Isolation And Characterization Of HbWRKY1, A Hevea Brasiliensis Transcription Factor That Negatively Regulates HbSRPP

Hevea brasiliensis has been the only commercial source of natural rubber mainly because of its high yield, its quality, and the ease of harvesting. So far, researches on natural rubber biosynthesis pathway have made significant progress. Studies on the molecular regulation mechanism of natural rubber biosynthesis remain largely unknown. The key enzyme of participate in natural rubber biosynthesis, including a series of IPP isomerase and prenyl transferase, such as rubber transferase enzyme (RT), FPP synthase, Small rubber particle protein (SRPP) and so on. SRPP plays a direct role in elongating the rubber chain in rubber biosynthesis. In the study, based on HbSRPP gene, we isolated and analyzed systemically the characteristics and function of HbWRKYl, which will lay theoretical foundation for illuminating the regulation mechanism of SRPP in natural rubber biosynthesisThe promoter region of HbSRPP was isolated from Hevea brasiliensis by Genome Walker method. TATA box, CAAT box, GATA-box and light-, hormone-, tissue-specific-, abiotic stress-responsive elements were found in the promoter region of HbSRPP. The promoter of HbSRPP was subcloned into the yeast bait vector. A full-length cDNA (HbWRKYl) was isolated from a cDNA library of the latex by yeast one-hybrid technology. Biochemical analysis demonstrated that the HbWRKYl protein was capable of binding to the HbSRPP promoter, and it demonstrated transactivation activity in yeast.HbWRKYl encodes a479amino acid protein containing two WRKY domains and a putative N-terminal Leu zipper; Bioinformatic analysis showed that the entire sequence of HbWRKYl has a high identity with those of other WRKY proteins; Analysis of the phylogenetic tree indicated that HbWRKYl protein belongs to Group I of WRKY transcription factor family. The Q-RT-PCR results showed that the expression of HbWRKYl gene was tissue-specific, with relative high transcription in somatic embryo, low transcription in flower, bark, latex and leaves.The results of subcellular localization indicated, in the transformed onion epidermal cells, the HbWRKY1-GFP fusion protein was localized in the nucleus, whereas the control GFP was distributed throughout the cell, demonstrating that HbWRKY1was targeted to nuclei. HbWRKYl was subcloned into pGEX-6p-1expression vector, the recombinant protein was induced4h with3mmol/L IPTG in the condition of37℃; Western-blot analysis revealed that the expression of protein was correct.The HbSRPP promoter was transformed into tobacco by Agrobacterium-mediated transformation. In the T] plants of SRRPpro::GUS (β-glucuronidase), the GUS activity analysis revealed that the HbSRPP promoter drives expression of the GUS gene. HbWRKYl was transformed into the T1plants by Agrobacterium-mediated transformation, the GUS activity quantitative analysis revealed, co-expression of the effector construct35S::HbWRKY1with a reporter construct SRRPpro::GUS greatly suppressed expression of the GUS activities in stably transformed tobacco. These results strongly suggest that the HbWRKYl transcription factor does negatively regulate HbSRPP, HbWRKY1may be a negative regulator of natural rubber biosynthesis in Hevea brasiliensis.