芹菜中6-磷酸甘露糖还原酶基因的克隆、进化和表达分析

Cloning,evolution and expression pattern analysis of M6PR gene in celery

以2个芹菜品种‘津南实芹’和‘美国西芹’为试材,采用RT-PCR技术分别获得6-磷酸甘露糖还原酶(M6PR)cDNA序列。序列分析表明:来源于2个芹菜品种的6-磷酸甘露糖还原酶基因核苷酸序列全长均为930 bp,编码309个氨基酸;预测其蛋白质相对分子质量为35×103,pI值为6.38。空间结构分析显示:M6PR蛋白由11个α-螺旋和13条β-延伸主链组成,中间形成1个疏水穴;催化四分体(Asp、Tyr、Lys和His)分布于疏水穴内部,具有催化活性。进化分析显示:芹菜M6PR与卷柏、小立碗藓、云杉等远古物种的醛-酮还原酶(AKR)相似性较高。实时定量PCR表达分析表明:M6PR基因主要在芹菜的茎中表达,具有明显的组织特异性。

Full-lengths of cDNA sequences encoding mannose-6-phosphate reductase(M6PR)were cloned from celery(Apium graveolens)cultivars'Jinnanshiqin'and'Meiguoxiqin'with reverse transcript PCR(RT-PCR).Sequence analysis showed that the length of the gene was 930 bp,containing a complete open reading frame,which encoded 309 amino acids residues.Its relative molecular mass was 35×103,and pI was 6.38.Three-dimension structure analysis showed that M6PRs from the two celery cultivars were composed by 11 α-helices and 13 β-strands,among which there was a hydrophobic cavity.Catalysis was promoted by a conserved tetrad of active site residues(Tyr,Lys,Asp and His).Amino acid sequence comparison indicated that the M6PRs from'Jinnanshiqin'and'Meiguoxiqin'had high similarity with the AKRs from Selaginella moellendorffii,Physcomitrella patens and Picea sitchensis.Quantitative real-time PCR analysis showed that M6PR gene was tissue-specific and was mainly expressed in the stem.