Identification Of TaSPX Family In Wheat And Functional Analysis Of TaPHO1;H2 Gene Related To Low Phosphorus Tolerance

Wheat(Triticum aestivum L.)is an important grain crop in China,which often suffers from various abiotic stresses including nutrition during its growth and development.Phosphorus(P)is an important component of nucleic acid,ATP,carbohydrate and other life molecules,and it participates in photosynthesis,signal transduction,energy metabolism and other processes in plants.The content of inorganic phosphorus directly absorbed by plants in soil is low.Phosphorus deficiency is one of the important limiting factors for crop yield.Under phosphorus stress,plant roots are underdeveloped and grow slowly,affecting plant quality and yield.SPX family is a family of proteins containing SPX domains,which plays an important role in phosphorus signal transduction and maintaining phosphorus homeostasis.At present,the whole genome of SPX gene family has been identified in rice,Arabidopsis thaliana and other plants,but there are few systematic studies on wheat SPX gene family.In order to enrich and deepen people’s understanding of the molecular mechanism of phosphorus uptake in plants and the regulatory mechanism of protein family genes containing SPX domains.In this study,we obtained the protein Ta SPXs family genes containing SPX domains related to phosphorus transport,absorption and utilization through transcriptome analysis technology,identified and analyzed the family genes using wheat database and related analysis software,analyzed the functions of the family genes in wheat using bioinformatics methods,and verified the functions of some genes using q RT-PCR and transgenic methods.In order to further study the action mechanism of genes,we searched for the interacting proteins of Ta SPX family genes Ta PHO1;H2 and upstream regulatory transcription factors through the validation of yeast library screening.The main research results are as follows:(1)Transcriptome sequencing and differential gene analysis under phosphorus starvationThe transcriptome technology was used to analyze the expression of differential genes in wheat seedlings under phosphorus starvation stress,analyze the expression mode of differential expression genes,verify the accuracy of transcriptome data by correlation analysis,compare and annotate differential genes by GO and KEGG databases,and found wheat Ta SPXs family genes related to phosphorus transport,absorption and utilization in the transcriptome data,The transcriptional expression profile of the gene family in response to phosphorus starvation was obtained.(2)Identification and Analysis of the Whole Genome of Ta SPXs Gene FamilyThe wheat genome database was retrieved using the hidden Markov model of the SPX domain,and 47 wheat SPXs genes,namely Ta SPXs family genes,were identified through screening.Phylogenetic analysis of the genes in this family shows that the Ta SPXs gene family can be divided into four subtypes(SPX,MFS,EXS,RING)and six clusters(I,II,III,IV,V,VI).Analyzing the distribution of the family genes in the genome,it is found that the family genes are basically evenly distributed in the A genome(16),B genome(13),and D genome(17),and the distribution in the genome is uneven.Among them,6D,7A,and7 D chromosomes have the most mapped genes,namely 5,7,and 6,respectively.No SPX family genes are found in chromosome 1 and 3.The analysis of the colinear relationship of genes in this family shows that most homologous genes have colinear relationship,and a few genes have colinear relationship in different chromosomal groups,indicating that the gene family may have chromosome translocation in the evolutionary process,and the loss of homologous gene fragments and gene loss occur at the same time.Gene structure and conservative motif analysis showed that Ta SPXs gene contained 2-15 exons and 1-14 introns.The 25 motifs were unevenly distributed in four subfamilies.The motif structure and number of the coding proteins of the same subfamily gene were similar.Using wheat RNA seq database to analyze the tissue expression of this family gene,the expression pattern belongs to constitutive expression.(3)Analysis of Ta SPXs gene expression pattern under phosphorus starvation stressThe expression level of Ta SPXs gene under phosphorus starvation stress was studied by fluorescent quantitative PCR.A total of 4 Ta SPXs genes were selected from each subfamily for analysis.The results showed that the expression patterns of these genes could be divided into two types.The expression levels of four Ta SPXs genes were up-regulated.After 7 days of phosphorus starvation treatment,normal treatment was carried out for 3 days.It was found that except for Ta SPX1-7D gene,other family genes were slightly up-regulated after normal phosphorus treatment,while Ta SPX1-7D gene was rapidly down-regulated to the pre stress expression level.The expression of Ta SPX1-7D gene was the highest in roots after 7 days of phosphorus deficiency stress.(4)Subcellular localization of Ta SPXs genes and Analysis of expression patterns in the promoter of the Ta SPX family gene Ta PHO1;H2Construction of fusion expression vectors Ta SPX1-7D-GFP and Ta PHO1;H2-GFP and Ta PHO1;H10-GFP was transformed into Agrobacterium tumefaciens by freeze-thaw method and transformed into Nicotiana benthamiana respectively.The subcellular location of protein expression in cells was observed under confocal microscope.The results showed that Ta SPX1-7D-GFP encoded protein was expressed in the nucleus,whereas Ta PHO1;H2-GFP and Ta PHO1;H10-GFP encoded protein are expressed on the cell membrane.The Ta PHO1;H2-p1304 recombinant plasmid was constructed,the upstream promoter of Ta PHO1;H2 was transformed into Arabidopsis,and different periods of Arabidopsis were stained using the gus staining kit to observe the expression site of the gene under a body microscope.The results showed significant Gus staining in the xylem vessels at the seedling stage in transgenic Arabidopsis,and also in veins;Color responses were observed in roots,rosette leaves,stem leaves,and inflorescences at the mature stage.(5)Functional Verification of Ta PHO1;H2 Gene of Ta SPX Family in Yeast MutantsThe growth of yeast mutant MB192 was inhibited in a low phosphorus environment due to the lack of a high affinity phosphorus transporter gene PHO84.The recombinant plasmid Ta PHO1;H2-p416 was transformed into yeast strain MB192.To a certain extent,the results showed that the MB192 strain that transformed Ta PHO1;H2-p416 could supplement the phosphorus transport capacity of yeast mutant MB192 on the solid medium with YNB(p H=6.0)of different Pi(0.02,0.06,0.1 m M K2HPO4)concentrations.This indicates that Ta PHO1;H2 gene is able to mediate phosphate transport in yeast.(6)Functional validation of the Ta SPX family gene Ta PHO1;H2 in ArabidopsisThe Ta PHO1;H2-p1300 recombinant plasmid was constructed,the Ta PHO1;H2 gene was transformed into Arabidopsis,and the overexpression lines were cultured with wt on MS medium with different phosphorus concentrations to study the function of this gene on phosphorus uptake in plants.The results showed that at phosphorus concentrations of 0 and 5μM,the root length and fresh weight of Ta PHO1;H2 overexpressing Arabidopsis plants were significantly increased compared with those of the wild-type Arabidopsis lines under m conditions.Meanwhile the Ta PHO1;H2 transgenic line also had more lateral roots and more obvious root hairs compared on lateral roots versus root hairs.(7)Screening and validation of the Ta PHO1;H2 interactor of Ta SPX family genesTa PHO1;H2-p BT3-N recombinant plasmids were constructed,and proteins Ta IAA14,Ta TPT1,Ta FBA1,Ta PRP1 which interact with Ta PHO1;H2 were selected by yeast two hybrid analysis.The interaction of the screened proteins with the bait proteins was verified by yeast one-to-one hybrid assay and dual luciferase complementation assay.The results showed that in the yeast one-to-one validation,NMY51 yeast harboring p BT3-N-Bait plasmid and Prey-p PR3-N plasmid were able to grow normally on the medium of SD/-Trp/-Leu/-His and SD/-Trp/-Leu/-His/-Ade.In dual luciferase complementation assay,there was obvious Luc signal in the injected area of the test group,and no fluorescence signal was detected in other negative control areas.These results indicated that the protein Ta PHO1;H2 interacted with the proteins Ta IAA14,Ta TPT1,Ta FBA1 and Ta PRP1.(8)Screening and validation of upstream regulators of the Ta SPX family gene Ta PHO1;H2Construction of p Ab Ai recombinant plasmids with the upstream promoter of Ta PHO1;H2,the upstream regulatory transcription factor of the gene Ta NAM21 and Ta DOF5.3 were obtained by yeast one hybrid screening.The regulatory relationship between transcription factors and promoters was verified by yeast one-to-one assays.For the yeast one-to-one assay,Y1 H yeast harboring the bait pabai plasmid and the prey ad plasmid were able to grow on the suppression deficient medium of SD/-Leu + ABA400.