Cloning And Function Analysis Of Phosphorus Transport-related Transcription Factor PmWRKY164 In Pinus Massoniana

Phosphorus?P?is a necessary nutrient element for plant growth and development.The deficiency of available P in tropical and subtropical soils is thus one of the most important limited factors to agricultural and forestry production.Thus,cloning and characterization of genes related to the low-P tolerance is an efficient approaches to solve the above problem via creating germplasm with high efficiency of P utility.Masson pine?Pinus massoniana Lamb.?,a gymnosperm genus conifer native species to southern China,is one of the most economically important forestry trees,which can be widely used for timber,pulp and resin production.However,the yields have been highly affected by the deficiency of available P in the soil of southern China.Therefore,it is essential to screening the key genes in response to low P stress,as well as to reveal the molecular regulation mechanisms of tolerance to low P,which will provide a theoretical basis for the molecular breeding and create novel germplasms with high quality and stress resistanceBased on the previous work,currently,a masson pine germplasm,which showed high tolerance to low P stress,was used as material,and the PmWRKYs in response to low phosphorus stress were preliminarily screened by RNA-Seq and quantitative PCR.Subsequently,the full-length sequence of a PmWRKY164 in response to low P stress was cloned by RACE strategy.To putatively identify the PmWRKY164 involving in low P stress,the temporal and spatial expression pattern of PmWRKY164 in different tissues were systematically established using qPCR analysis.Futher,the expression vector of PmWRKY164 was successfully constructed and identified by restriction enzyme digestion reactions,and the pBWA?V?HS-PmWRKY164 expression vector was introduced into tobacco through Agrobacterium-mediated procedure.The main contents and results in present study are as follows:1.With the combination of RT-PCR and real-time qPCR,the expression patterns of four WRKY transcription factors were characterized in response to low P₁ stress?Phosphorus content of 0.5 mg/L?.The expression of WRKY164 was persistantly up-regulated,thus it was selected to clone.2.The full-length sequence of WRKY164 was cloned by RACE strategy,which named as PmWRKY164.The sequence analysis showed that the total length of PmWRKY164 was 1,977 bp,containing 65 bp of 5'-UTR region and 748 bp of3'-UTR region,and the open reading frame?ORF?was 1,164 bp,encoding 387 amino acids.3.Bioinformatic analysis revealed that PmWRKY164 belonged to Group?b of WRKY family,which contained a single WRKYGQK conserved domain and C₂H₂(C-X_4-5-C-X₂₃-HXH)zinc-finger motif,and the homology analysis showed that PmWRKY164 had higher similarity with other plants in the conservative region of the WRKY family.Phylogenetic analysis demonstrated that PmWRKY164 was mostlyclose to the WRKY transcription factor of Taxus wallichiana var.chinensis and Picea abies.4.Tissue specific expression analysis showed that PmWRKY164 was a constitutive expression gene and expressed in root,stem and leaf,among which leaf showed the highest expression level,followed by root,and stem showed the lowest expression.In root and stem,PmWRKY164 showed the lowest and highest expression at the 36 d and 60 d after treatment,respectively;in leaf,PmWRKY164 demonstrated the lowest expression at the 12 d and the highest expression at the 60 d after the stress.Except for the increasing expression of leaves,the expression patterns of stem and root characterized in a up-down-up trend.5.Over-expression vector pBWA?V?HS-PmWRKY164 was constructed,subsequently,it was genetically transformed into tobacco“K326”via Agrobacterium-mediated method.Totally,20 transgenic lines were obtained by molecular detection.After qRT-PCR detection,three transgenic tobacco lines?Line-2,Line-3 and Line-8?with high expression level were selected for further experiments.6.The transgenic and wild tobacco were were subjected to different concentrations of low-phosphorus stresses.Except that the content of MDA,the content of phosphorus,Apase activity,POD activity and SOD activity in transgenic tobacco were significantly higher in comparison with those of the wild type.7.PmWRKY164-overexpressing lines had higher shoot Pi contents compared with wild-type plants under Pi-deficient condition;however under Pi-sufficient condition,there was no obvious difference between PmWRKY164-overexpressing lines and wild-type plants,indicating PmWRKY164 has a significant effect on PHO1in tabacco,which may promote PHO1 expression by positive regulation,thus promoting the plant's phosphorus absorption capacity and improving plant tolerance to low-phosphorus stress.