Abstract：WRKY transcription factors play important roles in various stress response. However, the phosphorus (P) deficiency in the tropical and subtropical forest soils has severely posed challenges for the productivity. Therefore, screening the related low-P stress genes, as well as to reveal the molecular regulation mechanisms of low phosphorus tolerance were necessary, which will provide a theoretical basis for the molecular breeding and create novel germplasms with high quality and stress resistance. WRKY transcription factors (TFs), a plant-specific TF family, play important and unique roles in biological regulations involving in stress defenses, development, and metabolite synthesis, however, little has been known about their roles in response to phosphorus starvation in masson pine (Pinus massoniana). To further understand their roles in low phosphorus (P) stress, currently, the full-length sequence of PmWRKY164 was cloned by rapid amplification of cDNA ends (RACE) methodology. Homologous analysis, multiple alignments, as well as related bioinformatics analysis were performed. Quantitative real-time PCR (qRT-PCR) was used to detect the temporal and spatial expression patterns of PmWRKY164. Finally, the pBWA(V)HS-PmWRKY164 expression vector was introduced into tobacco through Agrobacterium-mediated procedure. The result showed that PmWRKY164 (GenBank No. MH579749) was obtained, whose full-length cDNA was 1 977 bp and the corresponding lengths of open reading frames (ORF) was 1 164 bp, which encoded 387 amino acids, including a single WRKYGQK conserved domain and C2H2 (C-X4-5-C-X23-HXH) zinc-finger motif and belonged to GroupⅡb of WRKY family. Homology analysis showed that PmWRKY164 had higher similarity with other plants in the conservative region of the WRKY family. Phylogenetic analysis revealed that PmWRKY164 was mostly close to the WRKY transcription factor of Taxus wallichiana var. chinensis and Picea abies. Spatiotemporal expression analysis showed that PmWRKY164 constitutively expressed in root, stem and leaf, with the highest expression in leaf, followed by root and lowest in stem. In root and stem, PmWRKY164 showed the lowest and highest expression at the 36 d and 60 d after treatment, respectively; in leaf, the expression level was the highest at 60 d and the lowest at 12 d after the stress. Except that the expression level in leaves showed an upward trend, the expression levels of roots and stems showed a rising-decreasing-increasing trend. Further, twenty transgenic tobacco plants were obtained using the A. tumefaciens mediated. 3 different transgenic lines, i.e. Line-2, Line-3 and Line-8 as well as the wild-type lines, were subjected to different concentrations of low-P stresses. Except that the content of malondialdehyde (MDA), the content of phosphorus, Apase activity, peroxidase (POD) activity and superoxide dismutase (SOD) activity in transgenic tobacco were significantly higher in comparison with those of the wild type. This study provided new information and ideas for further exploring the function of PmWRKY164 and would provide a theoretical basis for the molecular breeding in prolonging of phosphorus stress of P. massoniana.
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