生物技术通报 ›› 2023, Vol. 39 ›› Issue (12): 179-186.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0694

• 研究报告 • 上一篇    下一篇

香蕉MaMC6的克隆及原核表达分析

滕梦鑫1(), 徐亚1, 何静1, 汪奇1, 乔飞2, 李敬阳2, 李新国1()   

  1. 1.热带作物生物育种全国重点实验室 海南大学热带农林学院,海口 570228
    2.中国热带农业科学院热带作物品种资源研究所,海口 571101
  • 收稿日期:2023-07-18 出版日期:2023-12-26 发布日期:2024-01-11
  • 通讯作者: 李新国,男,教授,研究方向:果树种质资源与栽培;E-mail: lixinguo13@163.com
  • 作者简介:滕梦鑫,女,硕士研究生,研究方向:果树分子生物学;E-mail: tengmx26@163.com
  • 基金资助:
    国家自然科学基金项目(32160679);国家自然科学基金项目(31760549)

Cloning and Prokaryotic Expression Analysis of MaMC6 in Banana

TENG Meng-xin1(), XU Ya1, HE Jing1, WANG Qi1, QIAO Fei2, LI Jing-yang2, LI Xin-guo1()   

  1. 1. National Key Laboratory of Tropical Crop Biological Breeding & School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228
    2. Chinese Academy of Tropical Agricultural Sciences, Tropical Crops Genetic Resources Institute, Haikou 571101
  • Received:2023-07-18 Published:2023-12-26 Online:2024-01-11

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 LaCl3 increased 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