杜氏盐藻DsKASⅢ基因的分离鉴定及其在氮胁迫下的表达分析
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摘要
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 Ds KASⅢ 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.
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 Ds KASⅢ 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.
引文
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[30]程蔚兰,邵雪梅,宋程飞,等.氮胁迫对埃氏小球藻生长及油脂积累的影响[J].生物技术通报,2017,33(11):160-165.
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[32]孙辉.盐藻的生长特性及逆境下β-胡萝卜素积累规律和机理研究[D].成都:四川大学,2005.
[33]华汝成.单细胞藻类的培养与利用[M].北京:农业出版社,1980,87-88,338-339.
[2]宋程飞,郝敬云,程蔚兰,等.杜氏盐藻电击转化体系的优化,山西农业大学学报:自然科学版,2018,38(3):36-42.
[3]汪本凡.杜氏盐藻纯化及生物学特性研究[D].安徽农业大学,2004.
[4]Lamers PP,Cc VDL,Kaasenbrood PS,et al.Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina[J].Biotechnology&Bioengineering,2010,106(4):638-648.
[5]Rad FA,Aksoz N,Hejazi MA.Effect of salinity on cell growth andβ-carotene production in Dunaliella sp.isolates from Urmia Lake in northwest of Iran[J].African Journal of Biotechnology,2011,10(12):2282-2289.
[6]朱松玲,王怡洁.盐度变化对杜氏盐藻的游离氨基酸和脂肪酸含量的影响[J].海洋科学,2005(3):8-11.
[7]Ahmed RA,He M,Aftab RA,et al.Bioenergy application of Dunaliella salina SA 134 grown at various salinity levels for lipid production[J].Sci Rep,2017,7(1):8118.
[8]Thakur,V.Biodiesel.An Alternative Method for Energy Crisis:AReview.Journal of Biological and Chemical Chronicles 2,2016,14-26.
[9]Cho K,Hur SP,Lee CH,et al.Bioflocculation of the oceanic microalga Dunaliella salina,by the bloom-forming dinoflagellate Heterocapsa circularisquama,and its effect on biodiesel properties of the biomass[J].Bioresour Technol,2015,202:257-261.
[10]Chisti Y.Biodiesel from microalgae[J].Biotechnol,2007,Adv.25(3),294-306.
[11]谷建勇.藻类对动物的营养和保健作用[J].饲料与畜牧,2002(4):22-24.
[12]Garg S,Jin H,Yandeau-Nelson M,et al.Materials and methods for characterizing and using a 3-ketoacyl-acyl carrier protein(ACP)synthase III(KASIII)for production of bi-functional fatty acids[J].US20180051263[P],2018.
[13]Fritzi M.Brück,Monika Brummel,Ricardo Schuch,et al.In-vitro evidence for feed-back regulation ofβ-ketoacyl-acyl carrier protein synthase III in medium-chain fatty acid biosynthesis[J].Planta,1996,198(2):271-278.
[14]Dehesh K,Edwards P,Byrne J.Overexpression of 3-Ketoacyl-AcylCarrier Protein Synthase IIIs in Plants Reduces the Rate of Lipid Synthesis[J].Plant Physiol,2001,125(2):1103-1114.
[15]Lai CY,Cronan JE.Beta-ketoacyl-acyl carrier protein synthase III(FabH)is essential for bacterial fatty acid synthesis[J].J Biol Chem,2003,278(51):51494-51503.
[16]蒋桂雄.油桐种子转录组解析及油脂合成重要基因克隆[D].长沙:中南林业科技大学,2015.
[17]Li J,Li MR,Wu PZ,et al.Molecular cloning and expression analysis of a gene encoding a putative-ketoacyl-acyl carrier protein(ACP)synthase III(KAS III)from Jatropha curcas[J].Tree Physiology,2008,28(6):921-927.
[18]González-Mellado D,Martínez-Force E.The role of beta-ketoacylacyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower[J].Planta,2010,231(6):1277-1289.
[19]Du X,Huang G,He S,et al.Resequencing of 243 diploid cotton accessions based on an updated A genome identifies the genetic basis of key agronomic traits[J].Nature Genetics,2018,50(6):796-802.
[20]Polle JEW,Barry K,Cushman J,et al.Draft nuclear genome sequence of the halophilic and Beta-carotene-accumulating green Alga Dunaliella salina strain CCAP19/18[J].Genome Announc,2017,5(43):e01105-e01117.
[21]Jensen A.Handbook of Phycological Metbods[M].Cambridge Univ Press,1978
[22]Díaz-Palma P,Stegen S,Queirolo F,et al.Biochemical profile of halophilous microalgae strains from high-andean extreme ecosystems(NE-Chile)using methodological validation approaches[J].Journal of Bioscience&Bioengineering,2012,113(6):730.
[23]Mogedas B,Casal C,Forján E,et al.beta-carotene production enhancement by UV-A radiation in Dunaliella bardawil cultivated in laboratory reactors[J].Journal of Bioscience&Bioengineering,2009,108(1):47-51.
[24]李璐,梁倩,安茜,等.紫苏β-酮脂酰ACP合成酶基因家族生物信息学分析[J].山西农业科学,2017,45(3):321-324.
[25]Li MJ,Li AQ,Xia H,et al.Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L.J Biosci.2009 Jun;34(2):227-38.PubMed PMID:19550039.
[26]Garg S,Yandeaunelson MD,Nikolau BJ.Substrate promiscuity ofβ-Ketoacyl ACP Synthase III(KASIII):Understanding the structural basis for functional diversity of KASIII enzymes[J].Biochemistry/Molecular Biology,2013,27(1):1.
[27]Waters N,Kopydlowski K,Guszczynski T,et al.Functional characterization of the acyl carrier protein(PfACP)and betaketoacyl ACP synthase III(PfKASIII)from Plasmodium falciparum[J].Molecular&Biochemical Parasitology,2002,123(2):85-94.
[28]宋亚楠,安茜,岳敏,等.杜氏盐藻DsFAD2基因的鉴定及缺氮胁迫下表达分析[J].山西农业大学学报:自然科学版,2018,38(3):23-29.
[29]姚杰.基于油脂及其它高价值副产物导向的杜氏盐藻培养研究[D].武汉:华中科技大学,2015.
[30]程蔚兰,邵雪梅,宋程飞,等.氮胁迫对埃氏小球藻生长及油脂积累的影响[J].生物技术通报,2017,33(11):160-165.
[31]Hu GR,Fan Y,Zhang L,et al.Enhanced lipid productivity and photosynthesis efficiency in a Desmodemus sp.Mutant induced by heavy carbon ions[J].PLoS One,2013,8(4):e60700.
[32]孙辉.盐藻的生长特性及逆境下β-胡萝卜素积累规律和机理研究[D].成都:四川大学,2005.
[33]华汝成.单细胞藻类的培养与利用[M].北京:农业出版社,1980,87-88,338-339.