Cloning And Functional Preliminary Analysis Of Fiber MATE Gene In Brown Cotton

Brown colored cotton is cotton whose fiber is endowed with brown color.It was reported that the chemical nature of pigment in brown colored fiber is quinone substances,which is fo rmed by polymerization oxidation of condensed tannins,that is,proanthocyanidins.The precursors of proanthocyanidins must be transmembrane transported into the vacuoles by MATEtransporter located on the vacuolar membrane to form oligomers of proanthocyanidin.MATE?multidrug and toxin extrusion protein?,a kind of multidrug and toxic Compound output protein,can be used to transport secondary metabolites of cationic dyes,glucosamine,antibiotics and drugs.In this study,the identification,cloning and functional analysis of theM ATE family genes were carried out,which provided a theoretical basis for exploring themolec ular mechanism of pigment formation and genetic improving the color quality of brown colored fiber.A reported MATE protein was used as a probe sequence to identify MATE family members in Upland Cotton.The qPCR was used to explore the expression profile of MATE genes in different cotton tissues.GhMATE13 and GhMATE23 genes were cloned and transformed into cotton and Arabidopsis for functional characterization.The results were as follows:1.A total of 91 GhMATE family genes were identified from genome database of Gossypium hirsutum L.through bioinformatics analysis method,and named as GhMATE1-GhMATE91,respectively.Bioinformatics analysis showed that the molecular weight of the proteins was 36.6⁶0.3 KD,encoding 374⁵48 amino acids.Phylogenetic relationship analysis revealed that all the MATE genes were classified into seven Groups.The chromosome mapping analysis showed that GhMATE genes were distributed with different densities over 25 chromosomes and clustered into five clusters.The qPCR results showed that GhMATE genes expressed in all of the cotton tissues with different expression patterns.Of these genes,Gh MATE13 and GhMATE23 were preferentially expressed in brown colored fibers,and showed higher expression level in brown colored fibers than in white cotton fibre,suggesting that they may play an important role in color generation of brown colored fibre.2.To explore the roles of MATE family genes,two MATE genes were cloned by using RT-PCR technique with cotton fiber cDNA at 15 DPA as a template,and named as GhMATE13?login number: NP₀01314298.1?and GhMATE23?Login number: XP₀16668081.1?,respectively.The plant expression vector was constructed using Gateway technology,and transformed into Arabidopsis wild-type and mutant tt12 by floral method,respectively.The results showed that overexpression of GhMATE13 in Arabidopsis increased the content of procyanidins in leaves.Additionally,the expression of GhMATE13 in the mutant tt12 rescued the low content of procyanidins in the mutant,making the procyanidins content back to normal.Finally,There was no significant change in the content of procyanidins in GhMATE23 overexpressing and complementing lines.These results suggested that GhMATE13 was more likely to be related to procyanidins biosynthesis compared with GhMATE23.3.GhMATE13 and GhMATE23 were genetically transformed into upland cotton YZ-1 and Xinluza33 by cotton tissue culture techniques.The resistant plants have been obtained through Kan screening,which will lay a foundation for exploring the functions of the GhMATE family genes in the color generation of brown colored fibers.