杜氏盐藻β-酮酯酰-ACP合酶(DsKASⅡ)基因的分离及功能分析  

Isolation and Functional Analysis of DsKASⅡ in Dunaliella salina

作　　者：高宇[1] 刘宝玲[1] 高慧玲 张飞 薛金爱[1] 李润植[1] GAO Yu;LIU Baoling;GAO Huiling;ZAHNG Fei;XUE Jin’ ai;LI Runzhi(Institute of Molecular Agriculture and Bioenergy, Shanxi Agriculture University, Taigu 030801, Shanxi, China)

机构地区：[1]山西农业大学分子农业与生物能源研究所,山西太谷030801

出　　处：《激光生物学报》2019年第2期114-122,113,共10页Acta Laser Biology Sinica

基　　金：国家自然科学基金项目(31401430);国家"948"项目(2014-Z39);山西煤基重点科技攻关项目(FT-2014-01);山西省重点科技项目(201603D312005);山西省留学归国人员科研基因项目(2015-064)

摘　　要：酮脂酰-ACP合成酶Ⅱ(KASⅡ)是催化棕榈酸(16∶0-ACP)延伸为硬脂酸(18∶0-ACP)的关键酶,其活性强弱决定着18碳脂肪酸含量的高低。本文以杜氏盐藻(Dunaliella salina)为试材,分离鉴定杜氏盐藻DsKASⅡ基因编码序列,采用生物信息学工具解析DsKASⅡ酶蛋白的亚细胞定位、高级结构、理化性质及系统发育等特性。检测氮胁迫下的DsKASⅡ表达量,以及藻细胞脂肪酸、叶绿素和β-胡萝卜素的含量。结果表明,DsKASⅡ编码的酶蛋白长度为476 aa,pI为6. 99,含叶绿体靶向肽和较多亲水区。二级结构主要由α-螺旋(22. 48%),β-片层(22. 06%)和无规则卷曲(55. 46%)组成。三级结构预测表明该蛋白整体呈紧密的心形结构,活性酶蛋白为同源二聚体。系统发育分析表明,DsKASⅡ氨基酸序列与莱茵衣藻CrKASⅡ同源性达99%,可能二者有着共同的进化祖先。qRT-PCR揭示,与正常培养的杜氏盐藻相比,DsKASⅡ在氮胁迫条件下的表达量明显上调,第3天时的表达量比正常培养的高4. 5倍。氮胁迫下藻细胞总油脂、油酸(C18∶1)和类胡萝卜素含量显著提高,然而棕榈酸(C16∶0)和叶绿素的含量明显降低。这表明,氮胁迫诱导杜氏盐藻DsKASⅡ基因上调表达,将更多的棕榈酸催化为硬脂酸,进而提高了单不饱和油酸的富集以及类胡萝卜素的积累。本研究为后续进一步解析杜氏盐藻氮胁迫条件下,油脂与胡萝卜素合成积累及藻细胞响应胁迫机制和优质富油藻种培育提供了科学参考。β-ketoacyl-ACP synthase II (KASII)is a key enzyme responsible for the conversion of palmitic acid (16 ∶0-ACP)to stearic acid (18 ∶0-ACP). KASII activity determines the level of C18 fatty acids. In this paper, Dunaliella salina was used to isolate and identify the DsKASII gene. Bioinformatics tools were used to analyze the subcellular localization, protein structure, physicochemical properties and phylogenetic characteristics of DsKASII enzyme. Detail examinations were conducted for DsKASII gene expression pattern, oil/fatty acid profiles, the contents of chlorophyll and β-carotene under nitrogen deficiency stress. The results showed that Dunaliella salina DsKASII enzyme protein had a length of 476 aa and a theoretical isoelectric point of 6.99. The protein contained plastid-targeting peptide and many hydrophilic regions. The main secondary structures included α-helix ( 22.48%),β-sheet ( 22.06%), and random coil (55.46%). The 3D structure simulation predicted that the DsKASII was compactly heart-shaped structure on the whole, with a homodimer as the functional form. Phylogenetic analysis indicated that DsKASII protein has the highest homology with Chlamydomonas reinhardtii CrKASII by 99%. It is possible that the two microalgae have a common evolutionary ancestor. Quantitative RT-PCR (qRC-PCR)analysis revealed that the expression level of DsKASII gene was up-regulated in algal cells under nitrogen deficiency stress compared with the normal culture controls. The expression level of DsKASII in the stressed cells was 4.5 times higher than that in the normal culture cells on the 3th day of cultivation. Moreover, under nitrogen stress, the total oil, oleic acid (C18 ∶1)and carotenoid content in algae cells increased significantly, whereas palmitic acid (C16 ∶0)and chlorophyll contents decreased. Taken together, the data indicated that nitrogen stress induced the up-regulation of DsKASII gene in Dunaliella salina , and then DsKASII catalyzed more palmitic acid into stearic acid, eventually increasing the accu

关 键 词：杜氏盐藻(Dunaliella salina) β-酮脂酰-ACP合酶II(KASII) 氮胁迫 油脂 胡萝卜素 

分 类 号：Q949.2[生物学—植物学]