北桑寄生内生真菌Penicillium sp. ZS108原生质体的制备与紫外诱变  

Preparation and UV mutagenesis of protoplasts from Loranthus tanakae endophytic fungus Penicillium sp.ZS108

作　　者：颜丽涵 赵博荣 杨虹[1] 邓丹丹 杨飞[1] 杨官娥[1] YAN Lihan;ZHAO Borong;YANG Hong;DENG Dandan;YANG Fei;YANG Guan’e(School of Pharmaceutical Science,Shanxi Medical University,Jinzhong 030600,Shanxi,China)

机构地区：[1]山西医科大学药学院,山西晋中030600

出　　处：《菌物学报》2024年第1期67-78,共12页Mycosystema

基　　金：中央引导地方科技发展项目(YDZX,20201400001443);山西省基础研究计划项目(202103021223241);山西省国际科技合作项目(201803D421065)。

摘　　要：鼠李素-3-O-鼠李糖苷(alpha-rhamnetin-3-O-rhamnoside, ARR)是从北桑寄生Loranthus tanakae中分离得到的黄酮类化合物,有良好的抗炎药理活性,具有较大的研究价值。从北桑寄生中分离出的内生菌能够产生与北桑寄生相同或相似的化学成分,其中有6种内生菌具备产鼠李素-3-O-鼠李糖苷的能力。由于微生物发酵具有高效、绿色可持续的优点,北桑寄生内生菌可以作为北桑寄生植物的替代资源。本研究以一株北桑寄生青霉属内生真菌ZS108为研究对象,利用酶解法制备其原生质体,探究了原生质体制备的适宜条件,采用聚乙二醇(PEG)转化法对不同酶解时间的原生质体进行初步转化,经DNA提取及PCR验证表明质粒成功整合到内生真菌ZS108基因组中。然后对原生质体进行紫外诱变,经过筛选最终得到14株鼠李素-3-O-鼠李糖苷产量比出发菌株高的突变菌株,正突变率为16.47%。其中突变菌株UV39产量最高,达到3.93 mg/L,比出发菌株高6.02倍。最后采用薄层色谱法和LC-MC法对突变菌株产鼠李素-3-O-鼠李糖苷的能力进行了验证。通过原生质体紫外诱变的方法选育高产鼠李素-3-O-鼠李糖苷菌株,为微生物发酵法生产鼠李素-3-O-鼠李糖苷早日实现工业化生产打下了坚实的基础。Alpha-rhamnetin-3-O-rhamnoside(ARR)is a flavonoid isolated from Loranthus tanakae,which has excellent anti-inflammatory pharmacological activity,and is highly valuable to research.Endophytic fungi isolated from Loranthus tanakae were able to produce the same or similar chemical components as Loranthus tanakae,and of which six species were able to produce ARR.Due to the advantages of high efficiency,green and sustainability to obtain ARR through microbial fermentation,endophytic fungi can be used as alternative resources of Loranthus tanakae.In this study,endophytic Penicillium sp.ZS108 was isolated from Loranthus tanakae,and the protoplasts of ZS108 were prepared by enzymatic hydrolysis.The protoplasts prepared at different enzymatic hydrolysis time were transformed by PEG/CaCl_(2) transformation method.DNA extraction and PCR verified that the plasmid was successfully integrated into the endophytic fungus ZS108 genome.Finally,the protoplasts were subjected to UV mutagenesis,and 14 mutant strains with increased ARR production were obtained after screening,and the positive mutation rate was 16.47%.Among the mutant strains UV39 produced the highest yield(3.39 mg/L)of ARR,being 6.02 times higher than the yield produced by original strain.The thin-layer chromatography and LC-MC methods were used to verify the ability of the mutant strain to produce ARR.This study provide reference for industrial production of alpha-rhamnein3-O-rhamnoside by microbial fementation.

关 键 词：内生真菌 制备条件 原生质体紫外诱变 鼠李素-3-O-鼠李糖苷 原生质体转化 

分 类 号：S567.3[农业科学—中草药栽培]