甜菜氮代谢相关蛋白BvAMT3-3基因的克隆及生物信息学分析

Cloning and bioinformatics analysis of nitrogen metabolism related gene Beta vulgaris ammonium transporter(BvAMT3-3)

AMT蛋白是一类铵转运蛋白,参与植物对铵态氮的吸收和运输,在植物生长发育、代谢以及胁迫应答等过程中起着重要作用。为进一步探究BvAMT3-3基因(XM_010669058)在甜菜中应答氮胁迫的分子机制,通过RT-PCR方法在甜菜体内克隆获得了Beta vulgaris ammonium transporter 3-3(BvAMT3-3)基因的全长序列。该基因CDS全长为1427 bp,分子量和等电点分别约为51.055 kDa和8.98,编码了475个氨基酸,属于稳定蛋白。系统进化分析显示,BvAMT3-3与SoAMT3-1亲缘关系最近。BvAMT3-3蛋白二级结构中的α-螺旋占比最大,属于稳定蛋白。跨膜结构域和保守结构域分析显示,该蛋白具有11个跨膜结构域,是一种含有铵转运结构域的膜蛋白。亚细胞定位显示其位于内质网膜上的可能性较大,可知该蛋白具有一定的转运功能。qPCR分析表明,缺氮(N0)和低氮(N1.5)逆境都不同程度地诱导BvAMT3-3基因的表达,同时随着胁迫时间的推移,这种诱导更为显著,即使恢复生长,植株中该基因的表达量虽然较处理时有所降低,但仍维持在较高水平。研究结果为进一步深入了解甜菜AMTs基因在氮逆境胁迫中的分子机制和提高甜菜的氮素利用效率提供了理论依据。

AMT proteins are a class of ammonium transporters,which participate in the absorption and transportation of ammonium nitrogen in plants,and play an important role in plant growth,metabolism and stress response.To further explore the molecular mechanism of BvAMT3-3(XM_010669058)in response to nitrogen stress in sugar beet,the full-length sequence of Beta vulgaris ammonia transporter 3-3(BvAMT3-3)gene was cloned in sugar beet by RT-PCR.Its full length CDS was 1427 bp,which encoded 475 amino acids.Its molecular weight was 51.055 kDa,the theoretical pI was 8.98,which belongs to stable protein.Phylogenetic analysis showed that BvAMT3-3 had the closest relationship with SoAMT 3-1.BvAMT3-3 is a stable protein with the largest proportion ofα-helix in its secondary structure.The analysis of transmembrane domain and conserved domain showed that the protein had 11 transmembrane domains,so it is a membrane protein containing ammonium transport domain.Subcellular localization showed that it was more likely to be located on the endoplasmic reticulum,indicating that the protein had a certain transport function.QPCR analysis showed that nitrogen deficiency(N0)and low nitrogen(N1.5)stress induced the expression of BvAMT3-3 gene in different degrees,and this induction was more significant with the passage of stress time.Even if the plant resumed growth,the expression of BvAMT3-3 gene in the plant was lower than that in the treatment,but still maintained at a high level.The results provide a theoretical basis for further understanding the molecular mechanism of AMTs gene in nitrogen stress and improving nitrogen use efficiency in sugar beet.