Cloning And Functional Analysis Of PpMYB60 Transcription Factor In Pennisetum Purpureum

Pennisetum purpureum belongs to Pennisetum,Poaceae,is a perennial herb grass with high biomass productivity.However,the presence of lignin greatly limits the degradation and utilization of the grass cell wall.Therefore,it is of great significance to use the grass plant lignin reasonably and appropriately to change the composition and content of the grass lignin by genetic engineering.In this study,a transcription factor was cloned from Pennisetum purpureum cv.Huanan,and its function was analyzed.The results of the study are as follows:(1)Primers MYB-F and MYB-R were designed according to the splicing sequence of transcriptome,and an 873 bp MYB transcription factor CDS sequence was amplified by using the c DNA of Pennisetum purpureum as the template,and named as Pp MYB60,Gen Bank ID: MG149570.(2)It was found by analysis that PpMYB60 contains 291 amino acids,and the total molecular weight is 31.81 KDa,which is an unstable protein with an instability coefficient of 51.42.The hydrophilic mean value(GRAVY) was-0.407.In the PpMYB60 protein structure,a highly conserved domain SANT was contained at the N-terminus,and a multiple sequence alignment and evolutionary analysis of its amino acids was carried out.It was found that PpMYB60 belongs to the R2R3 MYB transcription factor.(3)The yeast bait vector pGBKT7-PpMYB60 was constructed and transformed into yeast competent state.It was found to be able to grow on a lack of medium and rather than the three-deficient medium.This indicates that the PpMYB60 gene does not have strong transcriptional autoactivation.(4)The plant overexpression vector pCUbi1390-PpMYB60 was constructed and transformed into Agrobacterium,and the wild type tobacco was transformed by theleaf disc method.After identification,three transgenic lines with high expression levels were finally obtained.The lignin content of stems and petioles of transgenic tobacco and wild type tobacco was determined by phloroglucinol method.It was found that the lignin content of transgenic tobacco stems and petioles was significantly higher than that of wild type(P<0.01).The lignin content in the petiole and stem of OE5 plants was increased by 27.87% and 6.90%,respectively.Meanwhile,the lignin staining of stem lignin was carried out by hand slice.It was found that the number of xylem cells in the stem of the transgenic stem was more than that of the wild type,and the arrangement of the xylem cells was very close,while the number of xylem cells in the wild type tobacco was small,the diameter was small and the arrangement was loose.The results indicate that PpMYB60 gene can up-regulate lignin synthesis.(5)Real-time PCR experiments were carried out on transgenic tobacco to detect the expression levels of key enzyme genes CCo AOMT,CCR,CAD and 4CL in the lignin synthesis pathway.The results showed that the expression level of lignin synthesis related genes in transgenic tobacco was significantly higher than that in wild type(P<0.01).The expression levels of CCo AOMT,CCR,CAD and 4CL genes were 4.87,2.84,2.45 and 2.46 times of wild type,respectively.The results indicated that overexpression of PpMYB60 up-regulated the expression of downstream CCoAOMT,CCR,CAD and 4CL genes,thereby up-regulating the lignin levels of transgenic tobacco.In addition,the transgenic plants treated with 100 ?mol/L ABA and GA,respectively,showed that the expression of lignin synthesis related genes(CCoAOMT,CCR,CAD and 4CL)increased first and then decreased with the treatment time.(6)The dual luciferase reporter gene with LUC and REN as reporter gene was constructed to detect Pp CCo AOMTPro-LUC,Pp CCRPro-LUC,Pp CADPro-LUC,Pp4CLPro-LUC vector and Pp MYB60-SK vector,and the fluorescence value was determined by transient transgenic tobacco.The results indicated that the ratio of LUC/REN in the experimental group was significantly higher than that in the control group(P<0.01),especially the ratio of Pp MYB60-SK+PpCCoAOMTPro-LUC was 28.17 times higher than that in the control group.It was found that the transcriptionfactor Pp MYB60 interacted with PpCCoAOMTPro,PpCCRPro,PpCADPro and Pp4CLPro,and could activate the activities of PpCCoAOMTPro,PpCCRPro,PpCADPro and Pp4CLPro.This results indicated that the transcription factor PpMYB60 can specifically activate the activity of the key enzyme gene promoter in the lignin synthesis pathway,enhance the expression of key enzyme genes in the lignin synthesis pathway,and up-regulate the lignin level.