Cloning And Functional Study Of MFT-like Genes In Gossypium Hirsutum L.

Plant phosphatidylethanolamine-binding protein?PEBP?is comprised of three clades:FLOWERING LOCUS T?FT?-like,TERMINAL FLOWER1?TFL1?-like and MOTHER OF FT AND TFL1?MFT?-like.FT-like regulates identities of the determinate and TFL1-like regulates indeterminate meristems,and ultimately they affect flowering time and plant architecture.MFT is generally considered to be the ancestor of FT/TFL1,but its function is not well understood.Recent research has revealed MFT homologous genes play a very important role in regulating flowering and seed germination in plants.The flowering phenotype of MFT gene deletion mutant?mft-2?is not affected,but it is sensitive to ABA and has a low germination rate.However,the function of GhMFT-like genes in tetraploid upland cotton is still unclear.Objective:The role of GhMFT-like gene in regulating ovule development and seed germination of cotton was preliminarily determined through the analysis of bioinformatics and the expression pattern and the study on the transgenic plant.Methods:GhMFT-like genes were cloned by RT-PCR.The fusion vectors of GhMFT-like-GFP,BD-GhMFT-like and nYFP-GhMFT-like were constructed to observe the subcellular localization and protein interaction.The gene expression was analyzed by quantitative real-time PCR?qRT-PCR?.Plant expression vectors GhMFT-like were constructed and transformed into Arabidopsis by floral-dip.GhMFT-like overexpression vector,interference vector and CRISPR/Cas9 gene editing vector were constructed to carry out genetic transformation of cotton.Results:?1?The genome-wide analysis revealed that upland cotton genomes contained two MFT-like homologous genes,namely GhMFT1 and GhMFT1.?2?In this study,two cotton MFT-like homologous genes were cloned.The ORF length of GhMFT1 was 519 bp,and that of GhMFT2 was 528 bp.?3?GhMFT1 gene was highly expressed in leaves,ovules and fibers,while GhMFT2 gene was highly expressed in stems,flowers and fibers.The expression levels of GhMFT1 and GhMFT2 were higher during the development of ovule and fiber.During seed germination,both GhMFT1 and GhMFT2 expressions were decreased,and both were significantly up-regulated when ABA and NaCl were exogenously provided.?4?GhMFT-like is located in the cytoplasm and nucleus and interacts with GhFD protein.?5?The heterotopic expression of GhMFT1 and GhMFT2 in Arabidopsis did not affect the flowering of transgenic arabidopsis,but inhibited seeds germination of transgenic Arabidopsis.Moreover,the germination rate of transgenic Arabidopsis seeds was much lower when when exogenous ABA and NaCl were applied.Overexpression of GhMFT1 and GhMFT2 did not affect the growth of transgenic Arabidopsis seedlings.AtABI3,AtABI5,AtRGA and AtRGL2 were significantly upregulated in transgenic Arabidopsis seeds.?6?Through Agrobacterium-mediated transformation of cotton,we successfully obtained six 35S:GhMFT1overexpressing positive transgenic seedlings,nine 35S:GhMFT2 overexpressing positive transgenic seedlings and eight 35S:GhMFT2-RNAi transgenic T₀ generation plants.We also successfully obtained 13transgenic T₀ generation plants in GhMFT1-sgRNA1 editing site,five transgenic T₀ generation plants in GhMFT1-sgRNA2 editing site,18 ttransgenic T₀ generation plants in GhMFT2-sgRNA3 editing site and two transgenic T₀ generation plants in GhMFT2-sgRNA4 editing site.Conclusion:The upland cotton genome contains two MFT-like homologous genes.Two GhMFT-like genes were mainly expressed in fiber and ovule and regulated the germination of cotton seeds.GhMFT-like interacts with GhFD in the nucleus.The heterogeneous expression of GhMFT-like did not promote the flowering of transgenic Arabidopsis,but inhibited the germination of transgenic Arabidopsis seeds.Transgenic cotton with GhMFT-like overexpression,interference and CRISPR/Cas9 gene editing were obtained.The phenotype of transgenic cotton remains to be further verified.