转基因水稻中HPT蛋白质的检测及表达特征研究

doi:10.13523/j.cb.20150610

中国生物工程杂志

2015, Vol. 35

Issue (6): 61-67 DOI: 10.13523/j.cb.20150610

技术与方法

转基因水稻中HPT蛋白质的检测及表达特征研究

杨烁¹, 郝育杰², 兰金苹¹, 韦汉福², 魏健¹, 荣瑞娟¹, 武鹏程², 刘国振¹, 尹长城², 李莉云¹

1. 河北农业大学生命科学学院保定 071001;
2. 北京华大蛋白质研发中心有限公司北京 101318

HPT Protein Detection and Expression Pattern in Transgenic Rice

YANG Shuo¹, HAO Yu-jie², LAN Jin-ping¹, WEI Han-fu², WEI Jian¹, RONG Rui-juan¹, WU Peng-cheng², LIU Guo-zhen¹, YIN Chang-cheng², LI Li-yun¹

1. College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China;
2. Beijing Protein Innovation Co., Ltd. Beijing 101318, China

全文: PDF(791 KB) HTML

摘要：

hpt是转基因植物中最常用的筛选标记基因之一,建立了HPT蛋白质免疫印迹检测技术、了解其在转基因植物中的表达特征具有重要的理论意义和应用价值。在大肠杆菌中进行了HPT蛋白质的重组表达,利用纯化的HPT蛋白质制备了其特异性的单克隆抗体,采用免疫印迹学方法对转基因水稻4021中HPT蛋白质的表达情况进行了检测,该方法可检测出单粒稻米的10%(约1.5mg)所含的HPT蛋白质。定量分析发现水稻苗期HPT蛋白质含量约占鲜重的0.01‰。对CaMV35S启动子驱动下的转基因水稻中HPT蛋白质的表达谱进行分析,发现其在苗期和成株期的叶片中表达量较高,在灌浆期的种子中呈现不断增加的趋势,而在茎、分蘖节、茎节、叶鞘、叶枕和根等组织中表达量较低。建立了完整的利用免疫印迹方法检测HPT蛋白质的技术,同时系统分析了HPT蛋白质在转基因水稻中的表达谱。

关键词： 转基因水稻; HPT蛋白质; 免疫印迹技术; 筛选标记基因

Abstract:

Hpt as one of the most commonly selective marker gene was used in transgenic plants. To establish immunoblotting method for detectting HPT protein expression profiles in transgenic plants has important theoretical significance and application value. HPT recombinant protein was expressed in E. coli, and used to prepare specific monoclonal antibody. Western blot was used to detect the expression of HPT protein in transgenic rice of 4021, the results showed that in 10% of single grain of rice (about 1.5mg) contained HPT protein can be detected. Quantitative analysis revealed that the content of HPT protein in rice seedling account for about 0.01 ‰ of fresh weight. The expression profiles of HPT protein under CaMV 35S promoter-driven start in transgenic rice showed that the abundance of HPT was regulated higher levels in the leaves of the seedling and adult, showing a rising trend in the seed filling period, but HPT showing low abundance in the stems, tillers, node, shears, cushion, roots and other tissues. In short, an applicable immunoblot method for detectting HPT protein was established, while the HPT protein expression profiles were analyzed in transgenic rice.

Key words: Transgenic rice HPT protein Western blot Selective marker gene

收稿日期: 2015-03-18 出版日期: 2015-06-25

ZTFLH:

Q816

基金资助:

国家科技部转基因生物新品种培育重大专项(2009ZX08012-006B)、北京市自然科学基金重点项目(5121001)资助项目

通讯作者: 李莉云, 尹长城 E-mail: liliyun@hebau.edu.cn;Yincc@genomics.cn

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引用本文:

杨烁, 郝育杰, 兰金苹, 韦汉福, 魏健, 荣瑞娟, 武鹏程, 刘国振, 尹长城, 李莉云. 转基因水稻中HPT蛋白质的检测及表达特征研究[J]. 中国生物工程杂志, 2015, 35(6): 61-67.

YANG Shuo, HAO Yu-jie, LAN Jin-ping, WEI Han-fu, WEI Jian, RONG Rui-juan, WU Peng-cheng, LIU Guo-zhen, YIN Chang-cheng, LI Li-yun. HPT Protein Detection and Expression Pattern in Transgenic Rice. China Biotechnology, 2015, 35(6): 61-67.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150610 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I6/61

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