Differential Expression Profile Analysis Of Soybean Efficiency And Functional Verification Of Phosphorus Efficiency Related Genes

Phosphorus was one of the essential mineral elements required for plant growth and development.However,phosphorus easily reacted with iron ion and aluminium ion of the soil,which reduced the concentration of soluble phosphorus in soil.About 30%-40%of arable land in the world was facing a lack of available phosphorus.As an important cash crop,soybean[Glycine Max L.Merr.]played an important role in our national life.Soil phosphorus deficiency had become an important factor limiting soybean production in China.Under this situation,it was particularly important to dig the genes related to phosphorus efficiency in soybean,analyze the molecular mechanism of phosphorus metabolism and breed phosphorus efficient soybean varieties.In this study,we analyzed the expression profiles of a high-phosphorus-efficient soybean material C112 and a low-phosphorus-efficient material C73 treated with different phosphorus concentrations.Then a candidate gene GmGER12 with possible high phosphorus efficiency was selected for functional analysis.In addition,two PHR genes GmPHRa(Phosphate Response a)and GmPHRb(Phosphate Response b)homologous to AtPHR1 were cloned,and their roles in phosphorus metabolism in soybean were further expounded.The main research contents and results were as follows:With hydroponic experiments under different P treated conditions of 219 soybean accessions,we identified a low-P-efficient soybean accession C112 and a low-P-efficient accession C73.To further analyze expression changes of genes in genome level under low phosphorus condition,microarray experiments were performed on roots and leaves of C112 and C73 grown under different phosphorus concentrations for 10 days.257 up-regulated differentially expressed genes(DEGs)and 41 down-regulated DEGs were identified in the roots of C112.Only 11 up-regulated DEGs were found in the leaves of C112,and 3 and 7 down-regulated genes were found in the roots and leaves of C73,respectively.GO analysis showed the 317 DEGs were significantly correlated with wounding,response to jasmonic acid stimulus,oxidoreductase activity and cell wall.KEGG analysis enriched three significant metabolic pathways:phenylpropane biosynthesis,methane metabolism and phenylalanine metabolism.At the same time,we screened DEGs between different materials and different tissues,then 195 DEGs and 253 DEGs induced by low phosphorus stress were identified,respectively.Through comparative analysis,42 genes were identified to be closely related to the low phosphorus tolerance in soybean.According to the location of DEGs and the results of GO and KEGG enrichment analysis,we concluded that roots played a crucial role in P metabolism during seedling stage,massive genes involved in response to low-P stress in high-P-efficient accession.The responses of soybean to low-P stress were complex cross-talks;it not only depended on other nutrition(iron)and plant hormones levels(ethylene and jasmonic acid),but also on the metabolism of secondary metabolites.Roots played an important role in the adaptive response of soybean to low phosphorus stress.We selected GmGER12(Glyma20g36320)from 257 up-regulated DEGs in roots of C112 identified by gene expression profiles for subsequent functional analysis.The genome of the gene was 1062bp in length.It contained two exons and one intron.The full CDS length was 669bp,and encoded 222 amino acid residues.The gene was highly expressed in roots and nodules,especially in roots,and it was induced by low phosphorus stress.With the prolongation of phosphorus stress treatment time,its expression level in C112 showed an upward trend.Subcellular localization showed that the gene was mainly located in the cell membrane and a small amount in the cytoplasm.In order to verify function of GmGER12,we constructed plant overexpression vector pMDC83-GmGER12 and transferred it into wild Arabidopsis and soybean hairy roots.The length and number of root hairs both increased in transgenic Arabidopsis under low phosphorus condition.Then in transgenic soybean hairy roots,the root surface area,root hair density,root-shoot ratio,root dry weight and shoot dry weight all increased significantly under phosphorus deficiency condition.These results suggested GmGER12 might improve soybean tolerance to low phosphorus by changing root architecture,such as increasing root surface area and root-shoot ratio.The expression of acid phosphatase gene GmACP1,phosphate transporter genes GmPT1 and GmPT5 related to phosphorus uptake and utilization also changed in overexpressed soybean hairy roots.We also transfered RNAinterference vector pB7GWIWG2(Ⅱ)-GmGER12 into soybean hairy roots,and found that root dry weight and shoot dry weight decreased significantly under phosphorus deficiencycondition,shoot P concentration decreased significantly under normal phosphorus condition.We speculated that GmGER12 might play an important role in promoting phosphorus uptake by roots and accumulate in leaves.The candidate transcription factors GmPHRa and GmPHRb related to phosphorus efficiency in soybean were homologously cloned.Among them,the CDS of GmPHRa was 1455bp and encoded 484 amino acid residues,GmPHRb was 990bp and encoded 329 amino acid residues.GmPHRa was highly expressed in roots and root nodules,followed by flowers and leaves;GmPHRb showed higher expression in roots,root nodules and flowers,followed by leaves.GmPHRa and GmPHRb were induced by low-P stress,the expression level of GmPHRa reached a peak in 12h in soybean accession C112 under low-P stress,while expression level of GmPHRb peaked earlier in 0.5h.The transcriptional activation activity in yeast demonstrated the first 250 amino acids in the N terminal took part in the transcriptional activation activity of GmPHRa,while GmPHRb did not have transcriptional activation activity.In order to further study the function of GmPHRa and GmPHRb,Arabidopsis overexpressing GmPHRa,overexpressing GmPHRb and overexpressing GmPHRa/b,soybean hairy roots overexpressing GmPHRa,overexpressing GmPHRb and overexpressing GmPHRa/b,soybean hairy roots silencing GmPHRa,silencing GmPHRb were generated.Overexpressing GmPHRb in Arabidopsis increased the number of lateral roots.Under low phosphorus condition,root and shoot dry weight increased significantly in soybean hairy roots overexpressing GmPHRa and overexpressing GmPHRb,only a slight increase was observed of shoot dry weight in soybean hairy roots overexpressing GmPHRa/b.Meanwhile,the phosphorus concentration increased significantly in soybean hairy roots overexpressing GmPHRb and overexpressing GmPHRa/b,increased weakly in soybean hairy roots overexpressing GmPHRa.There was no significant difference in phosphorus concentration between RNA interference soybean hairy roots and the corresponding control.We also observed the six genes GmACP1,GmPAP14,GmPT1,GmPT5,GmSPX1 and GmSPX3 were all regulated by GmPHRb,GmPHRa was only related to the expression of GmACP1,GmPAP14,GmSPX1 and GmSPX3.GmPHRa and GmPHRb were both positive regulators for low phosphorus stress tolerance in soybean,but they might involve in responses to low phosphorus starvation through different pathways.