黄花苜蓿SKIP同源基因cDNA的克隆与生物信息学分析

Cloning and Bioinformatics Analysis of SKIP Homologous Gene cDNA from Medicago falcata L.

本研究利用RT-PCR方法从野生黄花苜蓿中克隆得到一个编码pre-m RNA剪切因子SKIP同源基因的c DNA片段,命名为Mf SKIP。对克隆片段的序列分析表明,该c DNA片段全长2 256 bp,包含一个1 836 bp的开放阅读框,预测编码一条含611个氨基酸的多肽,分子量约为69.1 k D,理论等电点8.78。对蛋白保守结构域的分析显示Mf SKIP具有SNW/SKIP蛋白特有SKIP的S-N-W-K-N序列,与水稻SKIP(NP_001048184.1)同源性高达85%。生物信息学分析表明,Mf SKIP属于定位在细胞核内的不含跨膜结构域且不含信号肽的亲水性蛋白,二级结构主要由无规则卷曲结构组成。系统发育分析表明,野生黄花苜蓿Mf SKIP蛋白与蒺藜苜蓿(Medicago truncatula)的Mt SKIP、鹰嘴豆(Cicer arietinum)Ca SKIP亲缘关系较近。本研究成功克隆野生黄花苜蓿Mf SKIP基因全长c DNA片段,为研究pre-m RNA剪切因子在黄花苜蓿响应非生物逆境胁迫应答中的作用奠定基础,并为抗逆牧草新品种培育提供候选基因。

In this research, a cDNA sequence of SKIP homologous gene which coding the pre-mRNA splicing factor was cloned from Medicago falcata L. by RT-PCR method. The sequence analysis showed that the full-lenghth of the cDNA was 2 256 bp, containing a 1 836 bp open reading frame （ORF）, which encoded 611 amino acids residues. The calculated molecular weight was 69.1 kD and iso-electric point was 8.78. Moreover, the analysis of protein structure revealed the S-N-W-K-N motif, which was absolutely conserved in SNW/SKIP protein family, which was also found in MfSKIP protein and shared 85% of similar nucleotide sequence with that of Oryza sativa （NP_001048184.1）. The bioinformatics analysis of MfSKIP showed that it was a hydrophilic protein, and located in the nucleus with no transmembrane structure and signal peptide. The secondary structure of MfSKIP was mainly composed of random coil. The phylogenetic tree analysis showed that the MfSKIP had a closely genetic relationsh- ip with MtSKIP and CaSKIP.