Expression and Functional Identification of Magnesium Transporter Gene CsMGT6 in Camellia sinensis

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (10): 2171-2182.doi: 10.16420/j.issn.0513-353x.2022-0506

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Expression and Functional Identification of Magnesium Transporter Gene CsMGT6 in Camellia sinensis

WEN Ting, LI Jing, ZHANG Jiaqi, WEI Yi, ZHU Wenrui, NI Dejiang, WANG Mingle^*()

National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops，College of Horticulture and Forestry Sciences，Huazhong Agricultural University，Wuhan 430070，China

Received:2023-03-21 Revised:2023-07-14 Online:2023-10-25 Published:2023-10-30

Abstract

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

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.

Figures/Tables 7

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

Fig. 1 The open reading frame of CsMGT6

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.

Fig. 3 The phylogenetic tree of CsMGT6 protein

Fig. 4 Subcellular localization of CsMGT6 protein

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.

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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Expression and Functional Identification of Magnesium Transporter Gene CsMGT6 in Camellia sinensis

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