Molecular Cloning And Functional Identification Of WUSCHEL Gene From Cotton (Gossypium Hirsutum L.)

Cotton is one kind of important economic crops and oil crops.And tissue culture techniques is crucial for the functional identification of cotton gene and bioengineering breeding.However,most cotton varieties are difficult to obtain regenerated seedlings through tissue culture.Therefore,the research on how to obtain cotton regeneration seedlings efficiently is particularly important.Many studies have shown that WUSCHEL(WUS)gene not only plays an important role in plant somatic embryogenesis,but also regulates the formation of shoot meristem and flower meristem.In this study,two GhWUS genes were isolated and cloned from Gossypium hirsutum,and the chimeric GhWUS inhibitors with SRDX were transformed into G.hirsutum ‘CCRI24' plants to analyze the role of GhWUS in cotton somatic embryogenesis.And then the inducible expression vector of GhWUS was transformed into Arabidopsis thaliana to analyze the effect of the GhWUS gene in somatic embryogenesis and shoot regeneration.The following results were obtained:1.GhWUS affects cotton somatic embryogenesis.Firstly,the expression profile of GhWUS gene was detected by qRT-PCR.The results showed that the expression level of GhWUS was higher in embryoid stage and lower in root,stem and leaf.Next,GhWUS was fused with an inhibitory domain,SRDX,to form a chimeric GhWUS-inhibiting transcription factor and transformed it into ‘CCRI24'.The chimeric suppressor was found to significantly inhibit the transformation from callus into embryogenic callus.2.Induced expression of GhWUS promotes the formation of embryoid and shoot in the root tip of Arabidopsis seedlingsWe constructed pER8 inducible expression vector of GhWUS and transformed it into Arabidopsis thaliana.It was found that the expression of GhWUS in Arabidopsis thaliana resulted in the formation of leaf-like structure and embryoid structure and the expression level of genes involved in somatic embryogenesis or shoot regeneration increased.3.GhWUS induces shoot formation without phytohormone and promotes embryoid formation with auxin from the excised root of Arabidopsis thalianaUsing transgenic Arabidopsis root explants for shoot regeneration experiments,we found that root explants can form shoot in SIM containing inducers without undergoing the pre-culture stage of CIM.Secondly,in the presence of auxin,induced expression of GhWUS can promote the formation of embryoid and adventitious shoots from excised root cultured on MS medium.Moreover,expression of GhWUS in excised root cultured on MS medium without exogenous phytohormones still promoted the formation of adventitious shoots in vitro.