基因重组与外源铁离子对大肠杆菌合成血红素的影响

【背景】大肠杆菌通过C5途径合成卟啉及血红素,5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)是C5途径中关键的前体物质,血红素由原卟啉IX (protoporphyrin IX, PPIX)螯合一个铁离子所形成,目前5-ALA与PPIX的外泌对卟啉的积累和血红素合成的影响尚不清楚。【目的】构建5-ALA外泌蛋白基因rhtA和卟啉外泌蛋白基因tolC双缺失的大肠杆菌以积累卟啉,同时外源添加铁离子,并过表达亚铁螯合酶基因hemH及参与铁摄取的基因efeB,促进卟啉向血红素的转化。【方法】通过Red同源重组敲除大肠杆菌BL21(DE3)的rhtA和tolC,并外源添加不同浓度的FeSO₄及Fe₂(SO₄)₃,同时构建重组质粒pEHE过表达hemH和efeB,检测卟啉和血红素含量,分析卟啉向血红素的转化。【结果】敲除rhtA和tolC对菌体生长无显著影响,与野生菌WT相比,敲除菌株WT-RT的卟啉含量增加,血红素合成略有提升。外源添加100μmol/L Fe²⁺     

春小麦对骤旱的响应特征及其阈值分析

与缓慢发展的干旱过程不同,骤旱具有发生速度快,短期内可致害的特点。目前,关于作物骤旱致害的临界阈值及其调控机制尚不清楚。以春小麦为供试作物,通过桶栽试验,模拟研究骤旱过程中小麦受旱致害的过程特征及其控制因素。结果发现,发生骤旱时土壤含水量下降呈先快后慢的变化趋势,叶片水分和叶水势则呈先慢后快的指数变化趋势。叶片光合生理指标对土壤水分的下降存在明显的阈值响应,且不同生理指标的阈值并不完全相同,其中净光合速率与表征叶片光合能力的指标（最大羧化速率）对土壤有效含水量的响应阈值为0.4,气孔导度和蒸腾速率对土壤有效含水量的响应阈值分别为0.5和0.4。而小麦光合生理指标对叶片水分和叶水势的阈值响应并不明显。同时依据各生理指标相关和通径分析结果得出,骤旱发生时引起小麦叶片净光合速率快速降低的主导因子为非气孔因素,而并不是以往作物受旱研究中的气孔因素。本研究结果有望丰富干旱影响认知,并可为科学应对干旱提供依据。     

珍稀濒危植物蒜头果种子萌发特征与幼苗类型的研究

以去除果皮后阴干的珍稀濒危植物蒜头果种子为材料,在温室大棚沙池内进行层积处理,从层积处理开始至子叶出土的不同萌发阶段,考察蒜头果种子发育形态、贮藏物质积累、酶活性以及幼苗类型等变化特征,初步探讨其种子休眠形成原因。结果显示：(1)蒜头果种子从解除休眠开始至萌发形成幼苗的过程约需195 d,其中幼胚的形态发育后熟约需75 d,随后30 d内是种子集中萌发的时期,其发芽率达到最高(53.33%);依据种胚发育形态的标志特征将此过程划分为7个阶段(S1～S7阶段):S1阶段种子未萌发,S2阶段种胚“露白”,S3阶段胚根突破种皮长超过1 cm, S4阶段下胚轴与胚根连接处形成弯钩结构,S5阶段“S”型胚形成及胚根前端膨大,S6阶段种子不仅具有膨大的胚根且已有侧根的分化,S7阶段子叶脱落,胚芽出土,真叶出现。(2)蒜头果种子在湿沙层积过程中,种胚胚长和胚率从S1阶段的(5.49±1.57)mm和(19.48±5.72)%分别增加至S6阶段的(67.92±2.94)mm和(240.75±15.29)%,胚率平均增加了12.4倍,显示蒜头果种子的胚需要经历后熟过程才能萌发,属于胚后熟型种子。(3)从...     

生物质多糖的高效降解与降解酶（系）的精确定制

自然界中多糖类生物质资源十分丰富,然而其复杂的抗降解屏障限制了生物转化的进程.近年来,随着生物质多糖结构的快速解析以及大量多糖降解酶的鉴定研究,针对不同底物结构或产物需求,仿制高效微生物多糖代谢途径,精确定制多糖降解酶系,促进生物质高效转化已成为可能.本文分析中性多糖(纤维素和木聚糖)、碱性多糖(几丁质和壳聚糖)以及酸性多糖(褐藻胶)的精细结构组成与基团性质,总结3类多糖主要降解酶的活性架构特征及其底物精确结合模式.文章还阐述蛋白质工程设计与定制策略,针对酶分子不同功能区的分析,可为酶分子的功能快速设计与改造提供靶点,以获得适宜于工业应用的高效酶分子,此外,根据微生物胞外降解酶系的降解次序与协同关系,可基于应用需求精确定制复杂多糖降解酶系,实现生物质的高效与高值降解转化.     

维生素C生物合成相关脱氢酶研究进展

维生素C是一种人体必需的维生素,在食品制药等领域拥有巨大的市场。工业上维生素C主要以微生物发酵生产的2-酮基-L-古龙酸为前体,然后通过内酯化反应获得。微生物发酵中,山梨糖途径和葡萄糖酸途径因为转化率高一直是研究的热点。文中从维生素C生物合成相关脱氢酶的角度阐述了:山梨糖途径和葡萄糖酸途径中关键脱氢酶在定位、底物谱、辅因子和电子传递上的特点;山梨糖途径和葡萄糖酸途径中面临的主要问题和改造策略等。最后讨论了维生素C生物合成中山梨糖途径和葡萄糖酸途径可能的研究方向。     

河南济源人工引水渠隧道蝙蝠冬眠生态学特征

2017-2020年期间,每年1月份对河南省济源市邵原镇布袋沟水库人工引水渠隧道内蝙蝠进行冬眠生态学特征调查,共发现2科5属7种蝙蝠在此冬眠,包括马铁菊头蝠（Rhinolophus ferrumequinum）、小菊头蝠（R.pusillus）、华南水鼠耳蝠（Myotis laniger）、白腹管鼻蝠（Murina leucogaster）、金管鼻蝠（Mu.aurata）、奥氏长耳蝠（Plecotus ognevi）和亚洲宽耳蝠（Barbastella leucomelas）。马铁菊头蝠是优势种（约52%-73%的冬眠个体）,其次是小菊头蝠（约19%-37%）、华南水鼠耳蝠（约5%-8%）,其余蝙蝠物种数量不足3%。2017-2020年冬眠蝙蝠个体总数呈增长趋势,但仍少于早期报道的数量。有42个隧道每年均有蝙蝠冬眠,而且不同年度冬眠数量也不尽相同。通过多元线性回归分析发现,隧道长度可能是影响蝙蝠冬眠栖息场所选择的主要影响因子（Adjusted R²=0.208,P=0.001）。每个隧道内,蝙蝠具有不同的冬眠栖点位置,约4/5的蝙蝠选择温暖且环境相对稳定的隧道深处（> 30 m）作为冬眠栖点,超过95%的个体选择长度> 60 m的隧道冬眠。蝙蝠具有不同的冬眠方式,绝大多数个体采用独栖方式进行冬眠（> 90%）,少数采用聚集方式。不同的冬眠栖点和冬眠方式可能有利于冬眠成本优化。此外,栖点温度与蝙蝠体温之间呈显著正相关（R²=0.98,P < 0.001）,而且蝙蝠冬眠期间的栖点温度具有种内和种间差异。研究结果为我国蝙蝠种群保护和冬眠场所管理提供科学依据。     

枸杞SWEET基因家族的鉴定与糖含量关系分析

【目的】糖外排转运蛋白（sugars will eventually be exported transporters, SWEETs）在植物生长发育过程中发挥重要作用,为解析SWEETs基因在枸杞（Lycium barbarum L.）果实发育过程中对糖积累作用,进一步为揭示SWEET基因在枸杞果实发育过程中作用提供参考。【方法】研究基于枸杞基因组数据,采用生物信息学方法对枸杞SWEET基因（LbaSWEET）进行全基因组鉴定,并利用已发表的转录数据分析了LbaSWEETs在果实发育时期的基因表达情况。【结果】结果表明,枸杞SWEET基因家族共有37个成员,随机分布于10条染色体上,分别编码152~621个氨基酸,蛋白质分子量为16.87~69.97 kD,等电点为4.96~9.86。亚细胞定位预测位于叶绿体或质膜,大多数含有7个跨膜螺旋。系统进化分析发现,37个LbaSWEET蛋白可分为4个亚群,每个亚群的基因结构和保守基序组成相似。启动子元件分析发现,LbaSWEET基因启动子富含大量激素响应、逆境胁迫和生长发育响应元件。转录组数据和qRT-PCR分析表明,LbaSWEET9和LbaSWEET29基因表达量随着果实成熟呈现显著增加。相关性分析结果表明,LbaSWEET9和LbaSWEET29基因表达量与果糖含量呈显著正相关。【结论】研究推测LbaSWEET9和LbaSWEET29基因是果糖积累的关键基因。     

AMPK regulates homeostasis of invasion and viability in trophoblasts by redirecting glucose metabolism: Implications for pre‐eclampsia

Pre‐eclampsia (PE) is deemed an ischemia‐induced metabolic disorder of the placenta due to defective invasion of trophoblasts during placentation; thus, the driving role of metabolism in PE pathogenesis is largely ignored. Since trophoblasts undergo substantial glycolysis, this study aimed to investigate its function and regulatory mechanism by AMPK in PE development. Metabolomics analysis of PE placentas was performed by gas chromatography–mass spectrometry (GC–MS). Trophoblast‐specific AMPKα1‐deficient mouse placentas were generated to assess morphology. A mouse PE model was established by Reduced Uterine Perfusion Pressure, and placental AMPK was modulated by nanoparticle‐delivered A769662. Trophoblast glucose uptake was measured by 2‐NBDG and 2‐deoxy‐d‐[³H] glucose uptake assays. Cellular metabolism was investigated by the Seahorse assay and GC–MS.PE complicated trophoblasts are associated with AMPK hyperactivation due not to energy deficiency. Thereafter, AMPK activation during placentation exacerbated PE manifestations but alleviated cell death in the placenta. AMPK activation in trophoblasts contributed to GLUT3 translocation and subsequent glucose metabolism, which were redirected into gluconeogenesis, resulting in deposition of glycogen and accumulation of phosphoenolpyruvate; the latter enhanced viability but compromised trophoblast invasion. However, ablation of AMPK in the mouse placenta resulted in decreased glycogen deposition and structural malformation. These data reveal a novel homeostasis between invasiveness and viability in trophoblasts, which is mechanistically relevant for switching between the ‘go’ and ‘grow’ cellular programs.

Pre‐eclampsia (PE) is associated with trophoblast AMPK hyperactivation, presumably due to LKB1 phosphorylation, and glucose uptake is consequently increased via trafficking of GLUT3 from the cytosol to the plasma membrane. Such translocation enhances glycolytic flux and redirects glucose metabolic intermediates into gluconeogenesis, resulting in PEP accumulation, which not only benefits cell survival but also suppresses invasion by repressing MMPs, and thus in turn modulates switching between the ‘go’ and ‘grow’ cellular programs.     

IL‐33‐ST2 pathway regulates AECII transdifferentiation by targeting alveolar macrophage in a bronchopulmonary dysplasia mouse model

Evidence points to the indispensable function of alveolar macrophages (AMs) in normal lung development and tissue homeostasis. However, the importance of AMs in bronchopulmonary dysplasia (BPD) has not been elucidated. Here, we identified a significant role of abnormal AM proliferation and polarization in alveolar dysplasia during BPD, which is closely related to the activation of the IL‐33‐ST2 pathway. Compared with the control BPD group, AMs depletion partially abolished the epithelialmesenchymal transition process of AECII and alleviated pulmonary differentiation arrest. In addition, IL‐33 or ST2 knockdown has protective effects against lung injury after hyperoxia, which is associated with reduced AM polarization and proliferation. The protective effect disappeared following reconstitution of AMs in injured IL‐33 knockdown mice, and the differentiation of lung epithelium was blocked again. In conclusion, the IL‐33‐ST2 pathway regulates AECII transdifferentiation by targeting AMs proliferation and polarization in BPD, which shows a novel strategy for manipulating the IL‐33–ST2‐AMs axis for the diagnosis and intervention of BPD.