Genetic Transformation Analyses And Functional Identification Of Tomato Phosphate Transporter SlMPT3;1 Gene In Rice

Phosphorus usually plays an important role in involving in various metabolic and biochemical processes in plants.Phosphorus deficiency in soils delays the plant growth and leads a decrease in yield of crops.The lack of phosphorus resources and overuse of phosphate fertilizer lead to a truth that scientists have to make more effort in cultivating crop varieties with highly efficient absorption of phosphorus.Therefore,the study of screening and validation of high efficiency phosphate absorption and utilization of phosphorus transporter genes has become an effective way to cultivate high-efficiency phosphorus absorption and utilization of crop varieties.Previous studies have found that mitochondrial phosphorus transporters play a role in energy regulation.The purpose of this study is to better understand the molecular regulatory mechanisms of the target SIMPT3;1 gene in promoting phosphate uptake under phosphorus starvation.In this study,the SIMPT3;1 gene was isolated from a cDNA library of tomato seedlings.Bioinformatic analysis showed that the SIMPT3;1 protein has two conserved domains of Mito-carri superfamily containing 6 trans-mebrane motifs,suggesting that the SlMPT3;1 gene belongs to a member of the MPT family.Subcellular localization certified that the SIMPT3;1 gene was only expressed in the mitochondria,and has a typical characteristics of mitochondrial phosphate transporters in the MPT family.Functional complementatry test showed that the SIMPT3;1 target protein could compensate the function in the uptake and transport of phosphate in yeast strain.Using the agrobacterium-mediated method,we constructed a construct overexpressing the PCAMBIA1304::SlMPT3;1 fusion gene and transformed this construct into the rice mature embryo callus.A totoal of 18 positive transgenic seedlings were obtained by resistance screening and PCR detection,and three homozygous lines were confirmed through continuously screening.GUS staining analysis revealed that the SIMPT3;1 gene was strongly expressed in the root epidermis and xylem.Yeast two-hybrid experiment screening preliminarily confirmed that the target SIMPT3;1 protein interacts with the SlGPI protein;BiFC experiment verified that this interaction is located to the protoplasmic membrane.Tissue culture results showed that total root length,projected area and surface area significantly increased more than those in the receptor,respectively which indicated that SIMPT3.1 gene maybe promotes root development by improving the acidic rhizosphere environment of rice.A hydroponic experiment showed that the heights of the transgenic rice notably increased comparing with that of the receptor under low phosphorus supply.The chlorophyll contents,soluble sugar contents,proline contents and the contents of phosphate in the transgenic lines were obviously higher than those in the receptor.Transcriptome sequencing showed that the expressions of partial transcription factors and genes associated with phosphate uptake were significantly upregulated in transgenic lines and receptor under low phosphate condition.The qRT-PCR confirmed that the expression patterns of these DEGs have similar trends to the results in transcriptome sequencing analyses.Biomass measurements showed that both the grain number and the grain yields in three transgenic lines were significantly increased compared with the receptor under low phosphate condition..This study shows that the SlMPT3;1 gene belongs to mitochondrial phosphate transporter,and is induced by low phosphate,and also involves in the phosphate transport pathway by up-regulating or down-regulating of the partial differentially expressed genes,thus promoting rice growth and enhancing grain yield.The data indicates that the SlMPT3;1 gene is an efficient candidate and provides a material support for molecular breeding of crops.