文献类型：期刊文献

中文题名：草莓果实外源基因瞬时表达系统的建立

英文题名：Establishment of transient expression system of foreign gene with strawberry fruit

作者：刘敏[1,2] 滕文静[3,2] 董清华[1] 晁慧娟[1] 李凤岚[4] 邱德有[3,2]

第一作者：刘敏

机构：[1]北京农学院植物科学技术系;[2]国家林业局林木培育重点实验室;[3]中国林业科学研究院林业研究所;[4]中国医学科学院中国协和医科大学药用植物研究所

年份：2009

卷号：26

期号：1

起止页码：60-65

中文期刊名：果树学报

外文期刊名：Journal of Fruit Science

收录：CSTPCD;;北大核心:【北大核心2008】；CSCD:【CSCD_E2011_2012】；

基金：国家高技术研究发展计划-"863"计划(2007AA10Z182);国家自然科学基金(30671698);中央级公益性科研院所基本科研业务费专项资金(200701);北京市教委植物遗传育种改良与种质资源创新平台(5075201029)

语种：中文

中文关键词：草莓;果实;瞬时表达;根癌农杆菌;基因功能

外文关键词：Strawberry; Fruit; Transient expresion; Agrobacterium tumefaciens ; Gene function

分类号：S668.4

摘要：将东北红豆杉紫杉烷13α-羟基化酶(Taxane13α-hydroxylase,13OH)基因全长cDNA正向插入到pCAMBI-A1304,构建其植物表达载体pCT13OH;将水稻小RNA169d(microRNA169d,miRNA169d)基因前体(precursor)全长DNA正向插入到pCAMBIA1305.1,构建其植物表达载体pCO169d。用电击法把它们导入根癌农杆菌GV3101,获得有关工程菌株。用1mL无菌注射器分别将这2种工程农杆菌悬浮液注射到草莓(Fragaria×ananassa)授粉后1周、2周、3周的果实中,发现授粉后2周果实适宜注射,可用于瞬时表达。对授粉后2周的果实进行不同注射部位、根癌农杆菌不同活化时期和根癌农杆菌悬浮液不同注射量试验,以蒂部注射、根癌农杆菌活化12h和0.5mL悬浮液注射量的效果最好,与结构基因13OH和调节基因miRNA169d相融合的GUS基因都得到表达,瞬时表达成功。
The amplified full-length cDNA for Taxane 13α-hydroxylase （13OH） from Taxus cuspidata was inserted into pCambia1304 and a recombinant contstrnct pCT13OH was produced. In addition, the DNA for precursor of rice microRNA 169d （miR169d） gene was inserted into pCambia1305.1 and a recombinant contstrnct pCO169d was obtained. The recombinant plasmids pCT13OH and pCO169d were transformed into Agrobacterium tumefaciens GV3101 by electroporation. Furthermore, the transformation of these two engineered A. tumefaciens strains with strawberry were studied by injecting their overnight culture into strawberry（Fragariaxancalassa）fruit. GUS expression was monitored by histochemical assay of the cross section of strawbelxy fruit. The effect of injection position, culture duration and injection volume of engineered A. tumefaciens on GUS expression were studied. Fruit at the stage of two weeks post-pollination were the best materials for transient expression study. The best injection position, culture duration and injection volume of engineered A. tumefaciens was postion near pedicel, 12 h and 0.5 mL respectively. We anticipate that this rapid and transient expression system will be useful for studying the function of plant structural genes and regulatory genes including 130H and miR169d.