| 番木瓜镁离子转运蛋白基因CpMGT1的克隆与功能分析 |
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| 引用本文： | 许迎港,邹智,郭静远,孔华,朱国鹏,郭安平. 番木瓜镁离子转运蛋白基因CpMGT1的克隆与功能分析[J]. 热带作物学报, 2022, 43(6): 1114-1121. DOI: 10.3969/j.issn.1000-2561.2022.06.003 |
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| 作者姓名： | 许迎港 邹智 郭静远 孔华 朱国鹏 郭安平 |
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| 作者单位： | 海南大学园艺学院,海南海口 570228;中国热带农业科学院三亚研究院,海南三亚 572024;海南省南繁生物安全与分子育种重点实验室/中国热带农业科学院热带生物技术研究所,海南海口 571101;中国热带农业科学院三亚研究院,海南三亚 572024;海南省南繁生物安全与分子育种重点实验室/中国热带农业科学院热带生物技术研究所,海南海口 571101;海南大学园艺学院,海南海口 570228 |
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| 基金项目： | 中央级公益性科研院所基本科研业务费专项（No.1630052022001,No.1630052017011）; |
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| 摘 要： | 镁是一种大量金属营养元素,其在植物的生长、发育、光合、胁迫响应等生物学过程中起重要作用。在高等植物中,Mg2+的吸收、运输、分布和再分配主要由镁离子转运蛋白（MGT/MRS2）和Mg2+/H+交换体（MHX）介导。其中,MGT/MRS2又名CorA,最先在鼠伤寒沙门氏菌中被发现,后在拟南芥、水稻等模式植物中进行了较为深入的研究。番木瓜（Carica papaya L.）是一种隶属于十字花目番木瓜科的重要热带果树,至今还未见有关MGT基因的报道。本研究基于番木瓜的基因组和转录组数据,采用RT-PCR技术成功克隆到一个MGT基因（CpMGT1）,应用生物信息学手段预测蛋白的理化特性和保守基序,运用qRT-PCR分析基因的表达模式,利用缺失CorA、MgtA和MgtB的沙门氏菌突变株MM281进行功能互补。结果表明：CpMGT1的编码区为1332 bp,预测编码443 aa,理论分子量为50.43 kDa、等电点为5.12;该蛋白含有2个疏水跨膜区（TM1和TM2）,其中TM1包含高度保守的GMN基序;进化及亚细胞定位分析表明CpMGT1与拟南芥中AtMGT1和AtMGT2的亲缘关系较近,定位于细胞膜;定量分析显示CpMGT1基因为组成型表达,其中在根、茎和果实中的表达量较高;在MM281中异源表达可显著提高工程菌在低Mg2+浓度下的生长速率,表明CpMGT1具有高效的Mg2+转运活性。CpMGT1的克隆与鉴定为进一步揭示番木瓜MGT基因在不同组织特别是在果实中Mg2+的积累机制奠定了坚实的基础。
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| 关 键 词： | 番木瓜 镁离子转运蛋白 表达分析 沙门氏菌 功能互补 |
| 收稿时间： | 2021-11-15 |
Cloning and Functional Analysis of CpMGT1, a Magnesium Transporter Gene from Carica papaya |
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| XU Yinggang,ZOU Zhi,GUO Jingyuan,KONG Hua,ZHU Guopeng,GUO Anping. Cloning and Functional Analysis of CpMGT1, a Magnesium Transporter Gene from Carica papaya[J]. Chinese Journal of Tropical Crops, 2022, 43(6): 1114-1121. DOI: 10.3969/j.issn.1000-2561.2022.06.003 |
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| Authors: | XU Yinggang ZOU Zhi GUO Jingyuan KONG Hua ZHU Guopeng GUO Anping |
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| Affiliation: | 1. College of Horticulture, Hainan University, Haikou, Hainan 570228, China2. Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, Hainan 572024, China3. Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions / Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China |
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| Abstract: | Magnesium is a macronutrient that plays essential roles in plant growth, development, photosynthesis, stress response as well as other biological processes. In higher plants, it has been established that absorption, transport, distribution, and reallocation of Mg2+ are mainly mediated by MGTs/MRS2s (magnesium transporters/mitochondrial RNA splicing2s) and MHXs (magnesium-proton exchangers). Among them, the family MGT, also known as CorA (cobalt resistance A) that was originally identified in Salmonella typhimurium, was most studied, especially in model plants such as arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). By contrast, little information is available in papaya (Carica papaya L.), an economically important tropical crop of the Caricaceae family within Brassicales. Based on mining accessible genome and transcriptome data of papaya, in this study, a magnesium transporter gene named CpMGT1, which includes an open reading frame of 1332 bp, was successfully cloned using RT-PCR (reverse transcription polymerase chain reaction). Furthermore, the protein physicochemical properties and conserved motifs were investigated using bioinformatics tools, the gene expression patterns were analyzed using qRT-PCR (quantitative real-time PCR), and functional complementarity was performed through heterologous expression in S. typhimurium mutant MM281 that lacks Mg2+ transporting systems (i.e. CorA, MgtA, and MgtB) and can’t grow on media containing low concentrations of Mg2+. Results showed that CpMGT1 was predicted to encode 443 amino acids with a theoretical molecular weight of 50.43 kDa and an isoelectric point of 5.12; the protein was shown to contain two hydrophobic transmembrane regions (i.e. TM1 and TM2), while TM1 harbored the highly conserved GMN motif; the subcellular localization prediction suggested that the CpMGT1 protein was located in the plasma membrane, whereas the evolutionary analysis revealed that it was closely related to AtMGT1 and AtMGT2, two high affinity Mg2+ transporters in arabidopsis; the expression analysis showed that CpMGT1 was constitutively expressed in tissues examined, with most abundance in roots, stems, and fruits; heterologous expression of CpMGT1 in the MM281 mutant could significantly improve the growth of engineering strain at low Mg2+ concentrations, implying its high Mg2+ transport activity. Taken together, this study presents the molecular cloning and characterization of the first MGT gene in papaya, including sequence features, physicochemical properties, evolutionary relationships, gene expression patterns as well as Mg2+ transport activity. These results could not only lay a solid foundation for further uncovering the accumulation mechanism of Mg2+ in different tissues especially in the fruit, but also provide a valuable resource for genetic improvement in papaya and other species. |
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| Keywords: | Carica papaya magnesium transporter expression analysis Salmonella typhimurium functional complementarity |
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