Analysis Of The Interaction Between PpMYB4 Transcription Factor And PpCCoAOMT Promoter In Pennisetum Purpureum

Elephant grass(Pennisetum purpureum),a new type of energy C4 herbaceous plants,belongs to Gramineae,and is the most common in southern China.The advantages of elephant grass are the developed root system,sturdy stems,and lush leaves.So it can be the best feed crops,the paper industry and biomass energy raw materials.But its high lignin content limits the energy development value of elephant grass.Therefore,it is important to explore the key enzyme regulation mechanism in lignin synthesis,and to change the composition and content of lignin by biotechnology.Caffeyl coenzyme A-O-methyltransferase(CCoAOMT),a key enzyme in the lignin biosynthesis,which controls the connection and reduction of downstream enzymes.PpMYB4,belonging to R2B3 type MYB subgroup 4,is an inhibitory transcription factor which has a negative effect on the key enzymes in lignin biosynthesis and can change the composition and content of lignin.In this paper,the interaction between PpMYB4 transcription factor and PpCCoAOMT gene promoter was studied.The main findings are as follows:(1)The result of three varieties of elephant grass qRT-PCR which included in P.purpureum cv.N51,P.purpureum cv.MT-1 and P.purpureum cv.Huanan,showed that there was a different relative expression between PpCCoAOMT and PpMYB4 in different parts of different varieties.For the same elephant grass variety,the PpCCoAOMT gene expressed higher in the stem middle and mature internode,and PpMYB4 gene in its upper internode have a lower expression.For the different grass varieties,the expression of PpMYB4 gene in the upper stem internode was higher than PpCCoAOMT gene,while theexpression of PpCCoAOMT gene in the middle and lower internode was opposite to that in the upper part.Among them,the expression level of PpCCoAOMT gene in elephant grass N51's middle internode was the highest,and the relative expression of PpMYB4 was the lowest.The results showed that PpMYB4 could inhibit the expression of PpCCoAOMT in the lignin synthesis pathway.(2)The 932 bp promoter sequence before PpCCoAOMT gene initiation codon ATG was obtained by cloning using P.purpureum cv.N51 as the material.The results of software analysis and prediction show that the sequence has the core structure of the typical promoter region,and contains a variety of response elements of light,abiotic acid,gibberellin and other endogenous hormone,and the cis acting elements of drought,low temperature,dehydration and so on.Moreover,there has many kings of transcription factor recognition sites and binding regions,like as BHLH,WRKY,bZIP and Dof transcription factor binding sites,especially the R2R3-MYB.(3)Transient expression showed that the tobacco leaves with the PpCCoAOMT promoter could be stained blue and the GUS activity was significantly increased,indicating that PpCCoAOMT promoter has the expression activity.(4)Electrophoretic mobility shift assay(EMSA)showed that the AC-II cis element(ACCAACC)in PpCCoAOMT promoter can specificly combined with PpMYB4 transcription factor,and the binding region located at the-344 bp position of the promoter upstream of TSS,which is consistent with the prediction results.(5)The GUS expression analysis of PpMYB4 transcription inhibited the expression of PpCCR ? PpCAD ? Pp4 CL and PpCCoAOMT promoter which actived in lignin biosynthesis by the transient transformation experiment of tobacco leaf,which resulted in the activity of GUS reporter gene became decreased significantly.