褶紋冠蚌论文褶纹冠蚌过氧化氢酶基因克隆原核表达及酶活性分析.docVIP

褶纹冠蚌论文：褶纹冠蚌过氧化氢酶基因克隆、原核表达及酶活性分析 【中文摘要】褶纹冠蚌(Crist aria plicata)是我国重要“淡水育珠蚌”之一,近年来褶纹冠蚌频繁受到病害的影响,对其育珠能力产生了极大的影响。本文对褶纹冠蚌过氧化氢酶(cpCAT)的cDNA进行了克隆与原核表达。本文利用RACE-PCR及同源克隆方法,克隆出cpCAT cDNA全长。该基因全长为4863 bp,且有3个外显子,2个内含子。cpCAT cDNA全长为2618 bp,其中编码区1503 bp,5’端不翻译区136 bp,3’端不翻译区979 bp,具有典型的腺苷酸信号序列AATAAA和PolyA尾。该基因序列开放阅读框编码为501个氨基酸,预测蛋白质分子量为56.86kDa,等电点为6.77。BLAST分析显示cpCAT氨基酸序列和动物,植物,细菌的CAT基因有很高的同源性。cpCAT无信号肽,存在过氧化氢酶近端活性位点(61FNRERIPERVVHAKGAG77)和过氧化氢酶近端血红素配体签名序列(351RLYSYSDTH359),两个糖基化位点(145NNTP148)与(436NFSQ439)。系统发育树结果表明褶纹冠蚌与栉孔扇贝(Chlamys farreri)过氧化氢酶基因的亲缘关系最近。利用RT-PCR分析检测cpCAT基因经注射嗜水气单胞菌后的表达情况,结果表明在注射嗜水气单胞菌后,在血液、鳃、外套膜、闭壳肌和肝脏中都有表达,且在肝脏中的表达量最高,且各个组织中cpCAT基因的表达明显增加,12h后达到最大值,随后表达有所下降,到48h后恢复至原有水平。结果表明褶纹冠蚌在受到病害后,其体内的过氧化氢酶具有防御作用。根据开放阅读框设计带酶切位点(KpnI和EcoRI)的引物,构建重组表达质粒,经IPTG诱导表达,利用SDS分析表达产物。结果表明重组cpCAT在大肠杆菌(Escherichia coli) Rosetta-gami (DE3)中获得了表达,产物为56.86 KDa。纯化的cpCAT的酶活力可达11194.4+40.4U/mg,该酶最适温度为25℃,有较高的热稳定性(60℃以下),cpCAT的pH值的最适范围在pH5.0-10.0,最适pH值为7.0。用变性剂Urea和SDS处理时酶活性有所改变,用1%～3%SDS和2～4mol/L Urea处理时,酶活性保持80%以上。随着变性剂浓度的增加,cpCAT酶活力逐渐降低。 【英文摘要】Cristaria plicala is one of the most important “Freshwater pearl mussel” in our country. But recently Cristaria plicata is vulnerable to disease frequently, which makes a great influence to the ability of the pearl. The catalase cDNA of C. plicata (cpCAT) was cloned and the full length was expressed with Prokaryotic expression in the paper.The cpCAT cDNA full sequence has been cloned using homologous cloning and RACE-PCR. The gene is 4863 bp long and has a total of two introns and three exons. In addition the full-length cDNA of cpCAT contained 2618 bp, The cDNA contained a 5’untranslated region (UTR) of 136 nucleotides, the 3’UTR of 979 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1503 bp, encoding 501 amino acid residues with 56.86 kDa predicted molecular weight. The theoretical isoelectric point was 6.77. BLAST analysis showed that the deduced amino acid sequence of cpCAT had s