Functional Study Of Soybean PHD-Finger Transcription Factor GmpHD3 In High Temperature And Humidity Stress

Soybean(Glycine max L.Merrill)is the main source of plant protein and the main oil crop.Soybean cultivation in China has a long history and there are many soybean varieties.Spring soybean production areas in southern China have high temperatures and rain in summer.This high-temperature and humidity(HTH)environment affects the development of soybean seeds,causing some seeds to undergo field deterioration,seed vitality decline,and seed use value.In the early stage of the laboratory,two-dimensional electrophoresis was performed on the pre-harvest seed deterioration resistance varieties Xiangdou No.3 and deterioration varieties Ningzhen No.1 to find the differentially expressed protein GmSBHl.The promoter region cis-acting element HSE was used as the bait.Hybridization experiments screened the PHD-finger family transcription factor GmPHD3.Based on this,a series of experiments were carried out.The main results are as follows:The target sequences isolated from the leaves of the pre-harvest seed deterioration resistance varieties Xiangdou No.3 and deterioration varieties Ningzhen No.l and they were completely identical.The full-length of ORF was 738 bp,encoding 246 amino acids and including two domains,Alfin and PHD-finger.The closest relationship with Glycine soja ALFIN-LIKE 4-like protein and subcellular localization results show that it is expressed in the nucleus.The expression of GmPHD3 gene was highest in the mature pods of Xiangdou No.3,followed by flowers,leaves and mature seeds,and the expression was higher in the flowers and mature seeds of Ningzhen No.1.After 6 h of HTH stress,the gene expression in Xiangdou No.3 was the highest,which was significantly higher than that of the blank control group,and then began to decline.After 24 h,gene expression continued to increase.In Ningzhen No.1,the highest expression level was 168 h under stress and the lowest expression level at 24 h.The testing the soybean germ viability after stress indicated that:the germ viability of Ningzhen No.1 variety decreased more than the blank control.The seedling gene expression of Xiangdou No.3 was the highest after 12 h under drought stress,and that of Ningzhen No.1 was the highest at 6 h.For the salt stress,Xiangdou No.3 had the highest gene expression at 6 h,and Ningzhen No.1 had the highest gene expression at 24 h.Both Xiangdou No.3 and Ningzhen No.1 seedlings had the highest gene expression when the plants induced by ABA for 24 h.Construction of a recombinant expression vector fused with the GmPHD3 gene and transformed the inflorescences of Arabidopsis by Agrobacterium-mediated.Eight candidate positive strains were obtained by Kan antibiotic screening of Arabidopsis seeds harvested after transformation.The candidate strains were continued to be screened to T3 generation,and PCR identification was performed to identify four positive strains,named PHD-1,PHD-3,PHD-4 and PHD-6.Observing the growth of the plants,the phenotypic differences between wild type and overexpressing lines appeared at the third week and the transgenic lines had more rosette leaves.At six weeks,transgenic Arabidopsis have more branches than wild-type.Under HTH stress,the antioxidant enzyme activities of SOD,CAT and POD of the overexpressing lines were higher than those of the wild-type,and the treatment group was higher than the blank group.The content of antioxidant MDA was also higher than that of the blank control,but the overexpressing lines were lower than the wild-type.At the same time,the Arabidopsis seeds under stress were collected for standard germination experiments.The overexpression Arabidopsis had higher seed germination rates and the decreased of seed viability less than wild-type.Observation of seed germination and seedling growth under stress found that the overexpression lines was more resistant to drought,salt stress and ABA treatment than the wild-type.The results of Arabidopsis seed vigor assay showed that the germination potential,germination rate,germination index and vigor index of overexpression Arabidopsis seeds were higher than those of wild type after artificial accelerated aging treatment.The GmPHD3 protein was used as a bait to screen the soybean cDNA library after HTH stress.Three candidate proteins were obtained by screening and sequencing,which were MIEL1(zinc ion binding protein),SUVR4(histone lysine N-methyltransferase)and PP2C 57-like(protein phosphatase).The interaction between GmPHD3 and GmMIEL1 was verified in vivo and in vitro by yeast two-hybrid rotation verification and BiFC experiment,respectively.The false positive of the yeast two-hybrid experiment was excluded,and the interaction position of these two proteins was found in the nucleus.