苹果GMP、GME和GGP基因的克隆与表达分析

Cloning and expression analysis of GMP,GME and GGP genes in apple

【目的】克隆苹果GDP-甘露糖焦磷酸化酶(GDP-mannose pyrophosphorylase,GMP)、GDP-甘露糖-3′,5′-表异构酶(GDP-mannose-3′,5′-epimerase,GME)和GDP-1-半乳糖磷酸酶(GDP-L-galactose-1-phosphate phosphorylase,GGP)的基因序列并分析其表达特性,探讨GMP、GME和GGP基因在抗坏血酸(Ascorbic acid,AsA)合成过程中的作用。【方法】以‘嘎啦’苹果幼果为材料,分别运用RT-PCR和PCR法克隆GMP、GME和GGP基因的cDNA和gDNA全长序列,对其序列及编码产物进行生物信息学分析,并采用实时荧光定量PCR,分析以上基因在苹果不同组织(幼叶、成熟叶、衰老叶、花、幼果、成熟果和根)中的表达情况。【结果】RT-PCR法克隆及序列分析显示,GMP基因的cDNA全长为1 280bp,编码361个氨基酸,gDNA序列中含有3个内含子;GME基因的cDNA全长为1 323bp,编码376个氨基酸,gDNA序列中含有5个内含子;GGP基因的cDNA全长为1 677bp,编码446个氨基酸,gDNA序列中含有6个内含子。基因表达的实时荧光定量PCR分析表明,GMP、GME和GGP在苹果不同组织中均有表达,在叶片中的表达量高于其他组织,且表达量随着叶片的生长逐渐升高;在花、幼果和成熟果中,GMP、GME和GGP的表达量差异不明显。【结论】克隆获得了苹果的GMP、GME和GGP基因,其在苹果不同组织的生长过程中有不同的表达特性,但GME的表达水平远远低于GMP和GGP,这在一定程度上表明,在苹果AsA的生物合成过程中,GMP、GGP较GME有更重要的作用。

[Objective] The gene sequences of GDP-mannose pyrophosphorylase （GMP）, GDP-man- nose-3r,5^-epimerase （GME） and GDP-L-galactose-l-phosphate phosphorylase （GGP） were cloned and their genetic variations in expression were analyzed, aiming to investigate the roles of these genes in synthe- sis of ascorbic acid （AsA）. [Method] The full-length cDNA and gDNA sequences of GMP,GME and GGP in young fruit of ＇Gala~ apple were cloned by RT-PCR and PCR,and their sequences and coding products were analyzed using bioinformatics methods. The dynamic expression differences of GMP,GME and GGP in different tissues （young leaf, mature leaf, senescent leaf, flower, young fruit, mature fruit and root） were assayed by real time PCR. [Result] The obtained cDNA sequence of GMP consisted of 1 280 bp and 361 a- mino acids,and the gDNA sequence contained 3 introns. The cDNA sequence of GME consisted of 1 323 bp and 376 amino acids,and the gDNA sequence contained 5 introns. The cDNA sequence of GGP consisted of 1 677 bp and 446 amino acids, and the gDNA sequence contained 6 introns. The mRNA expressions of GMP,GME and GGP were detected in all apple tissues. Leaves had higher mRNA expression levels of GMP,GME and GGP than other tissues. The mRNA expression levels of GMP,GME and GGP in leaves increased as the growth of leaves. The mRNA expression levels of the three enzymes were not significantly different in flower, young fruit and mature fruit. [Conclusion] GMP,GME and GOP genes were cloned and they were differently expressed during the development of different tissues. GME had much lower expres- sion levels than GMP and GGP. It was indicated that GMP and GGP played more important roles in AsA synthesis of apple than GME.