银杏GbASR基因的克隆、生物信息学及表达分析

GbASR gene from Ginkgo biloba:Cloning,Bioinformatics and Expression Analysis

为了明确银杏ASR(ABA-stress-ripening)在应答非生物逆境胁迫中的作用,以便为ASR基因功能、环境胁迫研究提供材料。本研究以银杏(Ginkgo biloba)为材料,利用分子生物学技术对ASR基因进行了克隆,构建pCAMBIA1300-ASR-GFP融合表达载体,进行亚细胞定位分析。采用序列比对方法分析GbASR蛋白结构域及系统进化;利用qRT-PCR方法进行组织特异性表达及非生物逆境胁迫下的基因表达分析。结果表明,银杏ASR基因编码区全长546 bp,共编码182个氨基酸;蛋白质等电点为5.33,分子量大小为20111.24 Da,为亲水性稳定蛋白;GbASR蛋白含有1个高度保守的ABA/WDS结构域,且与火炬松PtASR蛋白同源性较高;GbASR蛋白定位于细胞核。此外,GbASR基因在银杏幼苗根、茎、叶各组织中均有表达,且在叶部有较高的表达;在高盐、干旱和高温胁迫下,GbASR基因均受诱导,且在不同胁迫处理时间0 h、6 h、24 h和48 h的表达存在显著的差异。以上分析说明GbASR基因参与响应高盐、干旱和高温等非生物胁迫,这为植物的育种及解释ASR基因功能提供依据。

The aims are to clarify the role of ASR(ABA-stress-ripening)gene from Ginkgo biloba in response to abiotic stress,and provide materials for studying ASR gene function and environment stress.In this study,Ginkgo biloba was used as material,GbASR gene was cloned and the fusion expression vector pCAMBIA1300-ASR-GFP was constructed by molecular biology techniques,then the subcellular localization of GbASR protein was analyzed.Protein domains and phyletic evolution were analyzed by sequence alignment method.Tissue-specific expression and expression level of GbASR gene under abiotic stress were analyzed by qRTPCR method.The results showed that the total length of GbASR coding region was 546 bp,encoding a182 amino acid protein.The theoretical isoelectric point was 5.33 and the molecular weight of GbASR protein was 20111.24 Da,which was a stable hydrophilic protein.Sequence alignment and phylogenetic analysis showed that GbASR protein contained a highly conserved ABA/WDS domain and had high homology with PtASR protein.The subcellular localization analysis showed that GbASR protein was a nucleoprotein.In addition,GbASR gene was widely expressed in different tissues such as root,steam and leaf from Ginkgo biloba,and the GbASR gene was induced under salt,drought and heat stresses,and there were significant different expressions at 0 h,6 h,24 h and 48 h under salt,drought,and heat stresses.These results suggest that GbASR might participate in response to abiotic stresses such as salt,drought and heat stresses,and this study could provide a basis for plant breeding and explaining ASR gene function.