拟南芥根特异性启动子的鉴定

Jin Sun Kim, Sun-Hyung Lim, Sang-Ho Kang, Young-Mi Kim, and Jong-Yeol Lee
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引用次数: 1

摘要

组织特异性启动子的鉴定和应用是植物基因工程的主要挑战之一,目的是优化相关基因在合适组织中的高效表达。本研究利用拟南芥的开源数据库信息来确定根特异性表达启动子区域。从拟南芥基因组DNA中分离出7个可能作为根特异性启动子元件的序列。将7个启动子克隆到pBGWFS7中,其中β -葡萄糖醛酸酶(gus)和绿色荧光蛋白(gfp)基因在pBGWFS7中连接。采用组织化学GUS染色法和4 methylumbellliferyl β -D-glucuronide (MUG)荧光法测定转基因拟南芥不同组织中GUS的活性。为了确认根特异性表达,在转基因拟南芥植株上进行了gfp共聚焦显微镜分析。结果表明,与CaMV35S启动子相比,这5个启动子在根组织中表现出较强的GUS活性。为了验证作为根特异性启动子的作物应用有效性,将7个启动子引入番茄植株,并利用根结农杆菌ARqua1根结诱导菌株进行瞬时表达。两个启动子表明gus基因在转基因番茄植株的根中特异性表达。结果表明,这7个启动子在根系中具有特异活性,可用于作物改良。
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Identification of Root-specific Promoters Derived from Arabidopsis thaliana
: To identify and apply tissue-specific promoters is one of the major challenges in plants genetic engineering for optimizing efficient expression of interest genes in appropriate tissues. In this research, open-source database information of Arabidopsis thaliana was adapted to determine root-specific expressed promoter region. A total seven sequences that might function as a root-specific promoter element were initially isolated from Arabidopsis genomic DNA. Then seven promoters were cloned into pBGWFS7 in which β -glucuronidase ( gus ) and green fluorescent protein (gfp) genes were linked. The GUS activities were measured in different tissues of transgenic Arabidopsis by both histochemical GUS staining and fluorescent 4 methylumbelliferyl β -D-glucuronide (MUG) assay. To confirm root-specific expression, GFP-confocal microscope analysis was conducted in Arabidopsis transgenic plant. As a result, the five promoters showed strong GUS activity in the root tissue as compared with the CaMV35S promoter. To test crop application availability as a root-specific promoter, seven promoters were introduced into tomato plants and confirmed transient expression using Agrobacterium rhizogenesis ARqua1 root-nodule inducible strain. Two promoters showed that gus genes were specifically expressed in roots of transgenic tomato plant. Taken together, the novel seven promoters showed specific activity in root suggesting that it is applicable in crop improvement.
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