Cloning Of GhDAHP Homologous Gene And Preliminary Validation Of Verticillium Wilt Resistance Function In Gossypium Barbadense

Verticillium wilt has seriously affected the yield and quality of cotton in China.In this study,based on the preliminary laboratory research on St DAHP(GU479467.1)gene in land cotton in the early stage.We adopted the homologous sequence cloning method.Three DAHP gene fragments were cloned from the sea island cotton ‘Xinhai 16’.They were named as GhDAHP-1,GhDAHP-6 and GhDAHP-7.1.Bioinformatics analysis of target genes by online software: The full length of GhDAHP-1 gene is1755 bp,Encoding 539 amino acids;The full length of GhDAHP-6 gene is 1644 bp,Encoding 519 amino acids;The full length of GhDAHP-7 gene is 1554 bp,Encoding 517 amino acids.Bioinformatics analysis by online software revealed that the physicochemical properties of the GhDAHP proteins were similar.GhDAHP proteins are located in chloroplasts.GhDAHP protein is a hydrophilic protein,they does not have transmembrane transport domain,and no signal peptide.GhDAHP gene has high sequence similarity with Gossypium hirsutum,Gossypium raimindii and Theobroma cacao,The GhDAHP protein had the highest protein similarity and closest affinity to Gossypium hirsutum and Gossypium raimindii.2.Inoculation treatment of different sea island cotton,Detection of expression changes of GhDAHP gene by fluorescence quantitative PCR after infestation with Xanthomycetes.The results showed that: The expression of GhDAHP-1 gene was highest at 24 h of inoculation treatment in ‘Junhai 1’ and ‘Xinhai 16’.The expression of GhDAHP-1 gene in ‘Xinhai 20’ reached the highest level at 48 h of inoculation treatmen t.The expression of GhDAHP-6 gene in all three island cotton varieties reached the highest level at 48 h of inoculation treatment.The expression of GhDAHP-7 gene in "Junhai 1" reached the highest level at 24 h of inoculation treatment.The expression of GhDAHP-7 gene was highest in "Xinhai 16" and "Xinhai 20" at48 h of inoculation treatment.Comparing the expression changes of the three target genes in different tissues and different inoculation treatment periods of Sea Island cotton,we can see that: The expression of GhDAHP gene showed an increasing trend followed by a decreasing trend.indicating that GhDAHP genes were induced to be expressed by Verticillium dahliae.3.The GhDAHP gene was silenced by VIGS technology in Sea Island cotton "Xinhai 16".Inoculation of the silenced plants.Observation of phenotypic changes and disease finger survey,the results showed that:Wild-type plants and TRV2/GV3101 plants had less disease.In comparison,GhDAHP-1/GV3101 plants,GhDAHP-6/GV3101 plants and GhDAHP-7/GV3101 plants were severely affected,Even some plants have wilted as a whole.This indicates that plants silenced with the target gene are more susceptible to the disease than wild-type plants and null plants.The results of the disease index survey showed that: The disease indices of recombinant plants injected with the target gene were significantly higher than those of TRV2/GV3101 plants and wild-type plants not injected with recombinant bacteria.This indicates that the GhDAHP gene plays a role in cotton resistance to Verticillium wilt.