Cloning And Functional Analysis Of Almond Dehydrin Gene AcDHN1

Almond grows primarily in Nanjiang Areas, and periodic freeze injury has a great impact on the almond's industry, so cold resistance mechanism of Almond catch more attention all the time. Dehydrin is a prime gene associated with embryonic development and resistance, which has a significant role in abiotic stress, but there is no relevant reports about Almond dehydrin gene. In this study, we cloned Ac DHN1 genes from Xinjiang Almond cultivation, and make equence analysis and functional verification on it. The main results of this study are as follows:1. Cloning and Sequence Analysis of AcDHN1: The AcDHN1 gene was with an open reading frame ORF) of 924 bp and(GenBank accession number: KT949395) cloned from Xinjiang Almond 'ZhiPi'.Bioinformatics analysis showed that the sequence with protein molecular weight of 32.4 kD encod 308 amino acids, signal peptide prediction showed that it does not contain signal peptide and has no transmembrane domain, subcellular localization in the nucleus. Structural analysis showed that the sequence had two segments of Y, four K fragments, which belong to Y2 Kn type. The amino acid sequence results showed, K segment was high consistency between almond AcDHN1 and other dehydrin.Phylogenetic analysis showed that AcDHN1 has highest similarity with Prunus persica, and was close with Malus domestica and Pyrus bretschneideri.2. Expression Analysis: Quantitative PCR analysis showed that AcDHN1 was expressed at low temperatures, salt and drought stress, and induced stronger by the first one.3. Subcellular localization analysis: Construction of expression vector p1304-AcDHN1 and infection onion epidermal with, Agrobacterium. The subcellular localization analysis of AcDHN1 protein showed that green fluorescent protein can be detected in cell nucleus of onion epidermal, indicating Ac DHN1 located in the nucleus.4. Prokaryotic expression analysis: The prokaryotic expression vector was constructed and expressed a protein of approximately 52 kD in E. coli(DE3). The results showed that the optimized induction conditions of recombinant protein pET-AcDHN1 in IPTG concentration was 0.1 mmol/L, 3 h.5. Transgenic functional verification: Build pCAMBIA1303 vector and transformed into tobacco by Agrobacterium-mediated method. Kan confirmed and PCR detection showed AcDHN1 gene has been successfully transferred into tobacco. The physiological and biochemical indexes of transgenic tobacco and control tobacco under different stress conditions were determined. The results showed that the overexpression of AcDHN1 gene enhanced the tolerance to abiotic stress tolerance in tobacco.