雌核发育银鲫原肠期胚胎和尾芽期胚胎差异表达基因的呈现

构建了雌核发育银鲫原肠期胚胎和尾芽期胚胎间的抑制性差减杂交cDNA质粒文库。对原肠期’739个和尾芽期816个PCR阳性克隆进行斑点杂交,得到72个原肠期和98个尾芽期斑点杂交阳性克隆。测序和基因数据库比对结果表明：72个原肠期斑点杂交阳性克隆中,包括19个已知基因的cDNA片段和31个没有同源性的cDNA片段;98个尾芽期斑点杂交阳性克隆中,包括52个已知基因的cDNA片段和37个没有同源性的cDNA片段。采用虚拟Northern杂交和RT-PCR证实了部分基因在银鲫胚胎发育过程中的差异表达。这些差异表达基因的呈现为进一步研究银鲫胚胎发育的分子机制奠定了基础。     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

性逆转石斑鱼性腺差异表达基因的克隆和筛选

以 17α 甲基睾丸酮 (17α MT)饲喂 2～ 4龄赤点石斑鱼 (Epinephelusakaara) ,成功地促使其性转变为具有生殖功能的雄鱼 .应用抑制性差减杂交 (SSH)技术构建了石斑鱼性反转前后性腺组织的SMARTcDNA文库及其cDNA差减文库 ,从中随机挑取 12 0 0个克隆进行了PCR和斑点杂交筛选 ,得到 12 0个差异表达cDNA片段 .挑选 71个cDNA克隆测序 ,将所测序列经GenBank检索和生物信息学比较 ,发现有 5 1个cDNA片段序列无明显的同源性 ,2 0个片段与报道的基因有较高同源性 .在这 2 0个具同源性的片段中有 3个片段可能是与性别分化密切相关的重要功能基因 ,它们是钙调蛋白基因、活性蛋白激酶C受体基因和一氧化氮合酶蛋白抑制剂基因 .这 3个基因被分别命名为鱼钙调蛋白基因 (GenBankaccession :AY2 8136 3)、鱼活性蛋白激酶C受体基因 (GenBankaccession :AY2 8136 4)和鱼一氧化氮合酶蛋白抑制剂基因 (GenBankaccession :AY2 8136 5 ) .     

银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMART cDNA合成和RACE—PCR技术克隆到雌核发育银鲫(Carassius auratus gibelio)肌酸激酶M3-CK基因的全长cDNA。银鲫M3-CK cDNA全长1551bp，编码380个氨基酸，与普通鲤鱼(cyprinus carpio)M3-CK的氨基酸序列同源性高达95％。种系分析表明，银鲫M3-CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支，与鲤鱼的M3-CK聚在一起，与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern杂交显示银鲫M3-CK基因在胚胎发育中差异表达。RT—PCR表明，银鲫M3-CK基因在成熟卵母细胞和胚胎发育早期可检测到少量的转录产物，在胚胎发育期间从肌肉效应期开始转录，并一直持续表达。组织RT—PCR表明，银鲫M3-CK基因只在心脏和肌肉表达。     

银鲫原肠胚差异表达基因的筛选

胚胎发育是基因组中各个基因在时间和空间上选择性表达的结果。为了鉴定参与鱼类早期胚胎发育和胚层分化的调控因了,分别构建了银鲫(Carassiusauratusgibelio)原肠胚和成熟卵子的SMARTcDNA文库,并采用差异筛选的方法,从银鲫原肠胚SMARTcDNA文库中筛选不同于成熟卵子的差异表达基因。通过菌斑和PCR产物的两轮斑点杂交,从大约1500个克隆中筛选出131个阳性克隆,从中选择58个克隆测序并将测得的序列进行了数据库比对分析,结果显示这些差异表达基因大部分为参与转录和翻译的调控因子和核糖体蛋白以及一些在胚胎早期大量表达的参与脂类代谢的脂蛋白和一些未知的新基因。接着采用RTPCR技术,对其中6个基因在胚胎发育不同阶段的表达特征进行了分析,进一步证实这些基因在胚胎发育过程中存在表达差异;研究还从分析的基因中揭示出4类不同的表达模式。通过本研究,已筛选出一批在银鲫胚胎发育早期开始表达的调控基因,为开展鱼类胚胎发育早期表达基因的功能研究奠定了前期工作基础。       

金鱼雌核发育单倍体胚胎血液循环障碍产生机制

为研究鱼类单倍体血液循环障碍产生机制,人工诱导获得金鱼(Carassius auratus)雌核发育单倍体胚胎并进行活体观察及邻联茴香胺染色,结果显示金鱼雌核发育单倍体胚胎存在不同程度的血液循环不良和红细胞生成缺陷.为进一步探讨其发生的分子机制,利用反义RNA整胚原位杂交技术比较分析了原始造血和血管发生关键基因scl(...     

Early developing embryos affect the gene expression patterns in the mouse oviduct

Fertilization and development of mouse embryos occur in the ampullae of oviduct. We hypothesize that fetal-maternal communication exists in the preimplantation period, allowing optimal development of embryos. It is known that embryotrophic factors from oviduct affect the development of embryos. Although embryos affect their own transport in the oviduct, the mechanism of action is unknown. As a step toward understanding the action of embryos on oviductal physiology, we adopted suppression subtractive hybridization (SSH) to compare the gene expression in the mouse oviduct containing early embryos with that of oviduct containing oocytes. Ten to twelve 1-cell mouse embryos were transferred to one oviduct of a foster mother and similar number of oocytes were transferred to the contralateral oviduct. The animals were sacrificed after 48 h and their oviducts were excised for mRNA study. Using SSH, we screened out 250 putative positive clones from the subtracted embryo-containing oviduct library and 97 of them were screened positive by reverse dot-blot analysis. DNA sequence analysis identified genes that shared high homology with sequences in GenBank/EMBL database with unknown functions. Overall, 13 of the 90 high-quality sequences (14%) were homologous to 6 different genes previously described. Reverse Northern analysis confirmed that the expression of these genes were higher in the embryo-containing oviduct than in the oocyte-containing oviduct. About 12% of these clones (11/90) were novel. This article is the first to report identification of genes in the oviduct that are upregulated in the presence of embryos during the preimplantation period.     

Identification and characterization of genes expressed in early embryogenesis from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis>

To understand the mechanism in induction of embryogenesis from microspores of Brassica napus, we isolated exhaustively the genes expressed differentially during the early stage of microspore culture. A subtracted cDNA library composed of up-regulated genes during androgenic initiation was produced by suppression subtractive hybridization followed by differential screening by dot blot hybridization, and a total of 136 non-redundant expressed sequence tags were identified. Analysis of the potential functions of the genes showed that 64% of these genes were homologous to known genes, and the remaining ones have not been previously reported to participate in embryogenesis. Many embryo-specific genes were contained in the isolated genes, for example, genes cording lipid transfer protein, napin, cruciferin, oleosin, and phytosulfokine. Real-time RT-PCR analysis for 15 selected genes, which are understood to not be related with embryogenesis, demonstrated that all genes were expressed highly in the early stage of microspore embryogenesis. A few genes also showed higher expression in microspores cultured in non-embryogenic condition or in later stages of embryos. A principal component analysis based on expression profiles of the 15 genes demonstrated that these genes were classified into 2 groups, one characterized by their high expression in initiation of embryogenesis, and the other characterized by their expression in the early to middle stage of embryogenesis. The expressions of these genes were confirmed in zygotic embryos. The identification and characterization of the genes isolated in the present study provide novel information on microspore embryogenesis in Brassica.     

Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.     

Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutant

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.     

利用抑制差减杂交技术分离受水稻抗性调控的褐飞虱基因

为分离受水稻抗性调控的褐飞虱Nilaparvata lugens基因, 以取食感虫水稻台中1号和高抗水稻B5的2叶1芯秧苗24 h的褐飞虱4龄若虫为起始材料, 采用抑制差减杂交技术构建了两个群体间的正反向差减cDNA文库。通过斑点杂交从差减文库中筛选代表受水稻抗性调控的基因的cDNA克隆, 进行测序和功能分析, 挑选具功能的基因进行Northern杂交验证。结果表明, 通过斑点杂交筛选到的98个阳性克隆代表92个互不重复的单基因, 其中25个与动物的已知蛋白基因存在较高的同源性。Northern杂交表明, 这25个基因有11个表达上调, 8个表达下调, 提示它们可能在褐飞虱适应抗性水稻过程中发挥了重要作用。本研究结果为克隆上述新基因的全长cDNA序列及进一步研究其在褐飞虱与水稻互作中的功能奠定了基础。     

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps.

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis.     

金鱼etv2基因的克隆及在雌核发育单倍体和自交二倍体胚胎中的差异表达

为研究鱼类雌核发育单倍体循环系统发育异常的原因,从金鱼(Carassius auratus)中克隆了血管发生主调控基因etv2(Ets variant 2)的全长cDNA序列,并比较分析了该基因在雌核发育单倍体和自交二倍体中的表达。金鱼etv2 cDNA全长1531 bp,其开放阅读框为1116 bp,编码371个氨基酸。序列对比分析表明,金鱼ETV2蛋白的C端含有ETS(E26 transformation-specific)转录因子家族所共有的ETS DNA结合结构域,该结构域氨基酸序列与其他脊椎动物ETV2的同源性超过60%。RT-PCR和荧光实时定量PCR分析结果表明, etv2在自交二倍体金鱼成体的肝脏、心脏、肌肉、肾脏、精巢、脑和脾脏等多种器官组织中表达,但在卵巢和成熟卵子中不表达;在金鱼胚胎发育过程中, etv2从尾芽期开始表达,在体节形成后, etv2表达水平随胚胎发育而升高,在20体节期达到峰值,随后其表达水平降低。整胚原位杂交显示etv2特异性表达于自交二倍体金鱼胚胎的侧板中胚层、成血管细胞以及血管内皮细胞。在14体节期和20体节期,雌核发育单倍体胚胎中etv2在躯...     

链状亚历山大藻生长衰亡相关基因的筛选

采用抑制性消减杂交(Suppression subtractive hybridization,SSH)技术构建了衰亡期的链状亚历山大藻(Alexandriumcatenella)特异表达的cDNA文库。共获得800个克隆,利用巢式引物进行PCR筛选,最终确定阳性克隆556个。利用斑点杂交技术对这些阳性克隆进行差异筛选,获得差异表达克隆160个。测序后得到片段125个,经过归类,获得21种序列,其中6种在NCBI中经Blast,获得功能基因与其匹配,这些功能基因分别是CHK1类似检测点蛋白(checkpoint-like protein)、核酸外切酶复合体、谷氧还蛋白、Na+/K+ATPase、叶绿体中的一个开放阅读框和pG1蛋白,其他blast无同源序列,可能为新基因。推测链状亚历山大藻的衰亡过程可能涉及到DNA损伤、mRNA降解过程、氧化还原状态的变化、离子动态平衡的变化以及叶绿体一些生理状况的变化等过程。     

Identification of differentially expressed genes in seeds of two near-isogenic Brassica napus lines with different oil content

The regulation of seed oil synthesis in rapeseed is largely unknown. In this study, we compared the gene expression during seed development between two lines of Brassica napus with a 10% difference in oil content. We isolated the immature seeds 15 and 25 days after flowering at periods preceding and including the major accumulation of storage oils and proteins. The differentially expressed gene clones between the two rape lines were isolated by subtractive suppression hybridization (SSH). All SSH clones were arrayed and screened by dot blot hybridization, followed by RT-PCR analysis for selected clones. A total of 217 cDNA clones corresponding to 30 genes were found to have a high expression in seeds with high oil content. Six genes were highly expressed in seeds with low oil content. Northern blot and enzyme activity analysis demonstrated a change in expression pattern of several genes. The results provide information on gene-encoding factors responsible for the regulation of oil synthesis. The possible role of these genes in seeds is discussed. The genes in this study may be suitable as novel targets for genetic improvement of seed oil content and may also provide molecular markers for studies of rape breeding.