鸡球虫E.tenella叶酸合成途径关键酶PPPK-DHPS cDNA的克隆及其株间变异研究
摘要
本研究通过生物信息学方法对鸡E．tenalla叶酸合成途径关键酶PPPK-DHPS基因(pppk-dhps)进行了预测，用RT-PCR的方法获得鸡E．tenalla pppk-dhps的ORF。结果显示鸡E．tenella pppk-dhps基因ORF为2001 bp，G+C含量为51.92％，共编码666个氨基酸，大小约为74.3 Kda，无信号肽。通过生物信息学方法对鸡E．tenalla PPPK-DHPS进行结构和功能分析，结果显示，鸡E．tenallaPPPK-DHPS为一双功能酶，同时具有PPPK和DHPS功能区，并与其它原虫间具有高度保守性，序列相似性达到22.1％-38.5％，共有19个高度保守的序列模块，且顺序正确，无重叠，匹配度高，鸡E．tenalla PPPK-DHPS折叠类型也与其它生物相似。通过RACE方法，我们获得了鸡E．tenalla pppk-dhps全长cDNA，其长为2372 bp，5’和3’非编码区分别为244 bp和127 bp，与刚地弓形虫和恶性疟原虫pppk-dhps全长cDNA序列进行比对分析，结果显示三者非编码区存在较大差异，刚地弓形虫非编码区明显长于其它两种原虫，尤其在其3’端。
对鸡E．tenella不同磺胺药敏感虫株和抗性虫株PPPK-DHPS进行株间变异研究，结果显示不同虫株氨基酸序列相似性为99.1％-99.9％，在我们的鸡E．tenella磺胺药敏感株和抗性株的pppk-dhps基因多态性分析中发现，磺胺药抗性株甘肃株和上海株在其DHPS功能区403位点存在Asp→Asn的变异，这与刚地弓形虫在DHPS功能区407位点Asp→Asn的变异相似，因此推测DHPS功能区403位点的突变与鸡E．tenella磺胺药抗药性的产生有关。
In this paper, PPPK-DHPS gene sequence of E.tenella has beensuccessfully predicted by informatics. Then, based on the predictedsequence, pppk-dhps ORF of E. tenella is amplified from guangdong strain.The length of amplified pppk-dhps ORF is 2001 bp, which code a proteinwith 666 amino acid as same as the predict result. The G+C content ofpppk-dhps ORF is 51.92%. AA75-225 is PPPK conserve sequence, AA350-635is DHPS conserve sequence and the middle region is ligate region accordingto Protein Conserved Domains Blast analysis. Compared with otherprotozoans, the similarity of E. tenella PPPK-DHPS which have 19 highlyconserved blocks is 22.1-38.5%. When E. tenella compared with otherprotozoans, The PPPK-DHPS 3D structer is similarity by blast in PDBsumdatebase. The cDNA full length of pppk-dfips is 2372 bp. 5'-UTR and 3'-UTR of pppk-dhps is 244 bp and 127 bp, respectively. The similarity ofpppk-dhps UTR region among E. tenella, P. falciparum and T. gondii are lowby Sequence alignment analysis.
The similarity of chicken PPPK-DHPS amino acid sequence among thesulf-drug senstive strains and the sulf-drug resistance strains is from99.1% to 99.9%. The DHPS AA403 of shanghai strain and gansu strain whichare the sulf-drug resistance strains both are Asp instead of Asn. Themutation (Asp→Asn) of T. gondii DHPS AA407 was assciated with thesulf-drug resistance, so the mutation(Asp→Ash) of shanghai strain andgansu strain may be the reason of sulf-drug resistance in chicken
对鸡E．tenella不同磺胺药敏感虫株和抗性虫株PPPK-DHPS进行株间变异研究，结果显示不同虫株氨基酸序列相似性为99.1％-99.9％，在我们的鸡E．tenella磺胺药敏感株和抗性株的pppk-dhps基因多态性分析中发现，磺胺药抗性株甘肃株和上海株在其DHPS功能区403位点存在Asp→Asn的变异，这与刚地弓形虫在DHPS功能区407位点Asp→Asn的变异相似，因此推测DHPS功能区403位点的突变与鸡E．tenella磺胺药抗药性的产生有关。
In this paper, PPPK-DHPS gene sequence of E.tenella has beensuccessfully predicted by informatics. Then, based on the predictedsequence, pppk-dhps ORF of E. tenella is amplified from guangdong strain.The length of amplified pppk-dhps ORF is 2001 bp, which code a proteinwith 666 amino acid as same as the predict result. The G+C content ofpppk-dhps ORF is 51.92%. AA75-225 is PPPK conserve sequence, AA350-635is DHPS conserve sequence and the middle region is ligate region accordingto Protein Conserved Domains Blast analysis. Compared with otherprotozoans, the similarity of E. tenella PPPK-DHPS which have 19 highlyconserved blocks is 22.1-38.5%. When E. tenella compared with otherprotozoans, The PPPK-DHPS 3D structer is similarity by blast in PDBsumdatebase. The cDNA full length of pppk-dfips is 2372 bp. 5'-UTR and 3'-UTR of pppk-dhps is 244 bp and 127 bp, respectively. The similarity ofpppk-dhps UTR region among E. tenella, P. falciparum and T. gondii are lowby Sequence alignment analysis.
The similarity of chicken PPPK-DHPS amino acid sequence among thesulf-drug senstive strains and the sulf-drug resistance strains is from99.1% to 99.9%. The DHPS AA403 of shanghai strain and gansu strain whichare the sulf-drug resistance strains both are Asp instead of Asn. Themutation (Asp→Asn) of T. gondii DHPS AA407 was assciated with thesulf-drug resistance, so the mutation(Asp→Ash) of shanghai strain andgansu strain may be the reason of sulf-drug resistance in chicken
引文
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