缺刻缘绿藻ω3脂肪酸去饱和酶基因的特性及在氮饥饿过程中相对转录量的分析  

Characterization of a ω3 fatty acid desaturase gene from Myrmecia incisa and its relative transcription during the stress course of nitrogen starvation

作　　者：李春阳[1] 杜道海[1] 于水燕[1] 李慧[1] 刘凡[1] 周志刚[1,2] 

机构地区：[1]上海海洋大学农业部水产种质资源与利用重点开放实验室,上海201306 [2]上海市高校水产养殖学E-研究院,上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2010年第9期1343-1353,共11页Journal of Fisheries of China

基　　金：国家自然科学基金项目(30972243);国家"八六三"高技术研究发展计划(2009AA064401);教育部留学回国人员科研启动基金资助项目;上海市教育委员会科研创新项目(09ZZ167);海洋生物学重点学科资助项目(J50701)

摘　　要：根据莱茵衣藻和普通小球藻ω3脂肪酸去饱和酶氨基酸序列(GenBank登录号分别为ABL09485和BAB78717)的保守区(TMFWALF和HHDIGTH)设计简并引物,以缺刻缘绿藻H4301总RNA为模板,通过反转录PCR和cDNA末端快速克隆技术(RACE),克隆了缺刻缘绿藻ω3脂肪酸去饱和酶基因的cDNA全长序列(GenBank登录号:EU658930),它与莱茵衣藻的这个基因具有69%相似性。该基因cDNA序列长2330bp,其中5'-非翻译区长107bp;3'-非翻译区912bp,且具有明显的poly(A)尾巴;开放阅读框1311bp,编码一个436个氨基酸的蛋白质,分子量为49.06ku,等电点7.85;该基因编码的蛋白为膜结合蛋白,含有2个疏水区域和3个保守的组氨酸簇基序。将该基因的cDNA序列与其DNA序列比对后,发现该基因在其编码区存在6个内含子,剪切位点均符合"GT-AG"规则。实时荧光定量PCR结果显示,在氮饥饿培养过程中,缺刻缘绿藻ω3脂肪酸去饱和酶基因的相对转录量在4h时可能因休克显著提高(P<0.05),然后开始下调,12h开始显著下降(P<0.05),并在20h降到最低(约为完全培养基的11.6%),此后保持在低水平(为完全培养基的23.2%)波动(P<0.01)。脂肪酸组分的气相色谱结果表明,在氮饥饿培养过程中,花生四烯酸占总脂肪酸的百分含量逐步提高,而作为多不饱和脂肪酸ω3合成途径的产物,如α-亚麻酸和十六碳三烯酸(16∶3ω3)的百分含量在40h或96h后都显著降低(P<0.05)。这些结果表明缺刻缘绿藻ω3脂肪酸去饱和酶基因在氮饥饿过程中的相对低转录量,导致ω3合成途径相关产物百分含量的降低,确保了该藻沿着ω6途径来合成与积累花生四烯酸以提高其百分含量。另外,十六碳二烯酸(16∶2ω6)、亚油酸及花生四烯酸都可能是该克隆基因所编码ω3脂肪酸去饱和酶的反应底物。Based on the conserved amino acid sequences（ TMFWALF and HHDIGTH） of ω3 fatty acid desaturase from Chlamydomonas reinhardtii and Chlorella vulgaris（ GenBank accession Nos. ABL09485 and BAB78717,respectively） ,a degenerated pair of primers was designed. A gene was cloned from Myrmecia incisa Reisigl H4301 with these primers using reverse transcription-polymerase chain reaction（ RT-PCR） and rapid amplification of cDNA ends （ RACE） . Its homology with C. reinhardtii ω3 fatty acid desaturase reached 69% in amino acid sequence. The full-length cDNA sequence（ GenBank accession No. EU658930） was composed of 2 330 bp. It comprised a 107 bp 5＇-untranslated region（ UTR） ,a 912 bp 3＇-UTR with a typical poly A tail,and a 1 311 bp open reading frame（ ORF） encoding a 436-amino-acid protein with a putative molecular weight of 49. 06 ku and pI at 7. 85. The deduced amino acid sequence was characterized by a membrane-bound desaturase with two hydrophobic regions and three conserved histidine-rich motifs. Compared to the DNA sequence of this gene,its coding region was found to be interrupted by 6 introns with splicing sites well matching the GT-AG rule. Quantitative real-time PCR result showed that during the stress course of nitrogen starvation,the relative transcription of ω3 fatty acid desaturase gene increased（ P 0. 05） at 4 h possibly due to shock,subsequently declined and did so significantly from 12 h on（ P 0. 05） . The transcript level of this gene dropped to minimum at 20 h,where it accounted for 11. 6% of that at 0 h, thereafter fluctuated at a low level only 23. 2% of that at 0 h（ P 0. 01） . Composition of fatty acid analysis illustrated that arachidonic acid percentage of total fatty acids in M. incisa increased gradually during this stress course. On the contrary,the percentages of ω3 pathway products,such as α-linolenic acid and 16：3ω3, descended significantly （ P 0. 05） from 40 h or 96 h on. These results suggested that the lower relative transcription of ω3 f

关 键 词：缺刻缘绿藻 ω3脂肪酸去饱和酶基因 氮饥饿 实时荧光定量PCR 脂肪酸 

分 类 号：S917[农业科学—水产科学]