Effects Of Interaction Between MYB4 And CCoAOMT Promoter On Lignin Synthesis In Pennisetum Purpureum

Elephant grass(Pennisetum purpureum) is a perennial large herb of the genus Pennisetum.However,the high lignin content restricts the development and utilization of elephant grass.Lignin imparts functions to many types of cells in plant tissues and organs,and lignin metabolism involves plant growth and development.Lignin can resist lodging and prevent plant stems from bending or breaking,which is one of the most important characteristics affecting crop growth.Therefore,it is of great significance to use genetic engineering methods to reduce the lignin content of elephant grass or to change the utilization of lignin constituent grass.The MYB transcription factor and caffeoyl-Co A Omethyltransferase(CCo AOMT)promoter are involved in the regulation of lignin biosynthesis.This paper studies the elephant grass Pp MYB4 transcription factor(accession number: KU612217)and Pp CCo AOMT promoter(Pp CCo AOMTPro)interaction,in order to lay the foundation for the improvement of lignification of elephant grass.The main results are as follow:(1)Three different overexpressing lines(OE2,OE4,OE5) were obtained by screening and overexpressing Pp CCo AOMTPro-Pp MYB4 transgenic tobacco.Plants with dwarfing and leaf curling were observed in overexpressing plants.Among them,strain 4 was the most serious.Moreover,compared with WT wild type tobacco,the lignin content of stem and petiole was decreased by 29.91% and 34.96%.The experiment found that the third stem section was severely shortened,and the length,width,plant height and diameter of the leaves were affected to varying degrees.(2)After the double luciferase assay,the fluorescence value was determined to find a significant interaction between the grassy transcription factor MYB4 and the Pp CCo AOMT promoter.The ratio of the control group(LUC/REN)was 7.72,while the ratio of the experimental group was only 0.44.The control group was 17.55 times more than the experimental group.This indicates that the elephant grass transcription factor has a significant interaction with Pp CCo AOMTPro.This is an inhibitory transcription factor that inhibits the activity of specific promoters.(3)This study quantitatively measures GUS enzyme activity.The GUS enzyme activity of the experimental group was 260.60(pmol 4-MU/min·mg),while the GUS enzyme activity of the control group was 356.96(pmol 4-MU/min·mg).The GUS enzyme activity of the experimental group was reduced by 26.99% compared with the control group.The experimental results indicate that the elephant grass transcription factor can inhibit the activity of the promoter CCo AOMT.(4)The key enzyme genes(4CL、CCoAOMT、CCR、CAD) of lignin synthesis pathway in transgenic plants and wild-type plants were detected by Real-time PCR.The results showed that the expression level of the genes were significantly lower than that of control plants.It indicated that Pp CCo AOMTPro tissue-specific over-expression of Pp MYB4 resulted in a decrease in lignin content of the tobacco due to a decrease in the expression of related genes.Furthermore,after 100 μmol/L ABA,Me JA and GA treatment of transgenic plants,it was found that the expression of genes(4CL、CCo AOMT、CCR、CAD) in lignin synthesis increased,and then decreased with the increase of treatment time,and reached the maximum at 6h after treatment.