Studies On The Candidate Proteins Interacting With Small Rubber Particle Protein(SRPP)of Hevea Brasiliensis

Natural rubber(NR),a unique biopolymer of cis-1,4-polyisoprene,is an indispensable industrial raw material,and Hevea basiliensis(the para rubber tree)is the most important species that could produce quantities of latex used for commercially viable high-quality NR in the world.The latex is the cytoplasm of the laticifer where rubber biosynthesis occurs on a highly specialized organelle called rubber particle.Many rubber particle proteins are associated with rubber biosynthesis,including rubber elongation factor(REF),small rubber particle protein(SRPP),and rubber transferase(HRT),and they may form protein complex with other rubber biosynthesis-related proteins through protein interaction.SRPP is a pivotal protein that determines the ratio of rubber biosynthesis and the latex yield of rubber trees.Identification of the candidate proteins interacting with SRPP will not only facilitate to unveil the composition of the protein complex underlying the molecular mechanisms of rubber biosynthesis,but also provide the theoretical foundation for molecular breeding technology to increase the latex yield of rubber trees.In this research,the candidate interacting proteins of SRPP were screened and identified by yeast two-hybrid method.Gene expression pattern and subcellular localization of the SRPP interacting proteins were further analyzed using rubber tree clone Reyan 7-33-97 and Arabdopsis protoplasts as the experimental material,respectively.The main results were summarized as follows:1.Candidate proteins interacting with SRPP were identified by yeast two-hybrid system using SRPP as the bait.An expression vester SRPP-pGBKT7 containing the full coding region of SRPP was firstly constructed,and then was used to screen latex cDNA library.A total of 28 candidate SRPP-interacting proteins were obtained,and 12 were selected for further clarified by co-transformation method,among which 11 positive prey proteins were identified,including REF,thiamine thiazole synthase-like(TTS),caffeoyl shikimic acid esterase(CSE),F-box protein containing Kelch repeat motifs(F-box/kelch-repeat protein SKIP11,SKIP),proteasome subunit,auxin binding protein(ABP),UDP glucose transferase,and 60S ribosomal protein etc.2.Polyclonal antibody against SRPP was prepared.Western bloting detection showed that SRPP had the highest expression level in smaller rubber particles than in large ones,which is consistent with the highest activity of rubber biosynthesis of the small rubber particles.3.Subcellular localization was performed by using suspension cells of Arabidopsis for three SRPP-interacting proteins,i.e.,TTS,CSE and SKIP.Results demonstrated that TTS was located in both the plasma membrane and chloroplast,while CSE in the cytosol,and SKIP in the nucleus.4.Gene expression profiles of SRPP and its candidate interacting proteins were investigated by using quantitative real-time RT-PCR(RT-qPCR).The results showed that bark tapping(mechanical wound)can significantly increase the transcript levels of SRPP and its interacting proteins except for TTS and ABP.Ethrel and jasmonic acid(JA)treatments can dramatically induce the gene expression of SRPP,REF and HRT2,but inhibit the expression of TTS gene.