生命科学研究2011，Vol.15Issue(6)：476-484,9.
通路克隆入门载体pEN-L4*-PrbcS-*T-gfp-L3*的构建及其应用
Construction and Application of a Gateway Entry Vector pEN-L4*-PrbcS-*T-gfp-L3*
摘要
Abstract
In order to construct the plant expression vector that contains two expression cassettes for two target genes through a Gateway LR recombination reaction and allows the expressed proteins encoded by the target genes to be localized to the chloroplasts of transgenic plants, Hind III and Xho I sites were generated in the Gateway entry vector, pEN-L4-2-L3, through a quickChange site-directed mutagenesis technology. Then a DNA fragment containing the tomato Rubisco small subunit 3C promoter (PrbcS) and its transit pep-tide sequence (*T) as well as a GFP (Green fluorescent protein) reporter gene (gfp) was inserted in the modified entry vector between the Hind III and Xho I sites to develop the entry vector pEN-L4*-PrbcS-*T-gfp-L3*. The plant expression vector pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus containing the expression cassettes of gfp and gus genes could be constructed successfully with the developed entry vector pEN-L4*-PrbcS-*T-gfp-L3* and another Gateway entry vector (pENTR*-PrbcS-*T-gus) that contains attLl and attL2recombination sites as well as the plant destination vector pK7 m34GW2-8 m21GW3 through a Gateway LR reaction. Gfp and gus genes could be inserted in the genome of the transgenic plants and transcribed normally after the pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus vector was used to transform tobacco. The analysis of GUS staining and GFP localization indicated that the GUS protein was expressed in the transgenic tobacco leaves and the expressed GFP protein was targeted to the chloroplasts of the leaves. Using the expression vector can not only complete the genetic manipulation of two target genes through one transformation but also locate the expressed proteins regulated by PrbcS promoter to the transgenic plant chloroplasts via the transit peptide sequence (*T). As a result, the construction and application of pEN-LA*-PibcS-*T-gfp-L3* entry vector establishes a technique platform and provides the convenience for chloroplast genetic engineering.关键词
pEN-L4*-PrbcS-*T-gfp-L3*入门载体/通路克隆(Gateway)技术/光诱导型启动子/植物表达载体/叶绿体定位Key words
pEN-L4*-PrbcS-*T-gfp-L3/Gateway technology/light inducible promoter/plant expression vector/chloroplast location分类
生物科学引用本文复制引用
肖素勤,孙振,王莎莎,梁锋,李莉,年洪娟,李昆志,陈丽梅..通路克隆入门载体pEN-L4*-PrbcS-*T-gfp-L3*的构建及其应用[J].生命科学研究,2011,15(6):476-484,9.基金项目
国家自然科学基金资助项目(30670163) （30670163）
云南省中青年学术与技术带头人培养费资助项目(2004PY01-5) （2004PY01-5）
