Cloning And Functional Analysis Of PpMYB4 Transcription Factor And PpCCR Gene Promoter In Pennisetum Purpureum

Elephant grass(Pennisetum purpureum) belongs to Pennisetum, Poaceae, is a perennial herb grass. It is an important germplasm in tropical and subtropical regions. However, the high lignin content restricts the utilization of elephant grass. In order to take advantage of the germ plasm resource, it is vital to modify the lignification by genetic engineering. MYB transcription factors are involved in lignin synthesis at transcription level. The research cloned a MYB transcription factor as well as the promoter of cinnamoy-Co A reductase gene from elephant grass and carried out functional analysis on them, aiming to lay the foundation for lignification improvement. The main results are as follows.(1) In this study, a 756 bp MYB transcription factor CDS sequence from Pennisetum purpureum cv. Huanan was isolated by homologous cloning, named PpMYB4. The cloned sequences had been submitted to NCBI, with the nucleotide accession number of KU612217.(2) By bioinformatics analysis, PpMYB4 concluded 251 amino acids, and the protein formula of PpMYB4 was C1217H1954N362O356S17 with molecular weight of 27.89 KDa, theoretical p I of 8.81. It was predicted that PpMYB4 was an unstable and hydrophilic protein with the instability coefficient of 45.45 and hydrophilic average value(GRAVY) of-0.468. The PpMYB4 was located in the nucleus and contained two conserved domains. The phylogenetic tree analysis indicated that PpMYB4 belonged to the class of monocot R2R3-MYB transcription factor.(3) In this study, the vector of p AN580-PpMYB4 harboring e GFP reporter gene was constructed and transformed to elephant grass protoplast by PEG-mediated method. The result showed that PpMYB4 was located in the nucleus of the protoplast.(4) A sense vector p BA-PpMYB4 was constructed and transformed into tobacco via leaf disc method and seven positive transgenic tobacco lines were obtained. Compared with the wild-type plants, the transformants showed stunted growth due to reduced elongation of the stem internodes and premature of older leaves as well as abnormal leaf morphology. The lignin content of transgenic tobacco decreased significantly, while the OE6 line showed a reduction of 32.45 % compared with the control plants. It indicated that PpMYB4 was involved in the negative regulation of lignin synthesis.(5) The key enzyme genes in lignin biosynthesis were detected in transgenic plants. The relative expression of PAL, C4 H, CAD, CCo AOMT and HCT was 0.49, 0.38, 0.71, 0.30 and 0.45 respectively. The expression of PAL, C4 H, and CAD was significantly lower than that of wild-type plants. It indicated that overexpressed PpMYB4 in tobacco altered the key gene expression in lignin biosynthesis thus causing the change in lignin content.(6) A PpCCR promoter of 1368 bp was cloned by hi TAIL-PCR(from ATG). By prediction, the transcription initiation site of the sequence was T, which located at 261 bp upstream of the ATG. The PpCCR promoter contained TATA-box and CAAT-box. The cis-acting element can be divided into three categories. One category was related to plant growth and development, such as light-responsive elements, meristem expression associated elements(CAT-box), endosperm expression related elements(GCN4-motif), and circadian clock regulation elements(Circadian). The other category was some elements related to exogenous hormone responsiveness, such as the Me JA-responsive elements(CGTCA-motif and TGACG-motif), auxin-responsive element(TGA element) as well as ABA-reponsive element(motif IIb). The last category was some elements related to MYB transcription factor recognition sites.(7) The PpCCR promoter-GUS expression vector was constructed and transformed into tobacco by leaf disc method. GUS histochemical assay was carried out in the stems, petioles, leaves and roots of transgenic plants. The xylem and phloem vascular tissue cells in petiole and stem were stained blue while there was no blue signal in leave and root. The results indicated that PpCCR promoter was a typical vascular tissue specific promoter. Furthermore, the GUS activity and GUS expression level reached a maximum after 6 h under the treatment of 200 ?mol/L Me JA and 100 ?mol/LABA respectively. It showed that the PpCCR promoter respond to hormone induction. In other word, PpCCR promoter also has the characteristics of an inducible promoter. In summary, the PpCCR promoter is both a vascular tissue-specific and an inducible promoter, it can be developed as a dedicated promoter for lignin biosynthesis regulation.