紫花苜蓿抗逆基因MsDUF的克隆及其功能分析

【目的】克隆紫花苜蓿（Medicago sativa L.）抗逆新基因MsDUF，并对其进行序列特征分析，了解该基因在逆境胁迫下的表达模式。【方法】利用RACE法获得紫花苜蓿MsDUF全长的cDNA序列，对该序列进行生物信息学分析；用基因枪法进行MsDUF的亚细胞定位分析；采用实时荧光定量PCR研究该基因在高含量NaCl和PEG-6000的胁迫下，以及ABA和GA3诱导下的表达模式。【结果】测序结果显示，该基因cDNA全长714 bp，包含一个633 bp的完整开放阅读框，编码210个氨基酸，命名为MsDUF，GenBank登录号为JX183734。氨基酸BLASTP分析表明，MsDUF氨基酸序列与拟南芥、大豆和蒺藜苜蓿等具有53%—98%的相似性，属于DUF4228 superfamily。实时荧光定量PCR研究表明，该基因在逆境胁迫下上调表达。【结论】从紫花苜蓿中克隆了抗逆相关基因MsDUF，发现其定位于细胞质中，实时荧光定量PCR结果分析表明该基因在紫花苜蓿的抗逆反应中起着重要作用。

Isolation and Functional Analysis of the Stress Resistance Gene MsDUF in Medicago Sativa L.

【Objective】This study is aimed to characterize the stress resistance gene MsDUF in Medicago sativa by cloning and analysis of its sequence, and study the function of MSDUF under stress conditions.【Method】The full-length cDNA sequence of MsDUF was isolated by RACE. The obtained cDNA sequence and the deduced amino acid sequence were analysed. Using gene gun in subcellular localization. Real-time quantitative PCR was used to assess the expression of MsDUF in response to NaCl，PEG-6000，ABA and GA3.【Result】The sequencing result showed that the cloned cDNA (designated as MsDUF, GenBank accession No. JX183734) was 714 bp, contains an open reading frame (ORF) of 633bp, and encoding a protein with 210 amino acids. BLASTp search revealed 53% to 98% similarities between the MSDUF protein and those reported MSDUF from other species, such as Arabidopsis thaliana, Glycine max and M. truncatula. The RT-PCR result showed that MsDUF was upregulatedly in response to stress resistance.【Conclusion】The stress resistance related gene MsDUF was cloned from M. sativa and found that it localized in cytoplasm. RT-PCR analysis showed that the gene MsDUF plays an important role in response to the abiotic stress. It is a novel stress resistance gene in M. sativa.