日本沼虾cytMnSODmtMnSOD基因全长cDNA克隆及表达
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河南师范大学生命科学学院,河南师范大学生命科学学院,河南师范大学生命科学学院,河南师范大学生命科学学院,河南师范大学生命科学学院,河南师范大学生命科学学院

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国家自然科学基金项目(30940008);高等学校博士学科点专项科研基金(20094104110003);河南省高校科技创新人才支持计划(2009HASTIT022)


cDNA cloning and expression analysis of cytMnSOD and mtMnSOD from Macrobrachium nipponense
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College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University,College of Life Sciences,Henan Normal University

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    摘要:

    利用RACE 技术从日本沼虾肝胰腺中克隆了cytMnSODmtMnSOD基因cDNA全长序列。cytMnSOD基因cDNA全长1 233 bp,开放阅读框为858 bp,编码286个氨基酸,N端含有60个氨基酸残基组成的延伸区;mtMnSOD 基因cDNA全长1 113 bp,开放阅读框为654 bp,编码218个氨基酸,N端含有20个氨基酸残基组成的信号肽;cytMnSOD和mtMnSOD预测蛋白分子量及等电点分别为31.33、24.05 ku和5.62、7.12。日本沼虾cytMnSOD推导的氨基酸序列与其mtMnSOD的相似性为40%,二者均含有MnSOD的特征肽段(DVWEHAYY)、4个Mn2+结合位点和 2个N-糖基化位点。Real-time PCR结果表明,cytMnSODmtMnSOD在日本沼虾肝胰腺、肌肉、血细胞、大颚器官、卵巢和鳃等组织均有表达,其中肝胰腺表达量最高;肝胰腺cytMnSODmtMnSOD基因的表达量在蜕皮间期最高,蜕皮后期和蜕皮前期较低。嗜水气单胞菌刺激后3 h,肝胰腺cytMnSODmtMnSOD的表达量显著增加,推测MnSOD是参与机体免疫防御反应的一种重要分子。

    Abstract:

    The cytMnSOD and mtMnSOD genes were cloned from hepatopancreas of Macrobrachium nipponense using the rapid amplification of cDNA ends(RACE)method for the first time.The full-length cDNA of cytMnSOD was 1 233 bp with a 858 bp of open reading frame(ORF)encoding a 286 amino acids(aa)protein with a 60 aa leader sequence; whereas the full-length cDNA of mtMnSOD was 1 113 bp with a 654 bp of ORF encoding a 218 aa protein with a 21 aa signal peptide sequence in the N-terminus.The calculated molecular masses and estimated pI of translated cytMnSOD and mtMnSOD proteins were 31.33 ku,24.05 ku and 5.62,7.12,respectively.The deduced amino acid sequence of cytMnSOD had 40% of similarity to that of mtMnSOD. Both cytMnSOD and mtMnSOD contained a manganese superoxide dismutase domain(DVWEHAYY),four conserved amino acids responsible for binding manganese(H110,H158,D243,H247 and H48,H96,D180,H184),and two N-glycosylation sites(NHT,NMS and NHT,NLS).The constructed phylogenetic tree indicated that arthropod MnSODs could be classified into two distinct branches,cytMnSODs,and mtMnSODs.The MnSODs from M.nipponense were more closely related to Macrobrachium rosenbergii MnSODs than that from other crustaceans.Real-time RT-PCR analysis showed that both cytMnSOD and mtMnSOD from M.nipponense were expressed in hepatopancreas,muscle,haemocytes,ovarian,mandibular organ and gill,but relatively higher in the hepatopancreas.The levels of both cytMnSOD and mtMnSOD transcripts in hepatopancreas were the highest in stage C among the molt cycle.Injection of pathogenic bacteria Aeromonas hydrophila resulted in the significant increase of cytMnSOD and mtMnSOD expressions 3 h after treatment,indicating that MnSOD may act as an important molecule involved in immune defense against A.hydrophila.

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王文锋,关建义,吕黎,刘方,杨洪,宁黔冀.日本沼虾cytMnSODmtMnSOD基因全长cDNA克隆及表达[J].水产学报,2012,36(3):367~374

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  • 收稿日期:2011-09-29
  • 最后修改日期:2011-11-04
  • 录用日期:2012-02-24
  • 在线发布日期: 2012-03-15
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