| The latex of Hevea brasiliensis(the para rubber tree)is a white colloidal suspension liquid obtained from the rubber producing tissue(the laticifers),and is the main source of natural rubber,which is synthesized and stored in the rubber particle,a special organelle in the laticifers.Many rubber particle proteins are employed in rubber biosynthesis including several protein families of rubber elongation factors(REFs),small rubber particle proteins(SRPPs),and rubber transferases(cis-phenyl transferases,CPTs),and they may form protein complexes with other rubber biosynthesis-associated proteins through protein interaction.Identification of the candidate proteins that can interact with REFs,SRPPs or CPTs will facilitate to unveil the composition of the protein complexes underlying the molecular mechanisms of rubber biosynthesis.In this research,one gene of a new member(HbREF3)in the REF family was cloned from the latex of the rubber tree(clone Reyan 7-33-97).Gene expression profile and subcellular localization analysis of HbREF3 were carried out,and the candidate proteins interacting with HbREF3 were identified.The main results were summarized as follows:1.Based on the previous proteomic analysis of rubber particles,one gene for a new member of the REF family,HbREF3,was cloned.The full-length cDNA of HbREF3 gene was 1296 bp which contains an open reading frame(ORF)of 672 bp,encoding a protein with 223 amino acids,and the molecular mass(MW)of the putative HbREF3 protein is of about 24.7 kDa.2.The expression patterns of HbREF3 were investigated by real-time quantitative RT-PCR(RT-qPCR),and the results demonstrated that HbREF3 gene can express in different issues of the rubber tree with the highest expression level in the latex.Ethylene,methyl jasmonic acid and bark tapping can remarkably up-regulate the expression of HbREF3 in mRNA level.3.The prokaryotic expression vector pEASY-El-HbREF3 was constructed,and transformed into E.coli BL21 cells in which a protein with the molecular weight of about 25.0 kDa was induced.The recombinant protein of HbREF3 was used for polyclonal antibody preparation.Western-blot analysis demonstrated that the HbREF3 was expressed on both the small and the large rubber particles.4.The coding regions of HbREF3 and its ho mo logs AtREF in Arabidopsis thaliana and NtREF in Nicotiana tabacum were inserted into pCAMBIA1300 vector to generate fusion proteins of HbREF3:GFP,AtREF:GFP and NtREF:GFP.Subcellular localization was investigated by transforming the three recombinant vectors into the protoplasts of Oryza sativa,and he results showed that HbREF3:GFP,AtREF:GFP and NtREF:GFP were all located in the nucleus of rice protoplasts.5.The candidate proteins interacting with HbREF3 were identified through screening the latex cDNA library with the bait expression vectors that were constructed using the full coding region,the N-terminal region and the C-terminal region of the HbREF3,respectively.A total of 10 candidate proteins were identified by yeast two-hybrid assays using the generated bait proteins pGBKT7-HbREF3-F,pGBKT7-HbREF3-N and pGBKT7-HbREF3-C.Cotransformation assays of the bait and target proteins were further performed,which demonstrated that HbREFl,SRPP,RAB,HEV1,and glucosyl-transferase UDP can interact with both terminals of the HbREF3. |
