Molecular Mechanism Of Expression And Regulation Of HbWRKY1 From Hevea Brasiliensis

Natural rubber is found in the latex of the Hevea brasiliensis tree and consists of high molecular weight cis-polyisoprene. The research focuses on the separation and purification of key enzyme in the synthesis pathway, gene homology cloning and expression, enzyme property analysis, etc. Although some gnens controlling natural rubber biosynthesishave been cloned and characterized, the molecular and expression mechanism regulation of natural rubber biosynthesis remains unclear.HbWRKYl is one of the WRKY transcription factor family and the class I member. The transcription factor of HbWRKYl in latex is significant by resist the protein for JA signal pathway. Biochemical analysis demonstrated that the HbWRKY1 protein was capable of binding to the HbSRPP (small rubber particle protein) promoter’s W-box, and may be a negative regulator of natural rubber biosynthesis in Hevea brasiliensis. Molecular mechanism of expression and regulation of HbWRKYl remains unclear in the word.By this research, we are cloning and Function Analysis of 5’Regulatory Region for HbWRKYl from Hevea brasiliensis, isolation of the proteins interacting with HbWRKYl proteins and the transcription factors that regulate the HbWRKY1 gene, will lay the oretical foundation for illuminating the regulation mechanism of HbWRKYl in natural rubber biosynthesis.The 5’ regulatory region of HbWRKY1 gene from Hevea brasiliensis was cloned using Genome Walker strategy. The 5’ regulatory region is 854 bp longer.Bioinformatics analysis showed that TATA box, CAAT-box and other core configurations were found in this promoter region. Several sequences similar to eukaryotic cis-regulatory element were found in the 5’-UTR proximal 5’flanking sequence of HbWRKY1 gene. Several plant expression vectors with 5’regulatory region of HbWRKY1 gene from Hevea brasiliensis were constructed and transgenic Nicotiana benthamiana plants were obtained by Agrobacterium-mediated transformation. GUS activity analysis revealed that the fragment of 5’ regulatory region of HbWRKY1 drives expression of the GUS gene transgenic Nicotiana benthamiana plants.Through yeast one-hybrid screening, three transcription factor, designated as HbERF、HbMYC2 and HbRGZFl,were identified. The predicted molecular mass of HbERF is 22.47 kDa, with an isoelectric point of 8.74 and the deduced protein showed high similarity to the ERF protein from other plant species. The predicted molecular mass of HbMYC2 is 74.61 kDa, with an isoelectric point of 5.69 and the deduced protein showed high similarity to the MYC2 protein from other plant species. The predicted molecular mass of HbRGZFl is 19.24 kDa, with an isoelectric point of 6.33 and the deduced protein showed high similarity to the RING zinic finger protein from other plant species. Through yeast two-hybrid screening,7 interaction partners of the HbWRKY1, which are involved in plant growth, development, metabolism and signal transduction.