The plant NAD-dependent isocitrate dehydro-genase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thali...The plant NAD-dependent isocitrate dehydro-genase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added to-gether to the in vitro system with 36 靏/mL of protein disul-fide isomerase, the expressed products had NAD-IDH enzy-matic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.展开更多
目的利用生物信息学方法识别和分析曼氏迭宫绦虫异柠檬酸脱氢酶(SmNAD-IDH)核苷酸序列及生物学特征;克隆该基因并诱导蛋白表达,分析其在裂头蚴阶段的转录水平。方法利用生物信息学在线分析工具对SmNAD-IDH基因的核苷酸序列进行识别,预...目的利用生物信息学方法识别和分析曼氏迭宫绦虫异柠檬酸脱氢酶(SmNAD-IDH)核苷酸序列及生物学特征;克隆该基因并诱导蛋白表达,分析其在裂头蚴阶段的转录水平。方法利用生物信息学在线分析工具对SmNAD-IDH基因的核苷酸序列进行识别,预测分析氨基酸序列的理化性质。分别以曼氏迭宫绦虫裂头蚴cDNA、孕节cDNA和成节cDNA作为模板PCR扩增目的基因,扩增产物与表达载体pET-30a重组后转化至BL21细胞中诱导蛋白表达。利用real time PCR技术分析裂头蚴阶段三羧酸循环中3个关键代谢酶的转录水平。结果BLASTx分析SmNAD-IDH基因全长1095 bp,编码蛋白由356个氨基酸组成。该基因与其他寄生虫同源基因核苷酸序列一致性>70%,与人类同源基因核苷酸序列一致性为51%。该蛋白的理论等电点为6.84,分子质量为40 ku,并且氨基酸序列中无信号肽和跨膜区,为脂溶性稳定的胞内蛋白。SmNAD-IDH在裂头蚴、成节、孕节中均有表达,在裂头蚴阶段三羧酸循环中的关键酶柠檬酸合成酶、酮戊二酸脱氢酶、SmNAD-IDH均有转录,以SmNAD-IDH的转录水平较高。结论成功重组SmNAD-IDH基因并诱导SmNAD-IDH蛋白表达,该蛋白对于曼氏迭宫绦虫,尤其是裂头蚴,是具有潜在研究价值的重要代谢酶。展开更多
文摘The plant NAD-dependent isocitrate dehydro-genase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added to-gether to the in vitro system with 36 靏/mL of protein disul-fide isomerase, the expressed products had NAD-IDH enzy-matic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.
文摘目的利用生物信息学方法识别和分析曼氏迭宫绦虫异柠檬酸脱氢酶(SmNAD-IDH)核苷酸序列及生物学特征;克隆该基因并诱导蛋白表达,分析其在裂头蚴阶段的转录水平。方法利用生物信息学在线分析工具对SmNAD-IDH基因的核苷酸序列进行识别,预测分析氨基酸序列的理化性质。分别以曼氏迭宫绦虫裂头蚴cDNA、孕节cDNA和成节cDNA作为模板PCR扩增目的基因,扩增产物与表达载体pET-30a重组后转化至BL21细胞中诱导蛋白表达。利用real time PCR技术分析裂头蚴阶段三羧酸循环中3个关键代谢酶的转录水平。结果BLASTx分析SmNAD-IDH基因全长1095 bp,编码蛋白由356个氨基酸组成。该基因与其他寄生虫同源基因核苷酸序列一致性>70%,与人类同源基因核苷酸序列一致性为51%。该蛋白的理论等电点为6.84,分子质量为40 ku,并且氨基酸序列中无信号肽和跨膜区,为脂溶性稳定的胞内蛋白。SmNAD-IDH在裂头蚴、成节、孕节中均有表达,在裂头蚴阶段三羧酸循环中的关键酶柠檬酸合成酶、酮戊二酸脱氢酶、SmNAD-IDH均有转录,以SmNAD-IDH的转录水平较高。结论成功重组SmNAD-IDH基因并诱导SmNAD-IDH蛋白表达,该蛋白对于曼氏迭宫绦虫,尤其是裂头蚴,是具有潜在研究价值的重要代谢酶。
