关键词: 杜氏盐藻, β-酮脂酰-ACP合酶III（KASIII）, 氮胁迫, 油脂, β-胡萝卜素

Abstract: This study is conducted to isolate and characterize the gene DsKASIII（β-ketoacyl-ACP synthase III）in Dunaliella salina and investigate the physiochemical features and functions of this enzymatic protein. Homologous cloning was used to isolate the encoding gene sequence of DsKASIII gene in D. salina,bioinformatics to analyze the physical and chemical properties and functions of DsKASIII protein,RT-PCR to examine the DsKASIII expression files in D. salina under nitrogen-deficiency stress,as well as the changes of total fatty acid and β-carotene contents were also tested in algal cells under N-deficiency and sufficiency cultivations,respectively. The results showed that DsKASIII gene contained 9 exons and 960 bp of ORF encoding a mature protein of 319 amino acids. The theoretical isoelectric point（pI）and relative molecular weight of DsKASIII were 6.21 and 33.3 kD,respectively. The protein was predicted to be located in chloroplast by anchoring the inner membrane with a mosaic pattern,and such conformation of the protein benefited the high-efficient catalysis reaction. Secondary structure of DsKASIII mainly consisted of α-helix（35.11%）,β- sheet（25.71%）and random coil（27.90%）. Three-dimensional simulations for DsKASIII showed that this protein can form a homodimer to perform its physiological functions. Phylogenetic analysis indicated that DsKASIII protein had the closest relationship with CeKASIII from Chlamydomonas reinhardtii,implying that they may have common evolutionary origin. Expression analysis by qRT-PCR showed that N-deficiency stress induced the up-regulation of DsKASIII gene,and the expression level of DsKASIII gene on the third day of the stress was 1.1 times higher than that of N-sufficiency culture. Compared to the N-sufficiency cultivation,N-deficiency resulted in the increase of the contents of total fatty acids and β-carotene in algal cells by 49.05% and 33.20%,respectively. In conclusion,the N-deficiency stress can induce the up-regulation expression of DsKASIII gene,thereby promote the biosynthesis and accumulation of oil and β-carotene in D. salina. The present data provide the scientific information for elucidating the mechanism underlying lipid and β-carotene biosynthesis and their regulations in D. salina under nitrogen stress.

Key words: Dunaliella salina, β-ketoacyl-ACP synthase III（KASIII）, nitrogen stress, oil, β-carotene