Objective To clone the rare cold-induced 2 gene (AcRCI2A) in pineapple and explore its expression in pineapple resistance to blackheart disease, laying the foundation for genetic improvement of blackheart disease resistance of pineapple fruit by this gene.

Method The AcRCI2A gene was cloned from the pulp of 'Comte de Paris' pineapple and analyzed by bioinformatics. Then it was constructed into the pCAMBIA1300-35S vector with GFP fused at the C-terminus using homologous recombination, and its subcellular localization was analyzed. Semi-quantitative RT-PCR was used to detect the expression of AcRCI2A in 'Comte de Paris' pineapple fruits harvested in different months in 2022. Furthermore, qRT-PCR was employed to analyze the expression pattern of the AcRCI2A gene during the storage in pineapples treated with hot air, abscisic acid (ABA), low temperature, and control, to preliminarily clarify its response to abiotic stress and its relationship with the occurrence of pineapple blackheart disease.

Result The AcRCI2A gene was successfully cloned, and its coding region was 165 bp in length, encoding 54 amino acids. This sequence was not found on the pineapple genome website, and the sequence number was XM_020253166.1 on the NCBI website. Bioinformatics analysis indicated that AcRCI2A possesses a typical conserved domain PMP3 of the RCI2 protein, contains two transmembrane domains and lacks a signal peptide. Phylogenetic tree analysis showed that AcRCI2A belongs to Group Ⅰ family and was closely related to AtRCI2C and AtRCI2H, potentially playing an important role in maintaining Na⁺/K⁺ homeostasis and membrane potential balance. Subcellular localization analysis revealed that AcRCI2A was localized to the plasma membrane. The occurrence of blackheart is closely related to the expression level of AcRCI2A at harvest and the expression level of AcRCI2A pineapple fruits prone to blackheart after harvest is extremely low. Further Analysis of the incidence of blackheart disease and the expression of AcRCI2A gene in pineapple fruit after hot air, ABA, and low temperature treatment showd that all three treatments significantly reduced the incidence of blackheart disease in postharvest pineapple fruit. The hot air treatment had the best inhibitory effect, and the incidence of blackheart disease was only 0%, 0% and 1% at 3, 6 and 9 days of storage. The expression of AcRCI2A gene was induced by hot air and ABA treatment, but inhibited by low temperature. The high expression of AcRCI2A corresponded to the lower incidence and occurrence index of blackheart disease during postharvest ripening and senescence of pineapple fruit.

Conclusion AcRCI2A is a typical rare cold-induced 2 protein localized in the plasma membrane. Its expression is induced by hot air and ABA treatment but inhibited by low temperature, closely related to pineapple blackheart disease.