Cloning And Genetic Transformation Analysis Of The Gene Encoding PtWRKY36-1 Transcription Factor From Poplar

Poplar is an important tree species for papermaking. A novel and important strategy has been developed by reducing lignin content or altering its components to satisfy the yield and quality of wood pulp while cutting pollution.WRKY transcription factors are found in the plant in recent years. They have highly conserved WRKYGQK amino acid sequences in their N-termini. It is commonly accepted that WRKY proteins specifically interact with the W-box [(C/T)TGAC(T/C)], which is found in the promoter region of numerous plant target genes, to accommodate the expressions of downstream target genes. Recently, some researches have found that WRKY is involved in the regulation of lignin biosynthesis. According to the data from gene chips GSE13990, we found that Pt WRKY36 is highly expressed in the xylem. Subsequently, it was cloned from Nanlin 95(Populus deltoids cv. Nanlin95) and molecular sequence character, expression pattern, subcellular location, transcription activity were investigated. Overexpression of a specific transcription factor for function identification, lay the foundation for breeding low-lignin poplar. Through this research we obtained following results:1. According to the data of gene chips GSE13990, a candidate gene Pt WRKY36 highly expressed in xylem was screened. The gene was cloned from Nanlin 95 using RT-PCR, named Pt WRKY36-1.2. PtWRKY36-1 encoded a protein 558 amino acid residues with a molecular weight of 6.03 k Da and a theoretical isoelectric point of 6.32. Nine allelic variations(SNPs) in the CDS region between Pt WRKY36-1 and Pt WRKY36. Pt WRKY36-1 possessed two WRKY domains, and it has been assigned to group of WRKY I transcription factors.3. Tissue expression pattern analysis revealed that Pt WRKY36-1 were expressed in the roots, stems, young leaves, old leaves, xylem and phloem, with the highest expression in xylem.The result is in accordance with the gene chips GSE13990.4. To determine the subcellular localization of the Pt WRKY36-1 protein, we constructed Pt WRKY36-1-GFP fusion protein and injected it into the otobacco cells. The results of this experiment revealed that these fusion proteins were expressed in the chloroplast, cytomembrane and nuclear.5. A fusion protein containing a GAL4 DNA binding domain and the whole ORF of Pt WRKY36-1 was constructed.The result suggested that Pt WRKY36-1 may not be a transcriptional activator.6. The over-expression vector p RI-201-AN-GUS-Pt WRKY36-1 was constructed and transformed into “Nanlin 95”.