烟草NtNRAMP2基因的鉴定及其在镉吸收中功能分析  

Identification of NtNRAMP2 Gene and Its Function Analysis in Cadmium Absorption in Nicotiana tabacum

作　　者：杨园园 程岚 许佳迪 李燕燕 邓兆龙 郝浩浩 王平平 金维环 郭红祥[1] YANG Yuan-Yuan;CHENG Lan;XU Jia-Di;LI Yan-Yan;DENG Zhao-Long;HAO Hao-Hao;WANG Ping-Ping;JIN Wei-Huan;GUO Hong-Xiang(College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China;Zhumadian Branch of Henan Province Tobacco Company,Zhumadian 463000,China;Shaanxi Branch of China National Tobacco Corporation,Xi'an 710061,China)

机构地区：[1]河南农业大学生命科学学院,郑州450002 [2]河南省烟草公司驻马店市公司,驻马店463000 [3]中国烟草总公司陕西省公司,西安710061

出　　处：《农业生物技术学报》2023年第10期2035-2047,共13页Journal of Agricultural Biotechnology

基　　金：驻马店市烟草公司科技项目(2021411700200005)。

摘　　要：自然抗性相关巨噬细胞蛋白(natural resistance associated macrophage proteins,NRAMPs)在金属跨膜转运中起重要作用。为了研究烟草(Nicotiana tobacum)NRAMP基因在重金属镉(Cd)吸收和转运中的作用,本研究从栽培烟草’K326’中克隆到1个NRAMP亚家族基因,命名为NtNRAMP2(GenBank No.OP972862);对其进行生物信息学分析和亚细胞定位,并使用qPCR分析其组织表达特异性、外源Cd、脱落酸(abscisic acid,ABA)以及乙烯处理的表达情况。结果表明,NtNRAMP2基因CDS全长为1629 bp,编码542个氨基酸,包含4个外显子和10个跨膜结构域。进化分析显示其与番茄(Lycopersicon esculentum)和马铃薯(Solanum tuberosum)亲缘关系最近,亚细胞定位于内质网和液泡中。qPCR结果显示,NtNRAMP2基因在烟草不同组织中均有表达,但在根中表达量最高。在不同浓度和时间镉胁迫诱导下,NtNRAMP2表达量都显著升高(P<0.05),外源ABA和乙烯都能诱导烟草根中NtNRAMP2基因表达。同时,构建NtNRAMP2过表达载体,通过农杆菌(Agrobacterium tumefacien)介导转化烟草’K326’并获得18株NtNRAMP2过表达转基因烟株。本研究为进一步探讨Nt NRAMP2基因功能提供了参考。Natural resistance associated macrophage proteins(NRAMPs)play an important role in metal transport in the transmembrane.To investigate the role of tobacco(Nicotiana tobacum)NRAMP genes in heavy metal Cd uptake and transport,a NRAMP subfamily gene,named NtNRAMP2(GenBank No.OP972862),was cloned from cultivated tobacco'K326'in this study.Bioinformatics analysis and subcellular localization were carried out to clarify the biological function of the NtNRAMP2,and its expression level in tissue specificity and under exogenous Cd,ABA and ethylene treatment were analyzed by qPCR.Sequence analysis showed that the full-length CDS of tobacco NtNRAMP2 gene was 1629 bp,encoding 542 amino acids,containing 4 exons and 10 transmembrane structural domains.Evolutionary analyses revealed that NtNRAMP2 was most closely related to tomato(Lycopersicon esculentum)as well as potato(Solanum tuberosum),subcellularly localised in the endoplasmic reticulum and vesicles.qPCR results showed that NtNRAMP2 gene was expressed in different tissues of tobacco,but the expression was highest in the roots.NtNRAMP2 expression was significantly increased under different concentrations and times of Cd stress,and NtNRAMP2 gene expression was also triggered in tobacco roots by both exogenous ABA and ethylene.Meanwhile,NtNRAMP2 overexpression vector was constructed and 18 NtNRAMP2 overexpressing transgenic tobacco plants were obtained by the Agrobacterium tumefaciens mediated transformation method.This study provides a reference for further exploration to study the function of NtNRAMP2.

关 键 词：烟草 自然抗性相关巨噬细胞蛋白(NRAMP) 基因表达 镉吸收 转基因 

分 类 号：S572[农业科学—烟草工业]