野生黄花苜蓿冷诱导基因克隆及序列分析

Cloning and Sequence Analysis of Cold Resistance Gene from Wild Medicago falcata

根据紫花苜蓿抗寒基因cas15B(登录号:L12462)的cDNA序列,设计1对特异引物,以经过低温胁迫的黄花苜蓿总RNA为模板,采用RT-PCR方法克隆获得了550bp的cDNA片段。测序和序列分析结果表明,扩增片段长度为550bp,编码159个氨基酸。主要由Gly(甘氨酸)、Glu(谷氨酸)、His(组氨酸)、Lys(赖氨酸)这4种氨基酸组成,占总量的70%。含1个重复了5次的10肽基序,其序列为Lys-Gly-Glu-Gln-His-Gly-His(Phe)-Val(Leu)-Gly-Gly。经序列比较分析,该片段与紫花苜蓿冷诱导基因CAS15B的核苷酸、氨基酸的同源性均为90%,命名为MfCAS15-1。亚细胞结构定位分析结果显示,MfCAS15-1是一种定向到核的蛋白,在调节或维持核的结构与功能方面起作用。本研究在黄花苜蓿中成功获得了抗寒基因同源序列,为最终克隆黄花苜蓿MfCAS15-1抗寒基因全长奠定了基础。

According to cDNA sequence of cas15B （Accession number：L12462） of Medicago sativa,a pair of specific primers were designed,by which a new cDNA fragment of 550 bp was amplified by RT-PCR from total RNA of Medicago falcate that suffered from cold stress.Sequencing result and sequence analysis showed that this amplified fragment encoded 159 amino acids,four of which were Gly,Glu,His and Lys,accounting for 70% predicted.The polypeptide contains a decapeptide motif repeated five times at regular intervals with the following consensus sequence Lys-Gly-Glu-Gln-His-Gly-His（Phe）-Val（Leu）-Gly-Gly.The amplified fragment which of the homology of nucleotide sequences and deduced amino acids sequences were both 90% with the cold-induced gene cas15B of Medicago sativa,was named as Mf CAS15-1,and the encoded protein was directed to the nuclear due to the subcellular location analysis.Mf CAS15-1 may play an important roles in gene regulation and the stabilization of nuclear structure and function in the process of cold acclimation.In this study,homologous sequences of cold resistance gene were received from Medicago falcata,which could provide the foundation for cloning the full length of cold resistance gene（Mf CAS15-1） of Medicago falcata.