大白菜细胞质雄性不育系和其保持系的RAPD分析

利用RAPD技术对大白菜细胞质雄性不育系CMS341-7和其保持系3411-7的基因组DNA进行了比较分析,共使用了269个随机引物,其中有163个引物在两系之间都得到了扩增产物,79个引物扩增结果在两系之间表现出了遗传多态性。找到了不育系和保持系的特异扩增条带CMSOPL01670和MOPB04600。并对这些特异片段的来源及其在细胞质雄性不育中的作用进行了讨论。     

A new cell line from Spodoptera exigua (Lepidoptera: Noctuidae) and its differentially expressed genes

A new cell line, designated IOZCAS‐Spex XI, was established from the pupal ovaries of Spodoptera exigua (Lepidoptera: Noctuidae) in TNM‐FH medium containing 10% foetal bovine serum. The spherical cells were predominant among the various cell types. The population‐doubling time during the logarithmic phase of growth was 81.7 h. It was confirmed that the cell line originated from S. exigua by DAF‐PCR technique. Analysis of susceptibility to baculovirus showed that the new cell line was susceptible to S. exigua nucleopolyhedrovirus (SeNPV), Autographa californica multiple NPV (AcMNPV) and slightly susceptible to S. litura NPV (SpltNPV), while not permissive to Helicoverpa armigera NPV and Hyphantria cunea NPV (HcNPV). Real‐Time PCR analysis was carried out to compare some differentially expressed genes between the cell line and the primary culture. The result showed that marked significant differences were observed in the expression of the genes of SUMO‐1 activating enzyme, BCCIP‐like protein, 10 kDa HSP, CypA, receptor for activated PKC, PDI‐like protein ERp57, ALDH, DEAD box ATP‐dependent RNA helicase‐like protein (P < 0.01), while a significant difference was obtained in the expression of GST gene between the cell line and the primary culture (P < 0.05).     

In search of differentially expressed genes and proteins

A great challenge for modern cell biology is the successful examination of the co-expression of thousands of genes under physiological or pathological conditions and how the expression patterns define the different states of a single cell, tissue or a microorganism. Gene expression can be analyzed today on a large scale by advanced technical approaches for differential screening of proteins and mRNAs. The identification of differentially expressed mRNAs has been successfully applied to understand gene function and the underlying molecular mechanism(-s) of differentiation, development and disease state. Analysis of gene expression by the systematic mapping of thousands of proteins present in a cell or tissue can be achieved by the use of two-dimensional (2D) gel electrophoresis, quantitative computer image analysis, and protein identification techniques. In this article, we comment on some of these techniques and try to stress their advantages and drawbacks. We show how data from RNA/DNA mapping, sequence information from genome projects and protein pattern profiling can be linked with each other and annotated. These comprehensive approaches permit the study of differential gene and protein expressions in cells or tissues.     

Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-beta-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

差异表达基因的分离策略

生命活动的各个进程伴随着不同基因的选择性开启和关闭。如何有效地分离克隆各种差异表达的基因,成为分子生物学研究的一个努力方向,大量差异基因分离策略因之问世。本文扼要介绍了近年来发展的几种主要分离策略,并详细介绍一种新的差异基因分离方法--抑制差减杂交。     

转化的大鼠胚胎成纤维细胞系差异表达基因的筛选研究

来源于转化的大鼠胚胎成纤维细胞系的两株细胞,A1－5细胞与B4细胞相比表现出非常强的抗辐射性并伴随不同寻常强的G2延迟效应;用PCR选择性抑制消减杂交方法对这两株细胞进行差减,希望找到对A1－5细胞表现出的不同寻常的表型起关键作用的某一个或某一些基因。结果得到了160个差减转化子,逐个进行序列测定,并进行Dot blot杂交,共得到35个差异表达基因片段（EST）。通过对美国国家生物技术信息中心（NCBI）的非冗余序列库（NT）、鼠EST库及人EST库的BLAST进行同源检索,发现其中21个代表了尚未登录的新基因,另外14个分别与已知基因高度同源。     

肾透明细胞癌Caki-1细胞系差异表达基因的生物信息学分析

目的比较肾透明细胞癌Caki-1细胞系与正常肾上皮细胞系ASE-5063中的差异表达基因（DEGs）,寻找潜在的肾透明细胞癌特异性分子标志物。 方法利用GEO数据库自带的GEO2R在线分析工具分析基因芯片GSE78179,将筛选出的DEGs分别导入Metascape、STRING以及Cytoscape进行综合分析并筛选出核心基因。最后使用FunRich等软件对筛选出的核心基因进行GO和KEGG富集分析。 结果共筛选出562个DEGs,其中上调基因345个,下调基因217个。进一步使用MCODE筛选出36个关键基因,GO功能分析发现这些基因与细胞粘附分子活性、趋化因子活性、细胞通讯和信号转导等密切相关;KEGG通路富集结果则表明差异基因主要集中在趋化因子信号通路、TNF信号通路以及NF-κB信号通路等多种与肿瘤相关的通路上。 结论运用生物信息学方法筛选出肾透明细胞癌Caki-1细胞系中DEGs,其中数个核心基因广泛参与多种肿瘤的病理进程,但尚未在肾透明细胞癌有相关研究报道,提示其可能是治疗肾透明细胞癌的潜在靶点。     

Conversion of a rice CMS maintainer into a photo- or thermo-sensitive genetic male sterile line

A maintainer line of 3-line hybrid rice commonly presents a certain genetic distance to a 2-line restorer line, but in many cases, 2-line restorer lines present defects upon recovery of the object cytoplasmic male sterile (CMS) line of the maintainer line, which impedes the utilization of their heterosis. Here, we report a strategy and an example of converting a maintainer into a photoperiod/temperature-sensitive genic male sterile (P/TGMS) line with an almost identical genetic background, thus maximizing the heterosis. Firstly, through treatment of maintainer line T98B with ⁶⁰C_O-γ irradiation, we identified the TGMS line T98S, which is sterile at higher temperatures and fertile at lower temperatures. Secondly, the T98S line was proven to be identical to T98B with regard to genetic background via an examination of 48 parental polymorphous SSR markers and exhibited excellent blossom traits similar to those of T98B, with an extensive forenoon flowering rate of 75.92% and a high exertion rate of 64.59%. Thirdly, in a combination test, three out of six hybrids from T98S crossed with 2-line restorer lines showed a yield increase of 6.70–15.69% for 2 consecutive years. These results demonstrated that the strategy can generate a new P/TGMS line with strong general combining ability (converted from a maintainer line), thus helping to increase the genetic diversity of male sterile heterotic groups.     

Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.