不同低温胁迫下草莓叶片FaGR基因表达分析

Expression pattern analysis of FaGR gene of strawberry leaves under stress of different low temperatures

关键词:  草莓  低温胁迫  半定量PCR  荧光定量PCR

Key words:  strawberry  low temperature stress  semi-quantitative PCR  real-time quantitative PCR

摘要:

为进一步探明草莓谷胱甘肽还原酶(glutathione reductase,GR)基因在低温胁迫下的表达调控模式,以揭示其作用机理和抗逆机制,该研究以草莓栽培品种‘丰香'植株为对象,分别用0、2、4、6、8、10℃低温胁迫以及室温(25 ℃)作为对照处理24 h,从处理植株的新鲜幼嫩叶片中提取总RNA并反转录成cDNA,采用半定量PCR及荧光定量PCR两种定量方法,分析FaGR基因在不同低温胁迫下的表达差异。结果表明:半定量PCR方法与荧光定量PCR方法的结果基本一致。FaGR的相对表达量受到不同低温胁迫和不同程度的诱导,在8 ℃和10 ℃低温胁迫下表达量上调,均高于对照水平,且在8 ℃低温胁迫下相对表达量最高; 当温度为6 ℃时,FaGR表达量急剧下降,当温度低于6 ℃时,FaGR表达量随着温度的降低而逐渐降低,并低于对照水平; 在0 ℃相对表达量时降至最低水平,约为最大表达量的一半。这说明半定量PCR结果准确可靠,可广泛应用于基因表达研究,而且FaGR表达量受到低温诱导,但不同低温处理对FaGR的表达量诱导程度不同。FaGR相对表达量在一定范围内的低温胁迫下增加,但在此低温范围外的低温胁迫下则降低,该研究表明6 ℃低温为临界值。以上结果为进一步调控该基因表达提供了科学依据,同时为提高植物抗逆能力提供了基础资料。

Abstract:

In order to investigate the expression pattern of glutathione reductase from strawberry under low temperature stress,so as to further reveal its function in defensing stresses and the molecular mechanism of cold-resistant in plants. We treated strawberry plants(Fragaria×ananassa‘Toyonaka')with different low temperatures including 0,2,4,6,8 and 10 ℃ in artificial chamber for 24 h respectively,and we also treated plants with 25 ℃ for 24 h as control. Subsequently,total RNA samples were extracted from the treated fresh young leaves,and cDNA first strand for PCR reactions was synthesized using these RNA samples. When all cDNA samples were done,we also chose two methods including semi-quantitative PCR and real-time quantitative PCR to detect the expression differences of FaGR between different low temperature treatments. We chose two different methods to determine the expression of FaGR,on one hand to make sure the differences of expression between samples were exactly due to the different temperatures,not because of the different methods; on the other hand,it was expected to see whether semi-quantitative PCR was reliable as we thought for gene expression research. At last we combined the results came from two methods and analyzed the expression differences of FaGR gene between samples treated with different temperatures. The results were described as below: the result of semi-quantitative PCR was generally consistent with that of real-time quantitative PCR,both of them showed that the relative expression of FaGR was greatly modified in diverse grades by different low temperatures. Furthermore,the relative expression levels of FaGR under low temperature at 8 and 10 ℃ increased compared to the control which was at 25 ℃,and the highest relative expression level of FaGR was detected at 8 ℃. However,thereafter,the relative expression level at 6 ℃ dropped dramatically to a level which was slightly lower than the control at 25 ℃. When the temperature was below 6 ℃,the relative expression level of FaGR decreased accompanied with the decreasing of temperatures,each of the expression level at temperature below 6 ℃ was lower than that at 25 ℃. The lowest relative expression level was detected at 0 ℃,which was almost only the half amount of the maximum expression at 8 ℃. These results supported that semi-quantitative PCR was exactly reliable and it could be widely used for gene expression research. Also,these results indicated that FaGR gene was definitely induced by chilling stress but within a certain range of temperature,and the degree of changes under different low temperatures was also different. In a certain range,FaGR was up-regulated by low temperature,however it was slightly suppressed by low temperature out of this range,and 6 ℃ was showed as the critical value in this research. What's meaningful,these results would provide a scientific basis for the further regulation of the gene expression,as well as some basic data to improve the ability of plants to resistant stresses.