响应黄龙病侵染的柑橘乙醇脱氢酶基因CsADH1的克隆与表达分析

对前期在感染黄龙病的‘晚锦橙’中发现的明显差异表达的柑橘乙醇脱氢酶(CsADH1)基因进行克隆测序分析,结果表明,CsADH1的CDS序列长为1143bp,编码380个氨基酸。蛋白质结构预测显示,CsADH1含有乙醇脱氢酶Gro ES(ADH_N)和ADH_zinc_N保守结构域。进化树分析显示,CsADH1蛋白与拟南芥、蓖麻和中国莲的ADH蛋白亲缘关系最近。亚细胞定位结果显示,CsADH1蛋白定位在细胞质中。以感病‘晚锦橙’的根和叶脉为材料,q PCR分析显示CsADH1在感病根和叶脉中均上调表达,且根中的表达水平是叶脉中的10倍。离子胁迫试验和外源植物生长调节剂诱导试验表明,Zn、水杨酸(SA)、生长素(IAA)和脱落酸(ABA)均显著诱导CsADH1上调表达,并且根中的表达水平明显高于叶中。本结果表明,CsADH1可能在柑橘根响应黄龙病菌侵染中起着重要作用。     

抗弓形虫和新孢子虫药物体外筛选方法的研究进展

弓形虫和新孢子虫是2种在形态结构及生物学特征方面非常相似的细胞内寄生性原虫,二者引起的疾病对人和动物健康造成了严重危害。开发和筛选高效、低毒的抗弓形虫和新孢子虫药物一直是抗原虫药物研究领域的重要方向之一。药物筛选包括体外筛选和体内筛选,体外筛选的结果是进行体内筛选的依据和基础。对抗弓形虫和新孢子虫药物的体外筛选方法研究进展进行综述,以期为开发更加高效的抗弓形虫和新孢子虫药物体外筛选技术提供参考。     

Plant residues do not have an immediate impact on soil bacterial community composition and abundance

Plant residues are often used as soil amendments in laboratory experiments, but they can reportedly release compounds interfering with soil DNA extraction and subsequent molecular biological analyses. Theoretically, for accurate comparison of microbial community composition in soils with and without added plant residues after a period of incubation, no significant difference at the beginning of the experiment is required between the amended and unamended control soils. We mixed plant residue into soil and immediately (within 10 min) commenced DNA extraction, and then performed 16S rRNA gene sequencing and quantitative PCR (qPCR) to determine bacterial community composition and abundance. Soil without plant residue addition served as a control. Five commonly used DNA extraction kits, 16S rRNA gene primer pairs, and soils, and two types (rice straw and alfalfa shoots) and three addition rates (2%, 4%, and 6%; w/w) of plant residue, were tested. In all cases, we found no significant difference in measured bacterial community composition or abundance between the treatments with and without added plant residue.     

Soil microbiome biomass,activity, composition and CO2 emissions in a long-term organic and conventional farming systems

The implementation of environmentally friendly agricultural policies has increased the need to compare agricultural aspects of conventional (CON) and organic farming (ORG) systems. The objective of the present work was to compare the effects of an organic and conventional long-term experiment on bacterial and fungal biomass and activity, as well as soil CO₂ emission and readily available nitrogen forms in a soil cultivated with Helianthus annuus L. The microbial biomass was more active and abundant in ORG as well as soil CO₂ emission. Despite being less abundant, fungi were more active than bacteria in both ORG and CON experiments. 16S rRNA gene sequencing showed that the ORG treatment had a significantly greater bacterial richness than CON. Cyanobacteria, Actinobacteria and Proteobacteria were the most abundant phyla contributing more than others to the differences between the two systems. Moreover, the soil ${\mathit{NH}}_4^{+}$ and ${\mathit{NO}}_2^{-}$ content was not significantly different between ORG and CON, while ${\mathit{NO}}_3^{-}$ was less in ORG. ORG sunflower yield was significantly less compared with CON. While much remains to be discovered about the effects of these agricultural practices on soil chemical properties and microbial diversity, our findings may contribute to this type of investigation.     

胡椒PnCAD基因的克隆和表达分析

木质素在植物体中具有运输水分、支撑植株和加强植物体免受侵害等功能，是苯丙烷代谢途径的重要产物之一。其中肉桂醇脱氢酶（cinnamyl alcohol dehydrogenase，CAD）是该途径中重要的限速酶。本研究在胡椒转录组测序的基础上，用RACE法进行克隆，对PnCAD基因全长进行生物信息学分析，并对其蛋白进行理化性质、亚细胞定位和系统进化树等分析；最后运用实时荧光定量PCR进行分析。结果表明：克隆得到CAD基因的全长cDNA，长度为1364 bp，开放阅读框（open reading frame，ORF）为1071 bp，编码356个氨基酸，预测相对分子量为3.879 kDa，等电点为6.27，属于亲水性蛋白；含有3个N-糖基化特征序列和9个磷酸化位点，可能处于细胞质内。结构域分析发现，胡椒CAD蛋白含有NAD(P)结合位点、多个催化锌和结构锌结合位点。系统进化树分析表明，胡椒CAD与细辛CAD6亲缘关系最近，胡椒和细辛的同源性最高为76%，均隶属于比较原始的双子叶植物。通过荧光定量分析发现，黄花胡椒在辣椒疫霉菌侵染下表达量升高，在8 h达到最高值，约为对照的10倍；之后在24 h时出现小幅度升高，约为对照组的6倍，之后下降。‘热引1号’胡椒的表达量在侵染8 h时达到最低，之后缓慢上升。总体来说，所有时间下黄花胡椒基因表达量均高于‘热引1号’胡椒，且差异显著。本研究结果可为今后研究胡椒抗非生物胁迫功能提供参考，为PnCAD基因的功能研究提供理论依据。     

大口黑鲈蛙病毒分子流行病学及组织病理分析

为探讨大口黑鲈蛙病毒（LMBV）的分子流行规律及在感染后的组织病理变化，实验对2019—2021年采集的723份患病大口黑鲈样品进行荧光定量PCR （qPCR）检测。结果显示，LMBV的阳性率为63.62%，挑选阳性样品接种鳜脑细胞（Chinese perch brain cells,CPB），共获得93株LMBV。通过对分离株MCP、ATP酶、DNA聚合酶和甲基转移酶基因进行PCR扩增与测序分析，发现上述基因在LMBV分离株中均保守，其中MCP、ATP酶基因一致性均为100.0%、甲基转移酶基因一致性为99.7%～100.0%、DNA聚合酶基因一致性为99.8%～100.0%。选取1株LMBV毒株感染健康大口黑鲈，采用qPCR方法对不同组织中的病毒载量进行检测，结果显示，大口黑鲈蛙病毒在心脏、肝脏、脾脏、肾脏、胃、肠和脑等组织中均有分布，人工感染LMBV后的前5天病毒载量逐步升高；感染后第5天，心脏中病毒载量最高（9.5×10⁵个/mg拷贝），脑组织中病毒载量最低（2.9×10²个/mg拷贝）。组织病理结果表明，LMBV可导致大口黑鲈多种组...     

军曹鱼NHE3和NKAα1a基因克隆、表达模式及相关miRNA的靶向调控

为了解军曹鱼NHE3与NKAα1a的序列特征、盐度胁迫下的基因表达模式以及相关miRNA的靶向调控作用，实验应用cDNA末端快速扩增技术克隆了NHE3与NKAα1a的全长cDNA序列；应用实时定量PCR (qPCR)技术分析了NHE3和NKAα1a的组织特异性和盐度适应性表达模式；采用双荧光素酶报告实验检测了NHE3和NKAα1a与相关miRNA的靶向调控关系。结果显示，军曹鱼NHE3与NKAα1a基因的开放阅读框长度分别为2 718和3 075 bp，分别编码905和1 024个氨基酸；NHE3、NKAα1a在鳃、肠、心脏等9种组织中均有表达，其中以鳃组织中的表达丰度最高。随着盐度升高，NHE3在鳃中表达量下降，低盐和高盐适应后均呈显著差异。NKAα1a基因表达量随着盐度升高出现不同趋势，在鳃和肠中受低盐和高盐影响后均显著上调，而高盐环境下其在体肾中的表达水平显著下调。在不同盐度适应过程中，NHE3、NKAα1a均以鳃组织中的表达水平最高。NHE3-pmirGLO-WT与miR-1335-3p共转染时，较对照组相对荧光素酶活性下降，并存在极显著差异；NKAα1a-pmirGLO-WT...     

甘蔗MYB转录因子基因ScMYB52-1的克隆及表达特性分析

MYB转录因子家族成员广泛参与植物的各种生物学过程,在调控植物适应非生物逆境过程中起关键作用。为分离甘蔗（Saccharum spp.）逆境响应MYB基因并研究其功能,本文应用3°RACE的方法,从甘蔗品种ROC22中克隆到一个R2R3-MYB转录因子基因,命名为ScMYB52-1。生物信息学分析表明,该基因cDNA序列长度为1072 bp,开放阅读框（ORF）长度为696 bp,5°非翻译区长54 bp,3°非翻译区长322 bp。该ORF编码231个氨基酸,推导蛋白的分子量为26.65 kDa,含有2个串联的MYB结构域。ScMYB52-1推导蛋白与模式植物拟南芥中的MYB家族蛋白的进化树聚类分析结果表明,ScMYB52-1蛋白与AtMYB52和AtMYB54的亲缘关系较近;进一步的多序列比对分析结果表明,上述三者蛋白质N端的MYB结构域序列高度一致,且三者的预测蛋白质三级结构类似,表明它们可能具有类似的功能。实时荧光定量PCR结果显示,ScMYB52-1在甘蔗组培苗的根中对PEG处理总体上不敏感,在叶中仅在3 h时短暂上调;ScMYB52-1在叶中受外源ABA和NaCl胁迫的诱导,在处理0.5 h均迅速上调表达,表达量分别为对照的3.35倍和2.75倍,并在处理期维持高于对照的水平;在根中受外源ABA和NaCl胁迫的抑制,在处理0.5 h时就开始下调表达,在处理24 h时表达量分别下调为对照的12%和40%,并在处理周期内总体上维持低于对照的水平。亚细胞定位分析表明,ScMYB52-1-GFP重组蛋白定位在烟草叶肉细胞的细胞核中。酵母双杂交自激活活性鉴定结果表明,ScMYB52-1蛋白无自激活活性。以上结果表明ScMYB52-1参与了甘蔗对高盐胁迫的应答,并可能受ABA信号通路的调控,在叶和根中存在差异的调控机制。本研究结果为进一步理解该基因在甘蔗非生物逆境适应过程中的功能及分子调控机制提供了初步的信息。