cDNA富集文库,结合PCR技术,克隆了条斑紫菜编码锰超氧化物歧化酶(Mn-SOD)的cDNA及基因组DNA全长序列,并进行了序列特性相关分析.研究结果表明:该基因cDNA序列全长为958个核苷酸,包含一个完整Mn-SOD基因的ORF,编码224个氨基酸和终止密码子;该基因在基因组中序列长度为1416bp,包含4个外显子和3个内含子,这既不同于高等植物的6个外显子和5个内含子,也不同于人的5个外显子和4个内含子;该基因编码区密码子的平均GC含量为60.9%,第三位密码子的GC含量高达84.9%;序列中包含4个与Mn2 的结合位点及一个保守的金属结合结构域,预测分子量为24469.09Da,等电点为5.99;条斑紫菜Mn-SOD与人的相关蛋白具有类似的空间结构,都有6个跨膜α螺旋结构域;由cDNA序列推导的氨基酸序列与莱茵衣藻相似性为57.7%,系统发生分析表明条斑紫菜Mn-SOD与绿藻莱茵衣藻和硅藻海链藻的亲缘关系较近.这是该基因在红藻门中的首次报道.     

β-半乳糖苷酶基因(lacZ)在海藻裙带菜中的稳定表达

借鉴海带遗传转化模式，利用基因枪法将β-半乳糖苷酶基因(lacZ)导入裙带菜雄配子体中，一部分诱导形成营养增殖的丝状体，另一部分和雌配子体受精生成孢子体。五个月后组织化学染色结果表明5％的孢子体个体和30％的丝状体细胞表现了lacZ的稳定表达，利用PCR和PCR-Southem检测得到了预期的阳性信号。本文结果提示了利用基因枪转化方法．以雄配子体介导获得外源基因在裙带菜中稳定表达的可行性．     

基因枪法建立紫菜丝状体遗传转化模式

以坛紫菜和条斑紫菜的自由丝状体为材料,利用基因枪法分别转化CaMV35S、SV40、FCP、Amt、Ubi 5种启动子与报告基因组合,转化后48h进行原位组织化学染色检测,首次针对重要经济红藻紫菜的自由丝状体建立并优化遗传转化技术,为紫菜研究提供了新工具。结果显示,SV40启动子可以驱动laeZ报告基因(编码β-半乳糖苷酶)在紫菜丝状体中的瞬间表达,空白与阴性对照未检测到本底;其它4种启动子未检测到基因表达。进一步优化实验发现,在可裂膜650psi、轰击距离6cm下获得最高转化效率为8.0×10~(-5),经定量检测与双因子方差分析,是最佳转化参数,提示基因枪参数对外源基因转化效率具显著影响。     

褐飞虱核糖体蛋白S6激酶基因NlRPS6KA2的克隆及其表达分析

根据转录组提供的RPS6KA2核心序列信息,应用RACE技术获得了一个编码褐飞虱核糖体蛋白S6激酶的基因NlRPS6KA2的全长cDNA,编码的蛋白含706个氨基酸,具有保守的S_TKc和S_TK_X结构域.荧光定量PCR测定结果表明,NlRPS6KA2基因在褐飞虱若虫和雄虫中表达量均较低,但在怀卵雌虫中大量表达.同时,褐飞虱从感性水稻品种TN1到抗性水稻品种RHT的适应过程中,该基因表达量呈现明显的下降趋势,在适应后有所回升.研究结果为进一步研究NlRPS6KA2基因在褐飞虱中的功能和阐明褐飞虱致害性变异机制提供了依据.     

温差和水分对黄连生物碱合成基因表达的影响

探究昼夜温差和水分缺失对黄连生物碱合成关键基因表达的影响。以昼夜温差条件下以及水分缺失条件下黄连植株为实验组,无昼夜温差以及正常水分条件下黄连植株为对照组,每隔7 d对黄连根茎叶组织进行采样,提取其总RNA,实时荧光定量检测黄连生物碱合成途径中6个具有重要功能的关键基因表达水平。昼夜温差处理后,黄连根茎叶中关键基因的相对表达量均高于对照组,说明昼夜温差处理可诱导关键基因的表达。而水分缺失情况下催化四氢氧化物氧化的关键基因表达量显著高于对照组,在恢复浇水之后,其表达量反而降低。催化亚甲基二氧桥的4个关键基因,其表达量随着时间的推移均升高,但在经过水分缺失处理后其表达量均显著低于对照组,说明水分的缺失会使其表达量降低。昼夜温差处理对关键基因具有直接特异诱导的作用,而水分的缺失则会增加催化四氢氧化物氧化的关键基因的表达量,而使催化亚甲基二氧桥关键基因的表达量降低。     

牙鲆抗菌肽hepcidin基因的克隆及表达分析

采用同源克隆的方法设计简并引物从牙鲆(Paralichthys olivaceus)肝脏中克隆了牙鲆hepcidin抗菌肽基因.牙鲆抗菌肽hepcidin基因组DNA全长821bp,序列分析表明该基因具有3个外显子和2个内含子.cDNA全长588bp,包含一个270bp的开放阅读框,编码一个长89氨基酸的前体肽.RT-PCR分析表明:该抗菌肽基因在正常牙鲆的肝脏、头肾、鳃、脾脏中表达量较高,在心脏、小肠中表达量较低;受到病原鳗弧菌感染的牙鲆各组织该基因表达量明显上升.牙鲆抗菌肽基因的克隆为水产养殖等领域的抗耐病品种的选育提供了基因源,为开发新的生物工程药物提供了基础理论和实验数据.     

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.     

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。     

Screening of target-specific stress-responsive genes for the development of cell-based biosensors using a DNA microarray

In this study, we describe a straightforward strategy to develop whole cell-based biosensors using fusions of the bacterial bioluminescence genes and the promoters from chemically responsive genes within Escherichia coli, in which chemical target-responsive genes were screened by using the information of gene expression data obtained from DNA microarray analysis. Paraquat was used as a model chemical to trigger gene expression changes of E. coli and to show the DNA microarray-assisted development of whole cell-based biosensors. Gene expression data from the DNA microarray were obtained by time course analysis (10, 30, and 60 min) after exposure to paraquat. After clustering gene expression data obtained by time course analysis, a group of highly expressed genes over the all time courses could be classified. Within this group, three genes expressed highly for overall time points were selected and promoters of these genes were used as fusion partners with reporter genes, lux CDABE, to construct whole cell-based biosensors. The constructed biosensors recognized the presence of model inducer, paraquat, and structural analogue chemicals of paraquat with a high specificity, and the results were reconfirmed by using DNA microarray experiments for those structural analogues. This strategy to develop whole cell-based biosensors assisted by DNA microarray information should be useful in general for constructing chemical-specific or stress-specific biosensors with a high-throughput manner.     

五个紫菜品系间遗传差异的RAPD分析

应用随机引物扩增片段多态性（RAPD）技术对2种紫菜的5个品系进行遗传多样性分析。共筛选出21条随机引物,PCR反应得到147条扩增片段。根据共享扩增片段计算遗传相似性指数（F）和相对遗传距离,利用NJ法构建系统树。结果表明,条斑紫菜或坛紫菜的养殖品系首先聚类在一起,两个条斑紫菜养殖品系之间的遗传距离是0．32,两个坛紫菜养殖品系之间的遗传距离是0．31。条斑紫菜养殖品系CPY1－08A与坛紫菜养殖品系CPH8－83之间的遗传距离最大,达0．42。本文结果显示RAPD可以作为简便有效的分子工具应用于紫菜的遗传多样性和种质鉴定研究中。     

cDNA芯片重复探针检测值的一致性分析

通过SOURCE数据库对4套cDNA数据的探针进行了注释,分析了对应同一条Unigene的多个探针的检测值(即重复检测值)之间的相关性.采用两种常规方法处理了重复检测值,比较了这两种处理方法对筛选差异表达基因的影响.结果显示:Unigene的重复检测值之间存在一定比例的负相关;更新探针注释数据后的重复检测值之间的低相关比例减少,高相关比例显著提高;重复点样探针之间的相关性高于其它重复检测值,但是仍有很多低相关;两种处理重复检测值方法对于用基因表达差异显著性分析方法(SAM)与T检验方法筛选差异表达基因影响不大.     

Study on the dynamic behavior of a DNA microarray

A theoretical dynamic kinetic model was derived and a series of experiments were carried out using low-density microarrays in various concentrations of spotting probe ([P]) and labeling target ([T]). It has been shown that target and probe determined the signal intensity together. At a certain range of DNA concentration, the signal intensity was in proportion to spotting [P]. At the higher DNA concentrations, there was a decrease in hybridization signal intensity, especially in cDNA microarrays. Since the DNA microarray was constructed on a solid surface, steric hindrance, which is induced by the solid surface and the high [P], decreased the probe immobilization efficiency, leading to a decrease of the immobilized probe density. The decreased hybridization efficiency also caused the compression in signal intensity when the target increased. Nevertheless, the intensity ratio of Cy5 to Cy3 was not compressed within a microarray in the two-color system. The ratio of Cy5/Cy3 is only determined by the ratio of two targets and independent of the density and the types of probe. Therefore, the two-color fluorescent strategy is more reasonable and reliable in detection of differential gene expression. All these results indicate that the DNA microarray can be used to detect differently expressed genes, though it cannot be used to detect the absolute mRNA abundance.     

Polychromatic microarrays: simultaneous multicolor array hybridization of eight samples

High-throughput microscale platforms have transformed modern analytical investigations. Traditional microarray analyses involve a comparative approach, with two samples, a known control and an unknown sample, hybridized side-by-side and then contrasted for genetic differences. The samples are labeled with separate dyes and hybridized together, providing a differential expression pattern based on the reporter intensities. In contrast, the fiber-optic microarray platform described herein is analyzed with a microscope, thereby enabling the use of virtually any reporter, including quantum dots. The instrumentation takes advantage of the narrow emission bands characteristic of quantum dots to perform multiplexed detection of Bacillus anthracis. Advancing beyond the standard red/green microarray experiment, a panel of eight reporters were linked to eight B. anthracis samples and simultaneously analyzed in a microarray format. The ability to employ an assortment of reporters, along with the capacity to simultaneously hybridize eight samples confers an unprecedented flexibility to array-based analyses, providing a 4-fold increase in throughput over standard two-color assays.     

Cellular response to gene expression profiles of different hepatitis C virus core proteins in the Huh-7 cell line with microarray analysis

In the current investigation, we constructed recombinants of expression of HCV genotype 1b, 2a, and 4d whole core proteins and established a human hepatoma (Huh-7) cell line which expressed different core proteins constitutively. In the Affymetrix human gene chip, the HG-U133 A and B were employed for identification of variant core protein gene expression in the Huh-7 cell line. In data analysis, we applied a threshold that eliminated all genes that were not increased or decreased by at least a 3-fold change in a comparison between transfected cells and control cells. All of these genes were annotated by using NetAffx analysis through the Affymetrix website and categorized on the basis of their biological processes. The microarray analysis result suggested that the gene expression profiles caused by three kinds of core proteins were mainly shown in metabolism, signal transduction, protease activity, immune responses, etc. and that some pathogenesis/oncogenesis, apoptosis, or anti-apoptosis gene expression were up/down-regulated simultaneously in the Huh-7 cell line. In conclusion, the gene expression profiles of variant core proteins were implicated in HCV replication, pathogenesis, or oncogenesis in the Huh-7 cell line, which is useful for our understanding of HCV variant core protein biological function and its pathogenic mechanism.