枇杷EjWRKY27基因克隆及低温响应表达分析

Cloning and Expression Analysis of EjWRKY27 Gene in Loquat in Response to Low Temperature

WRKY是植物中所特有的转录因子家族,参与植物形态建成及非生物胁迫响应等多种生物学过程。本研究对枇杷EjWRKY27基因进行克隆、生物信息学分析,并对其在枇杷5个发育组织及低温胁迫下的表达模式进行探究。研究结果显示,该基因位于枇杷基因组14号染色体上,编码区长度2157 bp,编码719个氨基酸,氨基酸序列包含WRKY结构域,属于IIb亚家族成员。系统进化结果进一步显示该序列与同是蔷薇科植物包括苹果、梨、桃、樱桃、梅、杏的同源性较高。生物信息学分析结果表明,EjWRKY27蛋白不是分泌蛋白,也不含信号肽和跨膜区;该蛋白为亲水性蛋白,其分子量、等电点、脂溶指数、不稳定性指数、平均亲水性值依次为73.12 kD,6.90,54.85,56.63,-0.752;EjWRKY27有128个磷酸化位点,其中丝氨酸92个、苏氨酸30个和酪氨酸6个。Ej WRKY27蛋白的二级结构以无规卷曲为主,比例为67.45%,其次为α螺旋,比例为21.5%,而延伸链、β转角的比例仅为8.84%和2.21%。该蛋白的三级结构域主要由RVSVIVR、WRKYGQK、RAYYRC、RKQVQR、ILVTTYE共5个β-折叠组成。RT-qPCR分析表明,该基因在根和果实中特异高表达,并受低温胁迫诱导,在处理0.5 h时显著应激上调表达。亚细胞定位预测表明EjWRKY27定位在细胞核内,而烟草瞬时表达系统的验证结果也证实如此。本研究结果为进一步研究EjWRKY27在逆境胁迫下的响应机制提供了一定的理论基础。

WRKY is a unique transcription factor family in plants,which is involved in many biological processes such as plant morphogenesis and abiotic stress response.In this study,the EjWRKY27 gene of loquat was cloned and bioinformatics analyzed,and its expression patterns in five developmental tissues of loquat and under low temperature stress were explored.The results showed that the gene was located on chromosome 14 of loquat genome,and the coding region was 2157 bp long and encoded 719 amino acids.The amino acid sequence contained WRKY domain and belonged to IIb subfamily.The phylogenetic results further showed that the sequence had high homology with the same Rosaceae plants,including apple,pear,peach,cherry,plum and apricot.The result of bioinformatics analysis showed that EjWRKY27 was not a secretory protein,and it did not contain signal peptide and transmembrane region either.The protein was hydrophilic,and its molecular weight,isoelectric point,lipolysis index,instability index and average hydrophilicity were followed by 73.12 kD,6.90,54.85,56.63 and-0.752;EjWRKY27 had 128 phosphorylation sites,including 92 serine,30 threonine and 6 tyrosine.The secondary structure of EjWRKY27 protein was mainly random curl,accounting for 67.45%,followed by a-helix,accounting for21.5%.The proportion of extended chain andβ-corner were only 8.84%and 2.21%.The tertiary domain of EjWRKY27 protein was mainly composed of 5β-folding composition,RVSVIVR,WRKYGQK,RAYYRC,RKQVQR and ILVTTYE.RT-qPCR analysis showed that this gene was specifically highly expressed in roots and fruits and was induced by low temperature stress,the expression of EjWRKY27 was significantly up-regulated at 0.5 h.Subcellular localization prediction showed that EjWRKY27 was located in the nucleus,which was also confirmed by the verification results of tobacco transient expression system.The results of this study laid a theoretical foundation for the further study of the response mechanism of EjWRKY27 under different stresses.