摘要
【目的】克隆多鳞鱚林岛综合征基因(vhl)并分析其在低氧胁迫下的表达变化,为揭示多鳞鱚应对低氧的适应机制提供研究资料,也为今后开展多鳞鱚新品种选育提供候选基因。【方法】采用PCR克隆多鳞鱚vhl基因c DNA全长序列,利用EditSeq、SMART、Signal IP 4.1及ClustalX等在线软件进行生物信息学分析;并以半定量PCR检测多鳞鱚vhl基因在不同组织中的表达情况,采用实时荧光定量PCR分析低氧胁迫对多鳞鱚vhl基因在鳃组织和心脏中表达的影响。【结果】多鳞鱚vhl基因cDNA序列全长727 bp,包括120 bp的5’端非编码区(5’-TUR)、106 bp的3’端非编码区(3’-TUR)和504 bp的开放阅读框(ORF),共编码167个氨基酸残基,编码蛋白存在4个结构域,即Pfam:VHL(第12~93位氨基酸)、Pfam:VHL_C(第5~22、31~39和98~153位氨基酸)、ParB结构域(第100~162位氨基酸)和SOCS_box结构域(第104~140位氨基酸)。多鳞鱚vhl氨基酸序列与盲曹鱼vhl氨基酸序列的同源性最高(81%),但系统发育进化分析结果显示多鳞鱚与攀鲈的亲缘关系最近。多鳞鱚vhl基因在不同组织中均有表达,其中以鳃组织、卵巢和精巢的表达量较高,其次是心脏和肝脏,在肌肉和脑组织的表达量较低。经低氧胁迫后,多鳞鱚vhl基因在鳃组织中的相对表达量显著上调(P<0.05,下同);在心脏中表现为随低氧胁迫时间的延长显著上调,恢复氧气4 h后vhl基因的相对表达量虽然呈显著下调趋势,但仍显著高于正常水平。【结论】多鳞鱚vhl基因编码蛋白存在Pfam:VHL、Pfam:VHL_C、ParB和SOCS_box等4个结构域,且低氧胁迫前后其表达量存在显著差异,即多鳞鱚vhl基因在低氧应答信号通路中扮演着重要角色。
【Objective】The Von Hippel-Lindau syndrome gene(vhl)of the Sillago sihama was cloned and its expression changes after hypoxia stress treatment were analyzed,which represented the insights into the underlying adaptation mechanisms of hypoxia stress and provided candidate genes for breed selection of S.sihama.【Method】cDNA sequence of vhl gene from S.sihama was cloned by PCR.Bioinformatics analysis on the sequence was conducted by online softwares such as EditSeq,SMART,Signal IP 4.1 and ClustalX,expression of vhl gene in different tissues were detected by semiquantitative PCR,and effects of hypoxia stress on expression of vhl gene in gill and heart tissues were studied by realtime fluorescence quantitative PCR.【Result】The vhl cDNA sequence was 727 bp in length,including a 5'-end noncoding region of 120 bp,a 3'-end noncoding region of 106 bp and an open reading frame(ORF)of 504 bp encoding a polypeptide of 167 amino acids.Four domains of the vhl protein were found,namely Pfam:VHL amino acids 12 to 93,Pfam:VHL_C(amino acids 5 to 22,31 to 39 and 98 to 153),and ParB domain(amino acid 100 to 162)and SOCS_box domain(amino acids 104 to 140).Multi-sequence alignment analysis of the S.sihama vhl gene showed the highest(81%)homology with Lates calcarifer.Cluster analysis showed that the relationship between S.sihama and Anabas testudineus was the closest.vhl gene expressed in different tissues of S.sihama.Its expression in the gill was significantly up-regulated after hypoxia stress(P<0.05,the same below),the expression of vhl gene in heart significantly up-regulated as hypoxia stress prolonged,and significantly down-regulated 4 h after reoxygen treatment,bur is was still significantly higher than normal level.【Conclusion】There are four domains in vhl gene encoded protein of S.sihama,namely Pfam:VHL,Pfam:VHL_C,ParB and SOCS_box.The expression of vhl gene before and after hypoxia stress is significantly different,indicating that vhl gene plays an important role in the hypoxia response pathway in S.sihama.
作者
林星桦
潘炎杨
陈芳圆
黄志棚
黄洋
杜涛
朱春华
李广丽
田昌绪
LIN Xing-hua;PAN Yan-yang;CHEN Fang-yuan;HUANG Zhi-peng;HUANG Yang;DU Tao;ZHU Chun-hua;LI Guang-li;TIAN Chang-xu(Fisheries College,Guangdong Ocean University,Zhanjiang,Guangdong 524088,China;Guangdong Research Center on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species,Zhanjiang,Guangdong 524088,China;Southern Marine Science and Engineering Guangdong(Zhanjiang),Zhanjiang,Guangdong 524088,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2020年第5期1201-1208,共8页
Journal of Southern Agriculture
基金
广东省科技计划项目(2015A020209163)
广东省普通高校特色创新类项目(2019KTSCX060)
广东省大学生创新创业计划项目(CXXL2018048,CXXL2019138)。
关键词
多鳞鱚
VHL基因
低氧胁迫
表达变化
Sillago sihama
vhl gene
hypoxia stress
expression variation
