苹果果实中细胞质型苹果酸酶基因(NADP-ME)的克隆与表达分析

Cloning and Expression Analysis of the Cytosolic NADP-malic Enzyme Gene of Apple Fruit

【研究目的】从苹果果实中克隆细胞质型NADP-苹果酸酶(NADP-ME)基因,并且从基因转录水平上研究该基因与苹果果实酸度的关系。【方法】利用RT-PCR和RACE技术克隆NADP-ME基因,半定量RT-PCR分析该基因在高酸和低酸个体不同发育时期果实中的转录情况,并进行原核表达分析。【结果】成功克隆了细胞质型NADP-ME基因的cDNA全长序列,命名为Mal-ME(GenBank注册号:DQ280492);该基因表达一个约66kD的蛋白;半定量RT-PCR分析表明:不同发育时期的果实中Mal-ME的转录与苹果酸含量呈负相关,即随着Mal-ME转录水平的增强果实含酸量下降。【结论】成功从苹果果实中克隆了细胞质型Mal-ME基因,Mal-ME在果实发育过程中对苹果酸的积累起负调控作用。

[OBJECTIVE]The purpose of this study is to isolate a fruit-related gene encoding cytosolic NADP-dependent malic enzyme and reveal the correlation between transcription and malic acid content of fruits.[METHOD]A full-length cDNA was cloned by RT-PCR and RACE （Rapid Amplification of cDNA Ends） from apple fruits, and its expression was studied by Semi-quantitative RT-PCR analysis,The recombinant prokaryotic expression vector pET30a-ME was constructed and expressed in E.coli BL21. [RESULT]A fruit-related gene encoding cytosolic NADP-dependent malic enzyme was cloned for the frist time and designated as Mal-ME （GeneBank accession No. DQ280492）. The recombinant prokaryotic expression vector pET30a-ME expressed a 66kD fusion protein in E.coli BL21. Semi-quantitative RT- PCR analysis showed that its transcription had negative correlation with malic acid content of fruits at different developmental stages. [CONCLUSION]cytosolic NADP-dependent malic enzyme gene was successfully isolated from apple fruits. This gene can regulate the accumulation of malic acid in fruits negatively during the development.