从天山雪莲叶片低温诱导的EST文库中获得了1个胚胎发育晚期丰富蛋白基因(LEA) cDNA全长序列.序列分析表明,该基因含有1个468 bp编码155个氨基酸的开放阅读框.NCBI保守域预测此蛋白属于LEA_2家族,命名为SiLEA14.系统进化分析表明,该蛋白与北柴胡的LEA-2蛋白亲缘关系最近.荧光定量PCR结果显示,SiLEA14表达量在低温、盐和干旱胁迫条件下迅速升高.亚细胞定位结果表明,SiLEA14蛋白定位于细胞核中.利用农杆菌介导法将该基因导入烟草,测定并分析转基因植株在冷冻和盐胁迫处理下的生理指标,结果表明,SiLEA14基因在烟草中的过量表达提高了烟草的抗冻和耐盐能力.%For a better understanding of stress responses of Saussurea involucrata Kar.et Kir.,we reported molecular characterization and expression analysis of SiLEA14 gene from the S.involucrata Kar.et Kir.EST data under cold-stress.Sequence analysis showed that the cDNA of SiLEA14 contained a 468 bp ORF encoding a polypeptide of 155 amio acids.NCBI conserved domains analysis showed that SiLEA14 protein was a member of late embryogenesis abundant protein class 2.Phylogenetic analysis indicated that SiLEA14 protein was most closely related to Bupleurum chinense LEA-2 protein.Real-time quantitative PCR analysis showed that SiLEA14 was constitutively expressed,up-regulated by cold,salt and drought stress quickly.Subcellular localization assay indicated the SiLEA14-GFP fusion prortein was located in the nucleus.A overexpression vector was constructed,and transgenic tobacco plants transformed by Agrobacterium tumefaciens were obtained.Transgenic plants resistances to freezing and salinity were investigated.The gene function was analyzed via measuring the physiological stress indicators under different osmotic pressures.The results showed SiLEA14 gene can improve tobacco resistance to freezing and salinity.
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