Charaterization Of Hbczf1, A Hevea Brasiliensis Transcription Factor That Positively Regulates HbHMGR1

Natural rubber, cis-1,4-polyisoprene, is a strategically important plant-derived material used in thousands of industrial applications. Currently, Hevea brasiliensis is the sole source of natural rubber. Biosynthesis of natural rubber is known to take place biochemically a mevalonate pathway, but molecular regulation of natural rubber biosynthesis isn’t known. The final step in the mevalonate production is catalyzed by the branch point enzyme,3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), which shunts HMG-CoA into the isoprenoid pathway, leading to the synthesis of natural rubber. In previous studies we found that HbCZF1binds the HbHMGR1promoter and activates the transcription in yeast. In this study, HbCZF1was isolated from a rubber tree, and it encoded protein that could bind to the HbHMGR1promoter. The expression of HbCZF1increased the HbHMGR1promoter in transgenic tobacco plants, indicating that HbCZF1maybe a positive transcription regulator of HbHMGR1involved in rubber tree natural rubber biosynthesis. This work contributes to an increased understanding of molecular regulation of natural rubber biosynthesis.The sequence analysis showed that HbCZF1possessed1,351nucleotides and contained an open reading frame encoding a deduced protein of369amino acids with a predicted molecular mass of43.73kDa and an isoelectric point of8.69. The deduced HbCZF1protein contained a typical CCCH motif C-X7-C-X5-C3-H and ist3RNA combined domain. The amino acid sequence of HbCZF1was aligned with those of the other plant CCCH proteins that exhibit high sequence identities. To confirm the subcelluar location of HbCZF1, the HbCZF1::GFP fusion and the GFP control constructs were introduced into onion epidermal cells by particle bombardment and observed under a fluorescent microscope. HbCZF1::GFP fusion was targeted exclusively to the nucleus of the onion epidermal cells, but the GFP control protein showed GFP signal throughout the cell. The nuclear localization of HbCZF1was consistent with its predicted function as a transcription factor. In EMS A, recombinmant protein His-HbCZF1showed binding the HbHMGR1promoter. Thus, in vitro HbCZF1was able to specifically recognize and interact with the promoter of HbHMGR1.The HbCZF1transcript levels in different rubber tree tissues were examined using real-time RT-PCR. The result showed that HbCZF1was expressed in all the tested tissues at different levels, with the highest transcription occurring in latex and the lowest transcription occurring in leaves. Furthermore, the co-expression of pHbHMGR1::GUS with CaMV35S::HbCZF1significantly decreased the GUS activity in transgenic tobacco, indicating that HbCZFl significantly increased the HbHMGRl promoter. These results suggested that HbCZFl maybe a positive transcription regulator of HbHMGRl involved in natural rubber biosynthesis in H. Brasiliensis.