The Cloning And Functional Characterization Of Fiber Development-related Gene GhEXPA8 In Gossypium Hirsutum L.

Cotton is one of the most important economic crops in the world and cotton fiber is important raw material for textile industry.At present,upland cotton is one of the main cultivated species whose fiber yield is high;however fiber quality needs to be improved.High fiber quality is the major goal of the cotton breeding.It is of great significance to discover genes related to land cotton fiber development and to explore their action mechanism on fiber elongation.As one of the longest plant cells,Cotton fiber is developing from ovule outer epidermal cells.Expansin is cell wall proteins that involved in cell expansion and cell wall modification process.It is involved in almost the whole process of the plant development.The role of expansin in cotton fiber cells development is not clear.Gh EXPA8,an α expansin gene,was down-regulated significantly in pag1 mutant with reduced fiber lengthwhen analyzing the transcriptome data of rapidly elongating fibers.In order to reveal the role of expansin in cotton fiber cells development,Gh EXPA8 gene related to fiber development was clonedand transformed to Arabidopsis thaliana by inflorescence infiltration procedure to analyze its function characterization.The results are as follows:1.The Gh EXPA8 gene cloning and bioinformatics analysisBased on the nucleotide sequences（Cot AD₂9957,Expansin2）submitted by Institute of cotton research of CAAS,the c DNA of the gene were cloned by RT-PCR and renamed as Gh EXPA8.Bioinformatics analysis showed that the CDS of Gh EXPA8 is 762 bp long,encoding 253 amino acids.The 1-25 amino acids encode the signal peptide.It contains DPBB₁ and pollen_allerg₁ structural domain.The signal peptide and two conserved domains are typical features of α expansin.Evolutionary tree analysis further showed that Gh EXPA8 encodes an α expansin.2.The functionanalysis of Gh EXPA8 geneThe relative expression level of Gh EXPA8 in different tissue and fiber of different development phase was analyzed by q PCR.The q PCR resultsalso showed that Gh EXPA8 gene was lowly expressed in the root,stem,leaf and ovule,while highly expressed in the 10 DPA fiber.The transcript level peaked at 10 DPA and thereafter declined sharply at 15 DPA in the fiber development process.The expression of PROGh EXPA8: GUS fusion was detected in Arabidopsis thaliana.Histochemical staining of GUS can be detected in various organs of transgenic plants,especially in young developing organs such as root hairs,root tip and stem tip.GUS activity was also evident in vascular tissue such as the veins and petioles.Arabidopsis trichome has a similar development mechanism with cotton fiber.GUS activity of Gh EXPA8 promoter can be detected in root hairs of Arabidopsis which suggested that the gene may be play a role in cotton fiber development.The expression patterns of Gh EXPA8 gene in cotton and transgenic Arabidopsis suggested that it may play a role in fiber elongation.Gh EXPA8 gene was also overexpressed in Arabidopsis thaliana.Phenotypic analysis showed that it increases Arabidopsis root length significantly.It suggested that Gh EXPA8 is really a functionalα expansin which most likely play a role in fiber development.In addition,the expression pattern suggest that the gene play a role in the process of fiber elongation.These results indicate that Gh EXPA8 is likely to be involved in fiber development process.3.We chose the hypocotyls of CRRI 24 as explants and conduct the transformation experimentof Gh EXPA8 gene by the method of Agrobacterium-mediated genetic transformation.The transgenic plants of T0 generation of Gh EXPA8 gene were obtained,which benefit the further study of Gh EXPA8 gene function.