基于LC-MS代谢组学的植物乳杆菌发酵黄芪的代谢产物分析

试验旨在利用色谱-质谱联用（liquid chromatograph-mass spectrometer,LC-MS）代谢组学技术分析植物乳杆菌发酵黄芪的代谢产物,并探索其互作发酵机制。分别采取植物乳杆菌发酵黄芪（FT组）和未发酵黄芪固态粉末（CT组）,经样本前处理、LC-MS分析、生物学信息分析等探寻差异代谢物并分析代谢通路。结果显示,总离子流图峰图重现性良好,发酵黄芪主要代谢物共鉴定出183种代谢成分,正离子和负离子模式样品间关系PCA图均能良好区分。火山图分析表明,FT和CT组间的代谢物变化具有差异性,正离子模式上调的1 416个代谢物富集到83个代谢途径;下调的935个代谢物富集到83个代谢途径;负离子模式上调的1 040个代谢物富集到52个代谢途径,下调的809个代谢物富集到45个代谢途径。发酵黄芪差异代谢产物酸类、脂类、酮类等氨基酸显著增加,烯类等氨基酸显著下调,其中上调代谢物15个,下调代谢物2个,关键代谢产物主要为α-硫酸二乙酯、2-甲基柠檬酸、3-异丙烯基-6-氧代庚酸等,涉及到丙酮酸代谢、丙酸代谢、半乳糖代谢等途径。本研究结果为发酵黄芪的代谢产物、发酵互作机制和临床应用提供理论依据。     

黄芪多糖诱导SOCS3表达对鸡巨噬细胞炎症反应的抑制作用

试验旨在探究黄芪多糖（Astragalus polysaccharide,APS）对LPS诱导的鸡巨噬细胞（HD11）炎症模型的抗炎效果和作用机制。用不同浓度的LPS刺激鸡巨噬细胞（HD11）,通过CCK8法检测细胞活力,实时荧光定量PCR检测白细胞介素-1β（IL-1β） mRNA表达的变化,以确定构建细胞炎症模型的LPS最适添加浓度。将HD11分为对照组（C）、脂多糖（LPS）组（L）、黄芪多糖（APS）组（A）和黄芪多糖抑制脂多糖组（A+L）,在LPS刺激后的2、6、12、24、48 h用实时荧光定量PCR法检测IL-1β、肿瘤坏死因子-α（TNF-α）、核转录因子（NF-κBp65）、p38丝裂原活化蛋白激酶（p38MAPK）和细胞因子信号转导抑制因子3（SOCS3） mRNA表达的变化,Western blotting法检测NF-κBp65、p38MAPK和SOCS3蛋白含量变化,ELISA检测IL-1β和TNF-α蛋白含量变化。细胞活力和IL-1β检测结果表明,构建细胞炎症模型的LPS最适添加浓度为0.5 μg/mL。实时荧光定量PCR结果表明,与对照组相比,L和A组IL-1β、TNF-α、NF-κBp65、p38MAPK和SOCS3 mRNA表达均显著升高（P<0.05）;与L组相比,A+L组在LPS刺激后的IL-1β、TNF-α、NF-κBp65和p38MAPK mRNA表达含量均显著降低（P<0.05）,SOCS3 mRNA表达显著增加（P<0.05）。Western blotting结果表明,与对照组相比,A组P-NF-κBp65/NF-κBp65、P-p38MAPK/p38MAPK和SOCS3/α-tubulin比值均显著增加（P<0.05）;与L组相比,A+L组P-NF-κBp65/NF-κBp65和P-p38MAPK/p38MAPK比值均显著降低（P<0.05）;A+L组SOCS3/α-tubulin比值显著升高（P<0.05）。ELISA结果表明,与对照组相比,L组IL-1β和TNF-α蛋白含量在2~48 h均显著增加（P<0.05）;与L组相比,A+L组IL-1β和TNF-α的蛋白含量在LPS刺激后12~48 h均显著降低（P<0.05）。以上结果表明,在LPS诱导的HD11细胞炎症模型中,APS通过促进SOCS3的表达抑制NF-κBp65和p38MAPK信号通路的过度活化,从而发挥抗炎作用。     

Crude extract of Astragalus mongholicus root inhibits crop seed germination and soil nitrifying activity

Astragalus mongholicus has been of medicinal use within the traditional Chinese system for centuries. However, little information is available on its allelopathic effects on other crop plants and soil biochemical properties. Field experiment showed that the extracted residues of A. mongholicus root inhibited seed germination of wheat. Inhibition of seed germination was further confirmed in laboratory using the same crude extract. When the crude extract was applied to soil at various rates and incubated for 30 days, soil urease activity and denitrifying enzyme activity were significantly increased while soil nitrification rate was significantly decreased at 10% amendment rate as compared to the control. Soil respiration rate was significantly increased by the crude extract when measured at the start of incubation but returned to basal levels after 30 days of incubation. The crude extract supplemented to NB medium significantly decreased the colony numbers of Agrobacterium tumefaciens C58, Paraccocus denitrificans and soil bacteria. The stimulating effects of crude extract observed in the amended soil was attributed to the easily-available carbohydrates in the extract, which might served as external energy sources for heterotrophic microbial activities. It was concluded that A. mongholicus contained some compounds that inhibited seed germination, soil nitrification and bacterial growth in general. Possible links between allelochemicals responsible for the inhibitory effects observed in the present study and the medically bioactive compounds are discussed based on information reported in other fields. Further work is needed to specify and verify the allelochemicals produced by this herbal plant.     

紫云英与化肥配施对稻田土壤养分和微生物学特性的影响

在安徽沿江双季稻区,通过4年田间定位试验研究了不同用量紫云英绿肥与化肥配施对水稻产量、土壤肥力和土壤微生物数量及活度的影响。结果表明,施用紫云英绿肥比不施肥增产40.75%~92.10%,比单施化肥处理增产26.30%~36.49%;与纯施化肥相比较,施用紫云英处理的微生物总量增加了48.26%~115.78%,微生物活度增加了5.88%~29.41%,且施用紫云英土壤全N、碱解N、有效P、速效K和有机质含量均较高。简单相关分析表明,土壤微生物特性与土壤全N、碱解N之间具有极显著的相关性。施肥土壤有机质含量与细菌总数之间无直接相关性,而与真菌、放线菌数量及微生物活度正相关。土壤微生物及土壤养分分析发现,化肥量70%与紫云英22 500 kg·hm-2配施比较适宜于安徽沿江双季稻区早稻生产。     

Renodulation and characterization of Rhizobium isolates from cicer milkvetch (Astragalus cicer L.)

In 1993 and 1994, 12 bacterial isolates were isolated from root nodules of cicer milkvetch (Astragalus cicer). In the tests for nodulation of A. cicer by these bacterial isolates, five were found to form hypertrophic structures, while only two formed true nodules. These true nodules were formed in a sterilized soil system. This system might be able to act as a DNA donor to provide residual DNA to other microbes in the soil. The rhizobial isolates were thought to have lost genetic material crucial to nodulation during the isolation process. This hypothesis was supported by an experiment in which isolate B2 was able to nodulate A. cicer in vermiculite culture after being mixed with heat-killed rhizobia, Rhizobium leguminosarum bv. trifolii and R. loti. The nodulation would not occur in vermiculite culture system without the heat-killed rhizobia. Based on the biochemical data, the B2 and 9462L, which formed true nodules with A. cicer, were closely related. The rhizobia type cultures that nodulate A. cicer include Bradyrhizobium japonicum, Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viceae, and R. loti. All of these rhizobia were from different cross-inoculation groups. The B2 and 9462L isolates could only nodulate Medicago sativa, Phaseolus vulgaris, and Melilotus officinalis, but not these species within the genus from which they were isolated: Astragalus. The traditional cross-inoculation group concept obviously does not fit well in the classification of rhizobia associated with Astragalus. The rhizobia isolated from A. cicer can be quite different, and the rhizobia able to renodulate A. cicer also quite diverse. Received: 27 June 1996     

紫云英荚果分层成熟特性及其种子产量研究

紫云英是我国重要的冬季绿肥,具有无限花序的生长特性。对不同时期紫云英各枝位荚果开展调查,研究荚果的成熟规律及各枝位对种子产量的贡献。结果表明,紫云英荚果生物量随生育期延长逐渐增加,第1~2枝位荚果生物量显著高于第5及以上枝位。种子总产量表现为26 d≈33 d>19 d>12 d>5 d,最高产量为123.2 g·m^-2,其中80%来自第1~4枝位荚果。荚果成熟具有明显的分层特征,第1~4枝位荚果成熟指数随时间呈现“S”形变化,于盛花期后12~26 d快速成熟;第5及以上枝位荚果成熟指数呈指数型变化,在第19~33 d快速成熟。下层枝位最大荚果长和宽、荚果内粒数、种子千粒重均显著高于上层枝位。荚果成熟度由Ⅰ级至Ⅵ级,荚色由青绿色转变为黑色,花序最大荚果长度增长2倍,宽度增长4倍,种子千粒重增长6倍。第1~3枝位种子产量随成熟指数变化的速率是第4~6⁺枝位的2.58倍。综合荚果成熟和种子产量,1~4枝位荚果应作为紫云英种子收获的主要部位,顶层荚果成熟度达Ⅳ级后收割可获最高产量。     

黄芪总黄酮对小鼠佐剂性关节炎的治疗作用

[目的]研究黄芪总黄酮(TFA)对小鼠佐剂性关节炎的影响。[方法]采用弗氏完全佐剂建立小鼠佐剂性关节炎模型,设立空白组、模型组、阳性组及黄芪总黄酮高、中、低剂量组,测定小鼠足趾肿胀度和脾脏指数,通过Griess法测定血清中NO的含量,ELISA法测定血清中IL-1β的含量。[结果]黄芪总黄酮能减轻佐剂关节炎小鼠足趾肿胀度,下调脾脏指数,抑制血清NO和IL-1β的分泌。[结论]该研究表明黄芪总黄酮对佐剂性关节炎小鼠起到很好的治疗作用,为临床上的应用提供了可靠的试验依据。     

珍稀竹种巨龙竹的研究现状和展望

从生物学特性、繁育技术、遗传多样性及开发利用等方面综述了巨龙竹的研究现状,提出了今后巨龙竹的研究重点和发展方向。     

黄芪鲨烯合酶基因的克隆和序列分析

参照豆科其他植物的鲨烯合酶基因序列设计了引物,通过逆转录PCR方法,从荚膜黄芪（Astragalus mem-branaceus（fish.）Bge）中克隆了鲨烯合酶基因cDNA序列（GenBank登录号HQ829974）。通过BLAST发现黄芪鲨烯合酶推测的氨基酸序列与大豆鲨烯合酶（BAA22559.1）序列相似性最高,达到了88%。黄芪鲨烯合酶的克隆为以后通过基因转化提高黄芪皂苷含量亦或通过生物工程手段生产黄芪皂苷打下基础。     

紫云英SSR分子标记的开发及在品种鉴别中的应用

利用生物素标记的(AG)₁₅、(CT)₁₅、(AC)₁₅、(GT)₁₅探针及链霉素亲和磁珠,从紫云英的基因组中富集微卫星(SSR)序列。在用富集片段构建插入文库的950个转化子中,经PCR检测及测序共得到127个SSR序列,微卫星序列的富集效率达15.8%。除去重复或无效的序列,得到33个序列用于引物设计。对征集的9个紫云英品种进行多态性分析,有6对引物扩增出明显且稳定的多态性位点18个。在紫云英的品种水平上,这些SSR位点产生的多态率为50%~100%,有效等位基因数为1.19~1.64,Nei氏遗传多样性为0.13~0.38,Shannon多态信息指数为0.22~0.56;利用这6对引物可将参试品种完全区分开,证明这些SSR位点可用于紫云英品种的指纹鉴别。根据SSR位点的相似性系数,将 9个紫云英品种主要聚成两个类群, 与传统按生育期的紫云英品种划分结果无必然的联系。