Molecular Mechanism Of Powdery Mildew Responsive Gene Hbapcio In Hevea Brasiliensis Muell. Arg.

The ubiquitin proteasome system (UPS) regulates most of the metabolic processings by enzymes modifications and the substrate degradation. The APC/C is one of the most important ubiquitin ligase, and involves in cell division and differentiation. Even though, it widely proved and reported the important function of the UPS in varied plants, not in Hevea brasiliensis anyway.In this study, we found HbAPC10 gene was one of the up-regulated gene amoung the response genes with the treatment of powdery mildew in rubber tree, so the HbAPC10 gene was cloned and analysised, the expression pattern analysised in the method of Q-RT-PCR with the treatment of powdery mildew, exogenous hormone and stress. In order to reveal the function of HbAPC10, the rubber tree SCATC 7-33-97 latex used as material to construced a high quality Yeast-two-hybrid cDNA Library, and the conserved function was identified by confocal laser and re-function of yeast apclOts mutant KK1427. The bait reconstruction vector which named pBD-GAL4-HbAPC10 being convered into the yeast strain Y2H Gold and used as a bait strain to screening the protein which interacting with HbAPC10, and analysis expression patterns of the interacting proteins with powdery mildew infection, exogenous hormone and stress treatment, then we can draw the conclusion as follows:Firstly, HbAPC10 was in response to powdery mildew infection, exogenous hormones and stress treatment, it may be play a role in the disease resistance and stress resistance in Hevea brasiliensis. It was up-regulated with the treatment of powdery mildew, it was down-regulated in 0-3 hour, and up-regulated from 6 to 72 h and reached its highest in 72 hour; it was up-regulated with the exogenous hormones(MeJA, ABA, ET) treatment and up-regulated and reached its highest from 6 to 10 hour the same with the injury treatment; it up-regulated with MG 132 which is a kind of ubiquitin inhibitor, and reached its highest in 72 hour; it indicates that HbAPC10 gene plays roles in disease resistance in rubber tree.Secondly, the Openning Read Frame (ORF) of HbAPC10 contains 579bp,encoding a putative proteins contains 192 amino acids, and it showed that it conserved a DOC1 domain by bioinformatics analysis.Thirdly, HbAPC10 was found in the nucleus with the method of transient expression with which converd the fusion protein 35S: XXGFP-HbAPC10 in tobacco by confocal laser analysis.Fourth, HbAPC10 was transformed into the yeast apclOts mutant KK1427 which is sensitive to temperature, and the reconstructed mutant strain can grew and devison at 35? as normal. The results showed that the HbAPC10 is a subunit of APC/C and it's a conservative protein in function of the APC10 which has a conserved function in different species.Fifth, the HbSRPP and HbPRS4 which interact with HbAPC10 being confirmed by the mathods of Yeast-two-hybrid. HbSRPP is a key enzyme in the biosynthesis of natural rubber,and HbPRS4 encodes a ribose phosphate kinase.HbAPC10 interact with HbSRPP and HbPRS4 was verified with the method of yeast two-hybrid, it indicated that HbAPC10 interact with HbSRPP in regulating the synthesis and resistance of natural rubber thesis.Sixth, HbPRS4 belongs to class ? with its highly homologous with SoPRS4 in spinach and Arabidopsis AtPRS4 in homologous cluster analysis. The expression pattern analysis showed that HbPRS4 was specifically expressed in the bark,indicating that its function was mainly related to bark, and the expression of HbPRS4 was up-regulated with the treatment of powdery mildew, it was up-regulated in 0-3 hour and unchanged from 6 h to 24h, then reached its highest in 72 hour with 8-fold, down-regulated in 72 h in its' 4-fold level; it down-regulated by exogenous ABA and wounding treatment, it was up-regulated in the exogenous treatment of ET, MeJA and MG132, These results suggested that HbPRS4 was regulated by resistance and multiple hormones.In this study, it demonstrates that HbAPC10 was involved in the processing of disease and stress resistance, cell division and natural rubber synthesis, and it provides theoretical basis and practical guidance for the yield regulation and disease-resistance breeding in rubber tree.