Characterization Of HbNAC1 Transcription Factor Of Hevea Brasiliensis

Natural rubber is a kind of important strategic material. Laticifer is the main organ of biosynthesis and storage of latex. Differentiation and quantity of laticifers directly affect the production of latex. Laticifers can be induced by mechanical damage or tapping. In order to clarify the molecular mechanism beyond the differentiation of laticifers induced by wounding, a putative NAC transcription factor possibly involved in differentiation of laticifer was identified by differential expression analysis in previous work and was termed as HbNAC1. HbNAC1 is a putative transcription factor containing highly conserved DNA binding domain in N terminal and variable transcription activation domain in C terminal. To further explore the function of HbNAC1, following works were carried out in this work:(1) Transcript’onal activity of HbNACl was identified:Fragments of HbNAC1 with different lengths were amplified using PCR and inserted into pBD-GAL4 vector. Yeast one hybrid showed that full-length and C-terminal of HbNAC1 have transcriptional activity, suggesting that C-terminal of HbNACl is transcription active domain.(2) The interactions between HbNAC1 transcription factor and the promoters of latex synthesis related genes. The vector of pGADT7-Rec-HbNAC1 was constructed and cotransformed into yeast cells together with pHIS2.1 vector containing promoter of HMG reductase (HMGR), or rubber transferase (HRT), or farbesyl diphosphate synthase (FPP), or small rubber partical protein (SRPP), or HbMYC1. Through observation of the expression of His report gene, it was found that HbNAC1 might bind the promoter of SRPP.(3) Prokaryotic expression and purification of HbNAC1 protein:prokaryotic expression vector of pGEX-6p-1-HbNAC1 was constructed and transformed into BL21 cells. The expression of HbNAC1 was induced using IPTG and the protein was purified. This experiment laid a foundation to further verify the interactions between HbNAC1 and the promoter of SRPP using electrophoretic mobility shift assay (EMSA) technology.(4) The interaction between HbNAC1 and the promoter of SRPP would be tested in plants. Over-expression vector (35S-HbNAC1) and transcriptional suppression expression vector (35S-HbNAC1-RSDX) were constructed and transferred into tobaccos previously transformed with GUS reporter gene that was under regulation of SRPP promoter. Through the GUS report gene, the interaction between HbNAC1 and SRPP promoter could be identified. The experiment is still ongoing.Taken together, in this work, the transcriptional activity of HbNAC1 was preliminarily identified, and the transcriptional activity domain was determined in C-terminal. Furthermore, HbNAC1 was proven to interact with promoter of latex-synthesis-related gene, SRPP, in yeast one hybrid test. This result required a further validation using another method, prokaryotic expression and protein purification were therefore carried for HbNACl, which laid a foundation for EMSA assay. Finally, the plant expression vector of HbNACl was constructed and transformed into tabacco, which would be useful for further study the interaction in planta.