甘蔗渣磷酸烯醇丙酮酸羧激酶基因的分离与功能鉴定

Q1 Agricultural and Biological Sciences
Peichong Lin, Yatong Yao, Lijuan Lu, Yanhui Bi, Zhigang Zhou
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引用次数: 0

摘要

与其他大型藻类类似,Saccharina japonica具有co2浓缩机制(CCM),可以提高光合作用效率并提高生物量。磷酸烯醇丙酮酸羧激酶(PEPCK)是胞浆中CCM的重要组成部分。然而,目前还没有关于粳稻细胞质PEPCK的报道。本研究从粳稻配子体全长转录组中筛选了一个预测定位于细胞质中的PEPCK基因(SjPCK1)的全长cDNA序列,并通过RT-PCR进行了鉴定。SjPCK1全长2174 bp,其中开放阅读框(ORF)为1734 bp, 5′-未翻译区(UTR)为216 bp, 3′-UTR为224 bp。SjPCK1的基因组DNA长度为21 294 bp,具有11个内含子和12个外显子的特征。该蛋白编码577个氨基酸,分子量为63 kD,等电点为8.47。氨基酸序列比对表明,PEPCK的功能位点在所选种属中高度保守。系统发育分析和序列鉴定表明,SjPCK1是一个atp依赖性PEPCK。通过大肠杆菌原核表达系统表达SjPCK1基因,得到带His 6标签的SjPCK1蛋白(rSjPCK1)分子量为81.93 kD。酶活性测定结果表明,rSjPCK1羧化酶和脱羧酶的比活性分别为1.91±0.29 U/mg prog和11.3±1.97 U/mg prog。这些发现为进一步分析细胞质PEPCK在粳稻无机碳储存中的作用提供了有价值的分子和生化见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
The isolation and functional identification of a phosphoenolpyruvate carboxykinase gene from Saccharina japonica
Similar to other macroalgae, Saccharina japonica has CO2-concentrating mechanism (CCM) that allows high photosynthesis efficiency and elevates biomass. Phosphoenolpyruvate carboxykinase (PEPCK) in cytoplasm is an essential component of CCM. However, no reports on cytosolic PEPCK of S. japonica are available. In this study, the full-length cDNA sequence of a PEPCK gene (SjPCK1) predicted to be localized in cytoplasm, was screened from the full-length transcriptome of S. japonica gametophytes and identified by RT-PCR. The complete cDNA sequence of SjPCK1 was 2174 bp in length, which encompassed an open reading frame (ORF) of 1734 bp, a 5′- untranslated region (UTR) of 216 bp and a 3′-UTR of 224 bp. In parallel, the genomic DNA of SjPCK1 was 21 294 bp in length, characterized by the presence of 11 introns and 12 exons. It encoded a protein of 577 amino acids with a molecular weight of 63 kD and an isoelectric point of 8.47. Amino acid sequence alignment showed that the functional sites of PEPCK were highly conserved in the selected species. Phylogenetic analysis and sequence characterization showed that SjPCK1 was an ATP-dependent PEPCK. SjPCK1 gene was expressed by using Escherichia coli prokaryotic expression system, and the SjPCK1 protein with His 6 tag (rSjPCK1) was 81.93 kD in molecular weight. Enzyme activity assay results showed that the specific activity of carboxylase and decarboxylase of rSjPCK1 was 1.91 ± 0.29 U/mg prog and 11.3 ± 1.97 U/mg prog, respectively. These findings provide valuable molecular and biochemical insights for a further analysis of the role of cytosolic PEPCK in the storage of inorganic carbon in S. japonica.
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来源期刊
Aquaculture and Fisheries
Aquaculture and Fisheries Agricultural and Biological Sciences-Aquatic Science
CiteScore
7.50
自引率
0.00%
发文量
54
审稿时长
48 days
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