Functional Analysis Of P₄-ATPase During The Development Of Ovule And Fiber In Upland Cotton

Cotton fiber develops from an ovule epidermal cell,and its development process mainly involves four stages:initiation,rapid elongation,secondary wall thickening and dehydration maturity,of which the rapid elongation and secondary wall thickening mainly determine the fiber length and fineness.During the elongation phase of fiber development,vesicle transportation plays an important role in the secretion of polysaccharides and the formation of the primary cell wall.The putative aminophospholipid ATPase（ALA）referred to as P₄-ATPase.It affects the production of vesicles,and plays an important role in the growth and development of plants by influencing the transportation of vesicles as reversed phospholipid molecules.In order to explore the function of ALA in the development of the fiber primary wall or secondary wall,the ALA family genes of Gossypium hirsutum was identified and carried out the genetic characteristics of them by silico.Secondly,the Arabidopsis mutant ala3was used to characterize the biological function of Gh ALA.Finally,the effect of Gh ALA on cotton ovule and fiber development was anylasised through up-regulation and Cas9-Gh ALA2 mutants.1.Identification of P₄-ATPase family genes in upland cottonTotal 32 P₄-ATPase genes related to At ALA3 were identified from the whole genome database of upland cotton by homology comparison.Digital expression profile analysis showed that these genes expressed in all tissues and organs of cotton differentially,including fibers and ovules at different developmental stages.Six candidate genes that expressed in fiber development predominantly were selected for structural domain homozygous.The results showed that these genes all have the important P-type domain of P₄-ATPase.2.Gh ALA3-3 and Gh ALA3-4 cannot restore the Arabidopsis mutant ala3 to wild-type levelIn order to investigate the biological function of Gh ALA,based on the ala3 mutant,Gh ALA was used to restore the phenotype of the ala3 mutant,and it was found that Gh ALA3-3 and Gh ALA3-4 could not restore the root length of the mutant to the wild-type level.3.The expression patterns of Gh ALA3-1 and Gh ALA2 in different tissues and organs of upland cottonUsing q RT-PCR technology to identify the expression pattens,Gh ALA3-1 and Gh ALA2 expressed in all tissues of cotton,but Gh ALA3-1 highly expressed in petals,and predominantly expressed in the early stage of ovule development and late fiber development,Gh ALA2 predominantly expressed throughout the fiber development period.4.Up-regulation of Gh ALA2 inhibites the fiber length and accelerates the thickness of secondary wallThere was no significant change in the growth and development of transgenic cotton plants compared with the wild type by up-regulate the transcription level of Gh ALA2.Scanning electron microscopy showed that the initial number of fibers also did not change significantly.However,the transgenic cotton mature fibers were shorter in length and thicker in cell walls.5.Gh ALA2 mutation affects seed development and fiber developmentThe CRISPR/Cas9 was employed to obtain mutants of Gh ALA2.It was observed that the growth and development of the mutants did not change significantly,but the mature fiber became shorter,the cell wall became thinner,the seed kernel became larger,and the oil content in the mature seed kernel increased.6.Seed oil content increase after ALA2 mutation in ArabidopsisFurther more,the seed oil content increased after Arabidopsis homozygous mutant ala2 was tested,liked the results in cotton.Then,we anylazed the expression pattern of At ALA2 in various tissues and organs of Arabidopsis,and it was found that the one transcripts of At ALA2 had higher transcription levels in the later stages of pod development,another transcripts of At ALA2 expressed in seed particularly.