植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产g-氨基丁酸

γ-氨基丁酸可由谷氨酸脱羧酶(glutamate decarboxylase, GAD)催化谷氨酸一步合成,反应体系成分简单、环境友好。然而,绝大多数GAD酶催化pH偏酸性且反应范围狭小,需要加入无机盐维持最适催化环境,增加了生产附加成分。此外,随着产物γ-氨基丁酸的生成,溶液pH会逐渐上升,不利于GAD酶的持续转化。本研究首先从实验室保藏的一株高产γ-氨基丁酸的植物乳杆菌(Lactobacillus plantarum)中克隆得到谷氨酸脱羧酶LpGAD,基于酶蛋白表面电荷修饰,选择9个位点进行定点突变及组合突变,酶学性质表征结果显示三突变体LpGAD^{S24R/D88R/Y309K}在催化pH区间内酶活力整体提高,尤其拓宽了在偏中性pH 6.0下的酶活,为野生酶的1.68倍。接下来,通过分子动力学模拟解析了酶活提高的机理。此外,将Lpgad与Lpgad^{S24R/D88R/Y309K}突变基因分别在谷氨酸棒杆菌(Corynebacterium glutamicum) E01中过表达,通过优化确定了摇瓶最适转化条件为反应温度40 ℃,菌体量OD₆₀₀=20,底物L-谷氨酸100.0 g/L,5-磷酸吡哆醛添加量为100 μmol/L。5 L发酵罐中,不调节pH,通过分批投料底物L-谷氨酸,γ-氨基丁酸产量高达402.8 g/L,较对照菌株提高了1.63倍。本研究成功拓宽了LpGAD的pH催化范围及酶活,提高了γ氨基丁酸的转化效率,为实现其规模化工业生产奠定了基础。     

-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of -cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

利用荧光SSR标记鉴定茶树自然杂交后代遗传背景

为研究茶树自然杂交后代遗传背景,分析不同茶树自然杂交后代遗传差异,本研究利用24对EST-SSR标记对82个茶树自然杂交后代和34个福建主要栽培品种进行分子标记,分析了茶树自然杂交后代的亲缘关系、群体遗传多样性、亲本模拟分析。结果表明：(1)24对SSR标记共检测到157个多态性位点,平均等位位点数为6.542个,Nei’s多样性指数平均为0.588,Shannon’s 信息指数平均为1.182,平均观测杂合度和期望杂合度分别为0.577和 0.591。(2)遗传距离聚类将个供试样品划分为4类,群体1主要为‘丹桂’及其自然杂交后代;群体2主要为‘丹桂’、‘黄观音’自然杂交后代与福建省乌龙茶品种;群体3主要为‘白鸡冠’及其自然杂交后代;群体4主要为福建省绿茶品种。(3)‘丹桂’、‘白鸡冠’、‘黄观音’自然杂交后代群体与福建主要栽培品种的遗传距离分别为0.079、0.117、0.107。(4)群体1亚群b内‘丹桂’自然杂交后代模拟亲本准确率为77.8%,模拟父本主要为福建乌龙茶品种,与群体2(亚群a)的遗传相似度、遗传分化系数、基因流分别为0.899、0.043、5.480。(5)AMOVA分析结果显示,有88.52%的遗传变异来自群体内部的个体间,表明遗传变异主要发生在群体内。     

外显子与内含子序列分维的双碱基研究

引入碱基间的关联,研究了外显子和内含子序列以双碱基为单位的分维,我们发现在这种情况下,外显子和内显子序列在短程和中程存在自相似性并分别定义了这两个区域的分维。结果表明,短程的分维值Ｄｇ一般比中程的Ｄｍ大,外显子的两个分维值比内含子大。我们改变双联体的位相而分维却不变,这反映出在双联体基础上,外显子的不规则性大于内含子,短程的不规则性大于中程,外显子和内含子序列对以２为周期的结构没有位相的特异性。     

Effects of vitamin D3 on signaling by prostaglandin E2 in osteoblast-like cells

We investigated the effects of vitamin D₃ on the signaling pathways by prostaglandin E₂ (PGE₂) in osteoblast-like MC3T3-E1 cells. The pretreatment with 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), an active form of vitamin D₃, significantly inhibited cAMP accumulation induced by 10 μM PGE₂ in a dose-dependent manner in the range between 1 pM and 1 nM. This effect of 1,25-(OH)₂D₃ was dependent on the time of pretreatment up to 8 h. 1,25-(OH)₂D₃ also inhibited the cAMP accumulation induced by NaF, a GTP-binding protein activator, or forskolin which directly activates adenylate cyclase. On the other hand, 1,25-(OH)₂D₃ significantly inhibited PGE₂-induced IP₃ formation in a dose-dependent manner between 10 pM and 1 nM. However, 1,25-(OH)₂D₃ had little effect on NaF-induced IP₃ formation. The pretreatment with 24,25-dihydroxyvitamin D₃, an inactive form of vitamin D₃, affected neither cAMP accumulation nor IP₃ formation induced by PGE₂. These results strongly suggest that 1,25-(OH)₂D₃ modulates the signaling by PGE₂ in osteoblast-like cells as follows: the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase and the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between the PGE₂ receptor and GTP-binding protein, probably G_i2.     

Melatonin Promotes Rice Seed Germination under Drought Stress by Regulating Antioxidant Capacity

Drought stress is a serious threat to the germination of plant seeds and the growth of seedlings. Melatonin has been proven to play an important role in alleviating plant stress. However, its effect on seed germination under drought conditions is still poorly understood. Therefore, we studied the effects of melatonin on rice seed germination and physiological characteristics under drought stress. Rice seeds were treated with different concentrations of melatonin (i.e., 0, 20, 100, and 500 μM) and drought stress was simulated with 5% polyethylene glycol 6000 (PEG6000). The results showed that 100 μM melatonin can effectively improve the germination potential, rate and index; the vigor index of rice seeds; and the length of the shoot and root. In addition, that treatment also increased the activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and reduced the content of malondialdehyde (MDA). The grey relational grade between the shoot MDA content and the melatonin seed-soaking treatment was the highest, which could be useful for evaluating the effect of melatonin on drought tolerance. Two-way analysis of variance showed that the effect of single melatonin treatment on rice seeds was more significant than that of single drought stress and interaction treatment of drought and melatonin (p < 0.05). The subordinate function results showed that 100 μM melatonin significantly improved the germination and physiological indexes of rice seeds and effectively alleviated the adverse effects of drought stress on rice seedlings. The results helped to improve the understanding of the morphological and physiological involvement of melatonin in promoting seed germination and seedling development under drought stress.     

鸡源暹罗芽孢杆菌CML548的益生特性及全基因组分析

【背景】芽孢杆菌是用于动物微生态制剂的重要菌株之一。暹罗芽孢杆菌因具有较强的抑菌活性,近年来受到广泛关注。【目的】对实验室前期分离的鸡源暹罗芽孢杆菌CML548的益生特性进行评价,通过全基因组测序及生物信息学分析,探究其抑菌及潜在的益生机制。【方法】通过牛津杯法、稀释涂布平板法分别对菌株CML548的抑菌和产酶特性、酸和胆盐的耐受性进行研究。使用IlluminaHiSeq2500测序平台进行全基因组测序,并通过多种生物信息学工具和数据库对基因组进行注释和分析。【结果】体外实验表明该菌株具有优良的益生性状：能够有效抑制致病性大肠杆菌、鼠伤寒沙门氏菌、产气荚膜梭菌的生长;能够同时产生蛋白酶、淀粉酶和纤维素酶;具有较高的酸和胆盐耐受性。全基因组测序分析表明菌株CML548的基因组大小为4061741bp,GC含量为46.07%,预测到3 961个编码基因。分别有1 693、2 704、3 413、186、67、1、5个基因被GO、KEGG、COG、CAZy、DBAASP、CARD、VFDB数据库注释;通过antiSMASH预测到16个与次级代谢产物合成相关的基因簇。此外,还发现其含有抗菌活性...     

喙尾琵琶甲肠道来源链霉菌(Streptomyces sp.)BPA71次级代谢产物的分离鉴定及其生物活性评价

【背景】昆虫是世界上种类最多、肠道菌群资源最丰富且多样的动物类群之一。昆虫肠道微生物具有产生活性次级代谢产物的能力,是活性天然产物的重要来源。【目的】研究药用昆虫喙尾琵琶甲(Blaps rynchopetera)成虫肠道来源链霉菌(Streptomyces sp.) BPA71的次级代谢产物及其生物活性。【方法】利用正相硅胶柱色谱、葡聚糖凝胶Sephadex LH-20柱色谱等方法分离纯化该菌株的发酵粗提物,采用牛津杯法进行抗菌活性追踪,确定抗菌活性部位,通过ESI-MS、NMR等波谱数据分析对化合物结构进行鉴定,采用微量肉汤稀释法测定最低抑菌浓度(minimal inhibitory concentration, MIC),采用MTS法测定抗肿瘤活性。【结果】从Streptomyces sp. BPA71的固体发酵提取物中共分离得到4个已知化合物,通过对比核磁数据确定为糠酸甲酯(1)、吡咯甲酰胺A (2)、吡咯甲酰胺B (3)和吲哚-3-乙酸甲酯(4)。抗菌活性结果显示化合物2具有广谱抗菌活性。此外,化合物2对宫颈癌细胞HeLa、肺癌细胞A549、肝癌细胞SMMC-7721、乳腺癌细胞MDA-MB-231和结肠癌细胞SW480这5株肿瘤细胞均有明显的抑制活性。【结论】喙尾琵琶甲肠道来源Streptomyces sp. BPA71可产生丰富的生物活性物质,该研究结果为进一步挖掘喙尾琵琶甲肠道链霉菌的活性天然产物奠定了基础,同时丰富了人们对喙尾琵琶甲肠道微生物的认识。     

多酶恒温扩增核酸试纸条法快速检测肺炎支原体方法的建立及应用

【背景】肺炎支原体是导致儿童和青少年呼吸道感染的重要病原体,长期以来由于其临床表现不特异而容易错过最佳治疗时期。【目的】结合多酶恒温扩增(multienzyme isothermal rapid amplification,MIRA)技术和核酸试纸条建立一种快速检测肺炎支原体的方法。【方法】以肺炎支原体社区获得性肺炎呼吸窘迫综合征(community acquired respiratory distress syndrome, CARDS)毒素编码基因为靶基因设计引物和探针,对反应体系的温度、时间等进行优化,评估其敏感性,通过检测肺炎支原体和其余7种病原体分析其特异性,并对35份临床样本进行验证。【结果】MIRA核酸试纸条法在37℃条件下,15 min内便可完成对肺炎支原体的检测,最低检出限为10 copies/μL;除肺炎支原体外,其余7种病原体均不能扩增,特异性较好。以实时荧光PCR检测为标准,MIRA核酸试纸条法对35份临床样本检测后的诊断特异度为100.00%、灵敏度为96.15%、阴性预测值为90.00%、阳性预测值为100.00%。【结论】本研究建立了MIRA核酸试纸条法...