摘要
为了利用通路克隆(Gateway)技术构建一个含有两个目的基因表达盒的植物表达载体,并把目的基因编码的蛋白质定位到转基因植物的叶绿体中,通过定点突变技术,在含有attL4和attL3重组位点的Gateway入门载体pEN-L4-2-L3中产生HindⅢ和XhoⅠ的酶切位点,然后在这两个酶切位点之间插入一个含有1,5-二磷酸核酮糖羧化酶小亚基的光诱导型启动子(PrbcS)及其叶绿体基质定位序列(*T)和绿色荧光蛋白(GFP)报告基因(gfp)的DNA片段,获得pEN-L4*-PrbcS-*T-gfp-L3*入门载体.用该载体和另一个含有attL1和attL2重组位点的入门载体(pENTR*-PrbcS-*T-gus)与Gateway的目的载体pK7 m34GW2-8 m21GW3进行LR重组反应可以构建一个能串联gfp和gus两个报告基因表达盒的植物表达载体pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus,所构建的植物表达载体转化烟草后,gfp和gus基因能插入到转基因烟草的基因组中并正常表达,所表达的GFP蛋白可正确定位到转基因植物的叶绿体中,而GUS蛋白也可以在叶片中表达.利用此表达载体通过一次转化事件不仅可以完成两个目的基因的转化操作,而且还可以利用叶绿体基质定位序列(*T)把PrbcS控制表达的目的蛋白直接定位到转基因植物的叶绿体中.因此pEN-L4*-PrbcS-*T-gfp-L3*入门载体的应用进一步扩大了Gateway技术及植物表达载体的应用范围,为叶绿体基因工程操作提供了一个更方便的技术平台.
In order to construct the plant expression vector that contains two expression cassettes for two tar-get 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 modi-fied 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 attL1 and attL2 recombination 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 normal-ly 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 al-so 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-L4＊-PrbcS-＊T-gfp-L3＊ entry vector establishes a technique platform and provides the convenience for chloroplast genetic engineering.
出处
《生命科学研究》
CAS
CSCD
2011年第6期476-484,共9页
Life Science Research
基金
国家自然科学基金资助项目(30670163)
云南省中青年学术与技术带头人培养费资助项目(2004PY01-5)
