《植物生理学报》 2018, 54(10): 1546-1552

水稻GluB-4终止子的克隆与功能分析

李文静^*, 冯雪, 孙艳香

廊坊师范学院生命科学学院, 河北廊坊065000

通信作者：李文静；E-mail: liwenjing888@163.com

摘　要：

目前转基因常用的终止子大多为细菌或真菌来源, 有可能引起转基因食品安全性问题。本研究以水稻(Oryza sativa) ‘kitaake’为材料, 克隆了谷蛋白B-4基因(GluB-4)的终止子GluB-4-T (GenBank登记号: X14393), 分析了其在不同启动子驱动下替代根癌农杆菌(Agrobacterium tumefaciens)胭脂碱合酶终止子(nos-T)的可能性。β-d-葡糖醛酸酶(GUS)组织化学染色结果表明, GluB-4-T在GluB-3启动子驱动下, 未改变GluB-3启动子的组织表达特异性, 但GUS表达强度发生了改变; GUS活性分析显示GluB-4-T GUS活性是nos-T的2.4倍。为验证GluB-4-T增强外源基因表达的特性不依赖于上游启动子序列, 将其克隆到强的胚乳特异性启动子GluC和组成型启动子OsActin下, GUS活性分别是nos-T的2.0和2.2倍, 说明GluB-4-T增强外源基因表达的特性不依赖于上游启动子序列。以上结果表明GluB-4-T在水稻体内发挥正常的转录终止活性, 且比nos-T表达活性强, 可以作为有效终止子用于遗传转化, 避免多基因转化系统中由于转基因同源性过高引起的转基因沉默现象。该结果为实现外源目的基因在水稻胚乳中特异表达提供理论依据。

关键词：水稻; GluB-4终止子(GluB-4-T ); 胭脂碱合酶终止子(nos-T ); GUS组织化学染色; GUS活性分析

收稿：2018-07-25   修定：2018-09-15

资助：河北省高等学校科学技术研究青年基金(QN2018017)和河北省遗传学重点发展学科(201221)。

Cloning and functional analysis of GluB-4 terminator in rice

LI Wen-Jing^*, FENG Xue, SUN Yan-Xiang

College of Life Science, Langfang Teachers University, Langfang, Hebei 065000, China

Corresponding author: LI Wen-Jing; E-mail: liwenjing888@163.com

Abstract:

The bacterial- or fungal-origion terminator is commonly used in transgenic research, which raises great concerns about the safety of genetically modified foods. In this study, we cloned glutelin B-4 terminator (GluB-4-T, GenBank number X14393) and evaluated the potential that GluB-4-T function as terminators in enhancing the expression of the GUS reporter gene driven by glutelin GluB-3, GluC and OsActin promoters in stable transgenic rice (Oryza sativa) lines. The results show that GluB-4-T significantly enhanced the activity of GluB-3 promoter without changing its tissue specificity but altering its expression intensity in endosperm. With GluB-4-T, the expression of GUS was higher by 2.4 fold than with the nos terminator (nos-T ). GluB-4-T combined with GluC and OsActin promoters, also increased GUS levels by 2.0 and 2.2 folds, respectively, in stable transgenic rice line, which indicates that the enhancements were independent of the promoter sequences. The results suggest that GluB-4-T has normal transcriptional termination activity in rice and is more potent than nos-T. GluB-4-T might be an ideal candidate for genetic transformation and it could function as faithful terminator in mono- or multi-gene transformation avoiding homology-based gene silencing. The results above lay foundation for the specific expression in rice endosperm.

Key words: rice; GluB-4 terminator (GluB-4-T ); opaline synthase terminator (nos-T ); GUS histochemical staining; quantitative GUS assay