Cloning And Expression Analysis Of Ectomycorrhizal Pht1 Phosphate Transporter Gene In Pinus Massoniana

Phosphorus(P)is one of the most important macronutrients in growth and development of plants.The deficiency of soil available phosphorus is a major factor restricting forestry production in global forestry.In nature,many trees species form symbiotic associations with ECM fungi.This mutualism improves the plants’ efficiency for uptake of mineral nutrients,particularly phosphates,and in return fungi obtain organic carbon from the plant to complete the fungal life cycle.Masson pine(Pinus massoniana Lamb.),a gymnosperm genus conifer native to southern China,has been one of the most economically and ecologically important forest trees.Masson pine is grown mainly in the subtropical and tropical regions,many of which are extraordinary serious lacked Pi in soils,and it is also one of the main limitations to the productivity of masson pine in this area.It’s atypical ectomycorrhizal tree species,ectomycorrhizal fungi contribute tremendously to inorganic phosphate uptake in plants,however,the mechanism underlying this phenomenon has not yet been fully described.The whole process of genetic information regulation and transduction involved the genetic code transcription and translation and the correlative protein function of the phosphate transporters in masson pine during ectomycorrhizal symbiosis are not understood.Thus,explanation of the molecular regulation mechanism of phosphate up take,translocation and utilization under low-phosphorus stress,and validation of key phosphate transporter genes has great significance to select and cultivate new germplasm with high potency of phosphorus utility.Currently,the potted seedlings incoculated by Pisolithus tinctorius and Boletus edulis were used as trial materials.The influence in the germination rate,the growth and physiological parameters of ectomycorrhizal symbiosis were investigated as subjected to different low-P stresses.Also,four PmPTs were cloned RACE methods,subsequently,their expression patterns were characterized by quantitative PCR technology.In addition,PmPT1 was genetically transferred into tobacco via Agrobacterium-mediated method to further validate its function in phosphorus absorption.The main results were as follows.1)As inoculated by ectomycorrhizal fungus,the seedling survival rate,plant height,stem diameter,dry weight and root/shoot ratio were obviously increased.Also,the root activity,main root length,total root length,number of lateral root,as well as root surface area were significantly elevated in comparison with that of the inoculation-free(NECM).However,ectomycorrhizal fungus gave no statistically remarkable effect on the seed germination.Further,mantle and intracortical hartig net were observed from the ectomycorrhizal roots,the ratio of cortex/cross-section of the ectomycorrhizal roots were substantially higher than those of the control,and the plant height and stem diameter were positively correlated with the ratio of mantle/cross-section of the ectomycorrhizal roots.The content of MDA decreased,in addition,the antioxidase activity of CAT,SOD,POD and GSH-PX heightened and the poisonous effect of active oxygen and free radical on plants of masson pines could be alleviated by ECM.As exposed to low-P stress,the ectomycorrhizal fungal inoculation helped young plants produce more gibberellins.The contents of soluble sugar and soluble protein of ECM plants was significantly higher than that of the non-ECM seedlings(NECM),indicating the enhanced tolerance at least partially correlated with osmotic solute.ECM demonstrated the remarkably higher chlorophyll content in leaves.2)Four members of phosphate transporter protein family genes(PmPTs,GenBank accession: AMR43649.1,AMR43650.1,AMR43651.1 and AMR43652.1)were cloned by rapid amplification of cDNA ends(RACE).The full-length cDNA sequences of PmPT1,2,3 and 4 were 2,388 bp,2,053 bp,2,315 bp and 2,108 bp,and the corresponding lengths of open reading frames(ORF)were 1,647 bp,1,647 bp,1,608 bp and 1,608 bp,which contained 548,548,535 and 535 amino acid residues,respectively.PmPT1,2,3 and 4 containing the typical domain of the Pi:H+ symporter(PHS-transporter),which owned a typical structure of MFS family members.The bioinformatics analysis implied that these PmPTs were highly similar to phosphate transporters from other species at amino acid level with over 74 %-78 % identities.Multiple sequence alignment showed that the four PmPT proteins carried a 13-amino-acid consensus sequence(GGDYPLSATIMSE)containing the Pht1 signature region.Phylogenetic analysis indicated that they might be obviously divided into gymnosperms clade and demonstrated high homology with dicotyledons,reflecting the considerably strict conservation during the evolutionary process.PmPT1,2,3 and 4 had non signal peptide and transmembrane domain,thus,it was presumably localized in plasma membrane,as well as the polypeptides were characterized by high hydropathicity and contained 12 putative intra-membrane regions and 1 cytoplasmic loop.3)The RT-PCR and Real-Time PCR were employed to quantified the expression of PmPTs in leaf,stem and root after treatments with different P concentrations in the presence or absence of ECM colonization.The temporal expressions of PmPTs were measured in plants colonized by ECM fungi,compared with the 1st day,the expression levels of PmPTs in ECM-colonized plants increased slightly on the 6th to the 24 th days and these levels were still higher than those of the control.After the 24 th day,the expression levels of PmPTs were obvious up-regulated in ECM colonized roots.The expression of PmPT1,2,3 and 4 might be considerably induced by the ectomycorrhizal fungi(ECM),which were expressed in leaf,stem and root.Their expression levels of leaf were significantly higher than that of stem and root in ECM-free plants(leaf ? root ? stem),however,the high expression of PmPT1,2,3 and 4 in ECM-inoculated roots were investigated(root ? leaf ? stem).Additionally,expressions of these PmPT members shared a similar pattern and might be intensively activated by low-P stress or inhibited under P excess.Moreover,total P contents in ECM-colonized plants were higher than that of NECM plants under low-P condition.We inferred that PmPTs were differentially expressed in roots to efficiently transport soil P,and that the total plant P uptake might depend on the expression regulation of P transporter genes,whose expression might be mediated by the integrated signals from ECM colonization and P availability in soil.Detailed synthetic analyses suggested that the ECM-triggered growth enhancement in masson pine might be at least partially ascribed to the up-regulations of PmPTs expression,especially under low-P status.4)The plant expression vector pSH-35S-PmPT1 was constructed and then was transferred into tabacco(Nicotiana tabacum)through leaf disc method transformation.A total of 31 transgenic lines were obtained and T0 generation seeds were harvested.T1 generation plants(Line-N1 and Line-N2)were verified to be transcriptionally over-expressed as detected by RT-PCR and qRT-PCR methodology as subjected to different P concentrations.Under low-P condition,the expression levels of exogenous gene in roots were significantly higher than that of the wild type.The overexpression of PmPT1 modified the pattems of the host Pht1 phosphate transporter genes expression which further enhanced the Pi deficiency tolerance of transgenic tobacco caused by PmPT1 and the phosphate transporters itself,thus,PmPT1 and two members(NtPT1 and NtPT2)of tobacco Pht1 phosphate transporter protein family had synergistic effects on the phosphorus uptake and translocation.Under phosphorus deficiency,the phosphorus contents of root and shoot increased by33.3 % and 25.5 %,respectively in Line-N1,and 30.7 % and 23.9 %,respectively in Line-N2.The total contents of inorganic phosphorus in Line-N1 and Line-N2 were 1.75-and 1.73-fold higer than that of the wild type.Under low-phosphorus status,the dry matter,content of chlorophyΙΙ,soluble sugar,soluble protein and enzyme and activity of POD,SOD,CAT were significantly elevated,conversely,the content of MDA decreased as compared to wild type.The results suggested that overexpression of PmPT1 improved the low-P tolerance of tobacco plants.The available evidences per se facilitated the understanding of ECM role in promoting masson pine growth,which as least partially ascribed to the up-regulation of PmPT1.The PmPTs provided the basis for the creation of germplasms with high tolerance to low-phosphorusstress.