Cloning And Function Verification Of A Phosphate Transporter Gene CmPT1 In Cut Chrysanthemum

Cut chrysanthemum(Chrysanthemum morifolium Ramat.)is one of the most important ornamental plants in cut flower market all over the world.Phosphorus(P)is a principal limited factor for the growth of cut chrysanthemum,P deficiency finally effects the quality and ornamental value seriously of cut flower.At the same time,more than 90%of the added fertilizer P may rapidly be transformed to P forms that are not easily available to plants.Developing cultivars with tolerance to phosphorus deficiency has become main direction of crop breeding.Based on the above reasons,we conducted the cloning and function verification of phosphate transporter gene in cut chrysanthemum,so as to lay a foundation for cultivating high phosphate use efficiency cultivars of chrysanthemum using the way of genetic engineering.The main results are summarized as follows:1.Evaluation on tolerance to P deficiency of 32 cultivars of cut chrysanthemum.To assess genotypic variation of cut chrysanthemum for tolerance to phosphorus deficiency,32 cultivars of cut chrysanthemum were screened and evaluated for tolerance to low phosphorus.The results showed that there existed evident genotype differences in different cultivars of cut chrysanthemum in tolerant ability to low phosphorus stress.Among all the characters studied,relative plant dry weight(low phosphorus supply/normal phosphorus supply),relative phosphorus content and relative phosphorus accumulation demonstrated significant genotypic variation(the CV was 12.14%,20.99%and 26.41%,respectively).Moreover,correlation analysis showed that there were significant positive correlations between relative plant dry weight and relative phosphorus content,between relative plant dry weight and relative phosphorus accumulation,and between relative phosphorus content and relative phosphorus accumulation(P<0.01),the correlative coefficients are 0.3067,0.7391 and 0.8258 respectively.Therefore,relative plant dry weight,relative phosphorus content and relative phosphorus accumulation were suggested as screening indexes of cut chrysanthemum on tolerance to low phosphorus stress.A comprehensive evaluation of low phosphorus tolerance of 32 cultivars was made by using hierarchical clustering analysis.Clustering analysis showed that 32 cultivars could be divided into extremely low phosphorus tolerant,low phosphorus tolerant,moderately low phosphorus tolerant,low phosphorus sensitive and extremely low phosphorus sensitive group respectively.Among 32 tested materials,the tolerant ability to low phosphorus stress of ’Nannongyinshan’ is higher than other cultivars,belonging to extremely low phosphorus tolerant cultivar;the tolerant ability to low phosphorus stress of ’Nannonghongfeng’,’Nannongxiangbin’ and ’Youxiang’ are lower than other cultivars,these cultivars are extremely low phosphorus sensitive genotypes;the others are moderately low phosphorus tolerant cultivars.Efficient genotypes with the desirable characteristics can be used directly in advance field trials or in breeding programs to cope P deficiency.2.Cloning of a phosphate transporter geneIn this study,a Phtl homologue,named CmPTl,was cloned from ’Nannongyinshan’,a low P tolerant cultivar of cut chrysanthemum,using a strategy based on the rapid amplification of cDNA ends(RACE).The cloned full-length cDNA of CmPT1 was a 1875-bp sequence,consisting of an ORF of 1596 bp,a 5’-UTR of 87 bp and a 3’-UTR of 192 bp.The encoded peptides of the CmPTl gene belongs to a family of 12 transmembrane domain proteins and shows significant sequence identity to known high-affinity Pi transporters.The Phtl signature GGDYPLSATIxSE was discovered in the sequence of CmPT1,therefore,CmPT1 is a Phtl transporter.3.Functional and Biochemical Analysis in YeastCmPTl was able to complement the PH084-deficient yeast strain MB192 in the high-affnnity concentratio range.The CmPT1 transporting 32Pi velocities after subtracting the Pi was mediated following the Michaelis-Menten kinetics equation.The apparent mean Km value for Pi transport of CmPTl was 35.2μM Pi,as determined by three independent experiments.The complement assay suggests that CmPTl has a high Pi affinity,mediating Pi uptake in the micromolar range.4.Expression pattern of CmPTlTo examine the expression response of CmPT1 to Pi supply status,real-time quantitative PCR(qRT-PCR)analysis was used to detect the expression pattern of CmPTl.Our results showed that the CmPTl transcript is primarily in roots,weakly expressed in stems,not detected in leaves under both Pi-sufficient(300μM Pi)and Pi-deficient(0μM Pi)conditions.The CmPT1 expression levels increased greatly in Pi-starved roots.5.Overexpression of the CmPT1 gene in chrysanthemum enhances the adaptability to Pi-deficient stressThe plant height,root volume,dry weight and total P concentration of the transgenic chrysanthemum plants that over-express the CmPT1 gene were significantly increased compared with the wild type plants in Pi-deficient condition.The CmPT1 gene may participate in the Pi-starvation signal transduction pathways,Overexpression of the CmPT1 gene directly or indirectly caused Pi-dependent root architecture alteration with enhanced root elongation and proliferated lateral root growth.In summary,CmPT1 encodes a typical,root-expressed,high affinity phosphate transporter in chrysanthemum.Taking the flexible expression pattern into account,CmPT1 might function in a wide range of Pi environments and play a significant role in phosphate acquisition under natural conditions.