cDNA RDA在类似普通2型糖尿病大鼠肾脏组织基因差异表达筛查中的应用

应用代表性差异分析 (cDNARDA)技术 ,对类似普通 2型糖尿病大鼠肾脏组织基因差异表达进行筛查 ,初步探讨类似普通 2型糖尿病大鼠肾脏损害发病的分子机制 .首先以类似普通 2型糖尿病大鼠肾脏组织作为实验组 (Tester) ,正常大鼠肾脏作为对照组或驱动组 (Driver)通过cDNARDA进行基因差异表达筛查 ;最终的差异产物亚克隆到Puc 18载体 ,测序及并进行生物信息学分析 ;半定量RT PCR对筛查到新的基因进行初步的鉴定 .结果发现 9个新ESTs ,2个新基因 .这 2个新基因分别与人及小鼠的丝氨酸蛋白酶抑制因子F ,及真核细胞转录启动因子 3亚单位 5 (EIF 3epsilon)基因有高度的相似性 (>90 % )并在类似普通 2型糖尿病大鼠肾脏组织表达上调 .推测 2个新基因分别是大鼠的丝氨酸蛋白酶抑制因子F及真核细胞转录启动因子 3亚单位 5 .两个新基因在类似普通 2型糖尿病大鼠肾脏组织表达上调 ,可能与类似普通 2型糖尿病大鼠肾脏损害相关 .同时 ,对新基因RS91进行了全长cDNA克隆     

Expression of octopaminergic receptor genes in 4 nonneural tissues in female Nicrophorus vespilloides beetles

Octopamine regulates the function of many tissues and physiological processes in invertebrates. The expression of octopamine receptor genes has been examined in multiple tissue types in several different insect orders. However, little work has addressed this issue in Coleoptera. Most studies characterize individual genes in different tissue types, but here we describe the expression of 6 octopamine receptor genes in thoracic musculature, oviducts, Malpighian tubules, and fat body of female Nicrophorus vespilloides beetles to characterize both different genes and different tissues within a single study. We then compare the gene expression profiles found in this beetle to other insects to examine the extent to which expression profiles are conserved across insects. We also examine the relative involvement of octopamine verses octopamine/tyramine receptors based on receptor gene expression in each tissue to help elucidate if tyramine plays a role in the regulation of these tissues. We find a high degree of overlap in the expression profile of the 6 genes examined in the thoracic musculature, a moderate amount for the oviducts, and divergent profiles for Malpighian tubules and fat body. Based on expression difference in receptor subtypes, our results also support the suggestion that tyramine is a biogenic amine with physiological actions separate from octopamine.     

Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi‐purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens

The suppressive ability of several strains of cyclic lipopeptide‐producing Bacillus rhizobacteria to grey leaf spot disease caused by Magnaporthe oryzae has been documented previously; however, the underlying mechanism(s) involved in the induced systemic resistance (ISR) activity in perennial ryegrass (Lolium perenne L.) remains unknown. Root‐drench application of solid‐phase extraction (SPE)‐enriched surfactin and live cells of mutant Bacillus amyloliquefaciens strain FZB42‐AK3 (produces surfactin, but not bacillomycin D and fengycin) significantly reduced disease incidence and severity on perennial ryegrass. The application of the treatments revealed a pronounced multilayered ISR defence response activation via timely and enhanced accumulation of hydrogen peroxide (H₂O₂), elevated cell wall/apoplastic peroxidase activity, and deposition of callose and phenolic/polyphenolic compounds underneath the fungal appressoria in naïve leaves, which was significantly more intense in treated plants than in mock‐treated controls. Moreover, a hypersensitive response (HR)‐type reaction and enhanced expression of LpPrx (Prx, peroxidase), LpOXO4 (OXO, oxalate oxidase), LpPAL (PAL, phenylalanine ammonia lyase), LpLOXa (LOX, lipoxygenase), LpTHb (putative defensin) and LpDEFa (DEFa, putative defensin) in perennial ryegrass were associated with SPE‐enriched surfactin and live AK3 cell treatments, acting as a second layer of defence when pre‐invasive defence responses failed. The results indicate that ISR activity following surfactin perception may sensitize H₂O₂‐mediated defence responses, thereby providing perennial ryegrass with enhanced protection against M. oryzae.     

分析EST寻找小鼠胸腺基质细胞表达基因

网上生物信息量的膨胀导致新基因寻找手段的改变,借助mRNA差异显示技术获得的17个表达序列标签（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）和小鼠EST库的BLAST同源检索,发现有4个序列与已知基因高度同源,其中的HDPs和Eps8两个基因是首次在小鼠胸腺基质细胞中发现,它们可能与T细胞在胸腺内的发育分化有关,其它EST则为新基因,本结果也为下一步新基因的克隆及功能研究提供了有益的线索。     

Peanut Resistance Gene Expression in Response to Aspergillus flavus Infection During Seed Germination

Aspergillus flavus produces potent mutagenic and carcinogenic polyketide‐derived secondary metabolites known as aflatoxins. Development of host plant resistance in peanut and other crops is the most environmentally friendly and cost‐effective method to eliminate the serious problem of aflatoxin contamination in grains. To confirm that putative peanut genes identified in a previous microarray study were involved in peanut resistance to A. flavus infection, 14 genes were selected for further investigation through real‐time PCR. The results revealed diverse patterns of gene expression during seed germination after A. flavus inoculation. Based on the expression levels and the relative‐expression patterns over a 7‐day period, the 14 host genes could be classified into six different groups belonging to three main biochemical and genetic defence processes of lipid metabolism, oxidative signalling and cell‐wall synthesis during counter‐attack. A network of gene expression patterns was activated in sequential order in response to A. flavus invasion in both resistant and susceptible peanut lines during seed germination. Understanding gene expression patterns in peanut will be useful to breeders and other scientists interested in incorporating genetic resources of resistance against A. flavus into peanut germplasm and/or commercial cultivars via conventional and/or molecular methods.     

Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature

Quantitative real‐time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species‐specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial‐feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host.     

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR

Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results, the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities.     

Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage

We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real‐time PCR assay. The concentration of the bacterium was monitored at 5‐day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca. L. asiaticus‐infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus‐infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25‐, 360‐ and 130‐fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.     

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5' 端cDNA片段克隆及序列分析

研究高等生物基因表达与调控的一个重要方面是分离基因的编码区及其上游的调控序列（ＤｅＶｅｅｒ等１９９７）,这需要获得一个基因的ｃＤＮＡ全长及从植物基因组获取全基因。在前文（周建明等１９９９）中曾经分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段,但是运用ｍＲＮＡ差异显示技术分离的ｃＤＮＡ片段往往只有近ｍＲＮＡ３’端的一部分,难以反映基因的结构及功能特点,因此,必须进一步分离其５’端的部分才有可能比较全面地了解此基因的特点。ＲＡＣＥ（ｒａｐｉｄａｍｐｌｉｆｉｃａｔｉｏｎｏｆｃＤＮＡｅｎ…     

Standard guidelines for the publication of telomere qPCR results in evolutionary ecology

Telomere length has been used as a proxy of fitness, aging and lifespan in vertebrates. In the last decade, dozens of articles reporting on telomere dynamics in the fields of ecology and evolution have been published for a wide range of taxa. With this growing interest, it is necessary to ensure the accuracy and reproducibility of telomere length measurement techniques. Real‐time quantitative PCR (qPCR) is routinely applied to measure relative telomere length. However, this technique is highly sensitive to several methodological variables and the optimization of qPCR telomere assays remains highly variable between studies. Therefore, standardized guidelines are required to enable the optimization of robust protocols, and to help in judging the validity of the presented results. This review provides an overview of preanalytical and analytical factors that can lead to qPCR inconsistencies and biases, including: (a) sample type, collection and storage; (b) DNA extraction, storage and quality; (c) qPCR primers, laboratory reagents, and assay conditions; and (d) data analysis. We propose a minimum level of information for publication of qPCR telomere assays in evolutionary ecology considering the methodological pitfalls and sources of error. This review highlights the complexity of the optimization and validation of qPCR for telomere measurement per se, demonstrating the importance of transparency and clarity of reporting methodological details required for reliable, reproducible and comparable qPCR telomere assays. We encourage efforts to implement standardized protocols that ensure the rigour and quality of telomere dynamics studies.     

Induction of cytokine formation by human intestinal bacteria in gut epithelial cell lines

Aims: To investigate the effects of human gut micro‐organisms on cytokine production by human intestinal cell lines. Methods and Results: Quantitative real‐time PCR assays were developed to measure the production of pro‐inflammatory (IL‐1α, IL‐6, IL‐18 and TNFα) and anti‐inflammatory (TGF‐β1, TGF‐β2, TGF‐β3, IL‐4 and IL‐10) cytokines in HT‐29 and Caco‐2 cell lines. They were co‐cultured with a range of mucosal bacteria isolated from ulcerative colitis patients, together with lactobacilli and bifidobacteria obtained from healthy people. HT‐29 cells were also co‐cultured with Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli and Salmonella typhimurium. The majority of commensal bacteria tested suppressed the expression of anti‐inflammatory cytokine mRNA, increased IL‐18, reduced IL‐1α, and with the exception of nonpathogenic E. coli, reduced TNF‐α. All overtly pathogenic species increased both pro‐inflammatory and anti‐inflammatory cytokine mRNA. Conclusion: Commensal and pathogenic species induced fundamentally different cytokine responses in human intestinal epithelial cell lines. Significance and Impact of the Study: Interactions between commensal bacteria tested in this study and the innate immune system were shown to be anti‐inflammatory in nature, in contrast to the pathogenic organisms investigated. These data contribute towards our understanding of how potential probiotic species can be used to suppress the pro‐inflammatory response in inflammatory bowel disease.     

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.