Function And Regulation Mechanism Of GhPRE6 Transcription Factor In Fiber Elongation Development Of Cotton

Cotton is widely grown in China as one of the main economic crops in the world.Cotton fiber is the main product of cotton production.However,the molecular regulation mechanism of cotton fiber development is still not fully understood and needs to be further explored,and it is very necessary to use transgenic technology to improve cotton fiber yield and quality.Studies have shown that many transcription factors play an important role in regulating cotton fiber development,among which the Basic helix-loop-helix/helix-loop-helix(bHLH/HLH)transcription factor may play a key role in the development of cotton fiber elongation.In a previous study,our laboratory discovered a mutually antagonistic bHLH/HLH system that regulates cotton fiber elongation:GhACE1 protein forms a homodimer in combination with cis-elements of the promoters of downstream target genes,thereby promoting cotton fiber elongation development,while GhFP2 inhibits cotton fiber elongation by interacting with GhACE1 to inhibit the formation of the GhACE1-DNA complex.We screened another bHLH transcription factor,GhPRE6,which interacts with GhFP2 by yeast two-hybrid technique,and then studied the function of GhPRE6 in cotton fiber development.This paper further improves the three antagonistic system of GhACE1,GhFP2 and GhPRE6 in regulating cotton fiber elongation.The main research results are as follows:1.GhPRE6 is a bHLH transcription factor that functions in cotton fiber elongationThe yeast two-hybrid cDNA library of cotton fiber was screened with pGBKT7-GhFP2 protein as bait,and a GhbHLH/HLH protein-GhPRE6 was obtained by screening,which was verified by one-to-one yeast two-hybrid experiment.In addition,bimolecular fluorescence complementation and in vitro pull-down experiments further confirmed that GhPRE6 can interact with GhFP2.The homology alignment of GhPRE6 amino acid sequence showed that GhPRE6 contains a highly conserved HLH domain from amino acids 18 to 77,indicating that it functions by interacting with other proteins.The results of dual-luciferase reporter experiments indicated that GhPRE6 may play a positive regulatory role in cotton fiber elongation development.2.Research on the mechanism of GhPRE6 transcriptional regulationIn order to analyze the function of GhPRE6 protein,its subcellular localization and tissue expression pattern were analyzed.The results showed that GhPRE6 was located in the nucleus and expressed predominantly in cotton fiber elongation.These results indicate that-GhPRE6 is a bHLH/HLH transcription factor that plays a transcriptional regulatory role in cotton fiber elongation.The GhPRE6 protein was found to have no autotranscriptional activation activity by transcriptional activation assay.Yeast two-hybrid and bimolecular fluorescence complementation techniques verified that GhPRE6 itself can form homodimers,which further indicated that bHLH/HLH transcription factors exert their transcriptional regulatory functions by forming different forms of complexes.3.Overexpression of GhPRE6 in Arabidopsis promotes epidermal hair elongation and developmentCotton fiber and Arabidopsis thaliana trichomes have similar developmental mechanisms,both of which are derived from unicellular polar elongation.Due to the long growth cycle of cotton and the difficulty of transformation,we firstly expressed it heterologously in Arabidopsis thaliana.gene to explore its function.Phenotypic analysis of transgenic Arabidopsis seedlings grown for one week showed that the length of hypocotyls of GhPRE6 overexpressed Arabidopsis was significantly longer than that of wild type,and the petioles of GhPRE6 transgenic Arabidopsis seedlings were also significantly longer than those of wild type.The morphology of hypocotyl and mesophyll cells was observed,and the cell length was significantly increased compared with the wild type.The epidermal hairs on the leaves of wild-type and GhPRE6-overexpressing transgenic Arabidopsis grown for 4 weeks were eluted,and their morphology was observed with a microscope,and the length statistical analysis was performed.Compared with the wild type,the number of epidermal hairs with four branches was not significantly different from that of the wild type.This indicates that overexpression of GhPRE6 can promote the elongation of Arabidopsis trichomes.Further RT-qPCR analysis results showed that GhPRE6 promoted Arabidopsis epidermal hair elongation by promoting the expression of cell elongation-related genes.4.Obtainment and preliminary analysis of GhPRE6 transgenic cotton plantsIn order to explore the function of GhPRE6 in cotton fiber development,we constructed a GhPRE6 overexpression and knockdown vector,and obtained some cotton seedlings through Agrobacterium-mediated genetic transformation.At present,45 different GhPRE6-OE lines have been obtained,GhPRE6-RNAi also has 5 lines,and embryogenic callus on CB4 and CB5 solid medium are also emerging one after another,pending phenotypic analysis.