摘要
菌核青霉(Penicillium sclerotiorum)是青霉属中一种具有很高生物活性的内生真菌,其产生的次级代谢产物具有一定的生物防治潜力,已在医学、农业等领域有广泛的应用.本研究以马齿苋(Portulaca oleracea)植株中分离纯化而来的菌核青霉为材料,针对其特异性保守序列设计引物,将目的片段通过分子克隆技术连接至pBM73-T载体中构建重组质粒为阳性标准品,选用SYBR GreenⅠ染料法建立菌核青霉实时荧光定量PCR检测方法.结果显示,PCR扩增后只有菌核青霉产生条带,青霉属的波兰青霉(P.chrysogenum)和产黄青霉(P.polonicum)均无条带产生,且未检测到其熔解曲线和扩增曲线,说明该方法具有很强的特异性.以重组质粒作为标准品构建的标准曲线,其循环阈值(Ct)与模板浓度在2.35×103～2.35×1010 copies/μL间有良好的线性关系,所得到的标准曲线为y=-3.211x+39.42,相关系数R2=0.999 3,组内和组间变异系数均低于5%.土壤样品检测结果表明,该方法可以灵敏地检测到环境样本中的菌核青霉及其数量,具有较好的应用潜力.本研究建立的方法具有良好的特异性、重复性及实用性,可用于探究菌核青霉在植株和土壤中的定殖情况,量化植物不同组织及土壤中菌核青霉数量.本研究为菌核青霉促生及抗性机制研究提供一定的数据和理论支持.
Abstract
Penicillium sclerotiorum is an endophytic fungus in the genus Penicillium with high biological activity that produces secondary metabolites with certain biological control potentials and has been widely used in medicine,agriculture,and other fields.This study utilized P.sclerotiorum obtained from Portulaca oleracea,and developed primers for its specific conserved sequence.The target segment was inserted into the pBM73-T vector by molecular cloning to create a recombinant plasmid as a positive control.A real-time PCR method for P.sclerotiorum was developed using the SYBR GreenⅠdye.PCR amplification revealed that only P.sclerotiorum exhibited bands,and there were no melting curves or amplification curves for P.chrysogenum and P.polonicum.The findings demonstrated a good linear relationship between the cycling threshold(Ct)and the template concentration between 2.35×103 and 2.35×1010 copies/μL in the standard curve created using recombinant plasmid as the standard.The obtained standard curve was y=-3.211x+39.42 with a correlation coefficient R2=0.999 3.The findings from the soil detection showed that the method's ability to accurately count endophytic P.sclerotiorum.The method established in this study had good specificity,reproducibility and practicability,which could be used to investigate the colonization of P.sclerotiorum in plants and soil and quantify the number in different tissues of plants.This study provides some data and theoretical support for the study of the promotion and resistance mechanism of P.sclerotiorum.
基金项目
广东省烟草专卖局(公司)科技项目(粤烟科项 202203)
广西中烟有限责任公司产学研科技项目(GXZYCX2020B006)
国家科技期刊平台
NSTL
万方数据