The Molecular Mechanism Of GhMYC2 Involved In Regulating Gossypol Synthesis In Cotton

Cotton is one of the most important economic crops in the world.Cottonseed contains fiber,fat,protein,and starch,which is a good resource of oil and protein;However,gossypol in cottonseed seriously affects the development and utilization of cottonseed.Gossypol,a sesquiterpene lactone,is an important secondary metabolite in cotton that resists pathogen invasion and insect bites.Gossypol is mainly stored in the pigment glands under the epidermis in cotton roots,stems,and leaves.It has great potential applications in the fields of medicine,agriculture,and industry.In addition,it resists pests and pathogens,and can be used as anticancer and contraceptive drugs.Inducing the synthesis of secondary metabolites is one of the important reactions of JA signal that involved in plant resistance to stress.MYC2,a positive regulator in JA signal pathway,can interact with ERF,DELLA to participate in the regulation of plant secondary metabolite synthesis and accumulation.MeJA can induce the synthesis and accumulation of gossypol in cotton hairy roots,but the specific molecular mechanism is still unclear.Glands is an unique tissue in Hibiscus mutabilis,which is the main organ for storing gossypol in cotton,they are also called "oil glands","black glands" and exist in various tissues of cotton.Glands play an important role in cotton resistance to biotic and abiotic stresses.Since GoPGF silenced,cotton plants showed a typical glandless phenotype,and the content of gossypol is significantly reduced.The function of GoPGF provides an important reference value for the cultivation of low gossypol cotton.However,the disease resistance and insect resistance of glandless cotton are weakened.Therefore,glandless only in cottonseed plants are ideal breeding varieties,which can ensure the resistance of cotton and make full use of cotton seed resources.In this study,we cloned and identified the b HLH transcription factor GhMYC2,analyzed its expression mode and function.GhMYC2 involved in the gossypol synthesis of cotton,we indicated that the molecular mechanism of JA signal pathway in regulating synthesis of gossypol.At the same time,we also found that GhMYC2 can interact with GoPGF,which is the key transcription factor for gland formation,GoPGF interacts with GoPGF to regulate the the synthesis of gossypol.These results provide an important reference value for glandless and low-gossypol cotton variety cultivation.The research results are as follows:1.There are 8 homologous genes with At MYC2 in cotton.It was found that Gh_D12G2074 has the highest homology,that has been reported as GhMYC2.The functional domain of GhMYC2 was highly conserved with other MYC2 genes.GhMYC2 has transcriptional activity and mainly localized in the nucleus.The relative expression of GhMYC2 was higher in cotyledons and stems and lower in ovules and roots.The expression level of GhMYC2 was significantly induced by JA in cotton seedlings was significantly.It shows that GhMYC2 is functionally similar to other MYC2 genes and participates in the regulation of gossypol synthesis in cotton.2.The changes of GhMYC2 expression affect the gossypol synthesis in cotton.Since silencing GhMYC2 in cotton,the expression of gossypol synthesis genes CYP71BE79,CDNC,CYP706B1,DH1,CYP82D113 and 2ODD-1 decreased significantly compared with the control.And the content of gossypol in cotton leaves decreased significantly,which indicate that the silencing of GhMYC2 inhibited the synthesis and accumulation of gossypol.The hairy root cotton overexpressed GhMYC2 was further cultured.RT-PCR experiment showed that the expression of CYP71BE79,CDNC,CYP706B1,DH1,CYP82D113 and2ODD-1 increased significantly in the hairy root overexpressed,and the content of gossypol was also significantly increased In conclusion,GhMYC2 is involved in the regulation of gossypol synthesis in cotton and plays a positive regulatory factor.3.GhMYC2 activates the expression of CYP71BE79,CDNC,CYP706B1,DH1,and CYP82D113 to regulate the synthesis of gossypol.The cis element analysis of the promoter of gossypol synthesis gene showed that the promoter regions of CYP71BE79,CDNC,CYP706B1,DH1,CYP82D113 and 2ODD-1 contained obvious G-box and MYC binding sites.The results of Y1 H,Chip q PCR and Double Luciferase report experiments indicated that GhMYC2 could specifically bind to the G-box,which exists in the promoter region of CYP71BE79、CDNC、CYP706B1、CYP82D113 and DH-1 to activate their expression.We did not detect the activation of the 2ODD-1 promoter by GhMYC2.To sum up,CYP71BE79,CDNC,CYP706B1,DH1 and CYP82D113 are the target genes of GhMYC2.GhMYC2 regulated their transcription levels to participates in gossypol synthesis.JAZ is a negative regulator in JA signaling pathway.Previous studies have shown that Gh JAZ2 can interact with GhMYC2 to inhibit its function.Through the Double Luciferase report experiment in tobacco,we proved that the activation of GhMYC2 on CYP71BE79 promoter was significantly inhibited by GhJAZ2.4.GhMYC2 can interact with GoPGF.GoPGF is a key transcription factor for gland formation in cotton.The silence of GoPGF caused an obvious glandless phenotype in cotton,and the content of gossypol is significantly reduced.With the silence of GhMYC2 silencing,the gland density of cotton leaves and stems decreased significantly compared.We proved that the interaction between GhMYC2 and GoPGF through Y2 H,Co-IP,BIFC assay,which provided an important reference for the study of molecular mechanism of cotton gland development.