采用荧光原位杂交技术,对分属5个科的10种植物的分生细胞的18S-25S rRNA基因(45S rDNA)的组织模式进行了比较分析.45S rDNA探针在所有供试植物的间期核都产生了两种杂交信号:荧光强、位于核仁周边的纽和荧光较弱分布于核仁内的点,表明不同植物间期核的rDNA染色质的组织模式相似.在每种植物的部分间期细胞都观察到点与纽相连或从纽发出的情况,而且点的数目越多纽就变得越小,点的有无和数目的多少与细胞的活性呈正相关.这些事实表明,纽代表了处于凝缩状态的非活性的rDNA染色质,点是由纽解凝缩而来,rDNA异染色质解凝缩形成点是植物rRNA基因活跃转录的细胞学表现,在同一物种中点的多少代表了间期核rDNA转录活性的强弱.我们的结果支持点是核仁内活性rRNA基因组织的结构单位及rRNA合成发生地点的推论.我们的结果还显示,不同植物间期核的rDNA染色质的组织也存在一些差异,其中核仁内点的最大数目有较大的不同.在所有供试植物的有丝分裂前中期细胞,45S rDNA探针在rDNA位点都产生了松散的信号块和许多点,表明植物的rDNA位点在有丝分裂前中期还有较活跃的转录.

Interleukin (IL)-27 is a new member of the IL-6/IL-12 family, composed of two subunits, the Epstein-Barr virus-induced gene 3 (EBI3) and p28 chains (p28), and produced by activated monocytes and dendritic cells. IL-27 plays an important role in the regulation of differentiation of naive T helper cells and has diverse effects on innate immune cells. However, the pro-inflammatory mechanisms of IL-27 are still not well known. In this study, we investigated the effect of lipopolysaccharide (LPS) on the production of IL-27. We found that LPS-stimulated IL-27 production was in a dose-dependent and time-dependent manner in THP-1 cells. We have also shown that IL-27 induced PGE2 production and COX-2 gene expression at the level of mRNA as well as protein. Moreover, we found feed back effect of PGE2 on the production of IL-27 in THP-1 cells. The results suggest that PGE2 significantly inhibits LPS-induced IL-27 production, without affecting basal IL-27 expression. Further experiment suggests that PGE2 and LPS regulate IL-27 through NF-kappa B pathway. Our findings may have wide implication for IL-27 in inflammatory diseases.

In order to analyze the conservation of maize centromeric satellite DNA (CentC) and centromeric retrotransposon (CRM) in the subspecies and relatives of Zea mays, dual fluorescence in situ hybridization (FISH) was used to detect the existence and distribution of the above two repetitive sequences in Zea mays ssp. mexicana, Z. diploperennis, Z. perennis, Tripsacum dactyloides, Coix lacryma-jobi, and Sorghum bicolor. In Z. mays ssp. mexicana, Z. diploperennis, and Z. perennis, both CentC and CRM probes produced strong or relatively strong signals in the centromeric regions of all chromosomes. There was an obvious variation in the intensity of hybridization signals on different chromosomes, indicating that different centromeres have different amounts of CentC and CRM sequences. In some centromeres, the intensity of CentC signals differed from that of CRM signals and was free from overlapping. In T. dactyloides, only weak CentC and CRM signals were detected in the centromeric regions of most chromosomes, while in C. lacryma-jobi and S. bicolor only relatively strong or strong CRM signals primarily located in the centromeric regions were detected. This result indicates that CentC is highly conserved among the subspecies of Z. mays and the species of Zea, and has high conservation in Tripsacum, a genus that is most closely related to Zea, and CRM is conserved among the species of grass family either closely or distantly related to Zea.

玉米（Zea mays）只有1对45S rDNA位点并在分裂期染色体形成次缢痕,是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交（fluorescence in situ hybridization,FISH）、CPD（PI与DAPI组合）染色和银染技术,分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号:荧光强烈地位于核仁周边的纽,而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出;点的数目越多,纽变得越小;点的数目多少与细胞的活性呈正相关。研究结果表明,纽代表了处于凝缩状态的非活性的rDNA染色质,纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现;不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示,大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示,同一间期细胞的2个同源rDNA位点的表达水平存在差异,同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示,早中期细胞的rDNA染色质相对解凝缩,银染在所有早中期细胞和部分中期细胞显示了明显的核仁,表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录,其转录在晚中期才停止。

In this study, outcrossing rates and genetic diversity in natural populations of Nelumbo nucifera were investigated. The estimated multilocus outcrossing rate (tm) based on 28 ISSR loci was over 90%. Analysis of genetic diversity revealed that this index was high at the species level (Hs = 0.325, I = 0.514), but low within the individual study populations (Hs = 0.148, I = 0.212). Gst-B was 0.547 and Nm was 0.414. The results of AMOVA indicated that 54.6% of the variation was due to the difference between the regions and 45.4% to the variation within the region. Although the populations were predominantly outcrossing, most of the genetic diversity was attributed to geographical effects instead of their habitats because low sexual recruitment and clonal growth deeply reduced the genetic diversity within the populations. On the basis of the high tm, Gst-B and low Nm values, we recommended that any future conservation plans should include both in situ conservation and germplasm collection.

Mustard (Brassica juncea) is an important crop in both ancient and modern world. It has a broad resource of genetic diversity that is used primarily as oilseed but as vegetables, condiment and medicines also. Its superior tolerance to adverse environments, e.g., drought, high temperature and low fertility suggests its better adaptability in future possible harsh environments. Chinese vegetable mustard displays a wide spectrum of morphotypes. A collection of 95 accessions of B. juncea representing oil and vegetable mustards from China, France, India, Pakistan, and Japan were assessed to determine diversity at the molecular level using sequence-related amplified polymorphism (SRAP). Eight SRAP primer combinations identified a total of 326 scorable fragments of which 161 were polymorphic (49.39%). The percentage of polymorphism for each primer combination varied from 21.88 to 66.67%. Both Shannon-Weaver and Simpson genetic diversity index indicated that the level of genetic diversity within vegetable mustard is much higher than within oil mustard, and also winter oil mustards are genetically more diverse than spring oil mustards. Based on the Cluster and Principal Coordinates analysis, which were conducted on the similarity matrix of SRAP marker data, vegetable, spring oil and winter oil mustard were clearly divided into three distinct groups and among these three groups, spring and winter oil mustard are geneticlly closer than vegetable mustard. This suggests that bilateral gene exchange between oil and vegetable gene pools in the breeding program will effectively elevate the genetic potential in developing higher yields, more disease resistance, better quality and better adapted lines.

The chromosomal location of 45S rDNA in sixteen plant species belonging to six families was analyzed using combined PI and DAPI(CPD)staining together with fluorescence in situ hybridization(FISH)with 45S rDNA probe.Fifty three 45S rDNA sites in total were detected in the tested species.The 45S rDNA sites occurred mainly in the short arms instead of the long arms,and interstitial and terminal 45S rDNA sites appeared at similar frequency.Secondary constrictions appeared in most interstitial 45S rDNA sites,but the orientation of the cluster of rDNA repeats relative to the secondary constriction differed among these interstitial sites.The chromosomal distribution of 45S rDNA sites in the tested plants could be classified into 12 types based on the differences in the chromosomal arm in which 45S rDNA site reside,the distance from centromere to 45S rDNA site,the formation of secondary constriction,and the orientation of the cluster of rDNA repeats.The interrelation among 45S rDNA site,NOR(nucleolar organizing region),secondary constriction,and satellite was analyzed based on our results and other previous reports.

DNA silver staining has widely been used to detect DNA fragments in polyacrylamide gels with high sensitivity. We developed an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure can be completed within 10 min instead of over 20 min with the conventional methods. The sensitivity is significantly improved by the silver-ion sensitizer (Eriochrome black T (EBT)) and the minimum of 0.11 and 1.75 ng of DNA amount can be detected in denaturing and nondenaturing polyacrylamide gel, respectively. Compared with the conventional silver staining methods, the improved optimal method can save time and display high sensitivity, color uniformity, and long storage time of the staining gels.

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization (GISH) procedure, fluorescence in situ hybridization (FISH) with genomic DNA to their own chromosomes (called self-genomic in situ hybridization, self-GISH) was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions (NORs). The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with C(o)t-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

To explore a simple, reliable, and effective method of karyotyping Brassica oleracea L., Cot-1 DNA was isolated from its genome, labeled as probe with a Biotin-Nick Translation Mix kit, and in situ hybridized to mitotic spreads. Specific fluorescent bands appeared on each chromosome pair. 25S and 5S rDNAs were labeled as probes with a DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit, respectively, and in situ hybridized to mitotic preparations. Signals could be detected on two chromosome pairs for 25S rDNA, and on only one for 5S rDNA. Cot-1 DNA contains rDNA. The site identity of Cot-1 DNA and 25S rDNA on the chromosome was determined by dual-colour fluorescence in situ hybridization (FISH). It showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome markers is a superior alternative. A more exact karyotype of B. oleracea has been developed based on rDNA locations and Cot-1 DNA fluorescent bands.

Using genomic in situ hybridization with genomic DNA, high-order chromatin fibers were successfully exhibited under a light microscope through the cell cycle in barley, rice, maize and field bean. From the interphase to prophase and metaphase of mitosis, the fibers were basically similar. Each was estimated to be around 200 nm in diameter, but the strength of signals was not the same along the fiber length. Through the cell cycle a series of dynamic distribution changes occurred in the fibers. In the interphase, they were unraveled. At the early prophase they were arranged with parallel and mirror symmetry. During late-prophase and metaphase, the fibers were bundled and became different visible chromosomes. The parallel coiling and mirror symmetry structures were visible clearly until the metaphase. In anaphase they disappeared. During telophase, in peripheral regions of congregated chromosome group, borderlines of the chromosomes disappeared and the fibers were unraveled. This demonstrated that mitotic chromosomes are assembled and organized by parallel and adjacent coiling of the fibers and the fibers should be the highest order structure for DNA coiling.

Comparative genomic in situ hybridization (cGISH) technology has become a popular approach to study genomic homologies and can be used for the comparison between distant species. In this study, we used biotin-labeled Arabidopsis genomic DNA to hybridize the chromosomes from two dicots (Lycopersicon esculentum and Vicia faba) and three monocots (Oryza sativum ssp. indica, Zea mays and Hordeum vulgare) at 75% stringency, in order to evaluate the genomic homologies between Arabidopsis and distantly related plants. We found the fluorescent hybridization signals appeared to be dispersed within the chromosomes in all observed species, and displayed an increasing as the genome size of a species becomes larger. Strong hybridizations were observed at the nucleolar organizing regions (NORs) in all species studied, which indicates the corresponding Arabidopsis DNA probes could potentially be applied for NOR mapping in plants. In all analyzed species, hybridization signals primarily located at the interstitial sites or chromosomal ends, whereas only a few detected in centromeric or pericentromeric regions. The patterns of cGISH signals represented with a different profile as compared to the chromosome C- or N-banding patterns in a particular species. Therefore it might be used as alternative measurement to enable the discrimination of individual chromosomes in plants. Our results have demonstrated the existence of numerous homologous repetitive DNA sequences between Arabidopsis and distantly related plants besides rDNA and telomeric repeats, and a significant portion of them are presumably stabilized before the divergence of dicotyledon and monocotyledon during evolution. Moreover, the results also supported the discovery of considerable amplifications of ancient conserved repetitive DNAs as large plant genomes developed during evolution.

Despite the economic importance of Nelumbo nucifera, there have been no molecular studies on genetic relationships among cultivars in the species. In the present study 38 accessions were sampled including 37 accessions of N. nucifera or hybrids between N. nucifera and Nelumbo lutea and a single accession of N. lutea. In the ITS analyses, Chinese and Japanese lotus comprise a single cluster with a moderate bootstrap support 68% indicating there is very high similarity between them. Moreover, these ISSR and RAPD results also indicate that there is very close genetic relationship between Chinese and Japanese lotus. In the ISSR and RAPD analyses, although 38 accessions all are distinctly separately into two groups, viz. N. nucifera and N. lutea, there is a high Jaccard similarity coefficient (0.785 and 0.656) between the two species. In N. nucifera the two different groups of the species, viz. flower lotus and rhizome lotus accessions show clear genetic variations. Seed lotus accessions do not form a distinct cluster but are interspersed among the flower accessions indicating that seed lotus is phylogenetically close to flower lotus and they might originate from close wild lotus in genetic relationship. In flower lotus, big-flower type accessions and medium-small type accessions have obvious genetic variation, indicating height is an important criterion in the classification system of flower lotus.

The genomic structures of Oryza sativa (A genome) and O. meyeriana (G genome) were comparatively studied using bicolor genomic in situ hybridization (GISH). GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e. O. sativa-O. sativa (A-A), O. sativa-O. meyeriana (A-G) and O. meyeriana-O. meyeriana (G-G), were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

Fluorescence in situ hybridization (FISH) was carried out on mitotic chromosomes of Zea diploperennis (DP), maize inbred lines F107, GB57 and their hybrids using knob-associated tandem repeats, 180-bp repeats and TR-1 elements, together with 45S rDNA as probes.The distribution patterns of the three repeated sequences were analyzed on parental chromosomes.The hybrids of maize and DP were identified by FISH.According to the hybridization signals on chromosome positions, the origin of foreign idioplasm was confirmed.The application using knob-associated tandem repeats as excellent chromosomal markers on detecting the integration of foreign chromosome and the chromosomal activity at meiosis was also discussed.

Centromeres are the chromosomal domains necessary for faithful chromosome segregation and transmission during mitosis and meiosis in eukaryotes. In the last decade, centromeres in some plant species including Arabidopsis, rice and maize have been deeply studied at molecular level. Centromeric DNAs evolve rapidly and are little conserved among various plants, but the types of centromeric DNA sequences and their organization patterns within centromeres are basically similar in plants. Plant centromeres are usually composed of clusters of tandemly arrayed satellite repeats that are interspersed with centromere-specific retrotransposons. In contrast to centromeric DNA, structural and transient centromeric/kinetochoric proteins are conserved among eukaryotes including plants. As the cases in other eukaryotes, the presence of CENH3 (centromeric histone H3)-containing nucleosomes is the fundamental feature of plant functional centromeres, and CENH3 plays critical roles in the identity and maintenance of plant centromeric chromatin.

Mitotic chromosome spreads of 16 plant species belonging to six families were analyzed using an improved combined PI and DAPI (CPD) staining procedure. Fluorescence in situ hybridization (FISH) with 45S rDNA probe was conducted sequentially on the same spreads to evaluate the efficiency and sensitivity of the technique. Fluorochrome staining with chromomycin A3 (CMA)-DAPI also was conducted to clarify the properties of the sequences involved in the CPD banded regions. Our results revealed that all of the NORs (rDNA sites) in the species tested were efficiently shown as red bands by CPD staining, and the number and position of the bands corresponded precisely to those of the 45S rDNA FISH signals, indicating that the detection sensitivity of CPD staining is similar to that of FISH. In 10 of the species tested including Aegilops squarrosa, Allium sativum, Oryza sativum ssp. indica, Oryza officinalis, Pisum sativum, Secale cereale, Setaria italica, Sorghum vulgare, Vicia faba and Zea mays, CPD bands were exhibited exclusively in their NORs, while in other six species including Hordeum vulgare, Allium cepa, Psophocarpus tetragonolobus, Arabidopsis thaliana, Brassica oleracea var. capitata and Lycopersicon esculentum, CPD bands appeared in chromosomal regions other than their NORs. The CPD bands were in accordance with the CMA bands in all species tested, indicating GC-rich sequences in the CPD bands and that the improved CPD staining procedure is specific for GC-rich regions in plant genomes. Our investigation not only elucidated the banding mechanisms of CPD, but also demonstrated that the CPD staining technique, which may be preferable to CMA staining, is an effective tool for detecting NORs and other GC-rich chromosomal regions in plants.

Molecular banding undertaken with comparative genomic in situ hybridization(cGISH) is a simple approach which generates chromosome characteristic signals in heterologous FISH experiments at regions with conserved repeated sequences.In this study,cGISH was undertaken with labeled total genomic DNA of Tripsacum dactyloides(2n=72) to chromosomes of several species in genus Zea.Four kinds of bands including interstitial band,centromeric band,telomeric band and knob were detected on all species.Compared to DAPI banding,the distributional characteristics of conserved repeated DNA sequences were illustrated among tested species.

Fluorescence in situ hybridization(FISH) is an effective and accurate molecular cytogenetic tool for mapping specific DNA sequences on chromosomes,interphase nuclei and DNA fibers.Rapid advances in plant FISH technique have been made in the past two decades.Dual FISH,multicolor FISH and multiprobe FISH cocktail have been developed to increase the number of detected targets;BAC-FISH and tyramide signal amplification FISH(TSA-FISH) have been applied to increase the detection sensitivity of very small chromosomal targets; and high resolution FISH such as pachytene FISH, interphase FISH, fiber FISH and FISH on super-stretched flow-sorted plant chromosome have been developed mainly to improve the spatial resolution of signals derived from flanking sequences. FISH have played important roles in the analysis of plant genome. It can physically map specific DNA sequences, provide effective marks for chromosome identification within a genome, investigate the evolution of plant genome by comparative mapping of the same DNA sequences in related species, construct the physicalmap of plant genome, directly reveals the DNA molecular organization of a specific chromosome regions, analyze the organization pattern of interphase chromatin and the dynamic changes of chromosomes in cell cycle, and characterize transgenes in plant genomes.

The ribosomal DNA (45S rDNA) behaviors during the cell cycle were analyzed on interphase nuclei, prophases, metaphases, pachytene chromosomes and extended DNA fibers in rice (Oryza,sativa ssp.indica cv.Guangluai No.4) by using high-resolution fluorescent in situ hybridization (FISH). The results show that 45S rDNA is located at the ends of short arms of chromosomes 9 and 10. But the signals are much more intense on chromosome 9 than on chromosome 10 in metaphase. Pachytene chromosome has rDNA signal arrays on chromosome 9. Different phases are described and discussed. These results indicate that the activity of rDNA at individual loci may also vary through the cell cycle in rice. On extended DNA fibers, 45S rDNA signals appear as strings of numerous red spots, but some signals are missed in some regions, probably result from weak signals or intergenic spacers.