Cloning And Functional Verification Of A Mixta-Like Transcription Factor GhMML2

Plant epidermal trichome is a natural physical barrier of plants,which plays a protective role for plants,can promote plant nutrient absorption,prevent transpiration and water loss,and reduce insect damage.Cotton is an important economic crop worldwide and the main source of natural fibers in the textile industry.Fiber length is one of the important characteristics that determine fiber quality.The fiber is a trichome structure formed by protrusions of the outer epidermal cells of the ovule.At the initial development stage of the fiber,the fiber cells on the surface of the ovule rapidly expanded within 3 weeks,reaching 80%of the length of the mature fiber(2.5-3.5 cm).The rate and duration of fiber cell elongation are directly related to the length of mature fiber,and ultimately determine the quality and yield of cotton fiber to a certain extent.Although all cotton ovule epidermal cells have the potential to differentiate into fibers,in the end only 30%can differentiate into fibers.Fiber development is affected by various internal and external factors,thus forming a complex but valuable model of cell differentiation.Studies have shown that cotton fiber and Arabidopsis epidermis have the same origin and may have the same development pattern.Studies on Arabidopsis leaf trichomes indicate that MIXTA-like transcription factors induce the expression of downstream genes such as the HOX gene by forming the MYB-bHLH-WD40 transcription activation complex,thereby controlling the initiation and development of trichomes.There are 10 MIXTA-like genes in upland cotton,called GhMMLlGhMML10.Previous studies have shown that MIXTA transcription factors play an important role in the development of cotton fibers.For example,GhMML3 and GhMML4 directly participate in the initial differentiation of cotton fibers,and control the development of linters and fibers,respectively.In order to further study the role of MIXTA transcription factors in fiber and epidermal hair development,the predecessors cloned these 10 MIXTA transcription factors from cotton,constructed an overexpression vector of GFP,and transformed them into tobacco to obtain homozygous transgenic tobacco plants.Through the phenotypes of these 10 transgenic tobaccos,it was found that the leaf epidermis of the transgenic lines with GhMML1_D07 and GhMML2_D08 changed.The follow-up study of GhMML1_D07 has been completed by the predecessors.This study mainly focuses on the functional study of GhMML2_D08.Through technical methods such as gene expression activity,heterologous transformation of tobacco,and cotton genome interaction protein screening,we observe the expression pattern of GhMML2 in cotton fiber,Influence on plant leaf development,and build GhMML2 potential fiber cell development regulatory networkThe GhMML2_D08 gene is 891 bp in full length(coding region)and encodes 296 amino acids(AA).It is a member of the 9th subgroup of R2R3-MYB.Through subcellular localization experiments,the results show that the protein is expressed in the nucleus,indicating that GhMML2_D08 encodes a nuclear localization protein.By observing the transgenic tobacco T1 representative type(electron scanning electron microscope and freezing scanning electron microscope),it was found that the ectopic expression of GhMML2_D08 in transgenic tobacco plants changed the morphological characteristics and number of tobacco leaf trichomes.Into a trichome with a multi-branch structure,and the density of epidermal hair is significantly reduced.In order to further determine whether the phenotype of transgenic tobacco is caused by the expression of foreign genes,3 transgenic lines with phenotype and 2 transgenic lines with no change in epidermal hair were selected,and RNA was obtained by reverse transcription.cDNA,RT-PCR,and quantitative results showed that the expression of 3 positive clones was significantly higher than that of the two transgenic lines with no change in epidermal hair,proving that the ectopic expression of foreign genes caused changes in the phenotype of tobacco leaf epidermal hair.In order to further study the mechanism of GhMML2_D08 in epidermal hair development,we constructed a yeast bait vector and screened cotton yeast library.We screened for a clone with a high frequency,encoding a bZIP transcription factor family protein.In order to further verify the interaction protein(Gh_D02G082),we obtained the full length of the TM-1 cDNA by PCR and constructed it into the expression vector pGADT7 by homologous recombination.The results showed that the two did interact.Studies have shown that bZIP transcription factors affect the development of cotton fibers,which indicates that Gh_D02G082 may form a complex with GhMML2_D08 to regulate the development of cotton fibers.The results of this study prove that the MYB MIXTA transcribed gene GhMML2_D08 plays an important role in the development of epidermal hair,which will help reveal the molecular mechanism of epidermal hair branch formation.It further proves the importance of the genes of the 9th subgroup of MIXTA transcription factor R2R3-MYB in regulating the differentiation and development of trichomes.