茶树镁转运子基因CsMGT6的表达和功能鉴定

doi:10.16420/j.issn.0513-353x.2022-0506

摘要/Abstract

摘要：

为探究镁转运子（Magnesium Transporters，MGT）参与调控茶树镁元素吸收、转运的机制，从茶树品种‘鄂茶10号’中克隆了1个镁转运子基因CsMGT6（GeneBank登录号：ON934629），并分析其功能。CsMGT6位于茶树第3号染色体上，开放阅读框为1 368 bp，编码455个氨基酸。多序列比对分析发现，CsMGT6含有2个跨膜结构域和1个保守的GMN基序。系统进化树显示，CsMGT6属于第5亚族，与山梨猕猴桃（Actinidia rufa）ArMGT6亲缘关系最近。亚细胞定位结果表明，CsMGT6定位在细胞质膜上。实时荧光定量 PCR分析发现，镁缺乏条件下CsMGT6在茶树根和叶部的响应不同，且在不同茶树品种中的响应存在差异。沙门氏杆菌功能互补试验表明，CsMGT6属于低亲和镁转运子，可能在较高浓度镁存在的条件下参与镁元素的吸收和转运。结果表明CsMGT6可被镁缺乏诱导表达，可能参与维持茶树细胞质膜内外的镁稳态。

关键词: 茶树, 镁转运子, CsMGT6, 定位, 转基因

Abstract:

To understand how magnesium transporters regulate the uptake and translocation of magnesium，a magnesium transporter gene CsMGT6（GenBank Accession No. ON934629）was cloned from Camellia sinensis cultivar‘Echa 10’，which was distributed on the third chromosome of C. sinensis genome. The open reading frame of CsMGT6 gene was 1 368 bp，encoding 455 amino acids. Multiple alignment analysis indicated that CsMGT6 contained two transmembrane domains and a conserved GMN motif. Phylogenetic tree analysis showed that CsMGT6 belonged to the five subfamily，which had the highest homology with Actinidia rufa ArMGT6. Subcellular localization assays confirmed that CsMGT6 was located in the plasma membrane in tobacco epidermal cells. Moreover，quantitative real-time PCR analysis indicated that CsMGT6 showed various response to magnesium deficiency in roots and leaves among five different tea plant cultivars. In addition，functional complementation tests in Salmonella typhimurium mutant strain MM281 proved that CsMGT6 was a low-affinity magnesium transporter，which might mediate the uptake and translocation of magnesium under high concentration of magnesium. These results implied that the expression of CsMGT6 could be induced by magnesium deficiency，and CsMGT6 might be involved in the maintenance of magnesium homeostasis in tea plant plasma membrane.

Key words: Camellia sinensis, magnesium transporter, CsMGT6, localization, transgene

文婷, 李婧, 张家琪, 魏仪, 祝文睿, 倪德江, 王明乐. 茶树镁转运子基因CsMGT6的表达和功能鉴定[J]. 园艺学报, 2023, 50(10): 2171-2182.

WEN Ting, LI Jing, ZHANG Jiaqi, WEI Yi, ZHU Wenrui, NI Dejiang, WANG Mingle. Expression and Functional Identification of Magnesium Transporter Gene CsMGT6 in Camellia sinensis[J]. Acta Horticulturae Sinica, 2023, 50(10): 2171-2182.

图/表 7

表1 引物序列

Table 1 Sequences of primers

用途 Usage	引物名称 Primer name	序列（5ʹ-3ʹ） Sequence
克隆 Clone	CsMGT6	F：ATGGGGAAGGGGCCATTCTC
克隆 Clone		R：CGATCCAATCAGTTTCTTCC
实时荧光定量PCR qRT-PCR	CsMGT6-q	F：AGGTTGAGGTGGTGTCTCTG
实时荧光定量PCR qRT-PCR		R：GATTGCCCGAACTTGTCGAA
内参基因 Reference gene	CsTBP-q	F：AAGGGATCCAAAGACGACAG
		R：TGAAATCCTTGAATTTGGCA
	CsTIP41-q	F：CGAAAGAGCCCATTCTCTTC
		R：ACGTGTGTCCCTCAATCTCA
亚细胞定位 Subcellular localization	CsMGT6-pCAMBIA2300	F：TTTGGAGAGGACAGGGTACCATGGGGAAGGGGCCATTCTC
亚细胞定位 Subcellular localization		R：CCTCCTCCTCTAGAGGATCCCGATCCAATCAGTTTCTTCC
原核表达 Prokaryotic expression	CsMGT6-pTrc99A	F：AGACCATGGAATTCGAGCTCGGTACCATGGGGAAGGGGCCA
原核表达 Prokaryotic expression		R：GCCTGCAGGTCGACTCTAGAGGATCCTCACGATCCAATCAG

图1 CsMGT6的开放阅读框

Fig. 1 The open reading frame of CsMGT6

图2 茶树CsMGT6与其他植物MGTs蛋白的多序列比对 Cs：茶树；At：拟南芥；Cc：克莱门氏小柑橘；Ar：山梨猕猴桃；Zj：枣；Rc：蓖麻。

Fig. 2 The alignment of CsMGT6 protein sequences with MGTs from other plant species Cs：Camellia sinensis；At：Arabidopsis thaliana；Cc：Citrus clementina；Ar：Actinidia rufa；Zj：Ziziphus jujuba；Rc：Ricinus communis.

图3 CsMGT6蛋白的系统进化树

Fig. 3 The phylogenetic tree of CsMGT6 protein

图4 CsMGT6蛋白的亚细胞定位

Fig. 4 Subcellular localization of CsMGT6 protein

图5 不同茶树品种镁缺乏条件下根和叶中CsMGT6的表达水平不同小写字母表示不同处理时间差异显著。Duncan’s多重比较法，P < 0.05。

Fig. 5 The expression level of CsMGT6 in response to low concentration of Mg2+ in roots and leaves of five tea plant cultivars Different lowercase letters indicate significant differences among different time. Duncan’s test for multiple comparison，P < 0.05.

图6 沙门氏杆菌镁转运缺失突变体MM281中CsMGT6的功能分析 A：平板试验；B：液体培养试验。Vector：转入pTrc99A空载的MM281；CsMGT6：转入pTrc99A-CsMGT6的MM281。固体培养基上的菌液从左到右依次稀释10倍。

Fig. 6 Functional analysis of CsMGT6 in the magnesium transport mutant MM281 of Salmonella A：Functional complementary analysis of CsMGT6 in the spot assay；B：the growth curve of Salmonella MM281 in liquid medium. Vector：MM281 transformed with pTrc99A；CsMGT6：MM281 transformed with pTrc99A-CsMGT6. The strains was diluted 10 times from the left to the right in solid medium.

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