Analysis Of The GhAIF Family And Functional Elucidation Of GhAIF3 In Cell Elongation In G.hirsutum

Elongation of the cell plays a crucial role throughout the growth and development stage process in cotton.Regulating the cell elongation can regulate the plant height,which plays an important role in shortening cotton branches and reducing the initial node position of the fruit branch.In addition,cell elongation also plays an irreplaceable role in the fiber development of cotton.Using the GhAIFs family of genes as the research direction,the following conclusions can be drawn:1.Eight,eleven,sixteen,and fifteen AIFs genes were identified in G.arboreum,G.raimondii,G.hirsutum and G.barbadense,respectively.The phylogenetic tree classifies AIF genes into three subfamilies.Analysis of gene duplication events suggested that AIFs might have evolved asymmetrically,and the gene amplification of AIFs in G.raimondii and G.hirsutum might be mainly due to segmental duplication and tandem duplication.Whereas the AIFs gene amplifications in G.arboreum were almost all segmental duplications.Ka/Ks analysis indicates that the majority of genes are subject to purifying selection.2.Transcriptome data and q RT-PCR analysis of the expression pattern of GhAIFs in different developmental stages of cotton fibers further confirm the potential role of GhAIFs during early fiber development.Cis-acting element analysis of the promoter of GhAIFs revealed that the promoter region of GhAIFs contains multiple hormone responsive and cold stress regulatory elements.Two different cold tolerance materials treatment with 4°C,the analysis of the expression quantity of GhAIFs showed that most of the GhAIFs responded to cold treatment and that there were differences in their expression levels in cold resistant materials and cold sensitive materials.These results suggest that GhAIFs actively respond to cotton chilling stress.QRT-PCR analysis showed that GhAIFs were significantly responsive to hormonal treatment.3.Taking GhAIF3（named GhAIF9 in the family）as a research object.Tissue expression analysis found that GhAIF3 was dominantly expressed in flower organs as well as in the early stage of fiber development,it was speculated that GhAIF3 might be involved in flower organ formation,and affected the initiation process of cotton fiber.4.Overexpression of GhAIF3 in Arabidopsis found that transgenic Arabidopsis resulted in delayed flowering;Virus induced gene silencing（VIGS）experiments found that inducing the decreased expression of GhAIF3 in cotton caused a reduction in the shoot initiation segment in cotton.Thus this gene might involved in negatively regulating flowering.Overexpression of GhAIF3 in Arabidopsis resulted in shorter leaf epidermal hair length,plant dwarfism,and shorter root length phenotypes,indicating GhAIF3played a negative regulatory role in cell elongation.QRT-PCR analysis indicated that overexpression of GhAIF3 promoted BR and sterol synthesis in Arabidopsis,and treatment with different concentrations of exogenous BR found that for the35S::GhAIF3 plants,root growth inhibition of the WT plants treated with BL was greatly attenuated,10^-9 M BL did not inhibit their root length,and 10^-8 M BL was required to reduce root growth by 50%compared with the untreated mock control.These results indicated that overexpression of GhAIF3 reduces the susceptibility of plant roots to BR inhibition.5.Y2H experiments showed that GhAIF3 can interact with GhCIL1.Under long day conditions,plants transgenic for 35S::Myc CIL1 flowered significantly earlier than the wild-type parents.Likewise,GhAIF3 was expressed in higher amounts in floral organs and overexpressed GhAIF3 late flowering phenotype.CIL1（also known as ACE1）forms a triple antagonistic b HLH system with the PREs and IBH1 to competitively regulate cell elongation,and overexpression of GhAIF3 can also lead to plant dwarfism and other phenotypes.The genome-wide identification of the GhAIFs family in G.hirsutum with the functional analysis in cotton fiber development and cold stress response as well as the cloning and functional analysis of GhAIF3 laid the foundation for in-depth exploration of the role of GhAIF3 in cotton genetic improvement.