Cloning of LEA3 gene from two Festuca arundinacea cultivars Qiancao 1 and Qiancao 2 and their expression under drought stress

ABSTRACT

(Objective) The aim of this paper was to explore the function of LEA3 gene and the molecular mechanism of drought resistance of Festuca Arundinacea, for which LEA3 gene from two Guizhou native F. Arundinacea cultivars (Qiancao 1 and Qiancao 2) were cloned and the bioinformatics of LEA3 genes and their expression under drought stress at seedling stage were analyzed as well. (Method) In this experiment, we cloned LEA3 genes of Qiancao 1 and Qiancao 2 by RT-PCR, referring to the sequences recorded by NCBI. At the same time, the amino acid sequence was analyzed by relevant software, and the expression of amino acid under drought stress was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). (Result) In this study, we found that LEA3 genes in Qiancao 1 and Qiancao 2 consisted of open reading frame (ORF) of 609 by and 642 by respectively, which encoded proteins of 203 and 213 amino acids residues, respectively. Compared with Qiancao 2, the LEA3 protein of Qiancao 1 lacks the motif composed of 11 amino acids. At the same time, 11 amino acid differences were found between Qiancao I and Qiancao 2. Bioinformatics analysis showed that the molecular weight of Qiancao 1 LEA3 protein was 20.72 kDa, the isoelectric point was 8.55, and the grand average of hydropathicity (GRAVY) was -0.971. The LEA3 protein of Qiancao 2 has a molecular weight of 22 01 kDa, an isoelectric point of 8.99, and a GRAVY of - 1.069. The hydrophobicity analysis showed that both Qiancao 1 and Qiancao 2 LEA3 proteins had no transmembrane structure, and the hydrophilicity of Qiancao 2 LEA3 was stronger than that of Qiancao 1 LEA3. Alpha-helixes are dominant in Qiancao 1 and Qiancao 2 LEA3 proteins. LEA3 proteins of Qiancao 1 and Qiancao 2 have high homology with that of barley, goatgrass, wheatgrass and wheat. The phylogenetic tree showed that LEA3 proteins of Qiancao 1, Qiancao 2, wheat, ryegrass, barley, goatgrass, bromus inermis and wheatgeas were clustered into one group. The results of gRT-PCR analysis showed that the expression of Qiancao 1 LEA3 was higher than that of Qiancao 2 in the whole process of drought stress at seedling stage. Compared with mild stress, the response of LEA3 in Qiancao 1 and Qiancao 2 under severe stress was stronger. (Conclusion) It was found that the drought resistance of Qiancao 1 and Qiancao 2 was positively correlated with the expression of LEA3 gene. Under the same conditions of drought stress, the expression of Qiancao 1 LEA3 gene was higher than that of Qiancao 2. Therefore, it can be inferred that Qiancao 1 is more adaptable to arid environment than Qiancao 2.