玉米种子特异启动子GBSSⅠ的克隆及其功能分析  

Cloning and Functional Analysis of Seed-Specific Promoter GBSSⅠin Maize

作　　者：张国珍 范杰英[2] 韦正乙[2] 坚伟宁 左朋 郑大浩[1] 邢少辰[2] ZHANG Guozhen;FAN Jieying;WEI Zhengyi;JIAN Weining;ZUO Peng;ZHENG Dahao;XING Shaochen(College of Agriculture,Yanbian University,Yanji 133002,China;Institute of Agricultural Bio-technology,Jilin Academy of Agricultural Sciences,Changchun 130033,China;School of Life Sciences,Changchun Normal University,Changchun 130032,China)

机构地区：[1]延边大学农学院,延吉133002 [2]吉林省农业科学院农业生物技术研究所,长春130033 [3]长春师范大学生命科学学院,长春130032

出　　处：《吉林农业大学学报》2023年第5期531-538,共8页Journal of Jilin Agricultural University

基　　金：国家转基因植物新品种培育重大专项(2016ZX08003-001);吉林农业科技创新工程项目(CXGC2017TD004)。

摘　　要：玉米子粒被认为是生产外源蛋白的理想载体,而利用种子(胚乳)特异性启动子是提高外源蛋白表达水平最简单的途径。从玉米基因组中克隆获得长为999 bp的GBSSⅠ基因启动子pGBSSⅠ,该序列不但含有CAAT-box等启动子基本元件,而且还包含光响应、激素调控、胁迫诱导和发育等多个顺式调控元件。利用该启动子构建植物表达载体pCBG-Gus,并经农杆菌介导法转化玉米幼胚,通过筛选和分化获得抗性植株。经Bar蛋白基因金标免疫试纸条和PCR检测确认获得转基因阳性玉米植株。对转基因玉米不同组织中Gus和内源GBSSⅠ的转录情况进行实时定量PCR分析和Gus组织化学染色分析,结果表明:2个基因的表达水平并不一致。在转录水平上,除了叶片(包括叶鞘)之外,其他被检组织中的Gus的表达均低于GBSSⅠ;在蛋白表达水平上,除了茎和根之外,其他组织中均可检测到程度不同的Gus活性表达,其中成熟种子(包括胚和胚乳)中Gus蛋白积累的水平最高。综上,玉米子粒特异启动子pGBSSⅠ是一个具有潜在应用价值的启动子,将为提升玉米胚乳中外源蛋白的表达量提供理论依据。Maize kernels are considered to be an ideal platform for the production of foreign proteins,and the use of seed(endosperm)-specific promoters is the simplest way to increase the expression level of foreign proteins.In this study,pGBSSⅠ,a 999 bp promoter of maize GBSSⅠgene,was cloned.The sequence of GBSSⅠcontains not only the basic elements of CAAT-box promoter,but also multiple cis-regulatory elements including light response,hormone regulation,stress induction and development.A plant expression vector,pCBG-Gus,was constructed using this promoter to drive the transcription of Gus.Agrobacterium-mediated transformation of maize was carried out with immature embryos and resistant plants were generated.Transgenic plants were confirmed by Aurum labeled immuno-strip assay on Bar protein and PCR method.The results of Real-time quantative PCR and Gus histochemical staining analysis in different organs and tissues suggested that the expression of Gus and native GBSSⅠare incoordinate in transgenic plants.The transcription level of Gus was lower than that of GBSSⅠin all tissues except in leaves(including sheath).On the other hand,Gus activity was detectable in various levels in most tissues except for roots and stems.Moreover,the highest accumulation of Gus was found in seeds(including embryo and endosperm).These results of the research indicate that this seed-specific pGBSSⅠpromoter is a potential promoter for increasing exogenic protein expression in maize kernels.

关 键 词：玉米 GBSSⅠ启动子 转录活性 农杆菌介导法 GUS染色 

分 类 号：S513[农业科学—作物学]