Analysis Of Upstream Regulatory Factors Of GaHD1, A Key Gene For Fiber Development In Gossypium Arboreum

GaHD1,a member of the HD-ZIP IV transcription factor family,acts as signal transduction targeting to functional genes.Previous studies have proved it to be a key regulatory gene controlling the initiation of trichon and fiber in cotton and its expression induced by gibberellin.In order to explore the upstream regulatory genes for Ga HD1,we extracted the upstream regulatory regions of GaHD1 and analyzed the L1 box cis-acting element using bioinformatics method,then predicted and confirmed the upstream regulatory genes for GaHD1.The main work included the followings:1.First of all,the promoter of the GaHD1 gene and the potential cis-acting elements at the basic of the bioinformatics analysis were obtained.The promoters were cut into different lengths according to the location of L1 box,fused with GUS reporter gene and transformed into Arabidopsis thaliana.GUS activity analysis showed that there were differences in the expression of GUS driven by four promoter fragments,in which,the staining of HD1-Pro-R1 was light,and the stems and leaf pubescence were not stained.The HD1-Pro-R2 darker in color,leaves and stalks were stained,and the R2 fragment had an L1 box more than the R1 fragment,indicating that L1 box is a very important element expressed in the fuzz.HD1-Pro-Intron only had GUS staining on the leaf edge,indicating that the 5'-UTR region has elements specifically expressed at the edge of the leaf.HD1-Pro-full length GUS is extremely active and is expressed in almost all areas.It is illustrated that intron elements of the 5'-UTR part allow the promoter to be not only specifically expressed at the leaf edge,and also has a promoter enhancer effect.Subsequently,we tested the GUS activity of transgenic Arabidopsis leaves.The results showed that the activity levels of HD1-Pro-R1 and HD1-Pro-Intron GUS were lower,while the activity levels of HD1-Pro-R2 and HD1-Pro-full length high,consistent with the staining results.In addition,we transformed the Arabidopsis thaliana plants transformed with the HD1-Pro-full length promoter into paraffin sections and found that GUS activity was highly expressed in the stem and leaf epidermal cells.At the same time,we treated the transgenic Arabidopsis thaliana plants with 5 ?M GA3.The results showed that HD1-Pro-R1 and HD1-Pro-Intron GUS staining and its activity level were almost no changed after treatment with GA3,while HD1-Pro-R2 and HD1-Pro-full length GUS activity has increased due to L1 box.This show that GA3 can induce certain genes or transcription factors to play a role in binding L1 box regulateion of Ga HD1 gene expression.2.In order to verify the up-regulated transcription factor of GaHD1 in vitro and cloning theknown transcriptional regulators(Ga HD1,GaHOX3,Ga PDF2,GaMML9)which were related to the development of cotton fiber.Four kinds of genes were respectively fused with PCY vector to transform Arabidopsis protoplast,among them,GaPDF2 and GaMML9 are new genes.We conducted subcellular localization studies and found that both of these genes are expressed in the nucleus.3.To verify whether the four transfer factors of GaHD1,GaHOX3,GaPDF2 and GaMML9 are upstream regulation genes of Ga HD1 promoter,yeast single hybridization method was used in this study.As a result,it was found that the GaHD1 promoter can bind to GaHD1,GaPDF2 and GaMML9,but can not bind to GaMML7 and Ga HOX3,indicating that GaPDF2 and GaMML9 are Ga HD1 upstream genes.4.Use of tobacco instantaneous transformation experiments to verify the upstream regulation of GaHD1 transcription factor,The results showed that there was no significant difference in GUS activity initiated by HD1-Pro-R1 and HD1-Pro-Intron co-transformed Ga HD1,GaHOX3,GaPDF2 and GaMML9 genes compared with the promoter.The promoters HD1-Pro-R2 and HD1-Pro-full length contained L1 box,and there was also no significant change in GUS activity after they were co-transformed into GaHOX3 respectively.However,the GUS activity of tobacco after co-transformation of GaHD1,GaPDF2 or GaMML9 was significantly higher than that of the control,These results indicate that GaHD1,GaPDF2 and GaMML9 can bind to GaHD1 promoter and enhance the expression of Ga HD1 gene,further confirming the regulatory genes upstream of GaHD1.