LI Jiale,DENG Lili,XU Shiqiong,et al.Cloning and Expression Analysis of FaABI5 Gene in Strawberry[J].Northern Horticulture,2022,(01):31-39.[doi:10.11937/bfyy.20212123]
草莓FaABI5基因的克隆与表达分析
- Title:
- Cloning and Expression Analysis of FaABI5 Gene in Strawberry
- Keywords:
- strawberry; FaABI5 gene; clone; expression pattern
- 文献标志码:
- A
- 摘要:
- 以‘红颜’草莓为试材，采用同源克隆方法得到FaABI5基因，利用荧光定量PCR法获取该基因的表达模式，通过转录活性验证和亚细胞定位分析其蛋白的特性，利用大肠杆菌诱导表达该蛋白，以期为进一步解析FaABI5的基因功能提供参考依据。结果表明：草莓FaABI5基因的开放阅读框为1 329 bp，共编码442个氨基酸，预测分子量约为47.1 kDa，理论等电点为9.70，属于不稳定亲水蛋白。氨基酸序列比对显示该基因与其它物种的ABI5蛋白具有较高一致性，系统发育树表明草莓FaABI5与森林草莓、月季中的同源蛋白聚为一支，亲缘关系较近。获得该基因起始密码子上游1 500 bp长的启动子序列，分析显示除了有大量ABA响应元件ABRE外，还存在许多光、植物激素及非生物胁迫响应元件。qRT-PCR结果表明，FaABI5在草莓的根和茎中表达量最高，在果实中表达量较低。在酵母中，FaABI5不具有转录激活能力。亚细胞定位试验证明FaABI5为核定位蛋白。在大肠杆菌Rosetta（DE3）中，28 ℃自诱导24 h可以获得具有生物活性的FaABI5重组蛋白。
- Abstract:
- The FaABI5 gene was cloned from the cultivated strawberry ‘Benihoppe’ by homologous cloning,the expression pattern of FaABI5 was obtained by qRT-PCR,the protein was characterized by transcriptional activity and subcellular localization,and the protein was induced to expressed using Escherichia coli,in order to provide a reference for further elucidation of the gene function of FaABI5.The results showed that the open reading frame of strawberry FaABI5 gene was 1 329 bp,encoding 442 amino acids,with a predicted molecular weight of about 47.1 kDa and a theoretical isoelectric point of 9.70,which was an unstable hydrophilic protein.Amino acid sequence comparison showed that this proteins had high identity with ABI5 proteins of other species,and the phylogenetic tree indicated that strawberry FaABI5 clustered with homologous proteins in wild strawberry and Chinese rose into one branch.A 1 500 bp long promoter sequence upstream of the start codon of this gene was obtained.In addition to a large number of ABA response elements ABRE,the promoter of FaABI5 contains many light,phytohormone and abiotic stress response elements.qRT-PCR results showed that FaABI5 was most highly expressed in roots and stems of strawberry,and less expressed in fruits.In yeast,FaABI5 does not have transcriptional activation ability.Subcellular localization experiments demonstrated that FaABI5 was a nuclear localization protein.Bioactive FaABI5 recombinant protein could be obtained by self-induction at 28 ℃ for 24 hours in E.coli Rosetta (DE3).
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备注/Memo
第一作者简介：李佳乐（1995-），女，硕士研究生，研究方向为果树栽培理论与技术。E-mail:515699748@qq.com.责任作者：汤浩茹（1963-），男，博士，教授，博士生导师，现主要从事果树遗传育种、果树种质资源与分子生物学等研究工作。E-mail:htang@sicau.edu.cn.基金项目：国家自然科学基金资助项目(31872083)。收稿日期：2021-05-19