Identification Of SPX Gene Family In Wheat And Functional Analysis Of TaSPX3 In Phosphorus Regulatory Network

The gene Ta SPX3,which was strongly induced and upregulated by phosphorus stress,was screened by Affymatrix gene chip technique in our laboratory.The gene belongs to the plant SPX family which is closely related to phosphorus homeostasis regulation.It is very likely to play an important role in the mechanism of low phosphorus tolerance in wheat.In order to study the mechanism of this gene,the full-length c DNA sequence of Ta SPX3 gene was cloned from common wheat variety Zhengmai 9023.The SPX gene family of wheat was identified and analyzed by bioinformatics.The vector of overexpression of Ta SPX3 was constructed by using the model plant arabidopsis thaliana as a carrier to analysis gene function of Ta SPX3.The main results were as follows:1.Identification and evolutionary analysis of the whole genome of wheat SPX gene familyThe SPX gene was identified and classified in wheat by using the Hmmer-3.1b2 software accorinding to the information of Ensemble database and,Arabidopsis and Pfam database.Finally,64 wheat SPX genes were identified.the SPX gene family can be divided into 3 subfamilies,including SPX subfamily,SPX-EXS subfamily and SPX-MFS subfamily,respectively.A series of characteristic of SPX protein were analyzed by Expasy.The molecular weight range of wheat SPX protein is 8281.72~97182.09 k Da,and the isoelectric point was ranged from 5.19 to 10.17.The total average hydrophilicity of the protein was ranged from-0.967 to 0.201.Wolf Psort was used to predict the subcellular localization of SPX protein.The results showed that 12 wheat SPX genes were located in nucleus,42 wheat SPX genes were located in plasmids,6 were located in cytoplasm,only a few were located in chloroplasts,and 2 were present in both the nucleus and the plasmid.The predicted total length of Ta SPX3-6 gene was 789 bp,encoding 262 amino acids.The isoelectric point of the protein was 8.91 and the molecular weight of the protein was 29887.54.The Ta SPX3-6gene was positioned in the cytoplasm,with the total average of hydropathy value was-0.309.The predicted gene Ta SPX3-6 is same to Ta SPX3 which we will study in wheat.A phylogenetic tree was constructed for some of the related species SPX genes using the multi-sequence alignment generated by CLUSTALW and the neighbour-joining method with 1000 bootstrap replicates in MEGA software.It is confirmed that Ta SPX3 gene has the most recent evolutionary relationship with rice Os SPX3.These bioinformatics prediction and analysis can provide a theoretical basis for clarifying the function of Ta SPX3.2.Construction of overexpression vector of Ta SPX3 Gene and its transformation in Arabidopsis thalianaIn this study,the full-length c DNA sequence of wheat Ta SPX3 gene was cloned and transformed into Arabidopsis thaliana by means of Agrobacterium tumefaciens,using the modified p S1300 vector with GFP tag.The transgenic plants were obtained by hygromycin resistance screening and expression detection.T3 homozygous transgenic plants were obtained by the molecular identification and self-cross mating of generations.3.Physiological phenotypic detection and gene expression analysis of Ta SPX3 overexpressed arabidopsis thalianaWild type Arabidopsis thaliana and transgenic Arabidopsis thaliana were regulated by phosphorus stress at seedling stage and adult stage.And then,the physiological indicators of Arabidopsis thaliana were observed under conditions of phosphorus deficiency,low phosphorus(5 ?M)and high phosphorus(500 ?M).The results were as follows:(1)The leaf color of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana was significantly different at seedling stage.Under the condition of phosphorus deficiency and low phosphorus,the shoots of transgenic plants showed obvious green,while the wild type showed dark green.The number of lateral roots of transgenic plants decreased compared with the wild type.This indicated that overexpression of Ta SPX3 could alleviate the physiological effects of phosphorus deficiency stress in Arabidopsis thaliana at seedling stage.(2)There were great differences between transgenic Arabidopsis and wild type Arabidopsis in the growth process of adult plant.In the study of Arabidopsis thaliana at adult stage,almost all the transgenic plants were heading and flowering under the condition of low phosphorus and high phosphorus,while most of the wild type were not showed this symptoms.Under the condition of phosphorus deficiency,both transgenic and wild type plants were heading,but the growth of transgenic plants was better than that of wild type,which indicated that overexpression of Ta SPX3 could promote the growth of Arabidopsis under phosphorus deficiency stress.But under the condition of low phosphorus and high phosphorus,the effect of promoting biomass was not obvious,but all of them promoted the flowering earlier.The expression changes of phosphorous stress-responsive genes(At RNS1 At IPS1 At SPX1,At PAP2,At PAP17 and AT4)in Arabidopsis thaliana can be directly reflected through physiological phenotypes.4.Expression localization of Ta SPX3 Gene in transgenic Arabidopsis thalianaThe localization of Ta SPX3 gene in 7-days-old Arabidopsis thaliana under normal phosphorus were observed using confocal laser scanning microscopy.The transgenic plants carrying Ta SPX3 coding protein with green fluorescent protein(GFP)fusion protein were constructed in this study.The results showed that Ta SPX3 encoding proteins are mainly concentrated in the conduction tissue of the root apical meristematic region.,and its mechanism remains need to be further studied.In this experiment,the genome-wide identification and protein characteristic prediction of wheat Ta SPX family were studied according to biological information.The molecular characteristics of Ta SPX3 gene were clarified,and its physiological function,expression analysis and tissue location of transgenic plants were also performed by overexpression of the gene in Arabidopsis thaliana in this study.The results showed that Ta SPX3 was an important role in reducing phosphorus deficiency in plants and laid a foundation for elucidating the biological function of Ta SPX3.