木薯根组织特异性启动子的克隆及鉴定  

Cloning and identification of root tissue-specific promoter from cassava(Manihot esculenta Crantz.)

作　　者：李远超 李可[3] 王连南 陈新[2] 李有志[1] 王文泉 LI Yuan-chao;LI Ke;WANG Lian-nan;CHEN Xin;LI You-zhi;WANGWen-quan(College of Life Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;Institute of Tropical Bioscience and Biotechnology,ChineseAcademy of Tropical Agricultural Sciences/Key Laboratory for Biology and Genetic Resources of Tropical Crops of Hainan Province,Hainan Institute of Tropical Agricultural Resources,Haikou,Hainan 571101,China;School of Life Science/College of Tropical Crops,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]广西大学生命科学与技术学院,广西南宁530004 [2]中国热带农业科学院热带生物技术研究所/海南热带农业资源研究院海南省热带农业生物资源保护与利用重点实验室,海南海口571101 [3]海南大学生命科学学院/热带作物学院,海南海口570228

出　　处：《南方农业学报》2023年第7期1925-1932,共8页Journal of Southern Agriculture

基　　金：国家重点研发计划项目(2018YFD1000500);亚热带农业生物资源保护与利用国家重点实验室(广西大学)开放课题项目(SKLCUSA-b202004);国家木薯产业技术体系品种改良岗位专家项目(CARS11-HNCX)。

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。【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 and 319 bp were obtained through PCR amplification.The promoters were fused with theβ-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

关 键 词：木薯 根 启动子 组织特异性 顺式作用元件 

分 类 号：S533.035.3[农业科学—作物学]