森林草莓精氨酸脱羧酶基因的电子克隆与序列分析  

In silico cloning and sequencing of an arginine decarboxylase gene from Fragaria vesca

作　　者：王静[1] 赵密珍[1] 王壮伟[1] 吴伟民[1] 钱亚明[1] 

机构地区：[1]江苏省农业科学院园艺研究所,江苏南京210014

出　　处：《江苏农业学报》2011年第3期628-633,共6页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省科技基础设施建设计划项目(BM2008008);江苏省科技支撑计划项目(SBE200930356)

摘　　要：从森林草莓中分离精氨酸脱羧酶基因(FvADC),分析其表达模式、序列特征和进化关系。采用电子克隆结合RT-PCR分离FvADC;半定量RT-PCR检测其在盐、热、冷胁迫下的表达情况;生物信息学分析FvADC的结构与进化。结果获得FvADCcDNA全长,包括2 154 bp的编码区,RT-PCR验证编码区序列与电子克隆序列一致;FvADC基因编码的精氨酸脱羧酶与桃、苹果和海棠的同源性分别达87%、84%和83%,含信号肽,具有2个保守的氨基酸结构域:ADC家族2磷酸吡哆醛结合位点和ADC家族2标记2序列;进化分析表明森林草莓精氨酸脱羧酶与苹果、桃精氨酸脱羧酶关系最近;半定量RT-PCR证实FvADC在盐、热、冷胁迫下诱导表达。以上结果说明电子克隆能用于草莓基因的分离,而FvADC可作为逆境候选基因用于草莓遗传改良。In this study,an arginine decarboxylase gene was isolated from Fragaria vesca,named FvADC（Fragaria vesca ADC）,and its expression pattern,sequence characteristics and evolutionary relationship were investigated.FvADC was isolated by RT-PCR in combination with in silico cloning.Semi-quantitative RT-PCR was used to determine the expression pattern under salt,hot and cold stresses.Bioinformatics analysis was used to study the structure,evolutionary relationship of FvADC.The results showed that the full-length cDNA sequence was 2 905 bp,which contained a complete open reading frame（ORF） of 2 154 bp,with a 456 bp 5′-untranslated region（UTR） and a 293 bp 3′-UTR.The ORF encoded a putative protein containing 717 amino acids.The two single RT-PCR bands confirmed the results.FvADC amino acid sequence shared high identity with those from Prunus persica（87%,AB379849）,Malus × domestica（84%,AB181854） and Malus hupehensis（83%,EU431331）.A putative N-terminal signal peptide was predicted.FvADC contained two highly conservative domains,i.e.,the ADC family 2 pyridoxal phosphate binding site and the ADC family 2 signature 2 sequence.FvADC putative protein had a calculated molecular mass of 243 243.17 and an isoelectric point of 4.80.FvADC was located in the cytoplasm.There was a close relationship between apple FvADC and peach FvADC by phylogenetic tree analysis.Based on the database of ESTs,FvADC was expressed in response to stress such as hot,drought,salt,chilling and exogenous salicylic acid.Semi-quantitative RT-PCR results showed that FvADC expression was induced under salt,hot or cold stresses.The above results indicated that in silico cloning would be efficient in isolation of strawberry genes,and FvADC was a good stress-resistant candidate gene for strawberry genetic improvement.

关 键 词：森林草莓 ADC基因 电子克隆 生物信息学 RT-PCR 

分 类 号：Q78[生物学—分子生物学]