水稻亚种间杂种F_1低温敏感不育基因的差异显示

为研究水稻籼粳杂种低温下的育性特点和特异基因表达,以孕穗期白天24℃,夜晚20℃的低温和正常温度下生长的籼粳杂种02428/3037F1及其亲本幼穗为材料,开花期对亲本、处理和对照的花粉育性分别进行了观察,种子收获后考察了其结实率。结果表明,与对照相比,白天24℃夜晚20℃的低温处理使杂种的花粉育性和结实率明显降低,但对亲本的花粉育性和结实率影响不大。用12条锚定引物和18条随机引物共216对引物组合对总RNA反转录后的cDNA进行了PCR扩增,共有35条在处理和对照间可重复扩增的表达差异的条带。对克隆后的差异条带进行了测序和Blast,发现这些差异片段与一些幼穗发育的EST有很高的同源性,同源性均在99%以上。其中一个与位于水稻第一号染色体的过氧化物酶基因的部分片段有100%相似性,另一个与位于第十二号染色体上的蛋白磷酸化酶基因有99.99%的同源性,分别与两个LTSS基因所在的染色体一致。Southern杂交显示它们均为单拷贝片段。这些基因的表达与关闭与籼粳杂种低温敏感不育可能有密切关系。

差异显示技术在马铃薯基因功能研究中的应用

近年来,包括马铃薯在内的植物功能基因组学研究得到飞速发展。基因差异表达技术的日趋完善,为探讨动植物特定的遗传现象、规律及抗逆机制研究提供了极大的方便。文章论述抑制性消减杂交技术、cDNA-AFLP技术、cDNA微阵列(也称为cDNA芯片)等技术在马铃薯基因功能研究中的最新进展。

基因表达谱差异显示技术及其在植物对害虫取食诱导反应研究中的应用(综述)

基因表达谱差异显示技术是最近兴起的研究同种细胞在不同状态下基因表达差异的一种有效手段。目前已成功地应用于植物对害虫取食诱导防御的分子机制和发掘植物内源抗虫基因的研究领域,并显示出独特的优势。本文仅就植物被害虫取食前后基因表达谱差异研究中所涉及的技术、原理、问题和取得的进展进行综述和展望。

采用任意引物PCR技术进行基因组差异显示

任意引物 PCR技术是通过使用短的单一引物进行聚合酶链反应 ,能给基因组提供几百个多态性标记 ,它是分析基因组间关系差异的有力工具。它已被广泛用于物种的分类、鉴定、估计亲缘关系、混合基因组样本的分析及创造特异探针等。其主要优点是适用于任何生物、使用 DNA的量极少及其高效性和低成本。本文对这种技术的产生、原理、特点。

基因表达差异显示分析技术在创伤研究中的应用

基因是细胞增殖、分化、成熟等各项生命活动的调控中心,也是许多疾病发生、发展和转归的决定性因素。基因表达的变化必然导致细胞、组织、器官乃至整个机体的各种异常。包括创伤在内的各种内外刺激,都可不同程度地引起基因表达的变化,最终妨碍机体健康。随着生物信息学的逐渐兴起和分子生物学的不断发展并向其他学科的逐渐渗透,业已建立起一系列研究基因表达变化的切实可行的技术手段(即“基因表达差异分析技术”,如DNA微阵列),对捕获基因表达的种种变化具有重要价值。这些技术已经在肿瘤及其他疾病的研究中得到了广泛应用;近几年也逐渐进入创伤研究领域,在一定程度上推动了创伤研究的发展。

结肠癌组织中差异表达基因的筛选和鉴定

目的筛选和克隆结肠癌和正常结肠组织差异表达的基因片段,为探讨结肠癌的发病机制提供线索。方法应用基因差异显示技术(DD-PCR),比较结肠癌和正常结肠组织基因表达的差异,对其中一条有明显差异的基因片段进行克隆、测序,测序结果提交GenBank数据库中进行同源性分析,并用半定量RT-PCR进行初步鉴定。结果同源性分析表明,该片段与已知基因DDX32高度同源(99%)。RT-PCR结果显示,在结肠癌组织中该基因mRNA的表达水平显著高于正常结肠组织(P<0.05)。结论DD-PCR是筛选差异表达基因的有效手段;在结肠癌组织中DDX32基因的表达显著高于正常结肠组织,此结果为研究DDX32基因在结肠癌发病中的作用提供了线索。

基于DDRT-PCR研究茶树对茶尺蠖取食诱导的基因表达谱差异

利用DDRT-PCR技术初步研究了茶树被害虫茶尺蠖取食后基因表达谱差异。结果表明:接头引物A3、A4、A6、A7和A8分别与随机引物R1-R8,R12组合扩增后,总共得到222条差异片段,占总条带数的32.8%。在所鉴定的20个差异片段中,有8个片段序列没有发现同源序列;有5个片段序列与未知功能蛋白相关;其余7个差异表达的片段序列可分为6类,即分别与光合系统II色素蛋白(C15)、非生物抗性诱导蛋白(D22)、糖苷代谢酶(A45)、核酸和蛋白转录调控因子(A12,D63)、生长素调节蛋白(E94)和营养性抗虫蛋白(D73)。其中差异表达片段C15、A45、D22、D73和E94在植物与害虫相互作用相关分子机制研究中首次被发现。

差显技术分析结核杆菌H37Rv与H37Ra差异表达的基因

采用差异显示技术比较了结核分支杆菌强毒株H37Rv和弱毒株H37Ra在体外培养条件下的基因表达差异。通过20种引物组合进行mRNA差异显示,克隆到了两菌株间的20余个差异表达基因,经序列分析及杂交鉴定发现其中2个基因仅在H37Rv中表达。其中Rv1894 c基因编码的可能是H37Rv中的一个新蛋白。而在H37Ra的基因组中含有这2个基因的编码序列,但均未检测到基因的表达。

前列腺癌患者外周血差异显示编码3基因mRNA的表达

目的探讨前列腺癌（PCa）患者外周血中差异显示编码3基因（DD3）mRNA的表达及其与临床分期及Gleason评分的关系。方法采用逆转录．聚合酶链反应（RT—PCR）法检测63例PCa患者及61例良性前列腺增生（BPH）患者外周血中DD3mRNA表达，同时分析不同临床分期和病理分级与DD3mRNA表达的关系。结果PCa患者外周血中DD3mRNA表达率为82．5％，BPH组无表达，差异有统计学意义（P〈0．01）。不同临床分期的DD3mRNA表达差异有统计学意义（P〈0．05），不同Gleason评分的DD3mRNA阳性表达差异有统计学意义（P〈0．05）。结论外周血DD3mRNA表达和前列腺发生发展密切相关。

Differential Gene Expression Between Wheat Hybrids and Their Parental Inbreds in Primary Roots

To provide an insight into the molecular basis of heterosis, differential display of mRNA was used to analyze the difference of gene expression between wheat (Triticum aestivum L.) heterotic hybrid A, nonheterotic hybrid B and their parental inbreds in the primary roots. By using 5′ end random primers in combination with three one-base-anchored primers, it was found that 22.5% and 22.9% of 877 total displayed cDNAs were differentially expressed between hybrid A, B and their parents, respectively. Both quantitative and qualitative differences in gene expression between hybrids and their parental inbreds were obvious, indicating that the patterns of gene expression in hybrids alter significantly as compared to their corresponding parents. On the other hand, by using MADS-box gene specific 5′ end primer for DDRT-PCR, we found that nearly all of the displayed cDNA fragments were polymorphic between hybrids and their parents, and major difference occurred in qualitative level, in which hybrid specific-expressed and silenced genes are the major two patterns, suggesting that MADS-box gene may be important for manifestation of differential gene expression and wheat heterosis. In comparison with our previous results by using seedling leaves, it is indicated that differential gene expression between hybrids and parents is dependent on the tissues tested, and more differentially expressed genes were observed in the primary roots than in the seedling leaves. Therefore, it is concluded that the expressions of both randomly displayed cDNAs and transcription factor genes, such as MADS-box, alter significantly between hybrids and their parents, which might be responsible for the observed heterosis.

Relationship Between Differential Gene Expression and Heterosis During Ear Development in Maize (Zea mays L.)

Maize （Zea raays L.） is one of the most important crops because of the remarkable properties of its hybrid, which is responsible for the high commercial value of hybrid maize. The genetic basis of heterosis （hybrid vigor） is not well understood. A differential display technique was performed to identify genes with differential expression across twelve maize inbred lines and thirty-three hybrids during ear development. An incomplete diallel design was used to investigate the relationship between the global framework of differential gene expression and heterosis. It was found that the genes belonging to MONO pattern （i.e., genes expressed in both parental lines and in hybrid） was the highest in percentage among the total five patterns and illustrated that the properties of differentially expressed genes are not entirely responsible for heterosis. Furthermore,a larger number of differentially expressed genes in hybrid, which serves as a major reservoir for generating novel phenotypes that exhibit heterosis of certain agronomic traits during early development and differentiation of maize ear. Moreover, there were some silent genesin hybrids that are responsible for the arrest or abortion of spikelets and for the increase in kernels weight.

Application of mRNA Differential Display to the Identification of Genes Related to Embryonic Development

mRNA differential display was established by Liang and Pardee in 1992 for the purpose of displaying the mRNA differences between two tissues. The early embryonic development in animals is primarily controlled by the maternal RNAs stored in egg. These mRNAs are being degraded as the development proceeds. In some animals, such as fish and amphibian, new transcripts do not appear until the midblastula stage (midblastula transition, MBT). In other animals, for example in mouse, the zygotic genes are expressed during very early stages of development. The mRNA programmed synthesis and degradation during embryonic development controls the cell differentiation, germlayer formation and pattern formation. All these mRNA changes could be displayed side by side as cDNA band differences by mRNA differential display and the genes corresponding to these differential mRNAs could thus be obtained.

Molecular Cloning of Early-expressed cDNAs from Rice in Response to Infection by Rice Blast Fungus Magnaporthe grisea

Compatible and incompatible reactions in rice plants ( Oryza sativa L. cv. Shenxianggen No.4) were resulted from inoculation with two different virulent races of rice blast fungus ( Magnaporthe grisea (Hebert) Barr), and thus an effective infecting system was established between rice plants and the rice blast pathogen. Two cDNA clones that showed induced and temporal patterns in expression in the very early stage in response to infection of the fungus were obtained from the plants by use of differential display. Of the two cDNA clones, Fastresp_a was induced to express in both compatible and incompatible interactions although it was expressed earlier in the former reaction. The second one, Fastresp_b , was only expressed in incompatible interaction. Southern blot analysis of the rice genomic DNA indicated that both of the two clones were from genome of the plant. No significant homology to the two genes was found from the rice gene database. This suggested that they were novel genes in rice and may play important roles in rice resistant response to infection of rice blast fungus.

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.

The Study on the Gene Expression of Preimplantation IVF Bovine Embryos

The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technology of mRNA different display and one different fragment was found. The result suggested that this fragment displayed high homology (99%) to cattle mRNA for ribosomal protein L31. Then to detect the expression of RPL31mRNA in 8 cell and blastocyst stage embryos by real-time quantitative PCR,the result showed the relative amount of 8 cells was 3.2 times of blastocyst's.

Pathways related to PMA-differentiated THP1 human monocytic leukemia cells revealed by RNA-Seq

Previous analyses have reported that the human monocytic cell line THP1 can be differentiated into cells with macrophage-like characteristics by phorbol 12-myristate 13-acetate(PMA). However, little is known about the mechanism responsible for regulating this differentiation process. Here, we performed high-throughput RNA-Seq analysis to investigate the genes differently expressed in THP1 cells treated with and without PMA and examined those that may be responsible for the PMA-induced differentiation of monocytes into macrophages. We found 3,000 genes to be differentially expressed after PMA treatment. Gene ontology analysis revealed that genes related to cellular processes and regulation of biological processes were significantly enriched. KEGG analysis also demonstrated that the differentially expressed genes(DEGs) were significantly enriched in the PI3K/AKT signaling pathway and phagosome pathway. Importantly, we reveal an important role of the PI3K/AKT pathway in PMA-induced THP1 cell differentiation. The identified DEGs and pathways may facilitate further study of the detailed molecular mechanisms of THP1 differentiation. Thus, our results provide numerous potential therapeutic targets for modulation of the differentiation of this disease.