Cloning, Expression And Regulation Of Soluble Inorganic Pyrophosphatase Genes From Hevea Brasiliensis

Natural rubber is a kind of polyisoprene as the main component of natural polymers, its biosynthesis consists of three distinct biochemical processes:chain initiation, chain elongation, and termination of the polymerization reaction. Excess pyrophosphate released in the processe of termination will eventually inhibit rubber biosynthesis. PPi hydrolysised by pyrophosphatase timely has an important role in regulating rubber biosynthesis and rubber production. In this study, three soluble inorganic pyrophosphatase genes were isolated, molecular characterization and expression regulation of them were studied, which will lay theoretical foundation for illuminating the function and mechanism of pyrophosphatase in Hevea brasiliensis. The main results were summarized as follows:Three soluble inorganic pyrophosphatase genes coding for216,215and216amino acids, designated as HbSIP1, HbSIP2and HbSIP3, were cloned. There are8exons and7introns in the coding regions of three genes, and all of these7introns are in accordance with the GT-AG splicing formula. Bioinformatics analysis showed that, HbSIP1/2/3shared high identities to soluble inorganic pyrophosphatases from other plants, had the typical structure of plant soluble inorganic pyrophosphatase, containing24highly conserved amino acid residues, in which15amino acid residues corresponding to putative active site residues, and a binding sequence of divalent metal ions.Prokaryotic expression vectors for three genes were constructed, and the fusion proteins were expressed in Escherichia coli. The purified proteins GST-HbSIP1/2/3had the activity of hydrolysising PPi, and their activity was dependent upon the presence of Mg2+. The optimum pH of GST-HbSIP1/2/3activity was7.5,8.0and7.5respectively, and the Km value of GST-HbSIP1/2/3was98.4μM,194.7μM and138.6μM respectively.The Q-PCR results showed that three genes were expressed in all detected organs, in which, the transcripts of HbSIP1and HbSIP3were higher in latex than in other organs. The expression levels of HbSIPl were accelerated in response to ET, JA, high temperature, low temperature, PEG and H2O2, while decelerated in response to SA and ABA. The transcription levels of HbSIP2were enhanced in ET, JA, SA, PEG and H2O2-treated, declined in high temperature-treated, while not significantly altered by treated with ABA and low temperature. The transcripts of HbSIP3were up-regulated by JA, SA, PEG and H2O2, negatively regulated by high temperature and low temperature, and basically stable under ET treatment.The2297bp,1482bp and844bp5’regulatory sequences of HbSIP1/2/3were isolated, and there was a1499bp intron in5’UTR of HbSIP1. Many basal promoter elements of eukaryotes, elements in response to stress signals and hormones, and elements binding transcription factors were found in5’regulatory sequences of HbSIP1/2/3. Plant expression vectors of pCAMBI1381-P1/2/P3containing1196bp,791bp and844bp of5’regulatory sequences of HbSIP1/2/3were constructed. GUS activity detection in transgenic plants indicated that P2and P3could drive GUS expression.One transcription factor (HbZF-HD) regulating HbSIP1, three transcription factors (HbGATA、HbWRKY and HbbHLH) regulating HbSIP2and two transcription factors (HbZF and HbMyb2) regulating HbSIP3were screened by yeast one-hybrid. The Q-PCR results showed that six transcription factors were expressed in all detected organs.Forty-one interaction proteins involved in protein synthesis, protein modification and activity regulation, latex basal metabolism, rubber biosynthesis, material transport and signal transduction of HbSIP1/2/3were screened by yeast two-hybrid. Bimolecular fluorescence complementation studies indicated that HbSIP1/2/3could interact with rubber elongation factor.