菊苣质体同源片段的克隆及非抗生素标记的质体定点整合表达载体构建

Cloning of homologous targeting sequences and construction of antibiotic-free plastid site-specific integration expression vector of chicory

依据烟草、玉米、拟南芥和大豆质体基因组rps7基因和16S rDNA基因的保守序列设计引物,以菊苣质体基因组DNA为模板,PCR扩增到1段4.8 kb的片段。序列分析表明,该片段全长4 751 bp(GenBank登录号为GQ199478),包括449 bp的rps7基因5′端序列、793 bp的rps12基因、72 bp的trnV基因、1 315 bp的16S rDNA基因5′端以及大部分的基因间隔区,该序列与烟草对应区段的相似性为92%,与莴苣对应区段的相似性为97%。以此片段作为定点整合外源基因的同源重组片段,构建了非抗生素标记的菊苣质体定点整合表达载体pJBADH-GFP,酶切分析及PCR检测证明构建的载体符合预期设计。该载体的构建为建立高效、稳定的菊苣质体转化和无抗生素筛选体系奠定基础,为进一步通过质体基因工程手段将更多感兴趣的基因导入菊苣进行遗传改良,或以菊苣质体作生物反应器生产动物口服疫苗等搭建技术平台。

A pair of primers used in amplifying plastid fragments of chicory was designed based on the conservative sequences of both rps7 and 16S rDNA genes in the tobacco,maize,Arabidopsis thaliana and soybean plastid genomes.The 4.8 kb fragment was cloned from the chicory plastid genome.Sequencing analysis indicated that this fragment was 4751 bp in size（GenBank Accession Number GQ199478）,and consisted of 449 bp 5′-end of the rps7 gene,793 bp sequence of rps12 gene,72 bp sequence of the trnV gene,1 315 bp 5′-end of the 16S rDNA gene and most gene intergenic regions.The BLAST results showed that the similarity of this fragment with tobacco and lettuce were 92% and 97% respectively.Using this fragment as a homologous targeting sequence,we constructed the site-specific integration chicory-specific plastid expression vector pJBADH-GFP that carries the multicistron expression cassette of prrn-BADH-gfp-psbA-3′.The results of restriction analysis and PCR detection confirmed this.This plastid expression vector is desirable both for use in establishment of a chicory antibiotic-free plastid transformation system and traits improvement of chicory or for use as a bioreactor to produce edible vaccines for animals via plastid genetic transformation.