| 摘要: |
| 根据已构建的溶藻弧菌(Vibro alginolyticus)诱导的马氏珠母贝(Pinctada fucata)血淋巴cDNA差减文库得到的ESTs序列, 应用cDNA末端快速扩增(RACE)技术成功克隆了其组织蛋白酶L基因(PFCatL), 并对其进行了生物信息学分析; 应用实时荧光定量PCR (Real-time PCR)技术, 研究了PFCatL基因在溶藻弧菌刺激前后马氏珠母贝足、外套膜、鳃、闭壳肌等8个组织中的表达变化。结果表明, PFCatL基因cDNA全长2004bp, 其中5′非编码区(5′-UTR)50bp, 3′非编码区(3′-UTR)865bp, 开放阅读框(ORF)1089bp, 编码362个氨基酸, 其分子量计算值(MW)为40.52kDa, 理论等电点(IP)为5.20; 生物信息学分析表明, PFCatL含有16个氨基酸残基组成的信号肽序列以及组织蛋白酶前体抑制功能域I29; Clustalw2多重比对发现PFCatL氨基酸序列在催化三联体Cys-His-Asn、底物结合位点以及二硫键形成相关的半胱氨酸残基位点高度保守; Real-time PCR研究发现, PFCatL在马氏珠母贝各组织中均有表达, 但各组织间的表达量存在差异, 其中以肾和闭壳肌中的表达量最高; 溶藻弧菌感染4h后, 外套膜、鳃以及血淋巴中PFCatL基因的表达较感染前显著上调。 |
| 关键词: 马氏珠母贝 组织蛋白酶L cDNA末端快速扩增 实时荧光定量PCR |
| DOI：10.11693/hyhz201306029 |
| 分类号: |
| 基金项目:国家自然科学基金项目, 31202023 号; 国家科技支撑计划课题, 2012BAD17B00 号。 |
附件 |
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| CLONING AND EXPRESSION ANALYSIS OF THE CATHEPSIN L GENE FROM PEARL OYSTER PINCTADA FUCATA |
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WANG Zhong-Liang1,2, JIAN Ji-Chang1,3, LU Yi-Shan4, DING Yu1,3, WANG Bei1,3, CHEN Gang1,2, WU Zao-He3,5
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1.Fisheries College, Guangdong Ocean University;2.Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal, Regular High Education Institute of Guangdong Province;3.Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals;4.Fisheries College, Guangdong Ocean UniversityGuangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals;5.Zhongkai University of Agriculture and Engineering
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| Abstract: |
| To examine the expression patterns of the PFCatLgene in various tissues, including foot, mantle, gill, adductor, heart, kidney, hepatopancreas, and haemolymph of healthy and Vibrio alginolyticus challenged oyster Pinctada fucata, we cloned the cDNA of cathepsin L (PFCatL) in the oyster by rapid amplification cDNA ends (RACE) technique based on the ESTs that analyzed from SSH cDNA library, and employed fluorescent real-time quantitative PCR (RT-PCR). The results show that the full length of PFCatLcDNA was 2004bp, including a 5' 50bp UTR, a 3' 865bp UTR, and a 1089bp ORF encoding a polypeptide of 362 amino acids with an MW at 40.52 kDa, and a 5.20 IP. A signal peptide of 16 amino acids and a peptidase inhibitor domain I29 were detected in PFCatLby SignalP and Prosite analysis. The catalytic triad, substrate binding sites, and cysteine disulfide linkage sites were highly conserved in PFCatL, indicated by multiple alignment analysis. PFCatLtranscripts were expressed ubiquitously in all tested tissues, while the expression levels were significantly different from each other; and the highest expression was detected in kidney and adductor. Four hours after
injection with V. alginolyticus, the expression of PFCatLtranscripts in mantle, gill, and haemolymph of P. fucatawere significantly up-regulated relative to that of the control. |
| Key words: Pinctada fucata cathepsin L rapid amplification of cDNA ends (RACE) fluorescent quantitative
real-time PCR |
