Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid （ArA, C20： 4ω-6, △5, 8, 11, 14）, a long chain polyunsaturated fatty acid （PUFA）, especially under nitrogen starvation ...Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid （ArA, C20： 4ω-6, △5, 8, 11, 14）, a long chain polyunsaturated fatty acid （PUFA）, especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ. phage vectors and a 545 nt expressed sequence tag （EST） was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chretiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5＇/3＇ rapid amplification of cDNA ends （RACE）. The cDNA sequence was 1 331 bp long and included a 33 bp 5＇-untranslated region （UTR） and a 431 bp 3＇-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I （81 bp）, II （81 bp）, and III （67 bp） that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.展开更多
cDNA library of Myrmecia incisa H4301 was constructed using λ phage vectors.The library consisted of 1.5×10 6 clones with a recombination rate of 90%,and 1942 clones were randomly sequenced.All 1854 readable exp...cDNA library of Myrmecia incisa H4301 was constructed using λ phage vectors.The library consisted of 1.5×10 6 clones with a recombination rate of 90%,and 1942 clones were randomly sequenced.All 1854 readable expressed sequence tags(ESTs) were clustered into 596 non-redundant sequences(NRSs),among which 126 NRSs were from 1384 ESTs,showing a high redundancy.Among the 596 NRSs,30 were ribosomal RNA,and 152 significantly matched with those available in NCBI database and JGI Genome Portal,the latter were divided into nine subcategories.Overall,59 NRSs were involved in photosynthesis,the respiratory electron transport chain,ATP synthesis,oxidation reduction,fatty acid biosynthesis,glucose metabolism,protein metabolism,and small molecular metabolism,suggesting that these genes were abundantly transcribed during energy and substance metabolism.Acyl-carrier protein,ferrodoxin and fatty acid elongase genes obtained from this cDNA library enabled presumption of a possible biosynthesis pathway of ArA in M.incisa.Codon usage analysis of 142 NRSs with 17798 codons in the predicted coding regions showed that the average G+C content level of the total codons was 55.39%,and that of the third position in base trimers was 66.42%,indicating a strong bias toward cytosine and/or guanosine in this algal genome.Among all synonymous codons,NAG was most favored,while NUA was most avoided.Phylogenic trees inferred from ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes and the extra partial sequences of 18S rRNA obtained from this library demonstrated that M.incisa belonged to Trebouxiophyceae and was significantly clustered with M.incisa SAG 2007,Lobosphaera tirolensis,M.bisecta,and Parietochloris incisa,but was clearly distant from P.pseudoalveolaris and P.alveolar.Transmission electron microscopy revealed pyrenoids traversed by many parallel thylakoids membranes,while starch grains were only clearly observed when cells were grown under nitrogen stress.Based on combined investigation of the phylogeny and morphological characteristics,it is proposed that M.incisa be kept in the genus Myrmecia in which there might be two parallel groups,one living freely and another symbiotic species.展开更多
从缺刻缘绿藻的转录组数据库中搜索到5条编码该藻碳酸酐酶(carbonic anhydrase,CA)的contig序列,据此序列设计基因特异性引物,利用c DNA末端快速扩增(rapid amplification of c DNA ends,RACE)技术,克隆得到c DNA全长序列,分别命名为M i...从缺刻缘绿藻的转录组数据库中搜索到5条编码该藻碳酸酐酶(carbonic anhydrase,CA)的contig序列,据此序列设计基因特异性引物,利用c DNA末端快速扩增(rapid amplification of c DNA ends,RACE)技术,克隆得到c DNA全长序列,分别命名为M iαCA1、M iαCA2、M iβCA1、M iβCA2和M iγCA,开放阅读框(ORF)长分别为963、1 089、1 041、738和687 bp,相应编码由320、362、346、245和228个氨基酸组成的蛋白。这些蛋白均富含疏水性氨基酸,分别占氨基酸总量的41.25%、45.31%、43.35%、42.45%和43.42%。基于缺刻缘绿藻和其他物种CA的蛋白序列所构建的Neighbor-Joining系统演化树显示,这些CA很明显地被聚类成α-、β-和γ-CA等3支。缺刻缘绿藻的2个α-CA都存在与Zn2+结合的3个His残基,2个β-CA也具有与Zn2+结合的2个Cys残基和1个His残基,但MiγCA中的Zn2+结合位点分别为Arg、His和Asn,不同于报道中的3个His。MiαCA1与莱茵衣藻的CAH3亲缘关系较近,因具有2个信号肽,它可能位于叶绿体的类囊体腔中并发挥作用。MiαCA2与莱茵衣藻的CAH1亲缘关系较近,因具有1个信号肽,可能在细胞的周质空间起着与CAH1类似的功能。M iβCA1和M iβCA2与莱茵衣藻的CAH7和CAH8亲缘关系更近,可能在细胞质中参与CO2和HCO-3之间的转化。MiγCA则与高等植物的γ-CA聚在一起,可能位于线粒体内发挥作用。由此推测,自缺刻缘绿藻所克隆的5个CA基因应在细胞的不同部位协同作用,通过参与CO2和HCO-3之间的转化以调节p H并实现CO2在细胞内转运的目的。展开更多
基金Supported by the National Natural Science Foundation of China (No.30972243)the Creative Project of the Shanghai Municipal Education Committee (No.09ZZ167)+1 种基金the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry,the Key Discipline Project of the Shanghai Municipal Education Committee (No.J50701)the Excellent Thesis Grant from Shanghai Ocean University
文摘Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid （ArA, C20： 4ω-6, △5, 8, 11, 14）, a long chain polyunsaturated fatty acid （PUFA）, especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ. phage vectors and a 545 nt expressed sequence tag （EST） was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chretiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5＇/3＇ rapid amplification of cDNA ends （RACE）. The cDNA sequence was 1 331 bp long and included a 33 bp 5＇-untranslated region （UTR） and a 431 bp 3＇-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I （81 bp）, II （81 bp）, and III （67 bp） that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.
基金supported by the National Natural Science Foundation of China (30972243)the Creative Project of the Shanghai Municipal Education Committee (09ZZ167)+2 种基金the National High Technology Research and Development Program of China (2009AA064401)the Key Discipline Project of the Shanghai Municipal Education Committee (J50701)Excellent Thesis Grant from Shanghai Ocean University
文摘cDNA library of Myrmecia incisa H4301 was constructed using λ phage vectors.The library consisted of 1.5×10 6 clones with a recombination rate of 90%,and 1942 clones were randomly sequenced.All 1854 readable expressed sequence tags(ESTs) were clustered into 596 non-redundant sequences(NRSs),among which 126 NRSs were from 1384 ESTs,showing a high redundancy.Among the 596 NRSs,30 were ribosomal RNA,and 152 significantly matched with those available in NCBI database and JGI Genome Portal,the latter were divided into nine subcategories.Overall,59 NRSs were involved in photosynthesis,the respiratory electron transport chain,ATP synthesis,oxidation reduction,fatty acid biosynthesis,glucose metabolism,protein metabolism,and small molecular metabolism,suggesting that these genes were abundantly transcribed during energy and substance metabolism.Acyl-carrier protein,ferrodoxin and fatty acid elongase genes obtained from this cDNA library enabled presumption of a possible biosynthesis pathway of ArA in M.incisa.Codon usage analysis of 142 NRSs with 17798 codons in the predicted coding regions showed that the average G+C content level of the total codons was 55.39%,and that of the third position in base trimers was 66.42%,indicating a strong bias toward cytosine and/or guanosine in this algal genome.Among all synonymous codons,NAG was most favored,while NUA was most avoided.Phylogenic trees inferred from ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes and the extra partial sequences of 18S rRNA obtained from this library demonstrated that M.incisa belonged to Trebouxiophyceae and was significantly clustered with M.incisa SAG 2007,Lobosphaera tirolensis,M.bisecta,and Parietochloris incisa,but was clearly distant from P.pseudoalveolaris and P.alveolar.Transmission electron microscopy revealed pyrenoids traversed by many parallel thylakoids membranes,while starch grains were only clearly observed when cells were grown under nitrogen stress.Based on combined investigation of the phylogeny and morphological characteristics,it is proposed that M.incisa be kept in the genus Myrmecia in which there might be two parallel groups,one living freely and another symbiotic species.
