中华绒螯蟹（Eriocheir sinensis）Peroxiredoxin 6和Thioredoxin1基因的克隆及表达

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	中华绒螯蟹（Eriocheir sinensis）Peroxiredoxin 6和Thioredoxin1基因的克隆及表达
其他题名	Molecular cloning and recombinant expression of peroxiredoxins 6 and thioredoxin 1 from Chinese mitten crab Eriocheir sinensis
	母昌考
学位类型	博士
	2008-05-28
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	中华绒螯蟹固有免疫过氧化物还原酶硫氧还蛋白基因克隆基因表达菌刺激抗氧化活力过氧化物酶活力
摘要	中华绒螯蟹是我国重要的水产经济动物，近年来养殖规模不断扩大，产量持续增加。但是，伴随着养殖规模的扩大，养殖环境也日益恶化并导致了大量疾病的发生，严重制约了中华绒螯蟹养殖业的健康发展。因此，疾病预防和控制对中华绒螯蟹养殖业的可持续发展具有举足轻重的作用。与其他无脊椎动物一样，中华绒螯蟹的免疫系统没有免疫球蛋白和淋巴细胞，而是依靠由细胞免疫和体液免疫构成的固有免疫系统来对病原进行识别和清除。中华绒螯蟹的固有免疫机制的研究有助于推动中华绒螯蟹病害防治工作的开展。本研究采用大规模EST测序方法，结合末端快速扩增（rapid amplification of cDNA ends，RACE）技术从中华绒螯蟹血淋巴中克隆到了过氧化物还原酶（peroxiredoxin，EsPrx6）和硫氧还蛋白（thioredoxin，EsTrx1）基因的cDNA 全长序列；采用实时荧光定量PCR 技术检测了这两个基因在健康个体中表达的组织分布情况以及鳗弧菌刺激后血淋巴细胞中的时序表达规律；同时，将这两个基因的编码区克隆到pET 系列载体，并在大肠杆菌中实现了重组表达，并进行了体外活性检测。过氧化物还原酶是一个抗氧化蛋白超家族，在保护机体免受活性氧（reactive oxygen species，ROS）的伤害中发挥着重要作用。中华绒螯蟹Prx6（EsPrx6）基因的cDNA 全长为1076 bp，5` UTR（untranslated region，UTR）为69 bp，3` UTR 为347 bp，开放阅读框（open reading frame，ORF）为660 bp，编码219 个氨基酸的蛋白。mRNA 3`-端具有多聚腺苷酸加尾信号（polyadenylation signal）AATAAA 和polyA 尾巴。EsPrx6 的预测分子量为 24 kDa，理论等电点为6.21，具有一个保守的Prx 结构域、一个AhpC 结构域和过氧化物酶催化活性中心PVCTTE，表明EsPrx6 属于1-Cys 型Prx。在所检测的组织中均有EsPrx6 的表达，其中以肝胰腺表达量最高，为血淋巴细胞中表达量的17.4 倍。鳗弧菌刺激后，血淋巴细胞中EsPrx6 的表达下降，到12 h 时，实验组显著低于对照组（P<0.05）；随时间推移，表达水平逐渐回升，但在整个实验期间，都没有恢复到起始水平。将EsPrx6 进行体外重组并在大肠杆菌E. coli BL21（DE3）中实现表达，重组EsPrx6 具有预期的抗氧化活性和过氧化物酶活性，其中抗氧化活力为14.69 U/mg 蛋白，高于相同条件下GSH 的抗氧化力（P<0.05），过氧化物酶活力为23.46 U/mg 蛋白。结果表明，EsPrx6 作为一种重要的抗氧化剂，在中华绒螯蟹抵御ROS 可能引起的氧化损伤方面具有重要作用。硫氧还蛋白是广泛存在于生物体内的一种具有硫醇依赖性的具有还原活性的蛋白。中华绒螯蟹Trx1（EsTrx1）基因的cDNA 全长为641 bp，5` UTR 为17 bp，3` UTR 为306 bp，开放阅读框为318 bp，编码105 个氨基酸。EsTrx1 的预测分子量为12.2 kDa，理论等电点为4.8。EsTrx1 不含信号肽，其氨基酸序列与其他动物的Trx1s 具有高度相似性，如与地中海黄蝎的Trx1 相似度达到73%；而与其他物种Trx2 的同源性很低，相似度仅为14.3-22.8%，表明EsTrx1 属于Trx1 亚族。实时荧光定量PCR 检测发现，EsTrx1 在鳃、性腺、肝胰腺、肌肉、心脏和血淋巴细胞中都有表达。血淋巴细胞中EsTrx1 mRNA 的表达量在菌刺激后上升，刺激后6 h，实验组表达量显著高于对照组和空白组（P<0.05），然后逐渐恢复到刺激前水平。为进一步探讨其生物学功能，将EsTrx1 进行体外重组并在大肠杆菌E. coli BL21（DE3）得到表达，重组EsTrx1 具有预期的氧化还原调节活性，抗氧化活力为3.06 U/mg，且抗氧化活力高于GSH（P<0.05）。rEsTrx1 的二硫键还原活力为5.03，低于凡纳滨对虾的二硫键还原活力（10.44），接近于大肠杆菌（4.93），小牛胸腺（6.50）和小牛肝脏（5.09），而高于鲍鱼Trx2（1.83）活力。结果表明，EsTrx1 在生理条件下能够作为一种重要的抗氧化剂，参与对细菌感染的免疫应答反应。
其他摘要	The Chinese mitten crab Eriocheir sinensis is one of the most commercially important native species for aquaculture industry in China. With the development of intensive culture and environmental deterioration, various diseases caused by bacteria, viruses and rickettsia-like organisms had frequently occurred in cultured E. sinensis populations, which resulted in enormous losses to the crab aquaculture. Like other invertebrates, E.sinensis has no memory effectors such as immunoglobulins and lymphoid cells in the immune response system, and depends on the humoral and cellular components of the innate immunity system to eliminate foreign invaders. Better understanding of the knowledge on the immune defense system of crab will be beneficial to the development of health management and diseases control in crab aquaculture. In the present study, peroxiredoxins 6 (designated EsPrx6) and thioredoxin (designated EsTrx1) were cloned from E.sinensis by using rapid amplification of cDNA ends (RACE) approaches. Quantitative real-time RT-PCR was employed to assess the mRNA　expression of EsPrx6 and EsTrx1 in various tissues and their temporal expression in haemocytes of crabs challenged with Listonella anguillarum. In order to elucidate its biological functions, EsTrx1 and were recombined and expressed in E. coli BL21 (DE3). Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5` untranslated region (UTR) of 69 bp, a 3`UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12 h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E. sinensis. The rEsPrx6 possess peroxidase activity of 23.46 U/mg protein and antioxidant activity of 14.69 U/mg. The antioxidant capacity was higher than that of GSH. All these indicated that Prx6 could function as an important antioxidant to protect crab from oxidative stress. Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. The full-length cDNA of EsTrx1 was of 641 bp, containing a 5` untranslated region (UTR) of 17 bp, a 3` UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids with the predicted molecular weight of 12.2 kDa. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepatopancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post stimulation, and then dropped back to the original level gradually. The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. The antioxidant capacity of rEsTrx1 was 3.06 U/mg, wich was stronger than that of GSH. The dithiol-reducing enzymatic activity (5.03) of rEsTrx1 was lower than that of Trx1 in shrimp Litopenaeus vannamei (10.44) , comparable to the specific activity of Trx1 from E. coli (4.93), calf thymus (6.50) and calf liver (5.09), and much larger than the mitochondrial Trx2 from abalone (1.83). These results together indicated that EsTrx1 could function as an important antioxidant in physiological state, and perhaps involved in the responses to bacterial challenge.
页数	98
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/333
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	母昌考. 中华绒螯蟹（Eriocheir sinensis）Peroxiredoxin 6和Thioredoxin1基因的克隆及表达[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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