摘要
为了探究中华绒螯蟹自身脂肪酸合成能力,采用RACE技术,克隆获得中华绒螯蟹ELOVL6 c DNA序列全长(Gen Bank accession:KT779219)。该序列全长2247 bp,包括235、873和1139 bp的5'非编码区(5'-UTR)、开放阅读框(ORF)和3'非编码区(3'-UTR),编码290个氨基酸(AA),具有ELOVL家族的全部特征:6个跨膜区、高度保守的组氨酸簇HXXHH、多个保守区以及内质网滞留信号KXKXX。分析发育树表明,ELOVL2与ELOVL5聚为一支,ELOVL6聚为另一支,其中中华绒螯蟹ELOVL6与其他节肢动物ELOVL6聚为一支,与凡纳滨对虾聚为一小支。采用异源表达方法研究ELOVL6编码的蛋白质对脂肪酸的作用,GC-MS结果显示,转基因的酵母培养物C16:0和C16:1n-7含量下降,而C18:0和C18:1n-9的含量升高,同时有新产物C18:1n-7产生。通过荧光定量PCR(q PCR)技术研究ELOVL6基因在中华绒螯蟹不同组织及不同脂肪源喂养下的表达情况,显示该基因在肠道、胃、心脏、肝胰腺、胸神经节、肌肉、眼柄、鳃和脑神经节9个组织均有表达,其中在肝胰腺中表达量最高,显著高于其他组织;在不同脂肪源饲喂的结果中,肝胰腺、肌肉、卵巢和精巢4个组织中均有表达,并且各组织在全鱼油组饲喂下表达量最低,其中用全豆油组饲喂后,肝胰腺中的表达情况显著升高,明显高于全鱼油组和鱼油豆油混合组。以上结果综合表明,中华绒螯蟹中ELOVL6能够催化C16的饱和及C16的单不饱和脂肪酸延长,同时受到饲料中不同脂肪源的一定影响,这为探究中华绒螯蟹PUFA合成途径及脂肪酸调控机制提供了依据。
We cloned full length cDNA of ELOVL6 gene （GenBank accession KT779219） in Eriocheir sinensis using genetic homology and RACE methods.The full sequence was 2247 bp, including of 235, 873 and 1139 bp of 5＇-URT, ORF and 3＇-URT. ELOVL6 gene specified a protein of 290 amino acids which got all of ELOVL features： six transmembrane-spanning regions, one diagnostic histidine box HXXHH motif, several conservative regions and ER retention signal KXKXX.The phylogenetic tree estabilshed by the Neighbor Joining method showed that ELOVL2 is clustered closely with ELOVL5 and ELOVL6 is clustered by itself. At the same time, ELOVL6 of Chinese mitten crab is the closest to the ELOVL6 ofLitopenaeus vannamei. Its biochemical function characterized by heterologous expression showed that it caused reduction of C16：0 and C16：1n-7 and increase of C18：0 and C18：1n-9, while produced new product C18：1n-7. Relative gene expression levels ofELOVL6 mRNA in nine tissues and three kinds of different lipids formula feed by quantitative real-time PCR.The results suggested that ELOVL gene was expressed in all of these tissues and it was highest in the hepatopancreas, while lower in the cranial ganglia and thoracic ganglia, and trace in the other tissues. Real-time Q-PCR also revealed that on one side, levels were expressed in hepatopancreas, ovary, transcripts and muscle, on the other side, levels were highest in the hepatopancreas when fed the SO diet compared with the FO diet and FO/SO diet.The results above showed that E. sinensis ELOVL6 could catalyze the production of C16 long-chain fatty acids including both saturated and monounsaturated fatty acids which laid a base for PUFA biosynthesis and its function was influenced by different levels of lipids. Thus, it provided a foundation for fatty acids biosynthetic pathway and its regulation mechanism.
出处
《水产学报》
CAS
CSCD
北大核心
2016年第6期844-855,共12页
Journal of Fisheries of China
基金
国家自然科学基金(31472287)
国家"八六三"高技术研究发展计划(2012AA10A409-5)
水产动物遗传育种中心上海市协同创新中心(ZF1206)
科技部港澳台科技合作专项(2014DFT30270)
上海市科委优秀学术带头人专项(12XD1402700)
上海市科技兴农重点攻关专项(沪农科2013第5-7号)~~
