三个烟草防御素基因的克隆及序列分析

In Silicon Cloning and Sequence Analysis of Three Defensin Genes in Tobacco(Nicotiana tabacum)

为从烟草中克隆与植物抗病虫反应相关的防御素基因,以辣椒防御素基因为探针,通过电子克隆的方法从烟草栽培品种K326中克隆到3个防御素基因(NtDEF1、NtDEF2和NtDEF3),并对其进行了序列分析。结果表明,3个防御素cDNA序列均包含完整的开放读码框,分别编码78、77、76个氨基酸,具有8个半胱氨酸的高度保守结构等典型的植物防御素特征,成熟肽含有γ-硫素蛋白功能结构域。通过同源比对和进化分析发现,NtDEF1、NtDEF2和NtDEF3的氨基酸序列与辣椒的一致性最高为50%-87%,亲缘关系较近;但与已报道的烟草属植物防御素的一致性仅为33%-40%,亲缘关系较远,表明NtDEF1、NtDEF2和NtDEF3是烟草中未报道的3个防御素基因。

In silicon cloning,three distinct defensin genes（NtDEF1,NtDEF2 and NtDEF3） were obtained successfully from tobacco cultivar K326 by using pepper CaDEF1 gene sequence（AF442388） to be probe.Sequence analysis showed that the in silicon cloned cDNAs had a complete open reading frame（ORF） and encoded 78,77 and 76 amino acid residues,respectively,including a strictly conserved γ-thionin domain of eight cysteines which is characteristic in plant defensins.Multiple alignment analysis based on the deduced amino acid sequences of defensin genes from different pIants indicated that NtDEF1,NtDEF2 and NtDEF3 showed high similarity with 50%-87% identities to the defensins from Capsicum annuum.However,with 33%-40% identities to the defensins previously reported from Nicotiana,the evolutionary analysis showed that NtDEF1,NtDEF2 and NtDEF3 were very far related to them.In conclusion,this is the first time that the NtDEF1,NtDEF2 and NtDEF3 genes have been reported in tobacco.