【目的】研究香蕉Ca M在温度胁迫及香蕉果实后熟过程中的表达模式,了解Ca M在增强香蕉果实对温度胁迫的适应性作用,解释Ca M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Ca M氨基酸序列,设计兼并引物。采...【目的】研究香蕉Ca M在温度胁迫及香蕉果实后熟过程中的表达模式,了解Ca M在增强香蕉果实对温度胁迫的适应性作用,解释Ca M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Ca M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Ca M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Ma Ca M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Ca M,长648 bp,编码138个氨基酸,命名为Ma Ca M(登录号:HM061077),序列分析表明,Ma Ca M包含4个EF-Hand钙离子结合区域,与Ma Ca M、Os Ca M、Zm Ca M、At Ca M3、Ta Ca M1-2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,Ma Ca M表达迅速增强;香蕉果实在冷害温度(7℃)下放置10 d,Ma Ca M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,Ma Ca M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Ma Ca M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Ma Ca M的表达。而在EGTA处理的同时,利用30 mmol·L-1 Ca Cl2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Ma Ca M的表达。【结论】Ma Ca M能增强香蕉果实对温度胁迫的适应性;Ma Ca M作为一种调控因子参与了香蕉果实后熟的褪绿转黄过程。展开更多
Changes of intracellular Ca 2+ ,pH value and mitochondria membrane potential(△Ψ m) in the apoptosis of MGC 803 cells induced by water soluble constituents of Glycyrrhiza uralensis Fisch(WSCG) were investigated.MGC 8...Changes of intracellular Ca 2+ ,pH value and mitochondria membrane potential(△Ψ m) in the apoptosis of MGC 803 cells induced by water soluble constituents of Glycyrrhiza uralensis Fisch(WSCG) were investigated.MGC 803 cells were incubated with 0.5,0.75,1.0 and 1.5 g/L WSCG for 1,4,7,11,15,19 and 23 hours respectively.The percentage of apoptosis and intracellular Ca 2+ content increased in a dose and time dependent manner,except that the intracellular Ca 2+ content of 1.5 g/L WSCG treated cells started to decrease after 11 hours.The cells were alkalized after various treatments,except that 1.5 g/L WSCG treated cells turned to acidify after 11 hours.It was found that the mitochondria △Ψ m of all treated cells decreased drastically at the first hour,then kept decreasing slowly until the 23rd hour.The results indicated that intracellular Ca 2+ ,pH and mitochondria △Ψ m all played pivotal roles in the apoptosis of MGC 803 cells induced by WSCG.展开更多
文摘【目的】研究香蕉Ca M在温度胁迫及香蕉果实后熟过程中的表达模式,了解Ca M在增强香蕉果实对温度胁迫的适应性作用,解释Ca M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Ca M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Ca M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Ma Ca M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Ca M,长648 bp,编码138个氨基酸,命名为Ma Ca M(登录号:HM061077),序列分析表明,Ma Ca M包含4个EF-Hand钙离子结合区域,与Ma Ca M、Os Ca M、Zm Ca M、At Ca M3、Ta Ca M1-2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,Ma Ca M表达迅速增强;香蕉果实在冷害温度(7℃)下放置10 d,Ma Ca M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,Ma Ca M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Ma Ca M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Ma Ca M的表达。而在EGTA处理的同时,利用30 mmol·L-1 Ca Cl2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Ma Ca M的表达。【结论】Ma Ca M能增强香蕉果实对温度胁迫的适应性;Ma Ca M作为一种调控因子参与了香蕉果实后熟的褪绿转黄过程。
文摘Changes of intracellular Ca 2+ ,pH value and mitochondria membrane potential(△Ψ m) in the apoptosis of MGC 803 cells induced by water soluble constituents of Glycyrrhiza uralensis Fisch(WSCG) were investigated.MGC 803 cells were incubated with 0.5,0.75,1.0 and 1.5 g/L WSCG for 1,4,7,11,15,19 and 23 hours respectively.The percentage of apoptosis and intracellular Ca 2+ content increased in a dose and time dependent manner,except that the intracellular Ca 2+ content of 1.5 g/L WSCG treated cells started to decrease after 11 hours.The cells were alkalized after various treatments,except that 1.5 g/L WSCG treated cells turned to acidify after 11 hours.It was found that the mitochondria △Ψ m of all treated cells decreased drastically at the first hour,then kept decreasing slowly until the 23rd hour.The results indicated that intracellular Ca 2+ ,pH and mitochondria △Ψ m all played pivotal roles in the apoptosis of MGC 803 cells induced by WSCG.
