摘要
【目的】从大麦BAC文库中快速筛选包含目的基因HvGW2的阳性BAC克隆。【方法】构建BAC文库三维混合池,设计2对HvGW2特异性引物,进行BAC文库筛选,对含有目的基因HvGW2的阳性BAC克隆进行测序,获得编码大麦GW2蛋白的基因组DNA序列,并与禾本科中的GW2进行同源进化分析。【结果】构建了大麦BAC文库的三维混合池160个,文库空载率为0.18%;筛选到含HvGW2的阳性BAC克隆3个,其中,BAC克隆196M02包含完整编码该基因的序列;HvGW2包含8个外显子和7个内含子,具有与其它植物GW2相似的结构特征和保守的功能结构域;同源进化分析结果显示,大麦与小麦的GW2蛋白同源性最高;序列分析发现,HvGW2第6内含子上包含2个不同类型的反转录转座子插入,从而使大麦比其它作物的GW2长度增加11.5 kb以上。【结论】通过构建三维混合池快速筛选包含目的基因的单个阳性BAC克隆,建立了大麦目标基因克隆的辅助平台;HvGW2基因组中位于内含子区的反转录转座子插入导致基因长度增加,可能是影响该基因转录水平的关键变异。
[Objective] The objective of this study was to isolate HvGW2 gene from barley BAC library rapidly. [Method] Three-dimensional （3D） BAC pools were constructed and screened by using barley GW2 gene-specific PCR primers. Positive BAC clones were screened out and further sequenced, the obtained HvGW2 gene was used for homologous evolutionary analysis with those of other grasses. [Result] One hundred and sixty BAC DNA pools were prepared by 3D pooling strategy from barley BAC library with only 0.18% of empty clones. PCR-based screening demonstrated that only one of the three positive BAC clones （196M02） contained the complete coding sequence of HvGW2. Sequence analysis showed that barley GW2 gene contains eight exons and seven introns, which revealed that it has similar gene structure and conserved functional domain with other plant species. Furthermore, HvGW2 has the highest homologous with wheat GW2 protein. Moreover, the sixth intron ofHvGW2 gene contains two different retrotransposon insertions, which resulted in a 11.5 kb length increase of barley GW2 gene than the other crops. [Conclusion] By constructing three-dimensional BAC pools, and combining with PCR-based screening, positive BAC clones which contain target gene can be isolated quickly. Not only the quality of the barley BAC library was evaluated, but also an auxiliary platform for barley gene cloning was established. It was speculated that the retrotransposon insertions in gene intron might be the key variation which affected the gene transcription levels of HvGW2.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第1期9-17,共9页
Scientia Agricultura Sinica
基金
国家“863”计划项目(2012AA101105-4)
国家“十二五”科技支撑计划(2012BAD03B01-2)
国家现代农业产业技术体系建设专项资金(CARS-05)
