RNA Interference-Mediated Silencing of HbREF and HbSRPP Genes Reduces Allergenic Protein Content While Maintaining Rubber Production in Hevea brasiliensis

Allergenic proteins in natural rubber latex (NRL) pose significant health risks, particularly in rubber gloves. This study evaluated RNA interference (RNAi) technology for silencing <i>HbREF</i> (rubber elongation factor) and <i>HbSRPP</i> (small rubber particle protein) genes in <i>Hevea brasiliensis</i> to reduce latex allergen content. Double-stranded RNA (dsRNA) targeting these genes demonstrated high stability at 25-37 °C for 6 h and under UV/outdoor conditions for 72 h, but degraded rapidly above 50 °C. Among the three delivery methods tested, direct injection achieved the highest efficiency (>90% gene silencing within 12 h), followed by root drenching (54-84%) and foliar spray (46-70%). <i>HbREF</i> silencing achieved 98-99% expression reduction within 3 h, while <i>HbSRPP</i> showed dose-dependent responses (70-90% silencing) without off-target effects. Gene silencing affected downstream rubber synthesis genes <i>HbCPT</i> (cis-prenyltransferase) and <i>HbRME</i> (rubber membrane elongation protein) (37-58% reduction) while upstream genes remained unaffected. <i>HbREF</i> silencing reduced Hev b1 allergen by 64.04% and Hev b3 by 12.51%, whereas <i>HbSRPP</i> silencing decreased Hev b3 by 71.54% and Hev b1 by 13.48%. Both treatments caused only a 11-13% reduction in dry rubber content. This RNAi approach effectively reduces major latex allergens while maintaining rubber production, demonstrating commercial potential for developing hypoallergenic rubber products through precision agriculture biotechnology.