小鼠早期胚胎发育期间TGF-β_1免疫组织化学定位

用兔抗人血小板TGF-β_1 N末端1—29氨基酸残基人工合成多肽抗血清作探针以及免疫荧光和免疫酶染色技术,分析了1—12天小鼠早期发育期间胚胎的TGF-β_1物质分布。结果表明,着床前胚胎包括卵裂细胞,桑椹胚和胚泡的ICM及滋养外胚层等细胞均显示TGF-β_1阳性免疫荧光染色。免疫酶染色还证明,沿囊胚腔顶部单层排列的原始内胚层细胞比邻近的ICM细胞有较深的染色反应。随着胚胎着床和进一步发育,7天龄胚胎中胚层早期形成阶段,紧靠中胚层一侧的外胚层胞质中含有浓集的棕色颗粒;各胚层的部分区域也存在着染色强度上差别。8—12天龄胚胎中,体节,心壁、间质细胞和肠道以及卵黄囊的脏壁中胚层均有显著的TGF-β_1免疫酶阳性物质。这些结果表明,着床前小鼠胚胎富含TGF-β_1物质,着床后的胚外组织,例如卵黄囊也为胚胎进一步发育提供了富含TGF-β_1物质的微环境;同时也提示,小鼠早期胚胎发育期间的胚泡形成,ICM细胞分化出原始内胚层,卵柱期中胚层形成,以及以后的神经管、体节和肢芽形成阶段等一系列形态发生和器官形成过程中,TGF-β_1可能是参与重要作用的一种生长调节因子。     

Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.     

Discovery of novel DNA viruses in small mammals from Kenya

Emergence and re-emergence of infectious diseases of wildlife origin have led pre-emptive pathogen surveillances in animals to be a public health priority. Rodents and shrews are among the most numerically abundant vertebrate taxa and are known as natural hosts of important zoonotic viruses. Many surveillance programs focused more on RNA viruses. In comparison, much less is known about DNA viruses harbored by these small mammals. To fill this knowledge gap, tissue specimens of 232 animals including 226 rodents, five shrews and one hedgehog were collected from 5 counties in Kenya and tested for the presence of DNA viruses belonging to 7 viral families by PCR. Diverse DNA sequences of adenoviruses, adeno-associated viruses, herpesviruses and polyomaviruses were detected. Phylogenetic analyses revealed that most of these viruses showed distinction from previously described viruses and formed new clusters. Furthermore, this is the first report of the discovery and full-length genome characterization of a polyomavirus in Lemniscomys species. This novel polyomavirus, named LsPyV KY187, has less than 60% amino acid sequence identity to the most related Glis glis polyomavirus 1 and Sciurus carolinensis polyomavirus 1 in both large and small T-antigen proteins and thus can be putatively allocated to a novel species within Betapolyomavirus. Our findings help us better understand the genetic diversity of DNA viruses in rodent and shrew populations in Kenya and provide new insights into the evolution of those DNA viruses in their small mammal reservoirs. It demonstrates the necessity of ongoing pathogen discovery studies targeting rodent-borne viruses in East Africa.     

Antrodia camphorata ATCC 200183 sporulates asexually in submerged culture

Antrodia camphorata is a well-known Chinese medicinal mushroom that protects against diverse health-related conditions. Submerged fermentation of A. camphorata is an alternative choice for the effective production of bioactive metabolites, but the effects of nutrition and environment on mycelial morphology are largely unknown. In this study, we show that A. camphorata American Type Culture Collection 200183 can form arthrospores in the end of liquid fermentation. Different morphologies of A. camphorata in submerged culture were analyzed using scanning electron microscopy. The optimal carbon and nitrogen sources for sporulation were soluble starch and yeast extract. We found that a carbon-to-nitrogen ratio (C/N) of 40:1, MgSO₄ (0.5 g/l), KH₂PO₄ (3.0 g/l), an initial pH?5.0, and an inoculum size of 1.5?×?10⁵ spores/ml led to maximum production of arthroconidia. Our results will be useful in the regulation and optimization of A. camphorata cultures for efficient production of arthroconidia in submerged culture, which can be used as inocula in subsequent fermentation processes.     

Discovery and experimental analysis of microsatellites in an oil woody plant Camellia chekiangoleosa

Oil camellia trees are important woody plants for the production of high-quality cooking oil. On the contrary to their economic importance, their genetic and genomic resources are very limited, which greatly hamper the genetic studies on oil camellia trees. Microsatellites or simple sequence repeats (SSRs) have great value in many aspects of genetic analyses due to their high polymorphism and codominant inheritance. In this study, we report the large-scale development and characterization of SSR markers derived from genomic sequences of Camellia chekiangoleosa by high-throughput pyrosequencing technology. A total of 1,091,393 genomic shotgun reads were generated using Roche 454 FLX sequencer, the average read length was 319 bp, and the total sequence throughput was 347.9 Mb. These sequences were assembled into 35,315 contigs with total length of 14.8 Mb and the N50 contig size of 770 bp. By analyzing with microsatellite (MISA), a total of 5,844 perfect microsatellites were detected from the assembled sequences. Among them, tetranucleotide repeats were found to be the most frequent microsatellites in the genome of C. chekiangoleosa, and all the dominant repeat motifs for different types of SSRs were detected to be rich in A/T. Experimental analysis with 900 SSR primer pairs revealed that 66 % of them succeeded in PCR amplification. Further investigation with 345 SSR primer pairs showed that a relatively high percentage of primers amplified polymorphic loci (31.9 %). Experimental data also revealed that, overall, long microsatellite repeats (>20 bp) were more variable than the short ones (<20 bp) in the genome of oil camellia tree.     

Enhancing electrical outputs of the fuel cells with Geobacter sulferreducens by overexpressing nanowire proteins

Protein nanowires are critical electroactive components for electron transfer of Geobacter sulfurreducens biofilm. To determine the applicability of the nanowire proteins in improving bioelectricity production, their genes including pilA, omcZ, omcS and omcT were overexpressed in G. sulfurreducens. The voltage outputs of the constructed strains were higher than that of the control strain with the empty vector (0.470–0.578 vs. 0.355 V) in microbial fuel cells (MFCs). As a result, the power density of the constructed strains (i.e. 1.39–1.58 W m⁻²) also increased by 2.62- to 2.97-fold as compared to that of the control strain. Overexpression of nanowire proteins also improved biofilm formation on electrodes with increased protein amount and thickness of biofilms. The normalized power outputs of the constructed strains were 0.18–0.20 W g⁻¹ that increased by 74% to 93% from that of the control strain. Bioelectrochemical analyses further revealed that the biofilms and MFCs with the constructed strains had stronger electroactivity and smaller internal resistance, respectively. Collectively, these results demonstrate for the first time that overexpression of nanowire proteins increases the biomass and electroactivity of anode-attached microbial biofilms. Moreover, this study provides a new way for enhancing the electrical outputs of MFCs.     

Identification of regulatory variants of carboxylesterase 1 (CES1): A proof-of-concept study for the application of the Allele-Specific Protein Expression (ASPE) assay in identifying cis-acting regulatory genetic polymorphisms

It is challenging to study regulatory genetic variants as gene expression is affected by both genetic polymorphisms and non-genetic regulators. The mRNA allele-specific expression (ASE) assay has been increasingly used for the study of cis-acting regulatory variants because cis-acting variants affect gene expression in an allele-specific manner. However, poor correlations between mRNA and protein expressions were observed for many genes, highlighting the importance of studying gene expression regulation at the protein level. In the present study, we conducted a proof-of-concept study to utilize a recently developed allele-specific protein expression (ASPE) assay to identify the cis-acting regulatory variants of CES1 using a large set of human liver samples. The CES1 gene encodes for carboxylesterase 1 (CES1), the most abundant hepatic hydrolase in humans. Two cis-acting regulatory variants were found to be significantly associated with CES1 ASPE, CES1 protein expression, and its catalytic activity on enalapril hydrolysis in human livers. Compared to conventional gene expression-based approaches, ASPE demonstrated an improved statistical power to detect regulatory variants with small effect sizes since allelic protein expression ratios are less prone to the influence of non-genetic regulators (e.g., diseases and inducers). This study suggests that the ASPE approach is a powerful tool for identifying cis-regulatory variants.     

Genomic organization and evolution of the soybean SB92 satellite sequence

Repetitive DNA sequences comprise a large percentage of plant genomes, and their characterization provides information about both species and genome evolution. We have isolated a recombinant clone containing a highly repeated DNA element (SB92) that is homologous to ca. 0.9% of the soybean genome or about 10⁵ copies. This repeated sequence is tandemly arranged and is found in four or five major genomic locations. FISH analysis of metaphase chromosomes suggests that two of these locations are centromeric. We have determined the sequence of two cloned repeats and performed genomic sequencing to obtain a consensus sequence. The consensus repeat size was 92 bp and exhibited an average of 10% nucleotide substitution relative to the two cloned repeats. This high level of sequence diversity suggests an ancient origin but is inconsistent with the limited phylogenetic distribution of SB92, which is found an high copy number only in the annual soybeans. It therefore seems likely that this sequence is undergoing very rapid evolution.     

荧光探针在大鼠胃粘膜表面疏水性研究中的应用

采用１－萘胺－８－磺酸（ＡＮＳ）为疏水探针,对大鼠胃粘膜表面疏水性作了研究,结果表明：以ＡＮＳ（２５μｍｏｌ／Ｌ）与胃粘膜表面刮取物（胃粘液凝胶层）混合后的萤光强度（正常为１．２３±０．１９ＲＦＵ／胃）可代表该粘液层的疏水性;以不同浓度ＡＮＳ与胃粘液混合后的萤光强度呈饱和趋势,可用Ｓｃａｔｃｈａｒｄ作图法求得粘液中ＡＮＳ的最大萤光强度（２．４６７±０．６３８ＲＦＵ／胃）和相对亲和系数（０．０３２±０．０１６）,它们可分别代表胃粘液中疏水基团的总量和单个基团的疏水性,从而可阐明胃粘膜被盐酸损伤后凝胶层粘液的ＡＮＳ萤光减弱,系其疏水基团总量减少,而非单个基团的疏水性改变所致。