MaCaM在采后香蕉果实温度胁迫及后熟中的作用

【目的】研究香蕉CaM在温度胁迫及香蕉果实后熟过程中的表达模式，了解CaM在增强香蕉果实对温度胁迫的适应性作用，解释CaM参与调控香蕉果实褪绿转黄的机制。【方法】通过比对NCBI数据库中已有物种的CaM氨基酸序列，设计兼并引物。采用热硼酸法，从香蕉果皮中提取总RNA，通过RT-PCR与RACE方法扩增目的基因。利用DNAMAN软件和NCBI网站对CaM的氨基酸序列进行氨基酸比对和同源树分析。利用地高辛探针合成试剂盒（PCR DIG Probe Synthesis Kit）合成特异基因带有DIG标记的探针，使用Northern杂交法对MaCaM在采后香蕉果实温度胁迫及后熟中的表达规律进行分析。钙离子螯合剂EGTA及钙信号恢复处理采用香蕉果皮离体培养，采用真空渗透的方法对香蕉果皮进行试剂处理，利用色差计测定颜色h值。【结果】从香蕉果皮中克隆得到一个CaM，长648 bp，编码138个氨基酸，命名为MaCaM（登录号：HM061077），序列分析表明，MaCaM包含4个EF-Hand钙离子结合区域，与MaCaM、OsCaM、ZmCaM、AtCaM3、TaCaM1-2等基因同源性极高。Northern杂交结果表明，热激（52℃，3 min）处理香蕉果实0.5 h后，MaCaM表达迅速增强；香蕉果实在冷害温度（7℃）下放置10 d，MaCaM在冷藏的第7-10 d表达逐渐增强，当采后香蕉果实先经热激处理再放入7℃下贮藏，MaCaM表达在第4天和第7天强于7℃处理；乙烯催熟处理诱导香蕉MaCaM表达逐渐增强；30 mmol·L^-1钙离子螯合剂EGTA处理在一定程度上抑制了香蕉果实的后熟，同时也抑制了MaCaM的表达。而在EGTA处理的同时，利用30 mmol·L^-1 CaCl₂进行钙信号恢复处理，能一定程度地恢复香蕉果实的正常后熟，也恢复了MaCaM的表达。【结论】MaCaM能增强香蕉果实对温度胁迫的适应性；MaCaM作为一种调控因子参与了香蕉果实后熟的褪绿转黄过程。

The Role of MaCaM Gene in Temperature Stress and Fruit Ripening of Harvested Banana

【Objective】The objective of this experiment is to study the expression of CaM gene in temperature stress and fruit ripening of banana. Which is helpful to understand the effect of CaM gene in enhance the adaptation to temperature stress and reveal the mechanism of peel degreening during the ripening process of banana fruit.【Method】Degenerated primers were designed by comparative analysis of some species’ CaM amino acid sequence in NCBI database. The total RNA was extracted from banana peel by using hot borate method. In banana fruit, the rapid amplification of cDNA ends (RACE)-PCR techniques were used for 3’-UTR and fragment sequences cloning of the cDNA. The amino acid sequence of CaM was analyzed by using DNAMAN software and the NCBI website. Probe with DIG markers was made by using PCR DIG Probe Synthesis Kit. Northern blotting was performed to compare the expression of MaCaM gene in banana fruit under different temperature stresses and during ripening process. EGTA and calcium signal recovery treatment by using banana peels in vitro culture. The banana peel were treated by vacuum infiltration. The h values were measured by the color difference meter.【Result】A novel CaM gene, 648 bp, encoding 138 amino acids, named MaCaM (HM061077), was cloned from banana in this study. Sequence analysis showed that MaCaM gene contains 4 EF-Hand calcium binding regions. MaCaM gene has a high sequence similarity with MaCaM, OsCaM, ZmCaM, AtCaM3 and TaCaM1-2. Northern blotting results showed that the MaCaM gene expression was up-regulated rapidly at 0.5-24 h after heat shock (52℃ 3min). Banana fruits were stored at 7℃ (chilling injury temperature) for 10 days, the MaCaM gene expression increased gradually in the 7-10 d. When the banana fruits were stored at 7℃ after heat shock, MaCaM expression was higher in the 4 d and 7 d than those of at 7℃ alone. Ethylene was also observed to arouse the expression of MaCaM gradually. Furthermore, 30 mmol·L^-1 calcium ion chelator EGTA treatment inhibited fruit ripening and the expression of MaCaM to the same extent, while the EGTA combined with 30 mmol·L^-1 CaCl₂ treatment restored the normal ripening of banana and the expression of MaCaM gene to some degree.【Conclusion】These results showed that MaCaM gene takes an important role in enhancing the adaptation to temperature stress and MaCaM gene as a regulator is involved in the peel degreening during the ripening process of banana fruit.