Function Analysis Of Soybean BHLH-like Transcription Factors GmbHLH3 And GmPIF1

Soybean(Glycine max)is one of the most important economic crops in the world,providing humans with high-quality protein,vegetable oil and secondary metabolites,such as isoflavone.The growth and quality of soybeans are regulated by transcription factors.The bHLH transcription factor family is one of the largest transcription factor family,playing an important role in plant stress response,growth and development,synthesis of secondary metabolites,and hormone signaling.However,the functions of soybean bHLH transcription factors are less studied.In this study,two genes coexpressed with the seed development of soybean Jilin 32 were screened out,and through phylogenetic tree analysis,it was determined that these two transcription factors were homologous to AtbHLH3 and At PIF1 in Arabidopsis,and named GmbHLH3 and Gm PIF1.AtbHLH3 and At PIF1 were confirmed to participate in the JA signal response and light response pathways,and both participated in the phenylalanine metabolic pathway.But in soybeans,the functions of GmbHLH3 and Gm PIF1 are not clear yet.Therefore,this experiment explored the regulatory effects of GmbHLH3 and Gm PIF1 transcription factors on the synthesis of flavonoid compounds as well as the regulatory effects in JA pathway and the light response process,respectively.The main results are as follows:1.The GmbHLH3 gene was cloned from soybean cultivar Jilin 32.The CDS is 1521 bp in length.Protein sequence analysis revealed that it contains MYC domain,bHLH domain and nuclear localization signal.Phylogenetic tree analysis confirmed that GmbHHLH3 is closely related to AtbHLH3,AtbHLH13 and AtbHLH17 in Arabidopsis.Subcellular localization experiments show that GmbHHLH3 is localized in the nucleus.Real-time quantitative PCR(q RT-PCR)results showed that GmbHLH3 was expressed in all tissues of soybean cultivar Jilin 32,among which the expression level was the highest in flowers,and the expression level in immature embryos increased with the development of embryos.2.Using the soybean hairy root system,we analyze the effect of overexpression and RNA interference of GmbHLH3 and Gm PIF1 genes on soybean hairy root isoflavone accumulation.The results showed that,compared with non-transgenic hairy roots,overexpression of GmbHLH3 and Gm PIF1 genes can significantly increase the total isoflavones;interference with GmbHLH3 and Gm PIF1 genes can significantly reduce the accumulation of isoflavones.Among them,overexpression of GmbHHLH3 significantly increased the content of daidzein.Although overexpression of Gm PIF1 gene significantly increased the total amount of isoflavones,there was no significant difference between the content of isoflavones in the transformed hairy roots and the control.Using q RT-PCR to analyze the expression of genes related to flavonoid synthesis in the GmbHLH3 and Gm PIF1 transformed hairy roots,the results showed that the transcription levels of Gm PAL1,Gm4 CL,Gm C4 H,Gm CHI1B1,Gm IFS2,and Gm F3 H gene were increased,and transcription levels of Gm CHI1 A was decreased in the GmbHLH3-overexpressing hairy roots.The transcription levels of Gm PAL1,Gm CHI1B1,and Gm IFS2 genes were increased in the Gm PIF1-overexpressing hairy roots.Furthermore,GmbHLH3 and Gm PIF1 were overexpressed in Arabidopsis,and the flavonoid content was determined in the seeds by High Performance Liquid Chromatography(HPLC).The results showed that the total amount of flavonoids GmbHLH3-and Gm PIF1-overexpressing lines was significantly higher than that of the control in the Arabidopsis seeds.Based on the results of transforming hairy roots and Arabidopsis,it is concluded that overexpression of GmbHLH3 and Gm PIF1 transcription factors can promote the accumulation of flavonoids in Arabidopsis seeds.3.Using q RT-PCR to analyze the transcription patterns of GmbHLH3 transcription factors under ABA,Na Cl,ET,high temperature,low temperature,SA,PEG,and Me JA stress treatment conditions,the results show that GmbHLH3 responds differently to different treatments.Among them,the 24 h of Me JA treatment within,the transcription level of GmbHHLH3 was significantly higher than that of the control,and it had a maximum transcription peak at 3 h.4.Using the yeast two-hybrid analysis,it was confirmed that the GmbHLH3 transcription factor has no transcriptional autoactivation activity in yeast,and the GmbHLH3 protein and its homologous proteins AtbHLH3,AtbHLLH13 and GmbHLH3 a can grow on the SD/-Ade/-His/-Leu/-Trp/3-AT deficiency screening medium after being co-transformed into yeast;According to the analysis of bimolecular fluorescence complementarity analysis,the YFP fluorescence signal can be observed after the GmbHLH3 protein and AtbHLH3,AtbHLLH13 and GmbHLH3 a protein are co-transformed into tobacco for transient expression.These results indicate that GmbHHLH3 protein can interact with AtbHLH3,AtbHLLH13 and GmbHHLH3 a.5.Under 5 ?M and 25 ?M Me JA treatment conditions,the root lengths of GmbHLH3-overexpressing lines are significantly longer than those of the wild type in Arabidopsis.Under 25 ?M Me JA treatment conditions,the expression levels of pathogen response gene At PDF1.2,wounding response genes At VSP1,At LOX2,and At TAT1 were reduced GmbHHLH3-overexpressing lines in Arabidopsis.6.In vitro leaves of Arabidopsis treated with Me JA,it was found that relative GmbHHLH3-overexpressing lines slowed down leaf senescence,and the transcriptional levels of senescence promoting genes At SAG29,At AZF2,At WRKY22 and chlorophyll metabolic-related genes At SGR,At NYC1,At CLH1,At PPH,and At PAO decreased,while the transcriptional levels of senescence inhibiting factor At MKP2 increased.7.Analyze the phenotype of the Gm PIF1-overexpressing lines after treatment in the dark for 5 d and 7 d.It was found that the hypocotyls of the Gm PIF1-overexpressing lines were significantly longer than the wild type,and the cotyledon expansion angle and the apical hook angle was significantly reduced.The accumulation of protochlorophyllate(Pchlide)in the dark for 2-7 days was detected,and it was found that Pchlide increased with the increase of culture time,and the Pchlide content of the Gm PIF1-overexpressing lines was higher than that of the wild type.Using q RT-PCR to analyze the expression of chlorophyll synthesis genes in 5 d chlorophyll seedlings,the results showed that the transcription levels of chlorophyll synthesis genes and photosynthesis genes At HEMA1,At GUN5,At LHCB6,and At PSAE1 in the Gm PIF1-overexpressing lines decreased,while the transcription of Pchlide metabolism gene At PORC increased.8.The greening rate was investigated after 1-7 days of etiolated seedlings were transferred to white light for 3 days.The results showed that after 4 days in the dark,the greening rate of the Gm PIF1-overexpressing lines was higher than that of the wild type.The chlorophyll content of the 4-7 d etiolated seedling decreased with the increase of days in the dark,and the chlorophyll content of the Gm PIF1-overexpressing lines was significantly higher than that of the wild type.Using q RT-PCR analysis,when the 5 d etiolated seedlings were exposed to the light for 1 h,the chlorophyll synthesis gene and photosynthesis gene At HEMA1,At GUN5,At LHCB6,and At PSAE1 transcription levels in the Gm PIF1-overexpressing lines were higher than those of the wild type.9.Using the yeast two-hybrid system analysis,we analyze the interaction between the Gm PIF1 truncated protein and Gm PHYA and Gm PHYB truncated protein.The results show that the full length of the Gm PIF1 protein sequence can interact with Gm PHYA and Gm PHYB truncated protein,but Gm PIF1 truncated protein can no longer interact with Gm PHYA and Gm PHYB.In summary,GmbHLH3 and Gm PIF1 genes play a role in response to soybean JA and morphogenesis,and both participate in the accumulation of flavonoids.The research results can enrich people's cognition of soybean bHLH transcription factors and provide genetic resources for soybean molecular breeding.