‘金冠’苹果PP2C基因克隆及低温胁迫下的表达

Cloning and expression of PP2C genes response to low temperature in apple‘Golden Delicious’

【目的】为了鉴定并探究苹果MdPP2C基因对低温胁迫的响应机制。【方法】利用基因克隆技术,从苹果‘金冠’cDNA中克隆得到3个苹果MdPP2C基因,利用生物信息学技术对3个基因的理化性质、系统进化及保守基序等进行分析,并采用real time-qPCR技术检测其在4℃低温处理下的表达情况。【结果】序列分析结果表明,克隆得到的MdHAB1、MdHAI2和MdAHG3分别编码544、405和432个氨基酸,其预测的表观相对分子质量分别为58.26 kDa、44.29 kDa和45.80 kDa,理论等电点分别为5.03、6.63和5.01,均属于酸性蛋白;3个蛋白均属于不稳定蛋白和亲水蛋白,3个蛋白均无跨膜结构,主要定位于细胞核和叶绿体,且3个基因编码的氨基酸序列均具有PP2C超家族保守结构域;real time-qPCR结果表明,低温处理后,MdHAB1、MdHAI2和MdAHG3转录水平均呈现持续上调的趋势。【结论】从‘金冠’中克隆的3个PP2C家族基因均参与苹果‘金冠’对低温胁迫的响应,以上结果为探究苹果PP2C家族基因在苹果属植物对低温胁迫的响应过程及调控机制提供理论基础,为使用分子育种手段培育苹果耐寒性砧木提供新的基因。

[Objective]To identify and explore the response mechanism of MdPP2C genes to low temperature stress in apple.[Methods]Three MdPP2C genes were cloned from the cDNA of‘Golden Delicious’by gene cloning technology.Bioinformatics analysis technology was used to analyze physical and chemical properties,phylogenetic evolution and conserved motifs of the three genes,and the expression under low temperature stress was detected by real time-qPCR.[Results]Sequence analysis showed that the cloned MdHAB1,MdHAI2 and MdAHG3 encoded 544,405 and 432 amino acids,and their predicted apparent relative molecular weights were 58.26 kDa,44.29 kDa and 45.80 kDa,respectively,and theoretical isoelectric points were 5.03,6.63 and 5.01,which belonged to acidic proteins.All these three proteins belong to unstable protein and hydrophilic protein,have no trans-membrane structure and are mainly localized in the nucleus and chloroplast.Moreover,the amino acid sequences encoded by the three genes all have the PP2C superfamily conservative domain.The real time-qPCR results showed that the expression of MdHAB1,MdHAI2 and MdAHG3 genes were consistently up-regulated under 4℃low temperature treatment.[Conclusion]The three MdPP2C family genes cloned from‘Golden Delicious’were all involved in the response to low temperature stress.The results provide a insight into the molecular mechanism by which PP2C family genes regulate low temperature stress in apple,and provide candidate genes for breeding cold tolerance apple varieties.