Characterization And Functional Analysis Of Cotton Small G Protein Gene GhRab11d3 And Its Promoter In Gossypium Hirsutum

As one of the most important economic crops in the world,cotton provides a very important natural fiber for human beings.With the great progress of cotton genome sequence analysis,we tried to find the important genes related with fiber development in the whole genome of cotton and analysis function,and it will also improve the quality and yield of cotton.Rab protein family is the largest subfamily of small G protein family.As one of the most important families in cotton,the Rab gene family plays a very important role in the process of fiber development.We can gain an important basis for the cloning and functional analysis of related genes by whole genome analysis about sequence,classification,basicbiological information,gene structure,genome distribution and expression characteristics of Rab gene family members.So far,the identification of 57 members of the Rab gene family in Arabidopsis has been completed,but in cotton,the relevant gene family has never been reported.In this study,combined with the release information of cotton genome,the cotton Rab gene family members were analyzed.Further by differential expression analysis,we found a Rab gene which expressed different in fiber elongation.Combined with its expression pattern in the family,we found it was dominantly expressed in the fiber elongation stage.The gene was named GhRab11d3.We cloned this gene and its promoter,and studyed its characteristics and functions systematically.The main results are as follows:We got a total of 87 members of the Rab gene family by retrieving the whole genome database of diploid Gossypium raimondii.Through the naming rules of Arabidopsis and the naming rules of mammals,each member was named respectively.Biological information analysis showed that the number of amino acid is 200 to 300 aa among Rab gene family members in cotton and the protein molecular weight is between 20 Kda and 30Kda,which is consistent with the nature of the Rab protein itself.Chromosome distribution map showed that 2 genes were not found in corresponding physical location,and the remaining 85 Rab protein gene family members are distributed in all 13 chromosomes of Gossypium raimondii.The distribution of Rab gene on chromosome 10 was the most,there were 11 and only 2 genes were distributed on chromosome 4.Phylogenetic analysis showed that the 87 members were divided into eight categories,which correspond to the reported Rab family of Arabidopsis thaliana.Members of each class have a similar function.After analyzing the genetic structure of all the members,we found that the number of introns in the same class was basically the same,that is,the genetic structure of the same class members was basically similar.After analyzing the transcriptome data expression pattern of TM-1 members of corresponding Gossypium raimondii,it is found that about 161 genes were detected expression at least in a tissue and organ.It shows that most of the Rab family members will perform certain functionin in different growth and development stages.About half of the genes were expressed in the vegetative organs,flower organs,fiber and ovuleorgans of cotton,indicating that these genes are constitutively expressed genes and they will participate in the growing developmental process and have diversity in function.Only a small number of genes are expressed in one or some kind of tissues,indicating that these genes are functionally specific.The expression pattern of some genes is not clear enough and we have no found regularity.Li1 wild type has the normal developmental fiber,and the mutant has an ultra short fiber phenotype.Differential expression of a Rab gene in fiber development stages of Li 1 mutant and wild type was found by differential display analysis.Through the quantitative expression analysis of this gene in different fiber development tissues of Lil mutant and wild type,it was proved that the expression of 8DPA,10DPA,12DPA and 15DPA was significantly higher in the wild type than that in the mutant.It was proved that the gene has a positive regulatory effect on fiber elongation.Combined with the analysis results of Rab gene family,it was found that the gene was named as GhRab11d3.From the expression pattern,we can see that the gene belongs to constitutive expression,and it is dominantly expressed in the fiber elongation stage.It has 690 bp full-length cDNA of GhRablld3,encodes a protein of 229,with typical of four guanine nucleotide binding domain and two cysteine structure of C terminus,and pI was 6.84,molecular weight Mw 25.16KDa.This gene has a certain level of expression in all tissues and organs of TM-1,especially in the rapidlongation period of fiber.Subcellular localization was performed by means of the method of transformation of cotton protoplasts and injection of tobacco.The results showed that the protein was located in the cell membrane system,including the cell membrane,cytoplasm and nucleus.The red fluorescent signal of marker protein was colocated with the target protein,and it was found that there was a specific organelle involved in the endoplasmic reticulum.In order to further verify the function of this gene,the sense and antisense constructs of GhRablld3 driven by the constitutive promoter 35S and the fiber specific promoter RDL respectively were carried out through the genetic transformation of cotton.In addition to callus browning phenomenon of the 35S-ASC vector in the 50 days after the infection,can not get the transgenic progeny plants,the remaining three vectors were obtained a number of transgenic homozygous lines.Through transgenic progeny and receptor WO in the fiber elongation stage 5dpa,lOdpa and 15dpa qPCR analysis,we found expression of target gene in 35S-SC 303 line and RDL-SC 210 line was significantly increased,expression of target gene in RDL-ASC 98 line was significantly inhibited,so the choice of the three lines was performed to verify function.In comparison with the fiber length of three transgenic lines and receptor WO in 10DPA,15DPA,20DPA and mature stage of fiber development,it was found that the fiber length of the sense construct was significantly longer and the fiber length of the antisense construct was significantly shorted.Analysis of the size of the boll in 10 DPA and 15 DPA showed that,compared with the receptor W0,the boll of the sense vector of the transgenic plants became larger,and the boll of the antisense vector became smaller.Analysis of cotransformation of yeast two hybrid and bimolecular fluorescence complementation(BiFC)demonstrated that GhRablld3 interacted with four cell skeleton protein ACTa,ACTb,ACT2,ACT4 which are dominantly expressed in fiber elongation stage,and interacted stronger with ACTb and ACT2.This finding reveals that GhRab11d3 transported a component of the cell wall in the vesicle transport process along the cytoskeleton protein ACTIN.In the course of this process,it has some influence on the morphological structure of cytoskeletal protein actin filament F-Actin,and produces changes in the density or number of cytoskeletal protein actin filaments F-Actin.At the same time,the number of vesicles at the top of the fiber cell is changed,so that the transport capacity of the vesicles is affected.The pectin content of the final shipping to the cell wall will also be affected.We found that the pectin content of sense construct in fiber tissues was significantly increased,and the antisense vector was decreased,which may be the main reason for the change of fiber length.To better understand the function of this gene,we cloned a 1,546 bp upstream sequence of the GhRablld3 initiation codon ATG,which was used as the promoter of the gene and named it pGhRablld3 and then did promoter characteristic analysis.Prediction of the promoter transcription start site TSS is locatedthe base Tin the ATG upstream 312bp,which is marked as-312 bp.By plantCARE and PLACE online website of predicting promoter cis acting elements,wefound that TATA-box and CAAT-box,important transcription factor binding site elements,several hormone induced elements and the acting elements of regulating seed germination and pollen tube growth.The vector pBI121 with HindⅢ and BamHl cut 35S promoter.We constructed five segments deletion promoter vectorconnected with GUS reporter gene,respectively P1(196 bp),P2(385 bp),P3(546 bp),P4(746 bp)and P5(1,546 bp).We have carried out the stable genetic transformation of tobacco with each deletion promoter.In transgenic tobacco,pGhRab 11 d3 is mainly in the reproductive organs of tobacco,such as petals,anthers,peduncles and ovules.GUS staining and GUS enzyme activity assay showed that P1(196 bp)and P2(385 bp)were not significantly different from WT.From P3(546 bp),it could be observed that the GUS staining was significantly increased,and the activity of GUS was also significantly increased.These results indicate that the promoter has transcriptional activity starting from P3(546 bp),and the promoter length of the 546 bp upstream of the initiation codon ATG is essential for driving the expression of the downstream GUS gene.We can also infer core elementof pGhRablld3 is located between the P2(385 bp)and P3(546 bp),may be a MYB transcription factor biriding site(492 bp)or a MADS box transcription factor binding site(417bp).In addition,we found that the upstream sequence of the 546 bp also contains regulatory elements that enhance the expression of downstream genes.The promoter of GhRab11d3 can be induced by three hormones(including GA,IAA,and 6-BA),but not by ABA.Finally,cotton ovulesare bombarded for gene gunthrough the five deletion promoter plasmid vector.When the fibersappear,they are stained with GUS.It was found that P3,P4 and P5 vectors had a certain GUS staining,which once again proved that GhRah11d3 played a certain function in the development of fiber.