Cloning And Functional Verification Of A Transcription Factor GmPIF1 From Soybean

Soybean is one of the most widely planted cash crops in China and an important oil crop in the world.It has rich nutritional value and high economic value.Light signal is an important environmental factor to ensure the normal growth and development of soybean.Physins regulate plant growth and development by sensing light signals in plants and interacting with photosensitizing pigment interaction factors(PIFs).PIFs can not only integrate light signal transduction in plants,but also participate in hormone signal transduction.They also play a very important role in plant growth and development.At present,the research on PIFs is relatively thorough in Arabidopsis,and the research on GmPIFs gene family in soybean is relatively few.In this study,the GmPIF1 transcription factor was cloned from soybean variety Jilin 32,and its structural analysis and functional verification were performed.The analysis results are as follows:1.Using RT-PCR method to clone the CDS of GmPIF1 gene from soybean variety Jilin 32,a total length of 1530 bp,encoding 510 amino acids,containing APA,APB and bHLH conservative domains,belonging to the bHLH transcription factor family,subcellular localization Predicted in the nucleus.2.The expression of GmPIF1 gene in different tissues of soybean was studied using real-time quantitative PCR technology.The results showed that GmPIF1 was expressed in roots,stems,leaves,flowers,20 d,30d,and 40 d embryos,and the expression level was expressed.It is leaf> flower> 50 d embryo> root > 30 d embryo> stem> 20 d embryo.3.By transforming the prokaryotic expression plasmid pET28a-GmPIF1 into E.coli Rosetta,and induced by IPTG,SDS-PAGE electrophoresis results showed that GmPIF1 protein can be efficiently expressed in the prokaryotic system.4.The subcellular localized recombinant plasmid pHB-GmPIF1-YFP was transformed into Agrobacterium EHA105,and the tobacco epidermal cells were transformed by injection.The yellow fluorescence observed by fluorescence microscopy showed that GmPIF1 was localized in the nucleus.5.The yeast self-activation verification recombinant plasmid PGBKT7-GmPIF1 was transferred into yeast AH109.The growth on the defective medium showed that the GmPIF1 protein does not have transcriptional self-activation activity in the yeast system.6.The results of yeast two-hybrid experiments showed that the GmPIF1 protein could interact with the photochromic proteins of Arabidopsis AtPhyA and AtPhyB and soybean GmPhyA and GmPhyB under different light treatments.7.A plant expression vector recombinant plasmid pCHF3300-GmPIF1 was transferred into Agrobacterium EHA105,and Arabidopsis thaliana was infected by the inflorescence infection method to obtain a T3 GmPIF1 transgenic Arabidopsis homozygote.The wild-type,mutant and transgenic Arabidopsis were treated with red light and far-red light,and the germination rate,germination-related gene expression levels,and GA and ABA contents were measured.The results showed that GmPIF1 responded to PhyB-dependent Red light and PhyA rely on far-red light to regulate seed germination-related genes,thereby regulating GA and ABA content,and then regulating seed germination.