腾冲嗜热厌氧杆菌Cas2原核表达及生物信息学分析

为研究CRISPR/Cas系统及其相关蛋白Cas2(TTE2657)在腾冲嗜热厌氧杆菌热适应中的作用,应用PCR技术构建了原核重组质粒pET-28a::cas2,并在大肠埃希菌BL21表达Cas2蛋白;结合生物信息学软件对cas2编码蛋白的基本理化性质、氨基酸同源性、空间结构及蛋白质相互作用网络进行预测和分析。结果显示,成功构建了原核表达载体pET-28a::cas2并在大肠埃希菌BL21中得到表达,Cas2分子质量大小为9.9 ku,主要以可溶性形式存在;qRT-PCR显示cas2 mRNA在60℃和75℃高表达;生物信息学分析显示cas2基因其完整的ORF全长264 bp,编码88个氨基酸,其中Ile(14)、Ser(14)、Phe (12)含量较高,等电点为9.31,不存在跨膜结构。其蛋白质二级空间结构以α-螺旋、无规则卷曲、β-折叠为主,蛋白互作预测网络显示Cas2与Cas3、Cas5、Cas7等其家族大部分蛋白存在相互作用。进化树分析显示腾冲嗜热厌氧杆菌cas2基因与厌氧菌芽胞杆菌B7M1同源性最高(39.5%)。腾冲嗜热厌氧杆菌cas2编码蛋白是一种亲水性蛋白,在原核系统能高效表达。本研究为嗜热蛋白质的热稳定性机制的研究提供参考。     

被动游泳运动对小鼠抑郁样行为及其肠道菌群组成的影响

被动游泳运动可诱发小鼠抑郁样行为,游泳环境的改变已成为抑郁样行为严重程度影响因素之一。观察不同水质、水温及持续时间对被动游泳小鼠抑郁样行为的影响,并初步探讨肠道菌群组成与抑郁样行为的关系。通过不同条件下的被动游泳运动建立抑郁样行为小鼠模型。采用糖水偏好实验及强迫游泳实验评价其行为学变化;采用16S rDNA高通量测序技术及实时荧光定量PCR技术对小鼠肠道菌群进行分子生态学分析。被动游泳16周后,各模型组小鼠体质量及糖水偏爱度均较正常对照组降低,而不动时间则有所延长。其中,室温海水游泳15 min小鼠体质量及糖水偏爱度降低程度最大,不动时间最长,与正常对照组比较,均具有显著性差异（P<0.05）。各模型组小鼠肠道菌群Chao指数、Shannon指数及PCoA分析均较正常对照组具有显著性差异（P<0.05）,其从门水平到属水平的丰度也发生不同程度改变。其中,室温海水游泳15 min小鼠肠道菌群组成变化程度最大,并发生拟杆菌属、普氏菌属等多个菌属的富集以及乳杆菌属丰度的减少,实时荧光定量PCR实验也得到了较为一致的结果（P<0.05）。以上结果表明,被动游泳运动可导致小鼠抑郁样行为的发生,其肠道菌群组成也发生明显改变;同时,菌群组成的改变会随着抑郁样行为的严重程度而有所变化。     

红色诺卡菌固体培养基配方的优化

应用响应面优化设计法优化固体培养基配方,增大红色诺卡菌的固体培养细胞生物量。首先用Plackett-Burman法从现有培养基组分中找到影响红色诺卡菌细胞生物量的关键因素,再通过最陡爬坡法确定细胞生物量最大的配方,用作中心组合设计(Central Composite Design, CCD)实验的基础起始值,拟合数学模型方程,最后找到最优组分的组合。优化的配方转移至企业实施放大实验,对结果进行验证和比较。试验结果表明,培养基各组分中影响红色诺卡菌细胞生物量的关键因素为蛋白胨、NaCl、牛肉膏;最优固体培养基配方:蛋白胨42 g/L、牛肉膏8 g/L、NaCl 1.2 g/L、甘油10 mL/L、Na_2HPO_4·12H_2O 0.3 g/L、琼脂20 g/L。在细胞生物量方面最优固体培养基配方比原配方高104%。响应面优化设计可用于提高红色诺卡菌细胞生物量固体培养基的优化,也为红色诺卡菌培养条件、液体发酵的优化研究提供参考。     

野野村氏菌FIM02-765的分类鉴定及Simaomicin α的抗肿瘤活性

明确海洋沉积物来源的放线菌FIM02-765的分类地位,揭示其代谢产物Simaomicinα的抗肿瘤活性。通过形态学、生理生化特征、细胞糖分组成分析以及16S rRNA序列分析,对海洋放线菌FIM02-765进行菌种鉴定;通过大孔吸附树脂柱层析和Sephadex LH-20凝胶柱层析从该菌的发酵产物中分离得到稠环氧杂蒽酮类化合物Simaomicinα;通过MTT法对化合物Simaomicinα的体外抗肿瘤细胞活性进行了研究。结果表明,菌株FIM02-765属于野野村氏菌(Nonomuraea sp.),同时该菌产生的稠环氧杂蒽酮类化合物Simaomicinα具有较强体外抑制多种肿瘤细胞增殖的活性。研究表明1株经鉴定的海洋野野村氏菌FIM02-765产生稠环氧杂蒽酮类化合物Simaomicinα对肿瘤细胞具有较强体外抑制活性,为深入开展该菌的遗传育种以及Simaomicinα的生物合成研究提供参考。     

bFGF Promotes the Proliferation of Rat Peripheral Blood Mesenchymal Stem Cells Through β-Catenin Signaling

bFGF (basic fibroblast growth factor, bFGF) could promote the proliferation of bone marrow and cord blood mesenchymal stem cells. However, the effect of bFGF on the proliferation of peripheral blood mesenchymal stem cells (PBMSCs) needs further research. This study aimed to investigate the role of bFGF on the culture and expansion of PBMSCs in vitro. Firstly, arterial blood was collected from rats abdominal aorta. After mononuclear cells (MNCs) were separated with Ficoll separation fluid, MNCs were cultured in the DMEM medium without bFGF (served as control group) or with bFGF (10, 20 ng/mL, served as 10 or 20 ng/mL bFGF group). PBMSCs were obtained by adherent culture method. The third passage of PBMSCs was detected for the MSC surface markers and the effect of bFGF on the cell cycle of PBMSCs using flow cytometry. The effects of bFGF on colony formation, cell growth, and the expressions of cyclin D1, cyclin E, p21 and β-catenin were evaluated. PBMSCs showed no difference in morphology among the three groups. PBMSC clonies appeared 14 days after cultivation. Compared with the control group, the cell growth confluence of PBMSCs was obviously increased by 40% and 80% in groups treated with 10 ng/mL bFGF or 20 ng/mL bFGF respectively after culture of 21 days (all P<0.05). Compared with the group treated with 10 ng/mL bFGF, the confluence of PBMSCs in 20 ng/mL group was further increased by 28% (P<0.05). Cells of the third passage were positively stained for CD29 and CD90, while were negative for CD45. These results were consistent with the phenotypic characteristics of MSCs. Compared with the control group, the colony number of PBMSCs in the 10 ng/mL and 20 ng/mL bFGF groups was increased by 51% (P<0.05) and 92% (P<0.05), respectively. Compared with the 10 ng/mL group, the colony number of PBMSCs was further increased in 20 ng/mL group by 14% (P<0.05). The growth curve of PBMSCs showed that after 7 days of culture, the number of PBMSCs in 10 ng/mL bFGF group and 20 ng/mL bFGF group was increased by 41% (P<0.05) and 61% (P<0.05), respectively. Moreover, the cell number had a statistically significant difference between these two groups (P<0.05). Results from flow cytometry cell cycle showed that the numbers of PBMSCs in the G1 phase of experimental groups were significantly decreased as the concentration of bFGF increased when compared with the control group (P<0.05), whereas the number of PBMSCs in the S phase was significantly increased (P<0.05). Immunofluorescence experiments showed that, compared with the control group, bFGF significantly promoted the nuclear translocation and expression of β-catenin in PBMSCs. Compared with the 10 ng/mL group, the PBMSCs in 20 ng/mL bFGF group showed stronger nuclear translocation and expression of β-catenin. Western blot experiments showed that the levels of β-catenin and its target proteins cyclinD1 and cyclinE were significantly increased (all P<0.05), whereas expression of p21 was significantly decreased in PBMSCs in the bFGF groups in a concentration dependent pattern when compared with control group (P<0.05). The study firstly confirms that bFGF promotes the proliferation of PBMSCs by regulating the β-catenin signaling pathway, which may facilitate the aquisition of larger number of PBMSCs for stem cell engineering in vitro.     

Activation of Nrf2 by SFN Improves Hypoxic Exercise Endurance and Lactate Metabolism in Red and White Skeletal Muscles of Mice

Exercise endurance is closely related to the production and elimination of lactate in skeletal muscles. It has been confirmed in literature that nuclear factor erythroid-2-related factor 2 (Nrf2) plays an important role in exercise-induced skeletal muscle antioxidant, mitochondrial biosynthesis and energy metabolism. However, it is still unclear whether Nrf2 activation has any effect on lactate metabolism and exercise endurance under hypoxic environment. In this study, sulforaphane (SFN), the Nrf2 activator, was pretreated to mice and then the mice were subjected to an exhaustive exercise under hypoxia (11.2% O₂). The results showed that the exhaustive exercise significantly increased the blood lactate level in PBS-exercise group (P<0.05). However, this indicator in the SFN-exercise group was only 76% of that in the PBS-exercise group (P<0.05). Also, the running distance increased from 577.0 ± 52.9 m to 636.3 ± 101.4 m and the duration increased by 1.1 times as well (P<0.05). Western blotting was used to determine the relative expression of proteins in skeletal muscles. Protein expression levels of Nrf2 and p-Nrf2 in the SFN-control and SFN-exercise group were significantly increased in soleus (P<0.05). In extensor digitorum longus, SFN pretreatment induced the expression of p-Nrf2 increased by 38% and 52%, respectively (P<0.05). In addition, SFN pretreatment promoted MCT1 protein expression in SFN-Control/ SFN-Exercise group and decreased MCT4 protein expression level in SFN-Exercise group (P<0.05). Meanwhile, the expression of LDH-B proteins in soleus was increased by 62% (P<0.05). In extensor digitorum longus, expression of LDH-B in the SFN-control and SFN-exercise group was increased by 1.35 times and 1.31 times, respectively (P<0.05). These results suggested that activation of Nrf2 by SFN could improve the lactate transport and metabolism capacity of skeletal muscle, which probably was one of the potential reasons for the increased exercise endurance of mice in the hypoxic environment. This study provided some theoretical reference for the practical application of SFN in athletes, mountaineers and highland workers.     

湘西“蒿菜粑粑”原料植物鼠麴草总黄酮提取物体外抗氧化活性研究

为了解湘西特色食品“蒿菜粑粑”原料植物鼠麴草(Gnaphalium affine)总黄酮提取物体外抗氧化能力,采用DPPH、ABTS自由基清除实验,还原力实验和抑制β-胡萝卜素褪色实验等方法,测定鼠麴草总黄酮抗氧化活性。结果显示,鼠麴草总黄酮母液中总黄酮浓度为7.01 mg·mL–1mg/mL;总黄酮提取物对DPPH、ABTS自由基有较好的清除能力,其半数抑制浓度(IC50)分别为16.30 mg·L–1、30.16 mg·L–1,将胡萝卜素相对吸光度降为50%的时间延长至67.49 min,在还原能力、延缓胡萝卜素褪色和抑制脂质过氧化上也有较好效果。鼠麴草总黄酮提取物具有良好的体外抗氧化活性,可作为优质食用植物资源进一步开发与推广。     

九州虫草-半枝莲发酵产物不同极性部位与其抗A549细胞增殖活性的相关性分析

建立九州虫草-半枝莲双向固体发酵产物不同极性部位的HPLC特征图谱,并考察与其抑制肺腺癌A549细胞增殖活性的相关性。采用双向固体发酵技术制备九州虫草-半枝莲发酵产物;溶剂萃取法获得发酵产物的不同极性部位,并运用HPLC法建立不同极性部位的特征图谱;MTT法考察不同极性部位对A549细胞的增殖抑制作用;偏最小二乘回归法分析不同极性部位HPLC色谱峰与其抗增殖作用的相关性。抗增殖试验结果表明,不同极性部位对A549细胞均具有增殖抑制作用并且呈浓度依赖性;标示的9个共有色谱峰与抗增殖活性均呈正相关,其中色谱峰8、4、1、2、9分别为木犀草素、黄芩苷、对香豆酸、野黄芩苷、黄芩素。本研究建立了不同极性部位的HPLC特征图谱,极性部位中以二氯甲烷部位对A549细胞的体外抗增殖作用最佳,并借助谱效相关初步探究了发酵产物抗A549细胞增殖的药效物质基础,为双向固体发酵技术应用于菌种与传统中药提供了参考。     

常压室温等离子体诱变选育花脸香蘑新菌株

花脸香蘑作为一种营养丰富、菇型美观的珍稀食用菌,具有良好的开发利用价值。利用常压室温等离子体（ARTP）技术对野生花脸香蘑菌株610双核菌丝片段进行了诱变处理。利用拮抗试验进行了诱变菌株初筛。通过连续传代培养,自然淘汰劣势菌株并获得诱变效应稳定的48个菌株。综合平板培养和基于ISSR和SSR联合遗传多样性分析的结果,筛选出7个诱变菌株与出发菌株共同进行出菇比较试验。出菇试验及子实体蛋白和氨基酸营养分析的结果显示：诱变菌株Ls2和Ls3为筛选出的花脸香蘑优质高产新菌株。诱变菌株较出发菌株生物学效率分别提高10.27%和14.75%,总氨基酸含量、必需氨基酸含量以及氨基酸评分等指标也均显著高于出发菌株,品质提升效果极显著。     

木耳‘农黑1号'的选育报告

黑木耳新品种‘农黑1号'是以黑木耳‘Au5'和‘木耳黑龙3号'作为亲本菌株,通过单孢杂交配对选育而来。工厂化条件下的生产性试验结果表明：‘农黑1号'的第一潮平均鲜耳产量达到322.4g/袋,干耳产量平均为29.0g/袋,出耳率平均为93.3%,生产周期平均为71d。综合来看,‘农黑1号'具有产量高、周期短、出耳率高、一致性好等优点,可作为黑木耳工厂化栽培的优良新品种。