Study On Molecular Mechanism Of The Interaction Between Blue Copper Protein GhBCP4 And GhCrRLK Proteins Involving In Cotton Fiber Development In Gossypium Hirsutum

Cotton(Gossypium hirsutum)is one of the most important economic crops in the world,as the natural fiber,cotton fiber is the main raw material of textile industry.Cotton fiber is a kind of single-cell epidermal hair developed from the polar elongation of cotton ovule epidermal cells,it is an ideal biological material for studying the polar elongation of plant cells and the synthesis of secondary cell walls.Blue copper-binding protein(BCP)are a kind of copper ion-binding protein,which are widely involved in plant growth and development and stress response.In the previous work,we isolated a fiber dominant expression gene GhBCP4 from cotton,and studied its function in the development of cotton fiber,the results are as follows:1.Analysis of GhBCP4 protein subcellular localizationProtein structure prediction shows that GhBCP4 protein has a signal peptide(SP)at the N terminal,a GPI anchor site at the C terminal,and copper ion binding domain is located between the two.In order to study the cellular sublocalization of this protein,we constructed multiple GhBCP4 and eGFP fusion expression vectors and carried out cotton transformation experiments.The results showed that when eGFP reporter protein was placed at the C terminal of GhBCP4,no fluorescence signal was detected in cotton-positive callus cells;After removing the SP and GPI sites of the GhBCP4 protein,the remaining intermediate sequence was fused with eGFP for expression,and it was found that the fluorescence signal was mainly concentrated in the vacuole of cotton cells;The eGFP sequence was inserted between the copper ion binding domain of GhBCP4 and the anchor site of GPI,and it was found that the fluorescent signal was mainly concentrated between the cell membrane and cell wall of cotton cells.The above results indicate that the GhBCP4 protein is located between the cotton cell membrane and cell wall,and its SP and GPI sites play an important role in the accurate positioning of the protein.2.Phenotype analysis of GhBCP4 transgenic cotton fiber of T3 and T4 generationsThe expression amount and phenotype of T3 generation GhBCP4 transgenic cotton material were analyzed,two strains with low expression of GhBCP4 and shorter length of mature fiber than wild type(WT)in T3 generation RNAi transgenic plants were selected,two lines with high GhBCP4 expression and longer mature fiber length than the wild type in the overexpressed transgenic cotton plants were selected,they were planted to carry out follow-up analysis on T4 generation transgenic materials.After measuring the length of mature cotton fibers of transgenic plants found that the length of mature fibers of transgenic cotton overexpressing GhBCP4 increased compared with wild type,while the length of cotton fibers of GhBCP4 RNAi transgenic plants was significantly shorter.3.RNA-seq analysis of genes differentially expressed between GhBCP4 transgenic plants and wild-type plantsIn order to further study the regulatory mechanism of GhBCP4 involved in cotton fiber development,transcriptome sequencing was performed on 12 days post anthesis cotton fibers of GhBCP4 overexpressed lines,RNAi lines and WT lines.RNA-seq results showed that 1067 genes were differentially expressed in the overexpressed GhBCP4 plants,of which,610 genes were up-regulated and 457 genes were down-regulated.In RNAi plants,1305 genes were differentially expressed,of which 181 genes were up-regulated while 1,124 genes were down-regulated.In GhBCP4 OE fiber cells,up-regulated genes mainly related to the synthesis and signal transduction of ethylene,auxin,JA and other hormones,while the down-regulated genes were mainly related to secondary wall synthesis and transcription factors which involved in secondary wall development.The expression trend of these genes in GhBCP4 RNAi samples were reversed.The above results indicated that the overexpression of GhBCP4 could promote the synthesis of ethylene and auxin in fiber cells and stimulate the expression of ethylene and auxin response genes;at the same time,overexpression of GhBCP4 down-regulated the expression of genes related to the occurrence of secondary wall in some cotton fibers,which may affect the biosynthesis of secondary wall of fiber cells,and prolong the development time of cotton fiber extension,thus increasing the length of mature cotton fiber of GhBCP4 overexpressed lines.On the contrary,in GhBCP4 RNAi cotton fibers,the up-regulated expression of genes related to secondary wall synthesis and assembly of cotton fibers may cause cotton fiber cells to enter the secondary wall development stage in advance,and the elongation of cotton fiber cells is terminated in advance,which eventually leads to the shorter mature cotton fibers in RNAi lines.4.Analysis of cellulose content of GhBCP4 transgenic cottonTo further determine whether the change of GhBCP4 gene expression level would affect the content of secondary wall cellulose of cotton fiber,we collected 15 DPA,20 DPA and 25 DPA of transgenic cotton fibers,analyzed them by fixed fiber samples and paraffin sections,and extracted cell wall components of transgenic lines to analyze their crystalline cellulose content.The results showed that after the paraffin sections were stained with cellulose-specific dye S4B,the fluorescence in GhBCP4 overexpressing cotton fibers was weaker than that of wild type cotton fibers,and the cellulose content was reduced,while there was no significant difference of fluorescence between GhBCP4 RNAi cotton fiber and the wild type cotton fiber;However,when determinated the crystalline cellulose content in the cell wall components,it was found that the crystalline cellulose content of transgenic materials did not change significantly compared with that of wild type,and the experiment needs further verification.5.Screening and identification of GhBCP4 interacting proteinsGhBCP4 protein has higher sequence similarity to Arabidopsis ENODL14 protein.Previous studies had shown that ENODL14 interacts with receptor-like kinase FER in pollen tube development(Hou et al.,2016).Therefore,we selected GhCrRLK1,GhCrRLK11,GhCrRLK14,GhCrRLK25 and GhCrRLK27 proteins with high homology to FER protein and high expression in cotton fiber for further study,bimolecular fluorescence complementary(BIFC)was used to analyze whether they could interact with GhBCP4.The results showed that GhCrRLK1 and GhCrRLK25 could interact with GhBCP4.This result is of great value to elucidate the regulatory mechanism of GhBCP4 in cotton fiber and points out the direction for future research.