| Natural rubber is an important industrial raw material and the commercial natural rubber almost comes from rubber tree (H. brasiliensis). Natural rubber exists in rubber latex. Latex as the cytoplasm of laticifers, contains rubber particles (RPs),which is a class of unique organelles, and the place of rubber molecular extension and natural rubber storage. The results of the proteomics study of RPs showed that RPs contained more than 300 proteins, and the two most abundant proteins were rubber elongation factors (REF) and small rubber particle membrane proteins (SRPP),belonging to a same REF/SRPP family. Up to now, the study of REF and SRPP is not very clear. The study of the protein family could help reveal the natural rubber synthesis mechanism, provide a theoretical basis for follow-up study of how external stimulus regulating latex yield. In our study, the REF/SRPP gene family was comprehensively and systematically analyzed for the first time, and the possible biological functions of the family members were further explored.Nine new sequences of gene family were obtained by homologous cloning. The results showed that all members were more consistent in protein charges and hydrophobicity. The evolutionary analysis showed that most of the protein family members were more concentrated in the latex-producing plants, suspecting they are possibly involved in the synthesis of natural rubber. The REF and SRPP subfamilies were redefined by analysis of all member domains: the REF subfamily members had a variable N-terminal and a relatively short C-terminal beyond the REF domain,whereas the SRPP subfamily members had a short N-terminal and variable C-terminal.Structure analysis and mRNA clone showed that some members of the family existed alternative splicing forms. Subcellular localization showed that there were some differences between the two subfamilies of the protein, and the Southern blot showed that there was a high homology of REF 175 in the rubber genome. By analyzing the expression of the family members, it was found that almost all of the members at the transcriptional level responded to the stimulation of the tapping. However, except for REF 13 8, all the genes changed little or decreased under the ethylene treatment,suggesting that the regulation by ethylene may not be in the transcriptional level but in or after the translational levels. The protein isoforms of the main members of the family were analyzed by proteomics, and the accumulation of protein isoforms was compared among different rubber tree clones or between ethylene or tapping treatment. It was found that the total protein abundance of each REF/SRPP was up-regulated or unchanged after tapping, and down-regulated or unchanged after ethylene treatment, but the abundances of different isoforms of a same REF/SRPP were diverse from each other,for example,The isoforms 132, 134 of REF 175 and 236,270 of REF258 were not only predominant but also quite promoted by ethylene treatment in the high latex productivity rubber clone RY 8-79. It is presumed that these isoforms might play an important regulatory role in the process of ethylene promoted latex production of the rubber tree. In addition, polyclonal antibodies have been successfully prepared for further analysis through prokaryotic expression of the proteins and subsequent immunization of rabbits. The above results provide a theoretical and experimental basis for further revealing the the mechanism of nature rubber biosynthesis and the mechanism of ethylene to promote the latex yield. |
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