Establishment of a yeast two-hybrid cDNA library of Hevea brasiliensis and screening of HbEIN3-interacting proteins

【Objective】 This study aimed to establish a yeast two-hybrid library of Hevea brasiliensis， screen proteins that interacted with HbEIN3， and find out potential factors in ethylene-mediated HbEIN3 that regulated stress resistance in Hevea brasiliensis， so as to provide a theoretical basis for further elucidating the regulation mechanism of HbEIN3 in stress responses in Hevea brasiliensis. 【Method】 Total RNA was extracted from the leaves and latex of Hevea brasiliensis cultivar 7-33-97， followed by mRNA isolation and purification， after which cDNA was synthesized. A primary cDNA library was established using the Gateway method. The plasmids from the primary library were recombined with the se-condary vector pGADT7-DEST via LR recombination to generate a yeast two-hybrid cDNA library. A truncated HbEIN3 gene fragment （encoding amino acids 1-472， designated as HbEIN3_1-472） was cloned into the bait vector pGBKT7 to test its transcriptional self-activation activity. Using pGBKT7-HbEIN3_1-472 as the bait， candidate HbEIN3-interacting proteins were screened from the yeast two-hybrid cDNA library via the dual transformation approach. Yeast retransformation assays were used to further confirm the interactions between HbEIN3 and the candidate proteins. 【Result】 A cDNA library of Hevea brasiliensis was successfully established，with a titer of 4.00×10⁶ CFU/mL，a total library size of 1.60×10⁷ CFU，insert sizes ranging from 1000 to 2000 bp，and a recombination rate of 100%，meeting the requirements for library scree-ning. The yeast two-hybrid bait vector pGBKT7-HbEIN3_1-472 of the HbEIN3 gene was generated. The domain activated by HbEIN3 was localized at the C-terminal of the protein； HbEIN3_1-472 showed no self-activation， making it suitable for subsequent yeast two-hybrid library screening assays. A total of 20 candidate HbEIN3-interacting proteins were identified， including non-specific lipid transfer proteins （nsLTP）， ubiquitination-related proteins， and cinnamyl alcohol dehydro-genase （CAD）. There were twelve proteins enriched in signaling pathways： two proteins were enriched in the phenylalanine， tyrosine， and tryptophan biosynthesis pathway； one protein was enriched in the MAPK signaling pathway； and one protein was enriched in the NOD-like receptor signaling pathway， suggesting that these proteins might be involved in processes such as HbEIN3-mediated ethylene signal transduction and the regulation of stress resistance in Hevea brasiliensis. The remaining eight proteins were either unannotated or not enriched in any pathway. 【Conclusion】 The established yeast two hybrid cDNA library exhibits high quality， sufficient library capacity， and complete insert diversity， and it is successfully used to screen HbEIN3-interacting proteins. The 20 candidate interacting proteins identified in this study may interact with HbEIN3 to respond to ethylene signaling， thereby influencing the stress resistance of rubber trees. The results further refine the regulatory network of HbEIN3 and provide important genetic resources for understanding how Hevea brasiliensis copes with biotic and abiotic stresses through the ethylene signaling pathway.