Cloning And Expression Analysis Of Gossypium Hirsutum L.GhWRKY75

Objective:Cotton fusarium wilt seriously affects the yield and quality of cotton.Screening and functional studies of genes related to the resistance to fusarium wilt in cotton are important for cotton breeding for disease resistance.This study cloned GhWRKY75transcription factor gene from resistant cotton cultivars in upland,analyzed its expression characteristics,and constructed an overexpression vector to transform Arabidopsis thaliana,which laid a foundation for further study on the function of this gene and breeding cotton resistance to wilt disease.Methods:The WRKY gene was expressed as a probe in the gene expression profile of cotton induced by Fusarium oxysporum.The target gene was isolated by electronic cloning combined with RT-PCR.The gene sequence was analyzed by bioinformatics method and real-time fluorescent quantitative PCR was used.Technical analysis of the expression characteristicsofgenes,constructionofplantoverexpressionvectors,Agrobacterium-mediated transgenic technology will be plant overexpression vectors into Arabidopsis plants,screened transgenic Arabidopsis offspring.Results:1.A new gene was cloned from the root cDNA of the resistant cotton cultivar by using electronic splicing and RT-PCR technology and named GhWRKY75?accession number:MH 138002?.Analysis of the gene sequence revealed that the GhWRKY75 gene is 692 bp in length,has an open reading frame of 513 bp,contains an intron and encodes 170 amino acids,and has a WRKY domain and a typical C2H2 zinc finger structure,belonging to the II WRKY transcription factor family.Phylogenetic tree analysis of the homologous gene showed that the gene is closely related to Raymond's cotton.The total number of atoms encoded by the gene is2715,the molecular weight is 19.51 kd,and the molecular formula is C₈₅₇H₁₃₄₄N₂₄₆O₂₆₀S₈.In theory,the isoelectric point is 9.30.The stability index?II?is 32.05,and the protein is classified as a stable protein with a fat index of 60.12 and belongs to a hydrophobic protein.2.Analysis of the gene promoter sequence revealed that the GhWRKY75 gene promoter region contains regulatory elements involved in plant defense and stress response,and participates in the plant's response to ethylene,jasmonate,salicylic acid,and its response to low temperatures.The role of regulatory elements and the role of light response control elements.3.Real-time fluorescent quantitative PCR analysis of the expression pattern of GhWRKY75gene,GhWRKY75 gene can be induced by Fusarium oxysporum and hormones SA,ET,JA in resistant and susceptible varieties.In resistant varieties,the GhWRKY75 gene showed down-regulated expression after Fusarium oxysporum and hormone SA induction,and up-regulated expression after hormone ET and JA induction.Among susceptible varieties,GhWRKY75 gene was in Fusarium oxysporum,hormone SA,ET,After JA induction,they all showed up-regulated expression.4.Construction of the GhWRKY75 gene overexpression vector 35S:GhWRKY75,transformation of Arabidopsis thaliana by Agrobacterium-mediated floriculture,screening Arabidopsis transformed seeds,5 transgenic lines were obtained,and mature seeds were harvested for resistance The seeds of T2 generation Arabidopsis thaliana plants were screened to lay the foundation for further research on gene function.Conclusion:The GhWRKY75 gene was cloned in this study and belonged to the WRKY transcription factor family of type II.qPCR analysis showed that the gene was involved in the response of cotton to pathogenic bacteria and hormones SA,ET and JA.It was speculated that GhWRKY75 transcription factor may be related to the resistance to cotton wilt disease.