Expression pattern of CYC-like genes relating to a dorsalized actinomorphic flower in Tengia (Gesneriaceae)

Tengia has been called a "natural peloria" in the family Gesneriaceae because it exhibits an almost perfect actinomorphic flower from whorl one to whorl three. It would be especially interesting to know whether or how CYC-like gene activities are related to this type of perfect actinomorphic flower. To address this, we have isolated four CYC-like TCP genes and carried out an investigation on their expression patterns in Tengia. TsCYC1C and TsCYC1D have similar expression patterns with strong signals being detected in all five petals and stamens, whereas TsCYC2A and TsCYC2B are only transiently expressed in the very early floral meristem. Our results suggest that the expansion of the expressions of TsCYC1C and TsCYC1D from the dorsal to the ventral petals is likely responsible for the evolutionary formation of the fully dorsalized actinomorphic corolla, that is, an expanded functional domain of CYC-like gene dorsal identity in Tengia corolla. However, the expressions of TsCYC1C and TsCYC1D are not correlated with stamen abortion; therefore, TsCYC genes do not functionally repress the stamen development in Tengia flowers. This is probably due to changed cis-activities that result in the cell cycle-related genes uncoupling from the TsCYC regulatory pathway in Tengia.     

Altered expression patterns of TCP and MYB genes relating to the floral developmental transition from initial zygomorphy to actinomorphy in Bournea (Gesneriaceae)

The shift from zygomorphy to actinomorphy has been intensively studied in molecular genetic model organisms. However, it is still a key challenge to explain the great morphological diversity of derived actinomorphy in angiosperms, since different underlying mechanisms may be responsible for similar external morphologies. Bournea (Gesneriaceae) is of particular interest in addressing this question, as it is a representative of primarily derived actinomorphy characteristic of a unique developmental transition from zygomorphy to actinomorphic flowers at anthesis. Using RNA in situ hybridization, the expression patterns were investigated of three different Bournea orthologues of TCP and MYB genes that have been shown to control floral symmetry in model species. Here, it is shown that the initial zygomorphic pattern in Bournea is likely a residual zygomorphy resulting from conserved expression of the adaxial (dorsal) identity gene BlCYC1. As a key novel event, the late downregulation of BlCYC1 and BlRAD and the correlative changes in the late specific expression of the abaxial (ventral) identity gene BlDIV should be responsible for the origin of the derived actinomorphy in Bournea. These results further indicate that there might be diverse pathways in the origin and evolution of derived actinomorphy through modifications of pre-existing zygomorphic developmental programs under dynamics of regulatory networks.     

RNA原位杂交技术的一些应用技巧

目的:检测基因在动物组织或细胞中的时空表达模式。方法:转录反义RNA探针;利用RNA原位杂交技术检测人和小鼠牙原基中若干基因的表达。结果与结论:通过优化条件,转录出完整的反义RNA探针,并成功地利用RNA原位杂交技术在组织中检测到基因的表达;分析了一些在RNA原位杂交的过程中可能碰到的问题及其解决方法。     

植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...     

地高辛标记Ucp2基因RNA探针的制备和应用

目的:制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法:提取小鼠胚胎脑组织总RNA,设计引物,通过RT-PCR方法获取Ucp2基因片段,将其克隆到pGEM-T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果:成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。结论:成功制备了特异高效的地高辛标记Ucp2 RNA原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。     

应用小鼠整体原位杂交技术检测MDM2在鼠胚发育不同阶段的表达

整体原位杂交(whole-mountinsituhybridization,WMH)已经成为基因表达定位和表达分布模式研究的一种重要手段.该技术能在整体水平上精确地研究胚胎发育过程中基因表达的三维信息,而且为大规模筛选区域及组织特异性候选克隆提供了有利的技术手段.采用体外转录地高辛标记的RNA探针,检测已知基因MDM2在鼠胚胎发育不同阶段的表达模式.     

视黄酸对小鼠早期胚胎结构及0tx2基因表达的影响

以全反式视黄酸 ( all trans-retinoic acid,AT-RA)体外处理 EB( early allantoic bud)期和 LB( lateallantoic acid)期的小鼠胚胎 ,然后用洋地黄毒苷 ( digoxigenin)标记的 0 tx2反意义 RNA探针对整体胚胎进行原位杂交。以 4× 1 0 - 6 mol/ l的 AT— RA处理 ,抑制或减少了胚胎的前肠形成 ,经处理的胚胎的头褶没有对照的那样明显向腹部突出 ,其神经沟也不整齐 ;经处理的 EB期胚胎 ,其 0 tx2表达范围剧烈地向前退缩或只有很微弱的表达 ;但以同样浓度处理 LB期胚胎时 ,与对照相比 ,0 tx2的表达除了在极少数胚胎中变得弱一些以外 ,在绝大多数胚胎中其表达模式没有变化     

磷酸化酶mRNA在水稻雌蕊中表达的时空动态

利用ＲＮＡ原位杂交技术,对水稻受精前后雌蕊组织切片进行磷酸化酶基因表达的定位。结果显示,磷酸化酶ｍＲＮＡ在柱头、花柱、子房壁以及维管束中大量表达,而胚珠中除合点部位外表达很弱。受精前后的胚囊中各细胞磷酸化酶ｍＲＮＡ表达也很弱,并且没有明显的时空变化。在积累淀粉的胚乳细胞中磷酸化酶ｍＲＮＡ大量分布。原胚中的磷酸化酶ｍＲＮＡ表达较弱,到分化胚积累过渡性淀粉时才显著增加。本文首次对磷酸化酶ｍＲＮＡ在植物雌性器官发育过程中的时空分布动态作了初步研究。     

DNA and RNA within the nucleus: How much sequence-specific spatial organization?

Spatial organization of various nuclear components is often proposed as a means by which nuclei more efficiently carry out their various tasks. Such functional compartmentalization may involve a sequence-specific packaging and placement of DNA and RNA. Here we review recent insights, allowed primarily by advances in fluorescent in situ hybridization methodology, into the organization of nucleic acids within individual nuclei.     

苦苣苔科大叶石上莲CYC类基因RT-PCR表达模式研究

CYC类基因的分子系统学研究已经在苦苣苔科Gesneriaceae中展开,但是还缺乏对这些基因表达和功能的研究。因此,我们选择苦苣苔科大叶石上莲Oreocharis benthamii作为实验材料,分离出了CYC类基因的两个拷贝,经过分子系统学分析这两个基因分别属于苦苣苔科GCYC1和GCYC2两个分支,故命名为ObCYC1和ObCYC2。分区的RT-PCR实验结果显示这两个基因拥有不同的时间空间表达模式。ObCYC1与模式植物金鱼草Antirrhinum majus中的CYC基因类似,集中在花冠背部区域表达,这与它们拥有保守的功能区TCP和R相一致。但是,ObCYC1与CYC表达模式仍有区别,即,和CYC相比ObCYC1在花冠背部区域的表达提前减弱。这可能和大叶石上莲花冠微弱的两侧对称性相关。另外,由于大叶石上莲的背部花瓣较两侧和腹部花瓣小,因此,在功能上ObCYC1可能起抑制背部花瓣生长作用而CYC基因则促进背部花瓣生长。与ObCYC1不同,ObCYC2的保守功能区有更多的氨基酸位点变化,而且在RT-PCR实验中也没有检测到它的表达。因此,需要开展更深入的实验研究分析ObCYC2的基本功能,这将有助于了解GCYC2类基因在苦苣苔科及其近缘科中的功能和进化途径。     

Localization of mitochondrial large ribosomal RNA in the myoplasm of the early ascidian embryo

Mitochondrial large ribosomal RNA (mtlrRNA) is transferred out of mitochondria and associates with germinal granules in Drosophila and Xenopus embryos. It has been revealed that mtlrRNA outside of mitochondria is required for formation of the germ-line progenitor, or pole cells in Drosophila. In the present study, the distribution of mtlrRNA was examined in embryos of the ascidian, Halocynthia roretzi, during cleavage stages by whole-mount in situ hybridization. Until the 4-cell stage, the distribution of mtlrRNA coincided with that of mitochondria. which are localized to the cortical cytoplasm in the posterior region of the embryos. Both mitochondria and mtlrRNA were preferentially partitioned into muscle-lineage blastomeres during cleavage stages. After the 8-cell stage, a discrepancy in intracellular localization of mitochondria and mtlrRNA became evident. Mitochondria translocated into central yolkless cytoplasm, while mtlrRNA remained in the posterior cortex in the posterior muscle-lineage b astomeres. The significance of the cortical localization of mtlrRNA in muscle precursor cells in ascidian embryos is obscure. However, the results suggest that mtlrRNA is also transferred out of mitochondria in early ascidian embryos and may play some roles in developmental processes.     

应用引物原位标记技术检测21号染色体

应用原位引物标记技术（PRINS）检测了21号染色体着丝粒,在外周血和绒毛细胞的标记效率分别为91％和93％,实验过程可以在2h之内完成,证明这一检测方法是一种快速、灵敏、特异性良好的染色体数目检测方法,有可能用于21号染色体数目异常的快速诊断。     

A Review of Fluorescence in Situ Hybridization (FISH): Current Status and Future Prospects

Fluorescence in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells, tissues and tumors. This molecular cytogenetic technique enables the localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. FISH is quickly becoming one of the most extensively used cytochemical staining techniques owing to its sensitivity and versatility, and with the improvement of current technology and cost effectiveness, its use will surely continue to expand. Here we review the wide variety of current applications and future prospects of FISH technology.     

A Review of Fluorescence in Situ Hybridization (FISH): Current Status and Future Prospects

Fluorescence in situ hybridization (FISH) is a powerful technique for detecting DNA or RNA sequences in cells, tissues and tumors. This molecular cytogenetic technique enables the localization of specific DNA sequences within interphase chromatin and metaphase chromosomes and the identification of both structural and numerical chromosome changes. FISH is quickly becoming one of the most extensively used cytochemical staining techniques owing to its sensitivity and versatility, and with the improvement of current technology and cost effectiveness, its use will surely continue to expand. Here we review the wide variety of current applications and future prospects of FISH technology.     

Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris

In situ hybridization with a fluorescently labeled 16S rRNA-targeted probe was examined using Rhodopseudomonas palustris as a model organism, which had been grown at different rates and under different conditions of growth and starvation. The specific growth rate did not affect the percentage of hybridized cells in aerobically grown R. palustris cultures. However, significant changes in the percentage of hybridized cells occurred during extended periods of starvation. These changes were observed both in batch cultures grown and starved aerobically in the dark, and in cultures grown phototrophically and starved anaerobically in the dark. Aerobic growth in batch culture and subsequent starvation resulted in a complete lack of detectable hybridization after 20 days of starvation. In contrast, even after 30 days of starvation, 50% of all cells were still detectable in cultures grown aerobically at growth rates <0.06 h(-1) and then starved aerobically in the dark. The same was true for phototrophically grown cells that were starved anaerobically in the light. During starvation there was a clear, though non-linear, positive correlation between the percentage of hybridized cells and the RNA content. In contrast, no direct correlation was observed between the number of hybridized cells in a culture and the viability of this culture. Thus, in habitats with growing, non-growing, and starving bacteria, data on quantitative detection of populations based on 16S rRNA-targeted probing should be used with extreme caution as the detectability of the individual cells is strongly influenced by their physiological history and current physiological state.