Cloning and Functional Analysis of the Plasma Membrane Aquaporins Gene in Lolium perenne L.

【Objective】To provide an insight into mechanism of drought resistance and cultivar development in Lolium perenne L.,the sequence characteristics of the aquaporin gene LpAQP in L.perenne L.was analyzed and the expression profiling was studied.【Method】The full-length cDNA sequence of LpAQP was isolated by RACE.The obtained cDNA sequence and the deduced amino acid sequence was analyzed.In order to follow the intracellular localization of the protein,the GFP sequence was fused downstream to the LpAQP coding region and the fusion gene LpAQP::GFP was transferred into onion epidermal cells by biolistic method.The real time-PCR was adopted to study the expression profile of gene LpAQP.The southern blotting was used to analyze the copies number of the LpAQP.【Result】The potential open reading frame(ORF) of LpAQP(GenBank Accession No.JX569791) was 867 bp,encoding a protein of 288 amino acids with a predicted molecular mass of 30.7 kD.Bioinformatics analysis demonstrated that LpAQP exhibited a typical structure with an internal symmetry showing two highly conserved Asn-Pro-Ala(NPA) motifs and six membrane-spanning domains,and possessing the MIP family signal consensus sequence.The LpAQP amino acids showed high identity with other plant species PIP subfamily by NCBI homology comparison analysis.Phylogenetic analysis indicated that LpAQP was clustered with the HvAQP from Hordeum vulgare and TaAQP from Triticum aestivum.The result of transient expression showed that LpAQP gene was probably located in the membrane.Southern blotting analysis indicated that LpAQP was a single-copy gene in L.perenne L.Real-time PCR analysis showed higher expression of LpAQP gene was observed in shoot than other tissues.LpAQP expression level was up-regulated firstly,then down-regulated under drought stress.【Conclusion】 An aquaporin gene LpAQP was cloned by RACE from L.perenne L.This gene is regulated by drought stress.This study will provide important information for the future research on the gene-expression regulation during drought stress.