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2025年4月6日 星期日
农业生物技术学报  2024, Vol. 32 Issue (5): 1198-1205    DOI: 10.3969/j.issn.1674-7968.2024.05.020
  研究资源与技术改进 本期目录 | 过刊浏览 | 高级检索 |
菌核青霉SYBR GreenⅠ实时荧光定量PCR检测方法的建立与应用
宋嘉仪1, 张海霞2, 齐是1, 张纪利3, 陈东豪1, 韦建玉3, 黎平1, 陈泽鹏4, 颜健1,*
1 华南农业大学 资源环境学院,广州 510642;
2 广东烟草韶关市有限公司,韶关 512026;
3 广西中烟工业有限责任公司,南宁530001;
4 中国烟草总公司广东省公司,广州 510642
Establishment and Application of SYBR GreenⅠReal-time Fluorescent Quantitative PCR Method for Penicillium sclerotiorum
SONG Jia-Yi1, ZHANG Hai-Xia2, QI Shi1, ZHANG Ji-Li3, CHEN Dong-Hao1, WEI Jian-Yu3, LI Ping1, CHEN Ze-Peng4, YAN Jian1,*
1 College of Resources and Environment, South China Agricultural University, Guangzhou 510642, China;
2 Guangdong Tobacco Shaoguan City Co., Ltd, Shaoguan 512026, China;
3 China Tobacco Guangxi Industrial Co., Ltd, Nanning 530001, China;
4 China Tobacco Corporation Guangdong Province Company, Guangzhou 510642, China
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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。
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宋嘉仪
张海霞
齐是
张纪利
陈东豪
韦建玉
黎平
陈泽鹏
颜健
关键词 菌核青霉SYBR Green Ⅰ绝对定量    
AbstractPenicillium 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.
Key wordsPenicillium sclerotiorum    SYBR Green Ⅰ    Absolute quantification
收稿日期: 2023-06-30     
中图分类号: S476.19
基金资助: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)
通讯作者: * yanjian78@scau.edu.cn   
引用本文:   
宋嘉仪, 张海霞, 齐是, 张纪利, 陈东豪, 韦建玉, 黎平, 陈泽鹏, 颜健. 菌核青霉SYBR GreenⅠ实时荧光定量PCR检测方法的建立与应用[J]. 农业生物技术学报, 2024, 32(5): 1198-1205.
SONG Jia-Yi, ZHANG Hai-Xia, QI Shi, ZHANG Ji-Li, CHEN Dong-Hao, WEI Jian-Yu, LI Ping, CHEN Ze-Peng, YAN Jian. Establishment and Application of SYBR GreenⅠReal-time Fluorescent Quantitative PCR Method for Penicillium sclerotiorum. 农业生物技术学报, 2024, 32(5): 1198-1205.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.05.020     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I5/1198
 
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