杜氏盐藻DsKASⅢ基因的分离鉴定及其在氮胁迫下的表达分析

Isolation and Characterization of Gene DsKASⅢ from Dunaliella salina and Its Expression Under Nitrogen Stress

分离鉴定杜氏盐藻(Dunaliella salina)β-酮脂酰-ACP合酶Ⅲ(β-ketoacyl-ACP synthase Ⅲ,DsKASⅢ)基因并解析其编码蛋白理化特征及功能。依据同源克隆,分离杜氏盐藻DsKASⅢ基因编码序列,采用生物信息学方法解析DsKASⅢ编码蛋白的理化特性及功能,qRT-PCR检测缺氮条件下DsKASⅢ的表达谱,并检测细胞总油脂和β-胡萝卜素含量的变化。结果表明,杜氏盐藻KASⅢ基因含有9个外显子,ORF为960 bp,编码蛋白为319 aa。DsKASⅢ蛋白理论等电点pI为6.21,分子量为33.3 kD。亚细胞定位预测DsKASⅢ蛋白呈镶嵌状锚定在叶绿体内膜上,这种构象有助于利用能量高效率催化生化反应。DsKASⅢ蛋白二级结构主要由α-螺旋(35.11%)、β片层(25.71%)和无规则卷曲(27.90%)组成。三维模拟显示,DsKASⅢ蛋白以同源二聚体形式发挥催化功能的。系统发育分析表明,DsKASⅢ蛋白与莱茵衣藻KASⅢ蛋白亲缘关系最近,暗示其可能有共同的进化来源。qRT-PCR分析揭示,氮胁迫培养条件下,DsKASⅢ基因的表达显著升高,且在缺氮第3天时,表达量达峰值,比正常氮充足培养高1.1倍。氮胁迫培养的藻细胞总脂含量和β-胡萝卜素含量分别比正常氮充足培养的藻细胞提高49.05%和33.20%。氮胁迫能诱导DsKASⅢ基因的上调表达,进而促进杜氏盐藻细胞合成和积累高量油脂和β-胡萝卜素。研究为全面阐明氮胁迫条件下杜氏盐藻油脂及β-胡萝卜素合成及调控机制提供了科学依据。

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,RTPCR to examine the DsKASIII expression files in D.salina under nitrogen-deficiency stress,as well as the changes of total fatty acid andβ-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 ofα-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 andβ-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 andβ-carotene in D.salina.The present data provide the scientific information for elucidating the mechanism underlying lipid andβ-carotene biosynthesis and their regulations in D.salina under nitrogen stress.