Cloning And Functional Verification Of Genes Related To Fiber Development In Gossypium Barbadense L.

Cotton is the main source of renewable fiber in the world,mainly used in textile production,and plays an important role in the global textile industry.Cotton fiber is a single cell differentiated from the ovule epidermis and is an important model for studying cell elongation,doubling,and cell wall biosynthesis.With the acceleration of spinning speed and the improvement of people’s living standards,the demand for improving the quality of cotton fibers is becoming stronger.Therefore,mining cotton fiber development genes,understanding fiber cell development models,and elucidating the molecular mechanism of cotton fiber development and regulation are of great significance for making full use of cotton gene resources,improving cotton yield and fiber quality,and even developing artificial fibers.In this paper,based on the QTL loci associated with the specific strength of fibers in the laboratory in the early stage,combined with the functional annotation of Gossypium barbadense/HAU-AD2_genome_v2.0,and using the quantitative real-time PCR method,Based on 8 fiber materials with extremely different strengths,expression analysis was performed on the screened fiber differentially expressed genes.Finally,the fiber development candidate genes GbMYB5,GbRPL23A,and GbWDL2 were cloned and analyzed by bioinformatics.Using model crops Arabidopsis as reference Experimental methods such as gene-mediated silencing to verify the function of candidate genes,and to understand the expression patterns and molecular functions of candidate genes.The specific conclusions are as follows:1.Based on pre-labor fiber specific strength QTL locus annotation genes and differentially expressed genes enriched in RNA-seq at different stages of fiber development in Xinhai 21 and Xinluzhong 36,based on fiber development-related pathways and RPKM values,Fourteen differentially expressed genes were screened.2.Perform qRT-PCR analysis on the expression levels of 14 differentially expressed genes in fiber development.During the fiber development period of 0,5,10,15,20,25,30 DPA(days post anthesis),the expression of 9 genes,There are different differences in the amount of fiber strength extreme materials,including the expression of MYB5,AXS2,WNK8,SAP7,RPL23A genes during fiber development and elongation,and the secondary wall synthesis thickening stage shows significant or extremely significant differences;8 islands Correlation analysis between the fiber strength and the expression level of the material with extreme strength of cotton fiber shows that the expression level of MYB5,RPL23A,WDL2 and AXS2 genes is significantly or very significantly positive in fiber development and elongation and secondary wall synthesis thickening related3.Based on the results of real-time fluorescence quantitative analysis,clone the sequences of three gene coding regions of GbMYB5,GbRPL23A,and GbWDL2.GbMYB5 is an R2R3-MYB transcription factor and contains a highly conserved HTH-myb DNA-binding domain,which is in line with the characteristics of MYB transcription factors.GbRPL23A is a 60S ribosomal protein,which is a very hydrophilic stable protein,containing the ribosomal protein L14 protein domain;GbWDL2 is a new plant-specific microtubule-binding protein of the WVD2/WDL family,which is a hydrophilic unstable protein Contains a highly conserved TPX2 domain.All three genes are also expressed in different tissues.Except for GbMYB5,which is significantly expressed in petals,the expression levels of the three genes in other tissues are all lower than the stage of fiber development elongation and secondary wall synthesis in high FS materials.Genes play a major role in fiber development.4.GbMYB5,GbRPL23A successfully achieved gene silencing in island cotton,and the silencing efficiency of island cotton 5917 was significantly higher than that of Xinhai 21;qRT-PCR showed that in the leaves of cotton with two genes silencing successfully,the fiber elongation was inferior.The expression of thickening-related transcription factors and downstream genes in the living wall has changed significantly,indicating that GbMYB5 and GbRPL23A may affect the development of cotton fibers by regulating the expression of related genes during the elongation and thickening stages of fiber cells,and affect cotton fiber development.quality.