不同启动子调控外源基因在斑玉蕈中的表达分析  

Expression analyses of exogenous genes controlled by different promoters in Hypsizygus marmoreus

作　　者：杜莎莎 鲍大鹏[3] 马丽娟[1,3] 李晓玲 尚俊军 DU Sha-Sha;BAO Da-Peng;MA Li-Juan;LI Xiao-Ling;SHANG Jun-Jun(School of Chemistry and Life Science,Changchun University of Technology,Changchun,Jilin 130012,China;School of Chemical Engineering and Resource Recycling,Wuzhou University,Wuzhou,Guangxi 543002,China;National Engineering Research Center of Edible Fungi,Key Laboratory of Edible Fungal Resources and Utilization(South),Ministry of Agriculture,Institute of Edible Fungi,Shanghai Academy of Agriculture Science,Shanghai 201403,China)

机构地区：[1]长春工业大学化学与生命科学学院,吉林长春130012 [2]梧州学院化学工程与资源再利用学院,广西梧州543002 [3]国家食用菌工程技术研究中心,农业部南方食用菌资源利用重点实验室,上海市农业科学院食用菌研究所,上海201403

出　　处：《菌物学报》2021年第8期2065-2073,共9页Mycosystema

基　　金：上海市现代农业产业技术体系项目[沪农科产字(2021)第9号]。

摘　　要：启动子是控制基因转录的重要顺式元件,也是遗传转化实验中驱动外源基因表达的重要工具。在同一真菌中,不同启动子驱动外源基因表达水平可能存在明显差异。因此,选择合适的启动子是提高外源基因表达水平的关键。本研究分别应用花椰菜病毒35S RNA (cauliflower mosaic virus 35S RNA,CoMV35S)和斑玉蕈甘油醛-3-磷酸脱氢酶(Hypsizygusmarmoreus glyceraldehyde-3-phosphate dehydrogenase,HmGPD)基因的启动子构建了两个遗传转化质粒,在斑玉蕈中分别驱动外源的植物花青素合成基因表达,并利用来自刺芹侧耳的萎锈灵抗性基因进行转基因筛选。两个质粒通过农杆菌介导转化斑玉蕈单核菌株后,对具有萎锈灵抗性的转化子经PCR方法进行转基因验证,并运用实时荧光定量PCR对阳性转化子中外源基因的表达水平进行比较分析。结果表明,CoMV35S和HmGPD基因的启动子均成功驱动了植物花青素合成基因在斑玉蕈中转录,为增强基因表达而引入的内含子在转录过程中均被正确切割。其中,HmGPD启动子驱动外源基因表达水平比CaMV35S启动子驱动外源基因表达水平强22-36倍。Promoters are important cis-acting element that control gene transcription and are also important tools to drive the expression of exogenous gene in genetic transformation experiments.In the same fungus,there may be significant differences in the expression level of exogenous genes driven by different promoters.Therefore,choosing an appropriate promoter is the key to increasing the expression level of exogenous genes.In this study,two genetic transformation plasmids were constructed from the promoter of cauliflower mosaic virus 35S RNA(CaMV35S)and Hypsizygus marmoreus glyceraldehyde-3-phosphate dehydrogenase(HmGPD)gene,respectively,to drive the expression of exogenous plant anthocyanin biosynthesis genes in Hypsizygus marmoreus.The transformants were selected by carboxin resistance gene from Pleurotus eryngii.The two plasmids were transformed into monokaryotic strain of Hypsizygus marmoreus by Agrobacterium-mediated method.The carboxin-resistant transformants were verified by PCR.Quantitative real-time PCR were used to compare the expression level of the exogenous genes.Our results showed that the promoters of both CaMV35S and HmGPD genes could successfully drive the transcription of anthocyanin biosynthesis genes in Hypsizygus marmoreus,and the introns introduced to enhance gene expression were cut correctly during the transcription process.The expression level of exogenous gene driven by HmGPD promoter was 22–36 times stronger than that driven by CaMV35S promoter.

关 键 词：启动子 遗传转化 实时荧光定量PCR 

分 类 号：S646[农业科学—蔬菜学]