黄瓜液泡膜H^+-PPase基因CuPPase的cDNA克隆、序列分析与表达  

Cloning,Characterization and Expression of a H^+-PPase Gene CuPPase from Cucumis sativus L.

作　　者：洪艳艳[1] 史庆华[1,2] 王秀峰[1,2] 窦宏伟[3] 王双双[1] 刘泽洲[1] 

机构地区：[1]山东农业大学园艺科学与工程学院,山东泰安271018 [2]作物生物学国家重点实验室,山东泰安271018 [3]山东农业大学林学院,山东泰安271018

出　　处：《园艺学报》2010年第3期413-420,共8页Acta Horticulturae Sinica

基　　金：国家‘973’项目(2009CB119000);国家自然科学基金项目(30800751);国家博士后科学基金项目(20070421090)

摘　　要：利用GenBank登录的植物液泡膜焦磷酸酶氨基酸保守区序列设计简并引物,利用RT-PCR和RACE-PCR技术从黄瓜叶片中获得了一个液泡膜H+-PPase基因,命名为CuPPase,GenBank注册号为GQ223786。CuPPase蛋白与南瓜、绿豆同源性最高,分别为94%,92%。生物信息学分析表明,该基因全长2650bp,开放阅读框(ORF)2307bp,编码768个氨基酸;CuPPase蛋白大小约80kD,理论pI值为5.32。该基因有14个强的跨膜螺旋结构,PlantCare分析结果显示该基因序列具有脱落酸诱导、生长素诱导、赤霉素诱导、水杨酸诱导、低温诱导、干旱诱导的顺式作用元件。RT-PCR表明,CuPPase在叶和根中表达较高,茎中表达较低。CuPPas表达受盐胁迫以及高温、低温胁迫诱导,但与处理时间有密切关系,其表达均表现先升高后降低的趋势。Using PCR degenerate primers designed based on the conserved amino acid sequences of known tonoplast H＋-PPase to amplify cDNA fragments from cucumber by RT-PCR and RACE-PCR,a H＋-PPase gene named CuPPase was cloned.The CuPPase protein showed 94%,92% similarity to the H＋-PPases from Cucurbita moschata and Vigna radiata.Bioinformatics analysis indicated that the full-length cDNA sequence was 2 650 bp,which contained an open reading frame of 2307 bp and encoded a protein of 768 amino acid residues with a calculated molecular weight of 80 kD and isoelectric point of 5.32.There was fourteen strong inside to outside transmembrane helix.PlantCare analysis indicated that there were abscisic acid,somatotropic hormone,salicylic acid cis-acting responsive elements and gibberellin and low temperature,drought wound-responsive elements in the protein encoded by CuPPase.The result of RT-PCR analysis indicated that CuPPase expressions were different in roots,stems and leaves,and richer in leaves and roots,poorer in stems.The expression of CuPPase is induced by salt stress and low or high temperature stress,but is closely related to the processing time.Its expression trend increased firstly and then decreased.

关 键 词：黄瓜 CDNA克隆 序列分析 表达 

分 类 号：S642.2[农业科学—蔬菜学]