摘要
[目的]克隆忽地笑[Lycoris aurea (L’Hér.) Herb.]α-葡萄糖苷酶2编码基因。[方法]基于忽地笑的转录组数据库,分析功能注释为α-葡萄糖苷酶2的基因片段,通过设计特异性定量PCR扩增引物分析其在各组织中的表达丰度,利用RACE (rapid-amplification of c DNA ends)技术从高丰度组织中克隆获得忽地笑α-葡萄糖苷酶2编码基因。[结果]忽地笑α-葡萄糖苷酶2编码基因Lau Agl2 c DNA全长3 185 bp,开放阅读框为2 961 bp,编码含有986个氨基酸残基的Lau Agl2蛋白质。Lau Agl2蛋白质氨基酸序列具有α-葡萄糖苷酶Ⅱ特征性的保守结构域和天冬氨酸残基组成的活性中心,还具有一些蛋白质糖基化的潜在位点(N-X-S/T共有序列)。Lau Agl2蛋白质与油棕、陆地棉等其他植物的α-葡萄糖苷酶2的氨基酸序列一致性为73.0%以上。利用p ET表达系统,可以在大肠杆菌中实现Lau Agl2蛋白质的诱导表达。[结论]成功从忽地笑花葶和花瓣中克隆到α-葡萄糖苷酶2编码基因Lau Agl2并在大肠杆菌中对Lau Agl2进行了异源表达,为Lau Agl2蛋白质的功能鉴定及其在忽地笑种子萌发与生长发育中的作用研究奠定了基础。
[Objective]The gene encoding α-glucosidase 2 was to be cloned from Lycoris aurea( L’Hér.) Herb.[Method] Based on the transcriptomic data of L.aurea,the abundance of contigs annotated as α-glucosidase 2 was analyzed in different tissues by qRT-PCR,and then the α-glucosidase 2 encoding gene was cloned with the c DNA from the tissue with high gene abundance as the template via RACE technology.[Results]The full-length c DNA of Lau Agl2 is 3 185 bp with a 2 961 bp open reading frame,encoding a deduced polypeptide of 986 amino acids.Lau Agl2 protein contains the active center with aspartate and the conserved domains of α-glucosidase Ⅱ,and some N-X-S/T motifs for possible glycosylation.The amino acid sequence of Lau Agl2 protein shares more than 73.0% identity with α-glucosidase 2 from Elaeis guineensis Jacq.,Gossypium hirsutum Linn.and other plants.Lau Agl2 protein was successfully expressed in Escherichia coli on the p ET expression system.[Conclusion] The Lau Agl2 gene encoding α-glucosidase 2 was cloned from the scape and the petal of L.aurea. In this study,molecular cloning and prokaryotic expression of Lau Agl2 protein were investigated to facilitate further research in functional characterization and identifying the role in the seed germination,growth and development of L.aurea.
作者
李宜奎
李洁
程丽
钱彬彬
汪仁
LI Yi-kui;LI Jie;CHENG Li(Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing,Jiangsu 210014)
出处
《安徽农业科学》
CAS
2018年第36期78-82,89,共6页
Journal of Anhui Agricultural Sciences
基金
国家自然科学基金项目(31572151)
江苏省自然科学基金项目(BK20160599)
江苏省植物资源研究与利用重点实验室基金项目(JSPKLB201505)
关键词
忽地笑
α-葡萄糖苷酶2
分子克隆
原核表达
Lycoris aurea
α-Glucosidase 2
Molecular cloning
Prokaryotic expression
