Cloning and Prokaryotic Expression Analysis of MaMC6 in Banana

doi:10.13560/j.cnki.biotech.bull.1985.2023-0694

Abstract

Abstract:

Metacaspases（MCs）is a regulatory gene of programmed cell death in plants, and it participates in the response of plants to stress. MaMC6, a member of MaMCs family, was cloned from Brazilian banana（Musa AAA Cavendishcv. Brazil）cells by PCR, and its bioinformatics analysis, expression pattern analysis and functional verification were carried out. The results showed that MaMC6 was composed of 320 amino acids, was a stable protein, hydrophilic, of no transmembrane structure, contained a caspase-like domain, belonged to type II metacaspase protein, and had a defense and stress response. The results of RT-qPCR analysis showed that the relative expression of MaMC6 after 100 mmol/L NaCl treatment was 7.70 times higher than that of the control at 2 h, and the gene expression at 2 h after CaCl₂ treatment under salt stress was 3.99 times higher than that of the control, while the use of calcium chelating agent EGTA and calcium channel blocker LaCl₃increased the expression of MaMC6 under salt stress, reaching 9.92 and 11.20 times of the control at 2 h. The results of subcellular localization showed that MaMC6 was located in the cytoplasm and nucleus. The results of E. coli transformation showed that the growth of recombinant strain pET28a-MaMC6 was better than that of control strain pET28a under NaCl, mannitol, and high-temperature stress. To sum up, MaMC6 may be involved in the response of banana to abiotic stress. This study provides a reference for follow-up study on the role of MaMCs in banana stress resistance.

Key words: Musa acuminata, MaMC6, RT-qPCR, subcellular localization, prokaryotic expression

TENG Meng-xin, XU Ya, HE Jing, WANG Qi, QIAO Fei, LI Jing-yang, LI Xin-guo. Cloning and Prokaryotic Expression Analysis of MaMC6 in Banana[J]. Biotechnology Bulletin, 2023, 39(12): 179-186.

Figures/Tables 10

Table 1 Primers used in the experiment

引物名称 Primer name	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
Actin	CGTAGCACCAGAAGAACA	CATAAAGGGAGAGGACAG
MaMC6	GTGAGCAACAAGGAACTG	AGTAAAGGCAAGGGTGTT
pET28a-MaMC6	AAGAAGGAGATATACATGGAAGCGGGCAAGAAGCG	GTGGTGGTGGTGCTCCATGCAAGCACGTAGCTGCTG
1302-MaMC6-GFP	CGGGGGACTCTTGACATGGAAGCGGGCAAGAAGCG	TTCTTCTCCTTTACTCATGCAAGCACGTAGCTGCTG

Fig. 1 MaMC6 gel electrophoresis image

Fig. 2 Multiple comparison of amino acid sequences between MaMC6 and other plants At: Arabidopsis thaliana; Ma: genome A of Musa acuminata; Mb: genome B of Musa balbisiana; Os: Oryza sativa; Pd: Phoenix dactylifera; Eg: Elaeis guineensis; Ao: Asparagus officinalis; Mi: Mangifera indica; Cs: Citrus sinensis; Zo: Zingiber officinale

Fig. 3 Phylogenetic tree analysis of MaMC6 and other plants Mb: Musa balbisiana; Ma: Musa acuminata; Zo: Zingiber officinale; Os: Oryza sativa; Ao: Asparagus officinalis; Da: Dioscorea alata; Pd: Phoenix dactylifera; Eg: Elaeis guineensis; At: Arabidopsis thaliana; Pv: Pistacia vera; Mi: Mangifera indica; Cs: Citrus sinensis; Tc: Theobroma cacao; Dz: Durio zibethinus; Hs: Hibiscus syriacus; Ga: Gossypium austral; Gh: Gossypium hirsutum; Mc: Momordica charantia; Bh: Benincasa hispida; Ls: Lactuca sativa

Fig. 4 Prediction of the 3-three-dimensional structure of MaMC6 protein

Fig. 5 Analysis of cis-acting elements of MaMC6

Fig. 6 Gene expression of MaMC6 under NaCl treatment Different lower letters indicate significant differences at P<0.05 level. The same below

Fig. 7 Effects of different calcium effectors on the expression of MaMC6 under salt stress A：100 mmol/L NaCl + 10 mmol/L CaCl2；B：100 mmol/L NaCl + 10 mmol/L EGTA；C：100 mmol/L NaCl + 5 mmol/L LaCl2

Fig. 8 Subcellular localization of MaMC6 A, D: GFP fluorescence. B, E: Bright. C, F: Merge. Scale bar= 20 μm

Fig. 9 Functional verification of MaMC6 in Escherichia coli BL21 The upper row is the pET28 expressed strain, and the lower row is the pET28a-MaMC6 expressed strain

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