Cloning and expression analysis of PwPEBP gene and promoter sequence in Picea wilsonii

ObjectiveThrough the research of PwPEBP gene and its promoter expression characteristic and biological function in Picea wilsonii, this paper aims to explore PEBP genes in plant growth and development in the process of response to adversity stress function and mechanism of action.

MethodIn this study, we had obtained the cDNA sequence of PwPEBP by Picea wilsonii transcriptome data. Based on the bioinformatics analysis of PwPEBP protein by TMHMM, GOR4 and other online software, the open reading frame (ORF) sequence of PwPEBP was cloned by PCR technology. The expression level of the gene in different tissues, different adversities and hormone treatments was analyzed by RT-qPCR. The promoter sequence of PwPEBP was cloned by chromosome step method, and the prediction of the basic promoter region, transcription starting point and the action element of the PwPEBP promoter sequence was carried out by the on-line software BDGP and PlantCARE. Finally, the function of PwPEBP promoter was transferred into tobacco leaves by agrobacterium-mediated method.

ResultPwPEBP cDNA was 1 408 bp with an open reading frame flanked (ORF) of 585 bp encoding 194 amino acids．The protein molecular formula of PwPEBP was C₉₆₆H_{1 484}N₂₅₀O₂₉₉S₆. The protein had no peptide and transmembrane domain. Hydrophobicity analysis showed that the hydrophobic sites of PwPEBP were uniformly distributed, suggesting that the protein was hydrophilic．25 phosphorylation sites were found with NetPhos. The phylogenetic tree analysis showed that the PwPEBP and PEBP of the Picea sitchensis were clustered into a new PEBP protein. The tissue-specific expression analysis showed that PwPEBP was all expressed in stem, root, mature needle, young needle, pollen and seed. However, the expression level of PwPEBP in mature needle was the highest and the lowest in young needle. The expression of PwPEBP under stress and hormone treatments showed that the expression of PwPEBP was induced, respectively, but not by the salt stress．The sequence length of PwPEBP promoter was 903 bp. The online analysis showed that it contained cis-acting elements such as GA, ABA, MeJA and SA．The GUS color reaction and Luc quantification experiment further showed that PwPEBP promoter sequences with cis elements could respond to GA, ABA, MeJA and SA hormones.

ConclusionPwPEBP gene in Picea wilsonii widely responds to abiotic stress such as drought, low temperature and high temperature, especially in drought. Meanwhile, PwPEBP is also involved in the signaling pathways of ABA, GA, MeJA and SA hormones.