本氏烟草NbEHD1基因的生物信息学分析、CRISPR/Cas9载体构建及遗传转化

Bioinformatics Analysis of NbEHD1,Construction of CRISPR/Cas9 Gene Editing Vector and Genetic Transformation in Nicotiana benthamiana

为了研究本氏烟草NbEHD1基因的生物学功能,采用生物信息学方法对其基因结构、蛋白保守结构域、磷酸化位点、亚细胞定位及进化关系等多个方面进行预测。结果表明,本氏烟草NbEHD1编码序列全长1638 bp,其基因组序列包含16个外显子和15个内含子。NbEHD1蛋白大概率定位于细胞质中,无信号肽,无跨膜区,蛋白序列有42个磷酸化位点。NbEHD1属于P-loop_NTPase超家族,具有EHD家族特有保守结构域。系统进化关系结果表明,本氏烟草NbEHD1与马铃薯、番茄的EHD序列亲缘关系较近。经与SGN数据库中本氏烟草测序数据比对,获得NbEHD1预测的全序列并设计基因特异性引物,扩增得到其序列全长。在获得NbEHD1 CRISPR/Cas9基因编辑载体后,采用农杆菌介导的方法,将此载体成功转化到本氏烟草叶片,经鉴定成功获得T_(0)阳性植株16株,为进一步确定NbEHD1基因的生物学功能提供了研究材料。

In order to study the biological function of NbEHD1 in Nicotiana benthamiana,its gene structure,conserved domains,phosphorylation sites,subcellular localization and evolutionary relationship were predicted by bioinformatics methods.The encoding sequence of NbEHD1 was 1638 bp,and its genomic sequence contained 16 exons and 15 introns.NbEHD1 protein was predicted located in the cytoplasm,which had 42 phosphorylation sites and had no signal peptide and transmembrane region.NbEHD1 belonged to the P-loop_NTPase superfamily and had a conserved domain specific to the EHD family.Phylogenetic relationship showed that NbEHD1 was closely related to EHD sequences of Solanum tuberosum and Solanum lycopersicum.Blast analysis against N.benthamiana sub-data base in SNG was performed to obtain the predicted full length of NbEHD1,then its full nucleic acid sequence was amplified by gene-specific primers.After obtaining NbEHD1 CRISPR/Cas9 gene editing construct,the vector was successfully transformed into the leaves of N.benthamiana by Agrobacterium-mediated genetic transformation.Eventually 16 T_(0)-generation plantlets were successfully identified,which provided materials for further research to determine the biological function of NbEHD1.