Function And Regulatory Mechanism Analysis Of Cotton Somatic Embryogenesis Related Genes GhMYB44

Upland cotton is widely cultivated as an important fiber and cash crops.Establishment of rapid verification system for Upland cotton gene-editing vector urgently needed a stable and efficient somatic embryogenesis system.However,genetic transformation of cotton is still seriously limited by genotypes,low conversion efficiency and the conversionrate of seedlings deformities.In order to analyze the molecular mechanism of somatic embryogenesis.A differential expression of GhMYB44 gene was found in the RNA-Seq profile during cotton SE in our previous studies.GhMYB44 is differential expression in the embryogenesis during cotton SE.So we conducted the following research during the cotton SE.1.GhMYB44 regulates cotton somatic embryogenesis.To identify the function of GhMYB44 during cotton SE,overexpression and a suppressor domain SRDX to form a chimeric GhMYB44 inhibitor vectors were constructed and transformed into G.hirsutum CCRI24.The results revealed that overexpression of GhMYB44 restricted the uncontrolled callus proliferation and promoted the EC formation.The chimeric suppressor and ck explants were dedifferentiated vigorously but retarded EC formation.2.GhMYB44 affects auxin accumulation during cotton SEIn order to study whether GhMYB44 affects the concentration of endogenous auxin during cotton somatic embryogenesis,the IAAcontent of GhMYB44 transgenic and ck lines at 7、15、35 and 60 days post-induction was measured by ELISA kit.The results show that the concentration of endogenous IAA was increased in the GhMYB44 overexpression lines compared with CK.By contrast,it was decreased in the SRDX inhibitor vectors lines compared with ck duiring callus differentiation into embr yogenic callus stage.In order to investigate the function of GhMYB44 in auxin accumulation,the F1 hybrids of GhMYB44 transgenic lines with DR5::GFP line were generated to track the auxin accumulation.The GFP fluorescent signal was markedly increased in the shoot apical meristem(SAM)of the OE-GhMYB44/DR5::GFP hybrids compared with the CK/DR5::GFP hybrids.The results showed that there was a large amount of auxin accumulation in the GhMYB44 overexpression materials duiring the process of callus proliferation.It is speculated that callus with high auxin content is more likely to differentiate into embryogenic callus.3.The interaction protein of GhMYB44 was screen and then the function verification was conducted.In order to study the molecular mechanism of somatic embryogenesis regulated by GhMYB44 in depth,we have screened the interaction targets using effector GhMYB44 as bait in cotton somatic embryogenesis c DNA library.After that,we confirmed the interactions between GhMYB44 with Gh PYL8 using Y2H and Bi FC.The expression profile of Gh PYL8 gene was detected by q RT-PCR.The results showed that the expression level of Gh PYL8 decreased gradually in the early stage of callus induction,and with enhanced expression in embryonic tissues,then decreased gradually.That indicates that Gh PYL8 is closely related to the acquisition of embryogenic callus.The expression of the gene increased gradually in stage of the transformation from callus to embryogenic tissue in 6 highly differentiated materials.Furthermore,the function of Gh PYL8 during somatic embryogenesis was verified.The results revealed that over expression of Gh PYL8 restricted the callus initial dedifferentiation and promoted the EC formation.It is speculated that the interaction between Gh PYL8 and GhMYB44 decrease the transcriptional activity of GhMYB44 protein.Genetic and biochemical assays reveal the interaction between Gh PYL8 and GhMYB44 regulates callus morphogenetic patterns and then regulation the callus proliferation and embryonic callus formation.4.Screening of downstream target gene for GhMYB44 and functional identificationUsing MEME program,results showed GhMYB44 binding motif(TAACC).The Effector-Reporter assay proved that the expression of Gh LBD18 was inhibited by GhMYB44.Opposite patterns of expression were showed between GhMYB44 and target gene Gh LBD18 in the initial stage of callus dedifferentiation,indicated that GhMYB44 negatively regulates Gh LBD18 transcription.Overexpression of Gh LBD18 promotes callus formation and proliferation which is inversely related to the phenotype of GhMYB44.The above results indicated that GhMYB44 was able to bind to the―TAACC‖motif of Gh LBD18 promoter to inhibit the initial cellular dedifferentiation and callus proliferation of cotton...