荸荠淀粉合成酶AGPase的基因克隆及表达分析

Gene Cloning and Expression Analysis of ADP-Glucose Pyrophosphorylase in Eleocharis dulcis

【目的】克隆荸荠ADP-葡萄糖焦磷酸化酶(AGPase)小亚基基因EdAPS1的cDNA序列,并分析其序列特征及其在荸荠不同组织和球茎发育过程中的表达情况,为研究荸荠淀粉生物合成机制提供理论依据。【方法】通过RT-PCR技术从荸荠球茎中克隆EdAPS1基因的cDNA序列,采用生物信息学方法对其序列和所编码的蛋白进行预测分析,利用实时荧光定量PCR技术检测EdAPS1基因在荸荠不同组织和球茎发育过程中的表达情况。【结果】克隆获得的EdAPS1基因cDNA序列全长1716 bp,包含1个1521 bp开放阅读框(ORF),编码506个氨基酸。EdAPS1蛋白分子式为C_(2469)H_(3896)N_(672)O_(746)S_(23),相对分子量为55.67 kD,等电点为5.98,具有NTPtransferase和PbH1结构域。同源性和系统进化树分析结果表明,EdAPS1蛋白与不同植物相应蛋白的同源性在68.80%~86.91%,其中与油莎草相应蛋白(ALL29329.1)的亲缘关系最近。荧光定量PCR分析结果表明,EdAPS1基因在荸荠不同组织中均有表达,其中在球茎的表达量最高,与在其他组织的表达量差异显著(P<0.05,下同),尤其在球茎发育后期的表达量更高,且显著高于球茎其他发育时期。【结论】从荸荠中成功克隆了EdAPS1基因,可为后续阐明荸荠淀粉生物合成机制及培育高淀粉荸荠品种提供参考依据。

【Objective】To study the starch biosynthesis in Chinese water chestnut,we cloned and analyzed the cDNA sequence of ADP-glucose pyrophosphorylase(AGPase)small subunit gene(EdAPS1)from Chinese water chestnut.Using the sequence,the expression of EdAPS1 was further analyzed in various tissues and at different stages of corm development.【Method】RT-PCR(reverse transcription polymerase chain reaction)was used to clone the cDNA sequence of EdAPS1 from corm.The sequence and amino acid sequence of the protein was predicted by various bioinformatics software.The gene expression of EdAPS1 was profiled by real-time fluorescent quantitative PCR from various tissues and at different stages of corm development.【Result】The EdAPS1 cDNA sequence was 1716 bp in length,which contained a 1521 bp open reading frame(ORF).The predicted amino acid sequence of the ORF was 506 amino acids in size.The calculated chemical molecular formula of EdAPS1 protein was C_(2469)H_(3896)N_(672)O_(746)S_(23),with 55.6 kD in molecular mass and 5.98 in isoelectric point.NTPtransferase and PbH1 domains were found in the protein sequence.Using homology and phylogenetic tree analysis,the EdAPS1 protein sequence homology of AGPase between Chinese water chestnut and other plants ranged from 68.80%-86.91%.The most similar protein sequence of EdAPS1 was from Cyperus esculentus var.sativus(ALL29329.1).Analyzing by real-time fluorescent quantitative PCR,the EdAPS1gene expressed in all the selected tissues,and the corm had the highest expression and showed significant difference with the other tissues(P<0.05,the same as below).Especially,the expression level in the late development stage of corm was higher,which was significantly higher than that in other development stages of corm.【Conclusion】The EdAPS1 gene was successfully cloned from Chinese water chestnut,which provided a reference for further elucidating the starch biosynthesis mechanism of Chinese water chestnut and cultivating high starch varieties.