Cloning And Functional Analysis Of PmSPX Family Genes In Pinus Massoniana

Pinus massoniana(Pinus massoniana Lamb.)is a coniferous gymnosperm native to tropical and subtropical areas of southern China.Because of its fast-growing and high-yield characteristics,it has become an important economic tree species in China.However,the main producing areas of Pinus massoniana(P.massoniana)are severely deficient in phosphorus.In the long-term evolution process,P.massoniana obtained a great ability of resistance to phosphorus deficiency conditions.Therefore,studying the adaptive mechanism of P.massoniana in response to phosphorus deficiency provides a theoretical basis for cultivating high-quality P.massoniana germplasm resources with low phosphorus tolerance.SPX-domain proteins play a vital role in phosphorus homeostasis and signal transduction.However,there were few reported on the general information of SPX gene in P.massoniana and its response to phosphorus deficiency.Based on the previous transcriptome data of P.massoniana seedlings under low phosphorus stress,the basic information of SPX family members was identified in this study,the tissue expression pattern of PmSPX subfamily genes was analyzed,and the expression and localization of PmSPX subfamily members were further verified by prokaryotic expression and subcellular localization.In addition,the promoter sequences of PmSPX subfamily genes were isolated by chromosome walking method,and their cis-acting elements were predicted.The regulating function of PmSPX subfamily genes in response to phosphorus deficiency was analyzed by yeast one-hybrid,luciferase detection and genetic transformation,which provided a new information for the study of PmSPX genes in maintaining plant phosphorus homeostasis.The main findings are as follows:1.Transcriptome-wide identification and expression of SPX domain-containing members of P.massoniana.In this study,we screened the transcriptome data of P.massoniana in response to phosphorus deprivation.Ten SPX domain-containing genes were identified.Based on the conserved domains,the P.massoniana SPX genes were divided into four different subfamilies:SPX,SPX-MFS,SPX-EXS,and SPX-RING.The PmSPX proteins consist of 267～820 amino acids(aa),and the corresponding molecular weights range from 30.413 to 93.081 k Da,and the isoelectric point(p I)value of these PmSPX proteins ranged from 5.07 to 9.40.The subcellular locations of 10 PmSPX proteins were predicted,including the nucleus and plasma membrane.All proteins in the SPX-MFS and SPX-EXS subfamilies were located in the plasma membrane,and all the members in the SPX and SPX-RING were located in the nucleus.In addition,we analyzed the expression profiles of all SPX genes in P.massoniana under phosphorus stress using RNA-seq analysis.The results showed that all members of the PmSPX family were differentially expressed,and the expression of the PmSPX subfamily genes was up-regulated,indicating that the PmSPX family genes may play a key role in the response of P.massoniana to phosphorus deficiency.2.Cloning,prokaryotic expression,subcellular localization and expression pattern analysis of P.massoniana PmSPX subfamilies genes.In order to further understand the tissue expression pattern of PmSPX subfamily genes,the expression of PmSPX subfamily genes in P.massoniana seedlings was analyzed in different tissues(roots,stem and leaf).It was found that the whole PmSPX subfamily genes were significantly induced under severe phosphorus deficiency,and the relative expression levels of PmSPX4 were less changed than those of PmSPX1 and PmSPX2.Under the condition of phosphorus deficiency,PmSPX subfamily genes showed different patterns of expression in different tissues of seedlings.It was noteworthy that with the extension of stress time,the relative expression level of PmSPX subfamily genes in leaves gradually decreased,while that of PmSPX1 in roots gradually increased.Combined with the second and third generation transcriptomic data,RT-PCR was used to clone and verify the sequence information of PmSPX subfamily genes.Induced expression of PmSPX2 protein in Escherichia coli was found to be normal and basically consistent with the predicted protein.In addition,the localization of PmSPX1,PmSPX2 and PmSPX4 protein was also verified,and it was found that they were all located in the nucleus.3.Promoter cloning and regulatory pattern analysis of PmSPX subfamily genes.The promoter sequences of PmSPX subfamily genes were successfully isolated by genome walking method,and cis-regulatory elements were predicted.The results showed that these cisacting elements were composed of light response-related elements,hormone-responsive elements,stress response-related elements,metabolism-related element and transcription factor binding site.In addition,several phosphorus response elements have been identified(e.g.,P1 BS PHO and W-box).By comparing the cis-acting elements of PmSPX subfamily gene promoters,the results showed that PmSPX1 and PmSPX2 genes both contained cis-acting elements(e.g.,P1 BS PHO and W-box)and were close to the core regulatory region of the promoter,while PmSPX4 gene contained P1 BS and PHO cis-acting element,but far from the promoter core regulatory region.Based on the PmSPX1 and PmSPX2 promoters containing W-box and P1 BS elements,yeast single hybridization experiments showed that Pm WRKY75 protein may bind to the W-box element on the PmSPX2 gene promoter,and dual luciferase qualitative analysis showed that Pm PHR2 protein may interact with the P1 BS element on the promoters of PmSPX1 and PmSPX2 genes.In addition,luciferase protein interaction experiments showed that Pm WRKY75 protein could bind with PmSPX1 and PmSPX2 proteins.Subcellular localization experiments indicated that Pm WRKY75 protein might be localized in the nucleus.4.Genetic transformation analysis of PmSPX subfamily genes in P.massoniana.The transgenic Arabidopsis plants with PmSPX2 were obtained by floral dip method.Tobacco plants transformed with PmSPX1 and PmSPX4 were successfully obtained by leaf disc method.The transgenic Arabidopsis was induced to express PmSPX2 under Pi stress,and the root structure of transgenic Arabidopsis was more developed than that of wild-type,and the total root length and total root surface area of transgenic Arabidopsis increased significantly.The expression of Pi transport related genes At PHO1 and At PHT1;1 were significantly inhibited from overexpression of PmSPX2 plant.These results suggested that PmSPX2 might act as a negative regulator of phosphorus signaling to maintain phosphorus homeostasis in plants.