【摘要】 【目的】克隆鉴定木薯根组织特异性启动子，为深入解析和鉴定木薯块根发育及淀粉合成调控机制中关键基因的功能提供理论参考。【方法】根据木薯不同组织部位转录组数据，并结合实时荧光定量PCR检测结果，鉴定出木薯块根和初生根中特异高表达基因，使用PLACE在线网站分析该基因上游1464 bp启动子序列，依据其根特异顺式作用元件ROOTMOTIFTAPOX1(RMT1)的分布情况，对该序列进行截短分析，并设计其特异性引物，PCR扩增获得长度为1464、705和319 bp的启动子序列，与β-葡萄糖苷酶（GUS）基因融合并通过农杆菌介导转入拟南芥，对转基因植株进行GUS染色和GUS活力测定，从而鉴定启动子的组织特异性及启动活性。【结果】根据木薯不同组织的转录组数据，筛选出3个在初生根、块根和根尖分生组织中特异高表达的候选基因MeHPS(Phytozome13登录号Manes.01G078200)、MeSR2(Phytozome13登录号Manes.04G017600)和MeSR3(Phytozome13登录号Manes.14G006300)。结合实时荧光定量PCR检测结果，鉴定出木薯块根和初生根中特异高表达基因MeHPS。通过对MeHPS基因启动子分析发现，1464 bp启动子序列含有5个顺式作用元件RMT1。转基因植株的GUS染色结果显示，p1464启动子具有明显的根特异性，p705启动子具有输导组织和根部特异性，而p319启动子基本丧失组织特异性。根系的GUS活力测定结果显示，p1464、p705和p319驱动的GUS活力分别为8.33、7.87和10.52 U/g，均显著高于35S启动子驱动的GUS活力6.69 U/g(P<0.05)。【结论】MeHPS基因在根中特异高表达，其p1464启动子具有根特异表达活性，可用于在根中特异驱动目的基因高水平转录，p705具有较强的输导组织特异性，可用于驱动具有转运功能蛋白类基因的转录，而p319可视为组成型启动子，能部分替代35S启动子的应用。更多还原

【Abstract】 【Objective】Cloning and identifying cassava root tissue-specific promoters to provide reference for further analysis and identification of the functions of key genes in the development of cassava storage root and the regulation mechanism of starch synthesis.【Method】The specific high expression genes were identified in cassava storage root and primary root on the basis of transcriptomic data of different tissues in cassava and the results of real-time fluorescence quantitative PCR detection. PLA website was used to analyze the 1464 bp promoter sequence in the upstream region of the genes. According to the distribution of the root-specific cis acting element ROOTMOTIFTAPOX1(RMT1),the sequence was truncated for analysis,and its specific primers were designed,promoter sequences with the length of 1464,705 and319 bp were obtained through PCR amplification. The promoters were fused with the β-glucosidase(GUS)gene and transferred into Arabidopsis thaliana by the mediation of Agrobacterium tumefaciens. GUS staining and GUS activity determination were performed in transgenic plants to identify tissue specificity and promoter activity of the promoters.【Result】Based on transcriptomic data from different tissues in cassava,three candidate genes which specifically highly expressed in primary root,storage root and root apical meristem were selected:MeHPS(Phytozome13 ID:Manes.01G078200),MeSR2(Phytozome13 ID:Manes.04G017600)and MeSR3(Phytozome13 ID:Manes.14G006300). The specific highe xpression gene MeHPS in cassava storage root and primary root was identified by the results of real-time fluorescence quantitative PCR detection. The analysis of MeHPS gene promoter suggested that the 1464 bp promoter sequence contained five cisacting elements RMT1. The GUS staining results of transgenic plants showed that p1464 promoter had obvious root specificity,p705 promoter had conducting tissue and root specificity while p319 promoter basically lost tissue specificity. Determination results of GUS activity in roots demonstrated that the GUS activities driven by p1464,p705 and p319 were 8.33,7.87 and 10.52 U/g respectively,which were all significantly higher than that driven by35S promoter(6.69 U/g)(P<0.05).【Conclusion】MeHPS gene is specifically and highly expressed in roots,and its p1464promoter has root-specific expression activity,which can be used to specifically drive high-level transcription of target genes in roots. p705 promoter has strong conducting tissue specificity and can be used to drive the transcription of protein genes with transporting function. And p319 promoter,regarded as constitutive promoter,can partially replace 35S promoter.更多还原