榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因的克隆与表达

牻牛儿基牻牛儿基焦磷酸合酶是产紫杉醇内生真菌紫杉醇合成下游途径中的关键步骤之一,在榛子产紫杉醇内生真菌中进行紫杉醇生物合成研究时首先要确认GGPP合酶的存在。该研究通过RT-PCR方法克隆得到了榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因Pa GGPPS(Gen Bank登录号为KM881430)的c DNA开放阅读框(Open reading frame,ORF)。利用生物信息学方法,我们分析了该基因的序列,并对其编码的氨基酸序列进行了预测。结果发现该基因ORF长度为1 113 bp,预计蛋白分子量为40.98k D,等电点为6.168,表明该蛋白呈酸性。对其进行亲疏水性分析发现肽链整体呈现为亲水性。Pa GGPPS的主要二级结构元件为α-螺旋,并且包含一个异戊二烯类化合物合酶功能域。对榛子产紫杉醇内生真菌与其他物种的GGPPS进行氨基酸同源性分析,发现其与娄地青霉、曲霉菌和费氏新萨托菌的一致性较高,分别为94%、76%和76%。进化树分析表明来自动物、真菌和酵母的GGPPS聚为一类,来自植物的GGPPS聚为一类,其中榛子产紫杉醇内生真菌的GGPPS和青霉菌的进化关系最近,与植物的GGPPS的进化关系最远。对其进行基础生物信息学分析后,我们构建了原核表达载体,成功诱导其表达并得到可溶性蛋白。该研究结果为下一步深入研究GGPPS基因在内生真菌Penicillium aurantiogriseum紫杉醇合成途径中的作用及和构建高产紫杉醇基因工程菌株奠定了一定的基础。

南海红树林内生真菌Penicillium aurantiogriseum 299次级代谢产物研究

目的对南海红树林内生真菌黄灰青霉菌Penicillium aurantiogriseum 299的次级代谢产物进行研究。方法采用色谱技术对Penicillium aurantiogriseum 299的次级代谢产物进行分离纯化,根据化合物的理化性质、波谱分析数据以及与文献谱图对照的方法确定化合物的结构。结果从南海红树林内生真菌Penicillium aurantiogriseum 299的次级代谢产物中分离得到了4个化合物,分别鉴定为aurantiogrisidinol a/b(1a/1b)、4-(2′-羟基-丁炔氧基)苯甲酸(2)、(S)-penipratynolene(3)、aurantiamine(4)。结论化合物1a/1b为新化合物,化合物2是首次从青霉菌属真菌中分离得到。

Preparation, Characterization and in Vitro Evaluation of the Antitumor Activity of the Biologically Synthesized Silver Nanoparticles

This study was concentrated on the biosynthesis of silver nanoparticles from Penicillium aurantiogresium (IMI 89372) with a focus on its cytotoxicity in MCF-7 and MCT cancer cell lines as well as Vero (normal) cell line that was assessed by crystal violet assay after treatment with various concentrations (0.44 – 145 μg/ml) for 24 h. The cell morphology was examined by inverted light microscopy. Further, the radiosensitizing effect of silver nanoparticles (AgNPs) on MCF-7 was also demonstrated by assessing cell morphology, cell proliferation of MTT assay, LDH activity and induction of apoptosis through checking of some apoptotic genes that altered during carcinogenesis, including caspase-3, Bax and Bcl-2. Caspase-3 activity was also estimated. Synthesis of AgNPs was determined by UV-Visible spectrum and it was further characterized by TEM, FT-IR and X-Ray analysis (EDX, XRD). The biosynthesized AgNPs were spherical and of 12.7 nm in size as recorded by direct electron microscopy visualization. The biosynthesized AgNPs showed variation in cytotoxicity against MCF-7, MCT and Vero cell lines in a concentration dependant response with a varied degree of alteration in cell morphology. The result showed that AgNPs were highly toxic towards MCF-7 with IC50 value of 10.5 μg/ml. Treatment of MCF-7 (10.5 μg/ml) prior to irradiation improved the effect of irradiation dose (6 Gy) via increasing alteration of cell morphology, inhibition of cell proliferation, activation of the lactate dehydrogenase (LDH) and caspase-3 leading to induction of apoptosis which was further confirmed through increasing nuclear DNA damage and up regulation of caspase 3 and Bax genes and downrgulation of Bcl-2 genes. In conclusion, the present findings clearly indicated that AgNPs showed dose dependant cytotoxicity and verified that AgNPs acted as a potent radiosensitizer and could enhance gamma irradiation induced killing of MCF-7 breast cancer cells.

传统发酵豆酱中产脂肪酶菌株的筛选

脂肪酶属于羧基酯水解酶类,能将甘油三酯逐步地水解成甘油和脂肪酸。该研究从东北三省传统的家庭自制豆酱上获得纯培养真菌389株,通过三丁酸甘油酯平板法初筛获得具有脂肪酶活性的菌株41株,其中青霉属的真菌27株、曲霉5株、毛霉3株、犁头霉2株、酵母菌1株、镰孢菌2株、帚霉菌1株。结果表明,青霉属的真菌无论从数量还是酶活都高于其他属的真菌,其中橘灰青霉(Penicillium aurantiogriseum)数量最多。在分离到的菌株中,毛霉属的卷枝毛霉(Mucor circinelloides)酶活最高,是具有生物催化潜力的功能菌株。