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。     

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel,Anguilla anguilla

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

Thecate stem medusozoans (Cnidaria) from the early Cambrian Chengjiang biota

Cnidarians are phylogenetically located near the base of the ‘tree of animals’, and their early evolution had a profound impact on the rise of bilaterians. However, the early diversity and phylogeny of this ‘lowly’ metazoan clade has hitherto been enigmatic. Fortunately, cnidarian fossils from the early Cambrian could provide key insights into their evolutionary history. Here, based on a scrutiny of the purported hyolith Burithes yunnanensis Hou et al. from the early Cambrian Chengjiang biota in South China, we reveal that this species shows characters distinct from those typical of hyoliths, not least a funnel-shaped gastrovascular system with a single opening, a whorl of tentacles surrounding the mouth, and the lack of an operculum. These characters suggest a great deviation from the original definition of the genus Burithes, and a closer affinity with cnidarians. We therefore reassign the material to a new genus: Palaeoconotuba. Bayesian inference of phylogeny based on new anatomical traits identifies a new clade, including Palaeoconotuba and Cambrorhytium, as a stem group of sessile medusozoan cnidarians that are united by the synapomorphies of developing an organic conical theca and a funnel-like gastrovascular system. This study unveils a stem lineage of medusozoans that evolved a lifelong conical theca in the early Cambrian.     

嵌合抗原受体T细胞治疗的现状与展望

肿瘤严重威胁着人类健康,当前肿瘤传统的治疗方法有手术治疗、化疗、放疗和靶向药物治疗等。近年来,肿瘤免疫治疗,尤其是嵌合抗原受体（chimeric antigen receptor,CAR） T细胞免疫疗法在基础研究与临床应用中蓬勃发展,并在治疗血液系统恶性肿瘤方面取得了巨大成功。然而,大量研究显示,细胞免疫治疗后可出现不同程度的毒副反应,且部分患者缓解后再次复发。因此,了解细胞治疗面临的挑战与局限性,寻找解决的办法,对继续发挥细胞免疫疗法的潜能具有重要意义。本文就免疫细胞的CAR结构、病毒载体的选择、细胞治疗面临的挑战及前景进行综述。     

胰腺癌早期检测的生物标志物（英文）

胰腺癌症是最难诊断和治疗的恶性肿瘤之一,其特点是发病隐匿、进展迅速、预后差。目前,手术治疗仍然是首选治疗方法。然而由于缺乏早期症状,大约70%的患者在确诊时已经出现局部扩散或远端转移,从而无法进行手术治疗。由此看来,早期检测是提高患者治疗效果和预后的有效途径。临床上使用的成像方法 （CT、MRI、EUS等）通常无法检测早期病变,并且很容易受到操作员的影响。常规临床标志物如CA19-9、CA125、CA242和CEA受到限制,其敏感性或特异性不令人满意。因此,寻找新的具有高敏感性和特异性的标志物是实现胰腺癌早期检测的关键。近年来,对生物标志物的广泛研究主要集中在遗传学、转录组学和蛋白质组学上。特别是由microRNA(miRNA)、long non-coding RNA(lncRNA)和circRNA(circRNA)组成的非蛋白质编码RNA(non-protein coding RNA,ncRNA)为胰腺癌的早期检测提出了许多新思路。然而,其中绝大多数仍处于实验室研究阶段。而一项成熟的生物标志物研究应该整合基因组学、转录组学、蛋白质组学或代谢组学的数据,并结合患者的个体特征（如体重指数...     

基于拉曼技术的单细胞生长检测方法

目的 单细胞生长检测可以更加科学地揭示微生物代谢变化的规律,为后期微生物工程应用提供指导。针对微生物生长应用于食品安全期和最佳食用期的精准检测问题,本文提出一种基于拉曼技术的单细胞生长检测方法。方法 首先,通过同步培养实验采集了枯草芽孢杆菌两个批次共900个单细胞拉曼光谱（SCRS）数据,其中600个用于训练和测试,另一批次300个用于模型验证。其次,基于主成分分析的特征关系矩阵,提出CP-SP特征评估方法以筛选SCRS特征用于模型检测。再基于XGBoost构建检测模型,并应用网格搜索和交叉验证对检测模型进行调优。最后,应用混淆矩阵、ROC曲线评估模型对细胞滞后期、对数期和稳定期的检测准确率、敏感性和特异性。结果 选用CP-SP筛选的第一、第二和第四主成分较特征贡献率前3个主成分的分类性能提高了3.1%,调优后的细胞生长检测模型测试准确率为96.0%,验证准确率为92.3%。结论 基于拉曼技术的单细胞生长检测方法能准确识别单细胞生长状态且具有较高的泛化能力,可为食品安全和保鲜制定精准调控机制提供科学指导。     

谷子不育系及其相应可育系小孢子发育的细胞学观察

以谷子2个品种的不育系及其保持系为材料,对其小孢子发育的细胞形态学进行了观察研究。结果表明:昭盟A花药的外部形态及花粉母细胞的发育过程与昭盟B相似,能形成外观正常的三核花粉,但其花药不能开裂散粉。石炮A表现出了败育时间和方式的多样性,且不育系与保持系间在花药长度上有显著差异。此外,两种不育系的维管束都表现出不同程度的发育不良。不良程度与药囊退化程度相关。     

糜子黄、黑种皮遗传及转录组学分析

同源异型域-亮氨酸拉链(homedomain-leucine zipper,HD-Zip)转录因子广泛参与植物的生长发育和抗胁迫过程。该研究通过生物信息学方法对青稞HD-Zip基因家族进行全基因组分析鉴定,并采用qRT-PCR技术分析非生物胁迫下该基因的表达特性,为深入探讨青稞HD-Zip转录因子的生物学功能及其在高原作物抗逆育种中的应用奠定基础。结果表明：(1)成功从青稞基因组中共鉴定出41个HD-Zip基因家族成员,依次命名为i>HvvHD-ZipⅠ-1~Ⅳ-13,且这些基因在7条染色体上呈不均匀分布。(2)理化性质分析发现,HvvHD-Zip蛋白包含197～885个不等的氨基酸残基;分子量范围在19 914.36～94 014.87 Da;亚细胞定位表明HvvHD-Zip蛋白都位于细胞核。(3)根据多序列比对、系统进化、基因结构和保守基序差异将其聚为4个亚家族,各亚家族分类特征与系统聚类结果一致。(4)顺式作用元件预测分析发现,i>HvvHD-Zip基因启动子中含有11种植物激素和胁迫响应元件。(5)qRT-PCR结果显示,HvvHD-Zip Ⅰ、Ⅱ、Ⅳ亚家族基因对各胁迫响应明显;与根组织相比,多数i>HvvHD-Zip基因在叶组织中响应明显(上调或下调);与冷和盐胁迫相比,i>HvvHD-Zip各基因对旱胁迫响应较强。     

褪黑素调节烟草丁香假单胞菌抗性的功能研究

为分析褪黑素(N-乙酰-5-甲氧基色胺)在植物先天免疫中的功能及调控机理,研究以病原菌丁香假单胞杆菌(Pseudomonas syringae pv.tomato DC3000,Pst DC3000)—烟草互作系统为模型,检测了病原菌侵染对烟草褪黑素相关基因表达的影响,并探讨了褪黑素对植物叶片病原菌生长以及气孔开度和活性氧自由基(reactive oxygen species,ROS)含量的影响以及调控机理。结果表明:(1)Pst DC3000处理提高了烟草褪黑素合成(NtSNAT1)和受体(NtPMTR1)基因表达,且外源褪黑素处理降低了叶片中的病原菌含量。(2)与野生型植物相比,过表达大豆GmSNAT1基因显著提高了转基因烟草中内源褪黑素含量和NtPMTR1的表达,且转基因烟草叶片中的Pst DC3000菌落数显著下降。(3)外源褪黑素和细菌鞭毛蛋白多肽flg22处理诱导了野生型和转基因烟草保卫细胞中ROS产生和气孔关闭,且转基因植物对褪黑素和flg22诱导的气孔关闭和ROS产生比野生型烟草更加敏感。综上所述,研究表明褪黑素可能通过受体NtPMTR1介导的信号途径促进保卫细胞ROS产生,诱导气孔关闭,从而降低病原菌Pst DC3000的入侵。