Functional Verification Of Genes Related To Flavonoid Metabolic Pathways Against Blight In Gossypium Barbadense

Gossypium barbadense occupies an important position in the production of cotton in China,and blight has become an important reason restricting the high yield and quality of Gossypium barbadense.The research group has conducted in-depth research on the anti-blight of Gossypium barbadense using the recombinant inbred inbred lines and their parents constructed by Xinhai 14 and 06-146 as materials,and using RNA-seq,a number of candidate genes related to disease resistance were preliminarily screened,including disease-related protein genes,genes with detoxification function and related transcription factors,and it was found that flavonoid metabolic pathways play an important role in the process of anti-blight of Gossypium barbadense.Based on the previous research results,the whole genome identification and evolutionary analysis of the ANR gene family of flavonoid metabolic pathways were carried out,the candidate genes of the response were determined and cloned by expression analysis,and the function was verified by VIGS and genetic transformation technology,providing a reliable candidate for the anti-blight of sea island cotton.The main conclusions are as follows:1.Through the analysis and identification of the whole genome of sea island cotton,it was finally found that there were 69 ANR genes in the whole genome sequence,distributed on 19 chromosomes,and most of the genes were located in the cytoplasm.The results of evolutionary tree analysis show that the members of the ANR gene family can be divided into 5 large subgroups in Gossypium barbadense,which is relatively conservative in evolution.Through promoter analysis,it was found that the most cis-acting elements associated with Me JA were found.Through expression analysis,it is speculated that the Gb ANR-47 gene may be involved in the process of Gossypium barbadense against wilt bacterial stress.2.The gene Gb ANR-47 with a fragment size of 1011bp was obtained by cloning the disease-resistant material of Gossypium barbadense.Through expression analysis,it was found that Gb ANR-47 was significantly higher in disease-resistant materials than in disease-susceptible materials and was induced by methyl jasmonate and salicylic acid.Gb ANR-47was silenced by VIGS technology in disease-resistant material 06-146 and disease-susceptible material Xinhai 14,and the blight resistance and anthocyanin content of silent plants decreased significantly compared with negative controls.Compared with the wild type,the Blight resistance of T₃generation Arabidopsis plants overexpressing Gb ANR-47was significantly increased,while the blight resistance of Arabidopsis anr mutants was significantly reduced.This indicates that the Gb ANR-47gene is a positive regulatory gene for wilt resistance in island cotton.3.Successfully constructed Gb ANR-47 gene to tobacco mosaic virus silencing vector.After the Gb ANR-47 gene is silenced,the cotton seedlings with the Gb ANR-47gene are subjected to the infection test of cotton wilt bacteria.The results showed that the leaves of the silent cotton plants were more severe than those of the unstoppable plants,showing grid-like spots,and the plants died with the passage of the infestation time of the wilt bacteria.The same was seen after the anr T-DNA mutant Arabidopsis thaliana.4.GbDFR and Gb F3’H genes were silenced in the disease-resistant material06-146 and the disease-sensing material Xinhai No.14 by VIGS technology,and the blight resistance and anthocyanin content of the silent plants decreased significantly,and the expression of Gb ANR-47 also decreased significantly.Plant overexpression vectors for GbDFR and Gb F3’H genes were constructed and transferred to sea island cotton and Arabidopsis thaliana.Compared with the wild type,the blight resistance of transgenic T₃generation Arabidopsis plants increased significantly.This suggests that GbDFR and Gb F3’H genes may regulate flavonoid metabolic pathways upstream of Gb ANR-47 genes,which lays a certain foundation for studying the regulatory mechanism of genes related to wilt resistance and flavonoid metabolic pathways in Gossypium barbadense...