Functional Study On Gma-miR156 And Gma-miR166 Genes In Regulating Phosphorus Metabolism And Phosphorus Balance In Soybean(Glycine Max(L.)Merr.)

Phosphorus(P)is an indispensable macronutrient in plants,It is not only the main component of plant cells,but also participates in a series of physiological and biochemical reactions in cells.In nature,in order to increase the phosphorus uptake and utilization efficiency,the plants have evolved a series of adaptive mechanisms.MicroRNA(miRNA)regulates gene expression at the transcriptional level,it degrades the target genes or inhibits the translation of target genes to regulate plant development,biotic and abiotic stresses.In recent studies on plants response to phosphorus deficiency stress,miRNA has been found to play an important role in plant phosphorus metabolism and phosphorus balance,but few researches have been reported in soybean.The aim of this study was to explore the functions of gma-miR156 and gma-miR166 genes in regulating phosphorus metabolism and phosphorus balance in soybean.The main results were as follows:1.The effects of different soybean genotypes and concentration of Agrobacterium tumefaciens on genetic transformation efficiency of soybean cotyledon node mediated by Agrobacterium tumefaciens were studied,it was found that the two soybean varieties of Tianlong 1 and Williams 82 were susceptible to the Agrobacterium tumefaciens’ infection,The transient expression rate of GUS in explants was about 90%,when the concentration of Agrobacterium tumefaciens was 1.0(OD650),the transient expression rate of GUS in explants was the highest,they were 96.12%and 97.51%,respectively,at the same time,the efficiency of genetic transformation was higher,they were 4.37%and 3.88%,respectively.Combining the above findings found that Tianlong 1 was a good receptor material of soybean for Agrobacterium tumefaciens genetic transformation,and the genetic transformation efficiency was higher when the Agrobacterium tumefaciens’ concentration was 1.0(OD650).2.The results of qRT-PCR found that gma-miR156b and GmSPL9 genes under soybean phosphorus deficiency condition were induced to express in the different level,there was a negative regulation relationship between gma-m iR 156b and GmSPL9,suggesting that GmSPL9 may be a target gene of gma-miR156b.The inorganic phosphorus content of hairy roots of the overexpression of gma-MIR156b transgenic soybean had extremely significant higher than that of the control,the overexpression of gma-MIR156b transgenic Arabidopsis and soybean had more lateral roots,larger root-shoot ratio and higher content of inorganic phosphorus in roots,meanwhile the content of inorganic phosphorus in the leaves of the overexpression of gma-MIR156b transgenic Arabidopsis thaliana was increased,however,the content of inorganic phosphorus in the leaves of the overexpression of GmSPL9 transgenic Arabidopsis thaliana was decreased,they are negatively correlated.The above results showed that gma-miR156b and GmSPL9 may be involved in a series of physiological and biochemical processes in response to phosphorus deficiency stress of soybean.3.The results of qRT-PCR found that the gene of gma-miR166a was negatively expressed in soybean roots and leaves in response to phosphorus deficiency stress,the inorganic phosphorus content of hairy roots of the overexpression of gma-MIR166a transgenic soybean had extremely significant lower than that of the control,the overexpression of gma-MIR166a transgenetic soybean had less lateral root,smaller root-shoot ratio,lower content of inorganic phosphorus in root,and higher content of anthocyanins in leaves.The above results showed that gma-miR166a may play a role in the regulatory network of soybean response to phosphorus deficiency stress.