香蕉MaMC6的克隆及原核表达分析

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

摘要/Abstract

摘要：

Metacaspases（MCs）是植物程序性细胞死亡的调控基因，参与植物对胁迫的响应。利用PCR技术，从巴西蕉（Musa AAA Cavendish cv. Brazil）细胞中克隆到MaMCs家族成员MaMC6，对其进行生物信息学分析、表达模式分析和功能验证。结果表明，MaMC6由320个氨基酸组成，为稳定蛋白，具有亲水性，不含跨膜结构，含有一个caspase-like domain，属于II型metacaspase蛋白，具有一个防御和应激响应元件。RT-qPCR分析结果显示，100 mmol/L NaCl处理后MaMC6在2 h时相对表达量达到对照的7.70倍；盐胁迫下CaCl₂处理后，基因的表达在2 h时为对照的3.99倍；而钙离子螯合剂EGTA 和钙离子通道阻碍剂LaCl₃的处理提高了MaMC6在盐胁迫下的表达，2 h时达到对照的9.92和11.20倍。亚细胞定位结果显示，MaMC6定位在细胞质和细胞核上。转大肠杆菌结果显示，重组菌株pET28a-MaMC6在NaCl、甘露醇和高温胁迫下生长情况优于对照菌株pET28a。综上所述，MaMC6可能参与香蕉对非生物胁迫的响应过程。本研究为进一步研究MaMCs在香蕉抗逆中的作用提供参考。

关键词: 香蕉, MaMC6, RT-qPCR, 亚细胞定位, 原核表达

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

滕梦鑫, 徐亚, 何静, 汪奇, 乔飞, 李敬阳, 李新国. 香蕉MaMC6的克隆及原核表达分析[J]. 生物技术通报, 2023, 39(12): 179-186.

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.

图/表 10

表1 试验所用引物

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

图1 MaMC6凝胶电泳图

Fig. 1 MaMC6 gel electrophoresis image

图2 MaMC6与其他植物氨基酸序列多重对比 At：拟南芥；Ma：香蕉A基因组；Mb：香蕉B基因组；Os：水稻；Pd：海枣； Eg：油棕；Ao：石刁柏；Mi：芒果； Cs：甜橙；Zo：姜

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

图3 MaMC6与其他植物的系统进化树分析 Mb: 香蕉B基因组; Ma: 香蕉A基因组; Zo: 姜; Os: 水稻; Ao: 石刁柏; Da: 参薯; Pd: 海枣; Eg: 油棕; At: 拟南芥; Pv: 开心果; Mi: 芒果; Cs: 甜橙; Tc: 可可树; Dz: 榴莲; Hs: 木槿; Ga: 澳洲棉; Gh: 陆地棉; Mc: 苦瓜; Bh: 冬瓜; Ls: 莴苣

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

图4 MaMC6蛋白三级结构预测

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

图5 MaMC6顺式作用元件分析

Fig. 5 Analysis of cis-acting elements of MaMC6

图6 MaMC6在NaCl处理下的基因表达不同小写字母表示在P<0.05水平差异显著，下同

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

图7 不同钙效应剂对盐胁迫下MaMC6表达的影响

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

图8 MaMC6亚细胞定位 A、D：绿色荧光信号；B、E：明场；C、F：叠加；标尺：20 μm

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

图9 MaMC6在大肠杆菌BL21中的功能验证上排为pET28表达菌株，下排为pET28a-MaMC6表达菌株

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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