Abstract:

Objective: This study aims to clarify the bioinformatics of HrWRKY53 gene and its role in sea-buckthorn resisting Rhagoletis batava obseuriosa(RBO), so as to lay a foundation for the analysis of insect resistance mechanism and molecular breeding of Elaeagnaceae such as Hippophae rhamnoides. Method: Based on the transcriptome data of sea-buckthorn fruits before and after RBO infestation, the key differentially expressed gene HrWRKY53 involved in the damage of sea-buckthorn were explored from members of HrWRKY gene family. Bioinformatics analysis was carried out to analyze the secondary structure and hydrophobicity and to predict the phosphorylation sites. In the NCBI website, the protein sequences with high homology were searched by BLASTP for sequence alignment, and the phylogenetic tree was constructed. qRT-PCR was used to detect the tissue expression specificity of the gene and the gene expression pattern before and after RBO infestation and under the treatment of abscisic acid (ABA), methyl jasmonate (MeJA) and ethylene (ET). The expression levels of HrJA2, HrLOX1, HrAOS and HrAOC in the JA signal pathway in response to the damage of RBO were analyzed, and their correlation with the expression of HrWRKY53 was was analyzed. Result: The open reading frame (ORF) of HrWRKY53 gene obtained from sea-buckthorn transcriptome was 1 302 bp, which encoded 433 amino acid residues. HrWRKY53 protein included two WRKY conserved domains and C2H2 zinc finger structures, and belonged to the family of type I WRKY transcription factors. The secondary structure prediction showed that HrWRKY53 was mainly composed of random curl and contained 141 threonine phosphorylation sites, which could be closely related to phosphorylation. The hydrophobicity prediction showed that the protein belonged to a hydrophobic protein. Phylogenetic tree analysis displayed that the gene had the highest homology with FvWRKY25. qRT-PCR showed that the gene had obvious tissue expression specificity, and its expression was the highest in sea-buckthorn fruit. The gene expression increased significantly under RBO infestation, mechanical injury, treatments with ABA, MeJA and ET, among which the expression increased most significantly after JA induction. In addition, the analysis of key differentially expressed genes involved in JA signal pathway based on transcriptome data and fluorescence quantitative data showed that the expression of HrJA2 gene in JA signal pathway increased most significantly, and there was a significant positive correlation between HrWRKY53 and HrJA2. Conclusion: HrWRKY53 of Hippophae rhamnoides belongs to the family of type I WRKY transcription factors and has the highest homology with FvWRKY25 of strawberry in Rosaceae. This gene is involved in the response to RBO and hormone-induced. The expression of HrJA2 can be induced to promote JA synthesis, and then increase the expresses of HrWRKY53 in sea-buckthorn, to further improve the ability of seabuckthorn to resist RBO.

Key words: Hippophae rhamnoides, RBO, HrWRKY transcription factor family, HrWRKY53, hormone treatment