# Advances in Genome Sequencing and Natural Rubber Biosynthesis in Rubber-Producing Plants
## 4 Research Progress in NR Biosynthesis Regulation  
The genome assembly of _H. brasiliensis_ revealed 94 genes related to rubber biosynthesis which belong to 20 gene families [22]. Among these genes, 18 are associated with the mevalonate (MVA) pathway, 22 with the methylerythmitol phosphate (MEP) pathway, 15 with cytosolic initiator synthesis, and 39 with putative rubber particle-associated rubber elongation genes. Similarly, the genome assembly of _T. kok-saghyz_ revealed a total of 102 candidate genes related to rubber biosynthesis [15]. Among these, 40 genes are involved in all six steps of the MVA pathway, 23 genes are involved in all seven steps of the MEP pathway, 19 genes are associated with initiator synthesis, and 20 genes are associated with rubber-particle-associated rubber elongation proteins. The key proteins associated with rubber particles include rubber elongation factors (REFs), _cis_-prenyltransferases (CPTs), _cis_-prenyltransferase-like (CPTL) proteins, and small rubber particle proteins (SRPPs) [42, 51, 57, 60, 61]. Hence, these proteins have been widely identified and studied in rubber-producing plants, shedding light on the regulatory mechanisms of rubber biosynthesis. _cis_-prenyltransferases (CPTs), belonging to the prenyltransferase family, have been shown to possess the activity of incorporating IPP into polyprenyl chains, playing a key role in NR biosynthetic activity. HRT1 and HRT2 are two _Hewea_ rubber transferases predominantly expressed in latex. They possess all five highly conserved regions crucial for the catalytic function and substrate binding of _cis_-prenyl chain elongating enzymes [51, 52]. As mentioned earlier, HRT proteins purified from the _E. coli_ system exhibited no CPT activity. However, the heterologous expression of _HRT1_ and _HRT2_ in _Arabidopsis_ cell cultures resulted in long-chain CPT activity but failed to form NR [62]. Moreover, homologues of CPT have been isolated from other NR-producing plants such as _T. koksaghyz_, _L. sativa_, and _E. characius_, but none of them have been enzymatically identified as having rubber transferase activity [47, 53, 63]. These findings suggest that the CPT proteins require additional factors to exhibit their activity. The silencing of _CPTs_ in _T. brevicornicultatum_ through RNA interference (RNAi) resulted in a decrease in NR content, suggesting their involvement in NR biosynthesis [64]. Knocking out _LsCPT3_ via CRISPR/Cas9 in lettuce significantly reduced the rubber content in latex [13]. Notably, the growth and development of the mutants are unaffected, which indicates that NR does not have a significant physiological function in lettuce. Interestingly, both the native _LsCPT3_ and heterologous _CPTs_ from guayule (_PaCPT3_) and goldenrod (_ScCPT3_) proved the ability of complementing the mutants, resulting in the production of NR with a molecular weight (MW) exceeding 1 million Da in the _lscpt3_ background. In contrast, native goldenrod plants can only produce NR with a MW of 0.09 million Da. CPTLs are homologous to the human Nogo-B receptor (NgBR) and play a crucial role in rubber biosynthesis. They are primarily localized in the endoplasmic reticulum (ER) of non-rubber-producing eukaryotes and can also be found in both the ER and rubber particles of rubber-producing plants [53, 60]. Although lacking the conserved catalytic residues commonly found in CPT proteins, the C-terminal RxG motif of CPTL has been demonstrated to be essential for pentenyl-transferase activity [65]. The rubber transferase activator, TbRTA, in _T. brevicornicultatum_ interacts with TbCPTs located on the surface of rubber particles, thereby contributing to the formation of the rubber transferase complex [66]. Knocking out the _TbRTA_ resulted in a significant impairment of rubber synthesis, providing further evidence for the indispensability of _TbRTA_ (_TbCPTL_) within the rubber transferase complex.