HOU Hongmin,WANG Yaping,LIN Yanxiang.Molecular Cloning and Functional Analysis of VpSBP12 in Grape[J].Northern Horticulture,2019,43(03):35-43.[doi:10.11937/bfyy.20181926]
葡萄转录因子 VpSBP12 的克隆及功能分析
- Title:
- Molecular Cloning and Functional Analysis of VpSBP12 in Grape
- Keywords:
- SBP gene; salt stress; gene clone; over expression
- 文献标志码:
- A
- 摘要:
- 以华东葡萄‘白河-35-1’为试材，依据葡萄全基因组数据库(http://www.genoscope.cns.fr)，利用同源序列比对结果设计引物，克隆获得VpSBP12基因的DNA和cDNA序列，并分析其序列特征和表达特性，以期阐明其在葡萄抗盐胁迫中的作用，为葡萄抗逆育种提供参考依据。结果表明：VpSBP12基因DNA全长2 907 bp，其中含有3个外显子和2个内含子；该基因的完整开放阅读框序列全长1 137 bp，编码379个氨基酸，其中包括一个含有双向核定位信号的高度保守SBP结构域。经过亚细胞定位和转录活性分析表明，该基因可以定位到核里，且具有转录激活活性。构建过量表达载体将VpSBP12转化拟南芥后发现，转基因株系在盐胁迫培养基上的萌发率及根长显著高于野生对照。表明VpSBP12基因在拟南芥中的过量表达提高了植株的抗盐胁迫的能力。
- Abstract:
- Chinese wild Vitis pseudoreticulata clone ‘Baihe-35-1’ was used as material,the DNA and cDNA sequences of VpSBP12 were isolated by using the primers designed based on the Grape Genome Database (http://www.genoscope.cns.fr) to analyze its sequence characteristics.The bioinformatics and gene expression analysis of VpSBP12 could help to elucidate the functional characterization of VpSBP12 in salt stress responses,and further provide a theoretical foundation for grape resistant breeding.The results indicated that the DNA sequence of VpSBP12 comprised 2 907 bp,which contained 3 exons and 2 intron.The VpSBP12 cDNA sequence included a 1 137 bp open reading frame (ORF) which encoded a polypeptide of 379 amino acids including a highly conserved SBP-domain bearing a nuclear localization signal (NLS).It was confirmed that they were targeted to the nucleus and possessed transcriptional activation activity by subcellular localization and trans-activation assay.The over-expression vector pCAMBIA2300-35S-VpSBP12 was constructed and transformed into Arabidopsis thaliana by floral dip methods.The VpSBP12 transgenic lines showed higher germination rate and longer roots than the wild type under the salt stress.These results indicated that over-expression of VpSBP12 in Arabidopsis could improve the toleration to salt stress.
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备注/Memo
第一作者简介：侯鸿敏（1985-），女，山西榆次人，博士，副教授，现主要从事果树分子生物学等研究工作。E-mail：houhongmin@163.com.基金项目：国家自然科学基金资助项目(31801841)；青岛农业大学高层次人才科研基金资助项目（663/1114338，661/1116009）；国家现代苹果产业技术体系资助项目(CARS-28)；青岛市果树产业技术体系资助项目（QNJC201633）。收稿日期：2018-09-17