甜樱桃DELLA蛋白基因PaGAI的克隆与表达分析  

Cloning and Expression Analysis of PaGAI gene of DELLA Protein from Prunus avium

作　　者：钟翡[1] 沈欣杰[1] 刘芳[1] 袁华招[1] 刘兰英[2] 李天红[1] 

机构地区：[1]中国农业大学农学与生物技术学院,北京市果树逆境生理与分子生物学重点开放实验室,北京100193 [2]北京市海淀区植物组织培养技术实验室,北京100091

出　　处：《园艺学报》2012年第1期143-150,共8页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(30871696,31171938);公益性行业(农业)科研专项(201003021)

摘　　要：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。DELLA protein is a key negative regulation factor of gibberellins （GAs）signal transduction pathway. A whole cDNA sequence of DELLA protein gene, named PaGAI, was cloned from Prunus avium L. tender leaves using RTPCR and RACEPCR. The cDNA of PaGAI had a length of 2 310 bp and contain an openingreading frame （ORF） of 1 788 bp, which was supposed to encode a 595 aminoacid residues polypeptide of 64 kD. Bioinformatics analysis showed that the protein encoded by PaGAI had a conservative structural domain of DELLA protein. It had two strict Conservative amino acid domains DELLA and VHYNP in the N terminal and three repression regions ofVHVID, RVER and SAW in the Cterminal. PaGAI, with high homologous with the DELLA proteins in some other plants, and showed the highest homology of 84% with Malus domestica. The prokaryotic expression system of pGEX4T1/PaGAI was constructed and transformated into E. coli BL21, and the protein expression was induced by 0.5 mmol.L^-1 IPTG and a 91 kD fusion protein was detected by SDSPAGE. Semiquantitative RT-PCR analysis showed that PaGA1 widely expressed in flower, fruit, leaf and phloem, and PaGA1 in the expression of flower and phloem is much stronger than in fruit and leaf.

关 键 词：甜樱桃 DELLA蛋白 基因克隆 原核表达 

分 类 号：S662.5[农业科学—果树学]