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利用RT-PCR和RACE技术从甜樱桃(Prunus avium L.)嫩叶中克隆了赤霉素信号转导途径中的关键负调控因子DELLA蛋白基因cDNA全序列,将其命名为PaGAI。该基因cDNA全长2310bp,开放阅读框ORF为1788bp,推测其编码一个含有595个氨基酸残基的多肽链,蛋白质分子量约64kD。生物信息学分析结果表明,PaGAI编码蛋白具有DELLA蛋白保守结构域,其N端存在两个非常保守的酸性结构域DELLA和VHYNP作为信号感知区域,C端有VHIID、RVER和SAW结构域作为阻遏区域。PaGAI与其它植物的DELLA蛋白具有较高的同源性,其中与苹果MdRGL2b蛋白同源性最高,达到84%。构建pGEX-4T-1/PaGAI原核表达体系,并转化E.coli BL21,经0.5mmol·L-1IPTG诱导蛋白表达,SDS-PAGE检测获得了分子质量为91kD的融合蛋白,半定量RT-PCR分析表明,PaGAI在花、果实、叶片、韧皮部中普遍表达,在花与韧皮部中的表达远远强于在果实与叶片中的表达。
The full-length cDNA sequence of DELLA, a key negative regulator of gibberellin signaling pathway, was cloned from young leaves of Prunus avium L. by RT-PCR and RACE techniques and named as PaGAI. The full-length cDNA of this gene is 2310 bp in length and the ORF of the open reading frame is 1788 bp, suggesting that it encodes a polypeptide chain of 595 amino acid residues with a molecular weight of about 64 kD. The results of bioinformatics analysis showed that PaGAI encoded protein has the conserved domain of DELLA protein. There are two very conserved acidic domains DELLA and VHYNP at the N-terminus, and the VHIID, RVER and SAW domains at C-terminal as repressor regions . PaGAI has high homology with DELLA protein of other plants, and has the highest homology with apple MdRGL2b protein, reaching 84%. The prokaryotic expression system of pGEX-4T-1 / PaGAI was constructed and transformed into E.coli BL21. The protein was induced by 0.5mmol·L-1IPTG, and the fusion protein with the molecular mass of 91kD was obtained by SDS-PAGE. Semi-quantitative RT-PCR analysis The results showed that PaGAI was widely expressed in flowers, fruits, leaves and phloem, and its expression in flowers and phloem was much stronger than that in fruits and leaves.