花生铝响应类受体蛋白激酶AhPRK4的原核表达分析

Prokaryotic expression of aluminum associated receptor-like protein kinase AhPRK4 in peanut(Arachis hypogaea)

关键词:  花生, 铝胁迫, 花粉类受体蛋白激酶, 表达分析, 原核表达

Key words:  peanut(Arachis hypogaea), aluminum stress, pollen receptor-like protein kinase, expression analysis, prokaryotic expression

摘要:

花粉类受体蛋白激酶(pollen receptor-like protein kinase,PRK)是一类富含LRR结构域的类受体蛋白激酶,不仅在花粉发育和植物受精中发挥作用,也在胁迫响应中发挥作用。基于对前期花生根尖铝胁迫转录组数据的分析,我们发现了在转录水平响应铝胁迫的花粉类受体蛋白激酶基因AhPRK4,为探究AhPRK4在花生铝胁迫中的功能,该文进一步分析了铝胁迫处理下AhPRK4在花生耐铝品种‘99-1507'和铝敏感品种‘中花2号'(‘ZH2')根尖中的转录变化,通过序列分析、进化树构建等分析了AhPRK4蛋白的结构特点和亲缘关系,克隆了AhPRK4的胞内域序列(AhPRK4-CD),并通过原核表达和体外磷酸化体系分析了AhPRK4-CD的自磷酸化活性。结果表明:(1)不同铝处理时间及不同铝浓度处理后,AhPRK4的转录水平上调,显著响应铝处理,是铝诱导基因;(2)AhPRK4含有673个氨基酸,属于LRR-III蛋白激酶家族成员,具跨膜域和信号肽,且预测具有磷酸化活性位点;(3)体外诱导表达出约71 kD的可溶性蛋白(GST-AhPRK4-CD),经凝胶亲和层析纯化,得到基于蛋白印迹实验(Western Blot)验证正确的重组蛋白,重组蛋白可发生磷酸化修饰,但无明显的自磷酸化现象。综上认为,AhPRK4是一个铝胁迫应答基因,参与花生铝胁迫早期应答机制,且能发生磷酸化修饰。

Abstract:

The pollen receptor-like protein kinase(PRK)family, an LRR receptor-like protein kinase, not only played a role in pollen development and fertilization, but also played a role in stress response. Based on the analysis of transcriptome data that generated in our previous study, we found that AhPRK4 was an aluminum-responsive gene. To explore the role of AhPRK4 in response to aluminum stress, we analyzed the expression of AhPRK4 by qRT-PCR in ‘ZH2'(Al-sensitive)and ‘99-1507'(Al-tolerant), clarified the protein structure and genetic relationship of AhPRK4 by sequence analysis, phylogenetic tree construction and other genetic analysis, constructed the recombinant plasmid by homologous recombination, obtained the intracellular domain recombinant protein of AhPRK4 by prokaryotic expression technology and determined the activity of the recombinant protein by incubation with phosphorylated antibodys. The results were as follows:(1)The transcription level of AhPRK4 was up-regulated after different aluminum treatments time and different aluminum concentrations, indicating that AhPRK4 was an aluminum inducible gene;(2)The AhPRK4 protein had 673 amino acids with transmembrane domain, signal peptide and phosphorylation active sites, belonging to the LRR-III protein kinase family;(3)The GST-AhPRK4-CD recombinant protein was induced in vitro and verified by Western Blot. And the recombinant protein had phosphorylated on both serine/threonine and tyrosine residues, but had no significant auto-phosphorylation activity. In conclusion, AhPRK4 is an aluminum responsive gene, which participated in the regulation of short-term aluminum stress and is phosphorylated in vitro.