腊梅葡萄糖―6―磷酸脱氢酶G6PDH1)基因的克隆及表达分析.docVIP

腊梅葡萄糖―6―磷酸脱氢酶G6PDH1)基因的克隆及表达分析 　　[摘要] 葡萄糖-6-磷酸脱氢酶是磷酸戊糖途径中的关键限速酶。研究根据已经报道的G6PDH基因的保守序列引物扩增腊梅花中G6PDH基因的核心序列，利用RACE技术从腊梅中首次克隆得到一个G6PDH基因cDNA全长，命名为G6PDH1，并对G6PDH1蛋白质进行细胞定位预测、跨膜结构分析、三维结构模拟及聚类分析，预测G6PDH1的生物学信息；利用实时荧光定量PCR检测G6PDH1在腊梅不同组织中和冷处理条件下的表达差异分析。该研究中克隆获得的G6PDH1基因的全长为2 011 bp，开放阅读框1 551 bp，编码516个氨基酸。组织表达分析的结果显示，G6PDH1基因主要在花和根中表达，在叶和茎中表达量较低。通过冷处理，该基因在12 h时达到最高表达水平。研究通过对腊梅花中G6PDH1基因全长cDNA克隆和表达特异性分析，为以后深入研究腊梅的抗寒机制奠定了基础。 　　[关键词] 腊梅；G6PDH基因；表达分析 　　[Abstract] Glucose-6-phosphate dehydrogenase is main regulatory enzyme for pentose phosphate pathway. To amplify the core sequence of G6PDH gene from Chimonanthus praecox， the primers were synthesized， based on the conserved nucleotide sequence of other reported plant G6PDH genes. The specific primers were designed according to the major fragment. The full length cDNA of the G6PDH1 gene was isolated by the 3′ and 5′ rapid amplification of cDNA ends approach. Transcript levels of G6PDH1 isoform was measured by real-time quantitative RT-PCR in different tissues and in responds to cold treatment. The G6PDH1 subcellular localization， transmembrane domain， three-dimensional structure， and phylogenetic analysis were predicted by different software to analysis the bioinformatics of G6PDH1 protein. The G6PDH1 cDNA sequence was 2 011 bp in length and consisted of 1 551 bp Open Reading Frame （ORF）， encoding a protein of 516 amino acids. Expression analysis results in different tissues showed that G6PDH1 was primarily observed in flowers and roots， as opposed to the leaves and stems. Cold treatment experiments indicated that cold treatment caused a rapid increase in G6PDH1 expression in flowers within 12 h. The full-length cDNA of G6PDH1 and its expression analysis will play an important role for further study on cold stress responses in Ch. praecox. 　　[Key words] Chimonanthus praecox； glucose-6-phosphate dehydrogenase； expression analysis 　　doi：10.4268/cjcm 　　磷酸戊糖途径（pentose phosphate pathway， PPP）是植物体中糖代谢的重要途径，其主要生理功能是产生供生