Construction Of PpCCR Gene Regulating System And Its Effect On Lignin Synthesis In Pennisetum Purpureum

Elephant grass(Pennisetum purpureum),is a perennial large herb grass,and belongs to Pennisetum,Poaceae.It is an excellent forage grass and a new type of energy plant.However,the high lignin content restricts the development and utilization of elephant grass.Therefore,it is very important to reduce the lignin content or change the composition of lignin by means of genetic engineering.The MYB transcription factor and cinnamoy-CoA reductase(CCR)promoter are involved in the regulation of lignin biosynthesis.This paper studies the elephant grass Pp MYB4 transcription factor(accession number: KU612217)and PpCCR promoter(PpCCRPro)interaction,in order to lay the foundation for the improvement of lignification of elephant grass.The main results are as follow:(1)The PpCCRPro-LUC vector and PpMYB4-SK vector were detected by dual-luciferase assay system with LUC and REN as reporter gene.PpMYB4 was found to interact with PpCCRPro by transient detection of tobacco a nd able to repress the promoter activities of PpCCR.(2)The vectors of PpCCRPro-GUS and pBA-PpMYB4 were constructed,and transferred to tobacco.The activity of GUS was found to have a significant interaction with PpCCRPro,and the promoter activities of PpCCR were inhibited.(3)Electrophoretic mobility shift assay(EMSA)found that PpMYB4 can bind to AC-I components on PpCCRPro by using the expression vector p ET-PpMYB4 with the His tag protein gene and the PpCCR promoter probe.(4)The chromatin immunoprecipitation(ChIP)was constructed by expression vector of HA-pBA002-PpMYB4 with the HA-tagged protein.The results showed that PpMYB4 could interact with the promoters of lignin synthesis genes(4CL?CCoAOMT?CCR?CAD)in tobacco,and that binding capacity between PpMYB4 and CAD promoter was the highest.(5)A vector PpCCRPro-PpMYB4 was constructed and transformed into tobacco via leaf disc method,and three positive transgenic tobacco lines were obtained.Compared with wild-type tobacco,the transformants showed significant dwarfing and leaf curling.The lignin content of transgenic tobacco decreased significantly,and the OE3 line showed 35.31% and 36.54% reduction compared with the control plants,respectively.(6)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 PpCCRPro 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 ?mol/L ABA,MeJA and GA treatment of transgenic plants,it was found that the expression of genes(4CL?CCoAOMT?CCR?CAD)in lignin synthesis increased,and then decreased with the increase of treatment time.