棉花类表皮特异性分泌糖蛋白基因GhA01EP1的克隆和功能分析

Cloning and functional analysis of epidermis-specific secreted glycoprotein EP1-like gene GhA01EP1 in cotton

【目的】本研究对类棉花表皮特异性分泌糖蛋白基因GhA01EP1的克隆及功能分析,为进一步了解胞外蛋白抵抗环境胁迫的调控机制提供了基础。【方法】利用同源克隆法获得了冀2658中GhA01EP1的序列,通过实时定量聚合酶链式反应检测GhA01EP1基因的组织表达及干旱胁迫前后表达变化。对GhA01EP1蛋白的理化性质、结构、亚细胞定位等进行了生物信息学分析。用烟草瞬时转化试验对GhA01EP1蛋白进行亚细胞定位。利用农杆菌蘸花法获得转基因拟南芥,并对转基因植株进行抗旱鉴定。【结果】将克隆到的与抗旱相关的类表皮特异性分泌糖蛋白基因命名为GhA01EP1,位于A01号染色体。该基因无内含子,开放阅读框长1371 bp,编码456个氨基酸,包含B-lectin、Plant PAN/APPLE-like 2个结构域。GhA01EP1基因在根、茎和叶片中均有表达,在根中表达量最高。KEGG通路分析发现,GhA01EP1参与甘氨酸、丝氨酸和苏氨酸代谢以及苯丙氨酸代谢的可信度较高。烟草瞬时转化试验结果显示,GhA01EP1蛋白为分泌蛋白。抗旱鉴定发现,与野生型相比,转基因拟南芥在干旱胁迫下长势好、根系长、胁迫复水后恢复能力强。【结论】GhA01EP1基因在棉花和拟南芥抗旱中起积极作用。

[Objective]The aim of this study is to clone and analyze the epidermis-specific secreted glycoprotein EP1-like gene GhA01EP1 in cotton,which provided a basis for further understanding of the regulatory mechanism of extracellular protein in the resistance to environmental stress.[Method]The sequence of GhA01EP1 from Ji 2658 was obtained by homologous cloning strategy,the expression level of GhA01EP1 was analyzed by quantitative real-time polymerase chain reaction.The physical and chemical properties,structure and subcellular localization of GhA01EP1 protein were analyzed by bioinformatics method.Subcellular localization of protein was carried out by transient expression of tobacco transformation mediated by Agrobacterium.The drought resistance of transgenic Arabidopsis thaliana obtained by Agrobacterium-mediated transformation method was analyzed.[Result]We designated the epidermis-specific secreted glycoprotein EP1-like gene as GhA01EP1,without intron,which has an 1317 bp open reading frame and encodes 456 amino acids.GhA01EP1 is located on chromosome A01 and contains two domains:B-lectin and Plant PAN/APPLE-like.GhA01EP1 is expressed in root,stem and leaf,and with highest expression level in root.KEGG pathway analysis showed that GhA01EP1 is most likely involved in glycine,serine and threonine metabolism and phenylalanine metabolism.Subcellular localization analysis showed that the GhA01EP1 is a secretory protein.The result of drought resistance analysis showed that transgenic Arabidopsis grew better under drought stress than wild type,with longer root and stronger recovery ability after rehydration.[Conclusion]The GhA01EP1 plays a positive role in drought resistance of cotton and Arabidopsis.