# Biosynthesis of Natural Rubber: Current State and Perspectives
## 2 Biosynthesis of Natural Rubber and the Involved Genes/Proteins
### Genes/Proteins Involved in Natural Rubber Biosynthesis
#### 2.3.2 Rubber Elongation Factor and Small Rubber Particle Protein  
The rubber elongation factor (REF) and small rubber particle protein (SRPP) are homologous proteins originating from a common ancestor and belong to the stress-related protein superfamily [31]. They are the most abundant components of the rubber particle proteome [39; 40]. The 14.7 kDa HbREF (138 aa) and 22.3 kDa HbSRPP (204 aa) were cloned and investigated even earlier than HbCPTs [65; 66; 67; 68; 69]. They are the main allergens of latex proteins, so were also designated as Hevb1 and Hevb3, respectively [31]. Isoforms of REF and SRPP were identified and all REF/SPRR proteins have a similar \(\sim\)110 aa region called REF domain. REF subfamily members have a variable N-terminal and a relatively short C-terminal beyond the REF domain, while SRPP subfamily members have a short N-terminal and a variable C-terminal [70]. SRPPs are localized abundantly on SRPs, while different REF isoforms were found, either locating mainly on LRPs or expressing equally on both SRPs and LRPs [37; 71; 72]. HbREF interacts with HbSRPP, and was bound to the rubber particle lipidic monolayer more tightly than HbSRPP [31; 44]. Both of them have aggregative properties and may function in rubber particle stabilization and coagulation [71; 73].  
Both REF and SRPP were reported to be required for the natural rubber biosynthesis. They were demonstrated to have the activity of promoting the incorporation of IPP into whole latex or washed rubber particles, and this activity could be inhibited by the corresponding antibodies [67; 69; 74]. RNAi silencing lines of REFs or SRPPs often showed reduced latex content and rubber transferase activity, but the rubber molecular weight was not always changed. [PERSON] et al. reported a TbREF playing critical roles in rubber particle biogenesis and rubber production in _T. brevicornicultatum_[75]. Its knockdown mutants exhibited significantly reduced rubber content correlating with lower TbCPT protein levels and less TbCPT activity in the latex, while rubber molecular mass and the stability of rubber particles were unaffected, indicating TbREF is an important component of the rubber synthetic machinery [75]. [PERSON] et al. demonstrated that the abundance of REF mRNA transcripts in latex was 3- to 5-fold higher in high-yielding rubber tree clones than in low yielders, implying a positive correlation between REF gene expression pattern and latex yield [76]. TkSRPP3 RNAi lines showed significant decreases in root rubber content and produced dramatically lower molecular weight rubber than the control line, while TbSRPP RNAi lines showed 40-50% reduction in dry rubber content, but neither the rubber average molecular weight nor the polydispersity of the rubber was affected [47; 77]. In lettuce (_Lactuca sativa_), knockdown of LsSRPP4/LsSRPP8, which account for more than 90% of total transcripts of REF/SRPP homologs in latex, individually or simultaneously, had no effect on rubber content, molecular weight or polydispersity, suggesting that SRPP might not be a necessary component for the rubber transferase complex in lettuce [78].  
Although most studies proved REF/SRPP play positive roles in rubber biosynthesis, no catalytic domain was found in these proteins. [PERSON] et al. suggested that REF/SRPP family proteins are structural proteins with function in packaging/storage of synthesized natural rubber and stabilizing of rubber particles in _H. brasiliensis_, for all the identified REF/SRPP isoforms are acidic isoelectric point proteins which can maintain the negative charge and avoid the coagulation of rubber particles [72]. This view was also supported by the fact that the addition of REF did not activate the background activity of washed rubber particles in vitro [38]. The exact function of each REF/SRPP isoform needs to be identified by further study.