基于高通量测序分析盐角草根部内生细菌多样性及动态规律

【目的】探讨盐角草根部内生细菌群落多样性特征,揭示内生细菌群落结构在宿主关键发育期动态变化规律。【方法】通过罗氏454高通量测序获得内生细菌16S r RNA片段,然后进行生物信息分析。【结果】共获得20363条16S r RNA基因序列。各样品中可操作分类单元(operational taxonomic units,OTUs)在552–941之间。根部内生细菌群落主要包括4个门,其中Proteobacteri门占主导地位,其余依次是Firmicutes,Actinobacteria,Bacteroidetes。在Proteobacteria门中,Gammaproteobacteria是第一大纲,其后是Betaproteobacteria纲。宿主5个发育时期共同拥有7个细菌属,包括Azomonas,Serratia,Pantoea,Serpens,Pseudomonas,Halomonas,Kushneria。整体上看,Gammaproteobacteria纲在宿主5个发育时期呈现增长趋势。优势菌属在5个发育期存在差异,分别为Delftia,Kushneria,Serratia,Pantoea,Erwinia。所有文库总共含2108个特异OTUs,共同拥有5个相同OTUs。花期OTUs数量最多,结种期内生细菌多样性降低。在宿主的5个发育时期中,土壤p H、月均温和土壤盐含量这3个环境因子组成的集合对其内生细菌群落变化具有显著影响。【结论】盐角草内生细菌群落多样性丰富,宿主发育期决定了内生细菌群落结构。     

Diterpenoids from Wedelia prostrata and Their Derivatives and Cytotoxic Activities

One new ent‐kaurane diterpenoid, 11β,16α‐dihydroxy‐ent‐kauran‐19‐oic acid ( 1 ), together with eight known analogues 2 – 9 were isolated from the aerial parts of Wedelia prostrata. One of the acidic diterpenoids, kaurenoic acid ( 3 ), was converted to seven derivatives, 10 – 16 . All compounds were evaluated for their cytotoxic activity in vitro against human leukemia (K562), liver (HepG‐2), and stomach (SGC‐7901) cancer cell lines. Only four kaurenoic acid derivatives, 13 – 16 , with 15‐keto and substitutions at C(19) position, exhibited notable cytotoxic activities on these tumor cell lines with IC₅₀ value ranging from 0.05 to 3.71 μm . Compounds 10 – 12 , with oxime on C(15) showed moderate inhibitory effects and compounds 1 – 9 showed no cytotoxicities on them. Structure–activity relationships were also discussed based on the experimental data obtained. The known derivative, 15‐oxokaurenoic acid 4‐piperdin‐1‐ylbutyl ester ( 17 ), induced typical apoptotic cell death in colon SW480 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis.     

Distinct Promoters Affect Pyrroloquinoline Quinone Production in Recombinant Escherichia coli and Klebsiella pneumoniae

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiella pneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters—lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)—were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation.     

HIF-1α acts downstream of TNF-α to inhibit vasodilator-stimulated phosphoprotein expression and modulates the adhesion and proliferation of breast cancer cells

Metastasis is the leading cause of death in breast cancer patients. Recent evidence suggests that inflammation-related cytokine tumor necrosis factor-alpha (TNF-α) is implicated in tumor invasion and metastasis, but the mechanism of its involvement remains elusive. In this study, we employed MCF-7 breast cancer cells as an experimental model to demonstrate that TNF-α inhibits breast cancer cell adhesion and cell proliferation through hypoxia inducible factor-1alpha (HIF-1α) mediated suppression of vasodilator-stimulated phosphoprotein (VASP). We observed that TNF-α treatment attenuated the adhesion and proliferation of MCF-7 cells it also dramatically increased HIF-1α expression and decreased VASP expression. Through a variety of approaches, including promoter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified VASP as a direct target gene of HIF-1α. In addition, we confirmed that HIF-1α mediated the repression of VASP expression by TNF-α in MCF-7 cells. We also demonstrated that exogenous VASP expression or knockdown of HIF-1α relieved TNF-α induced inhibition of cell adhesion and proliferation. We identified a novel TNF-α/HIF-1α/VASP axis in which HIF-1α acts downstream of TNF-α to inhibit VASP expression and modulate the adhesion and proliferation of breast cancer cells. These data provide new insight into the potential anti-tumor effects of TNF-α.     

Century-Scale Responses of Ecosystem Carbon Storage and Flux to Multiple Environmental Changes in the Southern United States

Terrestrial ecosystems in the southern United States (SUS) have experienced a complex set of changes in climate, atmospheric CO₂ concentration, tropospheric ozone (O₃), nitrogen (N) deposition, and land-use and land-cover change (LULCC) during the past century. Although each of these factors has received attention for its alterations on ecosystem carbon (C) dynamics, their combined effects and relative contributions are still not well understood. By using the Dynamic Land Ecosystem Model (DLEM) in combination with spatially explicit, long-term historical data series on multiple environmental factors, we examined the century-scale responses of ecosystem C storage and flux to multiple environmental changes in the SUS. The results indicated that multiple environmental changes shifted SUS ecosystems from a C source of 1.20?±?0.56?Pg (1?Pg?=?10¹⁵?g) during the period 1895 to 1950, to a C sink of 2.00?±?0.94?Pg during the period 1951 to 2007. Over the entire period spanning 1895–2007, SUS ecosystems were a net C sink of 0.80?±?0.38?Pg. The C sink was primarily due to an increase in the vegetation C pool, whereas the soil C pool decreased during the study period. The spatiotemporal changes of C storage were caused by changes in multiple environmental factors. Among the five factors examined (climate, LULCC, N deposition, atmospheric CO₂, and tropospheric O₃), elevated atmospheric CO₂ concentration was the largest contributor to C sequestration, followed by N deposition. LULCC, climate, and tropospheric O₃ concentration contributed to C losses during the study period. The SUS ecosystem C sink was largely the result of interactive effects among multiple environmental factors, particularly atmospheric N input and atmospheric CO_2.     

Using pseudo-amino acid composition and support vector machine to predict protein structural class

As a result of genome and other sequencing projects, the gap between the number of known protein sequences and the number of known protein structural classes is widening rapidly. In order to narrow this gap, it is vitally important to develop a computational prediction method for fast and accurately determining the protein structural class. In this paper, a novel predictor is developed for predicting protein structural class. It is featured by employing a support vector machine learning system and using a different pseudo-amino acid composition (PseAA), which was introduced to, to some extent, take into account the sequence-order effects to represent protein samples. As a demonstration, the jackknife cross-validation test was performed on a working dataset that contains 204 non-homologous proteins. The predicted results are very encouraging, indicating that the current predictor featured with the PseAA may play an important complementary role to the elegant covariant discriminant predictor and other existing algorithms.     

家蚕色氨酸羟化酶(TRH)基因的克隆及表达特性分析（英文）

5-羟色胺(5-hydroxytryptamine, 5-HT)是生物界广泛分布的信号分子,涉及动物的重要行为。5-HT是色氨酸羟化酶(Tryptophan hydroxylase, TRH)将L-色氨酸羟化为5-羟-L-色氨酸,5-羟-L-色氨酸随即被多巴脱羧酶(Aromatic L-amino acid decarboxylase, DDC)脱羧而成。TRH作为5-HT合成的限速酶,在无脊椎动物神经调控中具有重要地位。鳞翅目昆虫中TRH的功能研究并不多。在家蚕中克隆了家蚕TRH (Bombyx mori TRH, BmTRH)的cDNA序列1667bp,其中包含1632bp的开放读码框(Openreadingframe,ORF)。人类TPH或者果蝇TRH(Drosophila TRH, DmTRH)与BmTRH有高度相似性,尤其BmTRH和DmTRH之间大多数氨基酸保守说明它们在系统发育上的密切关系并可能有相似功能。基因表达分析显示BmTRH主要表达于头部和中枢神经组织,免疫组织化学和Western blotting结果显示BmTRH只存在于神经组织中,即BmTRH可能仅参与家蚕的神经活动。此外,家蚕DDC(B.moridecarboxylase,BmDDC)和蛋白具有TRH活性的苯丙氨酸羟化酶基因(Phenylalanine hydroxylase, BmPAH)也在中枢神经系统中有表达,暗示家蚕神经系统5-HT的合成与果蝇中不同,可能有两种不同的调控机制。     

利用GeneHub软件分析人类染色体相邻基因的表达相关性

利用我们研制的基因信息融合分析软件GeneHub,按染色体开窗的方法将人类基因组沿着染色体划分为一系列基因群,用不同的相关测度(Pearson相关、Spearman秩相关、欧氏距离)考察这些基因群中所有基因间在二套独立的基因表达谱数据中的平均表达相关性。实验结果表明,与随机基因群相比,染色体上的邻近基因具有显著的表达相关性。约有40％的被检测基因包含于我们检测到的邻近相似表达基因群中。该现象提示真核生物中的基因存在模块化的共表达趋势。     

簇毛麦基因组特异性PCR标记的建立和应用

以普通小麦中国春、簇毛麦、中国春－簇毛麦二体附加系和代换系为材料进行RAPD分析,筛选出一个簇毛麦基因组特异性RAPD片段OPFO2757,该片段分布于簇毛麦所有染色体上。在对OPFO2757进行克隆、测序的基础上,设计一对PCR引物,建立了簇毛麦基因组特异性PCR标记。用这对PCR引物对不同普通小麦品种、不同硬粒小麦品种、不同居群的簇毛麦、中国春－簇毛麦二体附加系、中国春－簇毛麦二体代换系、普通小麦－簇毛麦双二倍体、硬粒小麦－簇毛麦双二倍体等材料进行扩增,凡具有簇毛麦染色体的材料都能扩增出一条长为677bp的DNA片段,而不具簇毛麦染色体的材料包括大麦、黑麦、长穗偃麦草、中间偃麦草等不能扩增出该片段。所以,该特异性PCR标记可用于快速跟踪检测小麦背景中的簇毛麦染色体。