褶纹冠蚌超氧化物歧化酶基因和溶菌酶基因的分子克隆及表达特征
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摘要
褶纹冠蚌(Cristaria plicata)是我国重要的“淡水育珠蚌”之一,开展其分子免疫的研究不仅具有理论意义,而且具有重要的应用价值。本文对褶纹冠蚌超氧化物歧化酶(CpSOD)和溶菌酶(CpLYS)的cDNA进行了克隆,并研究了这两种酶的表达特征。
利用RACE-PCR及同源克隆方法,克隆出CpSOD cDNA全长和CpLYS的部分cDNA。CpSOD基因全长891bp,其中编码区468bp,5'端不翻译区83bp,3'端不翻译区340 bp(含ployA尾24 bp)。该基因序列开放阅读框编码155个氨基酸,预测的分子量大小为15.8kDa,等电点为5.80。通过多序列比对结果发现,CpSOD和许多动物的Cu/Zn-SOD相似,均含有保守的4个铜结合位点和4个锌结合位点,信号肽预测未发现信号肽,表明CpSOD是胞质Cu/Zn-SOD。BLAST分析显示CpSOD与其它动物的Cu/Zn-SOD有很高的同源性,利用不同动物的胞质Cu/Zn-SOD氨基酸序列构建系统发育树,结果表明褶纹冠蚌与太平洋牡蛎、栉孔扇贝、紫贻贝共同形成一个小分支。CpLYS的部分cDNA包括530bp,3'端不翻译区259 bp(含ployA尾29bp),共编码89个氨基酸残基。BLAST分析发现CpLYS的部分cDNA序列与其它动物的i-型溶菌酶有较高的同源性,所以推测CpLYS为i-型溶菌酶。
利用RT-PCR分析检测CpSOD和CpLYS基因在注射嗜水气单胞菌后的表达情况,结果表明经注射嗜水气单胞菌后,血细胞中CpSOD基因的表达明显增加,到12h后达到最大值,随后表达下降,至48h后恢复至原有水平;而CpLYS基因在注射嗜水气单胞菌后表达量一直增加,至48h后达到最大值,结果说明褶纹冠蚌在受到病原体侵害后,其体内的超氧化物歧化酶和溶菌酶在免疫中均具有防御作用。
利用RT-PCR分析检测CpSOD和CpLYS在各组织中的表达情况,结果表明,CpSOD基因在血液、外套膜、鳃、肝胰腺和闭壳肌中均有表达,CpLYS基因在血液、外套膜、鳃、肝胰腺和闭壳肌中也均有表达。
Cristaria plicata is one of the most important "Freshwater pearl mussel" in our country. The understanding its immune system is essential for aqualculture. The present study is to clone superoxidase (CpSOD) and lysozyme (CpLYS) from Cristaria plicata, and to study on the expression characteristics of them.
The CpSOD cDNAs full sequence and CpLYS partial cDNAs have been cloned using homologous cloning and RACE- PCR. The full cDNA of CpSOD is 891bp, which contains an open reading frame of 468 bp, 5' untranslated region 83 bp, 3' untranslated region 340 bp(including a polyA tail of 24 bp). The open reading frame encodes a protein of 155 amino acid residues, the predicted molecular weight is 15.8 kDa, isoelectric point is 5.80. The CpSOD contains four amino acid residues required for binding copper and four amino acid residues required for binding zinc, which are highly conserved in other Cu/Zn-SODs. There were no signal peptide found showed that the CpSOD are icCu/Zn-SOD. Blast analysis showed the CpSOD has highest similarity with Cu/Zn-SODs of other bivalves, and Neighbor-joining phylogentic tree of CpSOD and icCuZnSODs amino acid sequences from different species showed that CpSOD were clustered with Chlamys farreri, Crassostrea gigas and Mytilus edulis. The partial cDNAs of CpLYS is 530bp, which contains an 3' untranslated region 259 bp(including a polyA tail of 29 bp), encodes a protein of 89 amino acid residues. Blast analysis showed the CpLYS has highest similarity with i-type lysozymes of other bivalves, so the CpLYS was suspected to be an i-type lysozymes.
Expression of CpSOD and CpLYS in hemocyte after injected by Aeromonas hydrophila 3h、6h、12h、24h、48h were determined by RT-PCR. The results showed that after injected by Aeromonas hydrophila, the expression of CpSOD were increased at first 12h, and decreased thereafter; hile the expression of CpLYS were increased from the first to 48h, which proved the CpSOD and CpLYS were important immune system in Cristaria plicata.
Expression of CpSOD and CpLYS in different tissues of Cristaria plicata were determined by RT-PCR and the results showed the expression of both CpSOD and CpLYS were detected in mantel, conductor muscle, hepatopancreas, sexual gland and hemocyte.
利用RACE-PCR及同源克隆方法,克隆出CpSOD cDNA全长和CpLYS的部分cDNA。CpSOD基因全长891bp,其中编码区468bp,5'端不翻译区83bp,3'端不翻译区340 bp(含ployA尾24 bp)。该基因序列开放阅读框编码155个氨基酸,预测的分子量大小为15.8kDa,等电点为5.80。通过多序列比对结果发现,CpSOD和许多动物的Cu/Zn-SOD相似,均含有保守的4个铜结合位点和4个锌结合位点,信号肽预测未发现信号肽,表明CpSOD是胞质Cu/Zn-SOD。BLAST分析显示CpSOD与其它动物的Cu/Zn-SOD有很高的同源性,利用不同动物的胞质Cu/Zn-SOD氨基酸序列构建系统发育树,结果表明褶纹冠蚌与太平洋牡蛎、栉孔扇贝、紫贻贝共同形成一个小分支。CpLYS的部分cDNA包括530bp,3'端不翻译区259 bp(含ployA尾29bp),共编码89个氨基酸残基。BLAST分析发现CpLYS的部分cDNA序列与其它动物的i-型溶菌酶有较高的同源性,所以推测CpLYS为i-型溶菌酶。
利用RT-PCR分析检测CpSOD和CpLYS基因在注射嗜水气单胞菌后的表达情况,结果表明经注射嗜水气单胞菌后,血细胞中CpSOD基因的表达明显增加,到12h后达到最大值,随后表达下降,至48h后恢复至原有水平;而CpLYS基因在注射嗜水气单胞菌后表达量一直增加,至48h后达到最大值,结果说明褶纹冠蚌在受到病原体侵害后,其体内的超氧化物歧化酶和溶菌酶在免疫中均具有防御作用。
利用RT-PCR分析检测CpSOD和CpLYS在各组织中的表达情况,结果表明,CpSOD基因在血液、外套膜、鳃、肝胰腺和闭壳肌中均有表达,CpLYS基因在血液、外套膜、鳃、肝胰腺和闭壳肌中也均有表达。
Cristaria plicata is one of the most important "Freshwater pearl mussel" in our country. The understanding its immune system is essential for aqualculture. The present study is to clone superoxidase (CpSOD) and lysozyme (CpLYS) from Cristaria plicata, and to study on the expression characteristics of them.
The CpSOD cDNAs full sequence and CpLYS partial cDNAs have been cloned using homologous cloning and RACE- PCR. The full cDNA of CpSOD is 891bp, which contains an open reading frame of 468 bp, 5' untranslated region 83 bp, 3' untranslated region 340 bp(including a polyA tail of 24 bp). The open reading frame encodes a protein of 155 amino acid residues, the predicted molecular weight is 15.8 kDa, isoelectric point is 5.80. The CpSOD contains four amino acid residues required for binding copper and four amino acid residues required for binding zinc, which are highly conserved in other Cu/Zn-SODs. There were no signal peptide found showed that the CpSOD are icCu/Zn-SOD. Blast analysis showed the CpSOD has highest similarity with Cu/Zn-SODs of other bivalves, and Neighbor-joining phylogentic tree of CpSOD and icCuZnSODs amino acid sequences from different species showed that CpSOD were clustered with Chlamys farreri, Crassostrea gigas and Mytilus edulis. The partial cDNAs of CpLYS is 530bp, which contains an 3' untranslated region 259 bp(including a polyA tail of 29 bp), encodes a protein of 89 amino acid residues. Blast analysis showed the CpLYS has highest similarity with i-type lysozymes of other bivalves, so the CpLYS was suspected to be an i-type lysozymes.
Expression of CpSOD and CpLYS in hemocyte after injected by Aeromonas hydrophila 3h、6h、12h、24h、48h were determined by RT-PCR. The results showed that after injected by Aeromonas hydrophila, the expression of CpSOD were increased at first 12h, and decreased thereafter; hile the expression of CpLYS were increased from the first to 48h, which proved the CpSOD and CpLYS were important immune system in Cristaria plicata.
Expression of CpSOD and CpLYS in different tissues of Cristaria plicata were determined by RT-PCR and the results showed the expression of both CpSOD and CpLYS were detected in mantel, conductor muscle, hepatopancreas, sexual gland and hemocyte.
引文
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