Abstract: INTRODUCTION: Waxes are protective substances found on the surface of plants, especially on fruits and leaves, which help prevent non-stomatal water loss from plant tissues. Lycium barbarum is a characteristic economic forest species of Ningxia. Its fruit is a berry with a high water content, and the surface of the fruit skin is covered with wax, which hinders the normal loss of moisture during the drying process. Alkanes are the main components of the wax on L. barbarum, and the CER3 gene is a key gene involved in alkane biosynthesis. Therefore, an in-depth study of the function of CER3 in L. barbarum provides a theoretical basis for revealing the metabolic synthesis of wax in goji fruits and the molecular mechanism of LbCER3.  RATIONALE: Research on the waxy coating of L. barbarum fruit skin has mainly focused on the structure and composition of the epicuticular wax, with relatively little attention given to its molecular mechanisms. This studyusing the main cultivated varieties of L. barbarum, Ningqi No.1 and Ningqi No.5, as test materials, this study employed methods such as GC-MS, transcriptomics, and molecular biology to investigate the changes in the content and composition of cuticular waxes on the outer and inner layers of the fruit at different developmental stages. It also involved the screening, cloning, and functional validation of key genes related to fruit wax.   RESULTS: During the development and maturation of the fruit, the content of inner and outer cuticular waxes in Ningqi No.1 and Ningqi No.5 gradually decreased, with the total wax content in Ningqi No.1 being significantly higher than that in Ningqi No.5. The outer and inner wax components of both varieties were the same, primarily consisting of alkanes, esters, and acids. Transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) identified LbCER3 as a key gene. The full-length CDS of the LbCER3 gene is 1 884 bp, encoding 628 amino acids, and contains a C-terminal WAX2 domain (Wax2_C) and a fatty acid hydroxylase domain (FA_hydroxylase). An LbCER3 overexpression vector was constructed and successfully transformed into tomato, resulting in a significant reduction in chlorophyll leaching rate in the T1 generation transgenic lines, along with a notable increase in leaf wax content and significant increases in the wax components of alkanes and acids.  CONCLUSION: These findings indicate that LbCER3 is a key gene in the synthesis of wax in L. barbarum.

Key words: functional verification, LbCER3, Lycium barbarum , wax