盐芥GRP7基因的克隆和逆境表达分析

Cloning and stress expression of GRP7 in Thellungiella salsugineum

【目的】从抗逆植物盐芥中分离Gl尺P7（glycine-richRNA-bindingprotein7，GRP7）基因，为进一步揭示GRPs基因在盐芥逆境应答中的作用机制及生物学功能奠定基础。【方法】利用盐芥GRP7基因的EST序列设计特异引物，克隆GRP7基因的全长序列，对其保守结构域和系统进化树进行分析，并对TsGRP7基因的启动子区进行预测；利用qRT-PCR技术，检测GRP7基因在不同逆境胁迫、不同组织中的表达情况。【结果】TsGRP7基因编码区全长522bp，编码173个氨基酸；TsGRP7蛋白的N端第9-82位氨基酸之间有1个RNA识别基序（RRM），RRM中含有保守的RNP-1和RNP-2亚结构域；多重序列比对和系统进化树分析表明，盐芥和拟南芥亲缘关系较近；启动子序列分析表明，盐芥GRP7启动子区含有HSE热激响应元件、LTR低温应答元件等多个逆境相关的顺式作用元件，表明盐芥GRP7基因参与逆境响应。qRT-PCR分析表明，盐芥GRP7基因在不同逆境胁迫条件及不同组织中表达不同。【结论】盐芥GRP7基因参与低温、高盐、PEG等逆境响应。

[Objective] The glyeine-rich RNA-binding protein7 （GRP7） gene was isolated from Thellungiella salsugineum to understand the role of GRPs and lay the foundation for further function analysis of GRPs in stress response. [Method] In this experiment,specific primers of TsGRP7 were designed based on EST sequence and full-length cDNA of TsGRP7 gene was isolated. The conserved domain and potential cis-element in promoter region were analyzed and phylogenetic analysis was performed. Quantitative realtime PCR analysis was carried out under different stresses in different tissues. [Result] The 522 bp coding region of TsGRP7 encoded 173 amino acid residues. The region between residues 9 and 82 was a typical RNA-recognition motif,containing conserved RNP 1 and RNP-2 subdomains. Multiple sequence alignment and system evolution analysis of TsGRP7 protein reveled that Thellungiella salsugineum had higher identity with homologous protein in Arabidopsis. Promoter sequence analysis by PlantCARE showed that mul tiple ciselements were associated with abiotic and biotic stress localized in promoters, such as HSE, LTR,ABRE,Box-Wl and P-box,indicating that the TsGRP7 gene involved in stree response. Quantitative realtime PCR analysis revealed that the expression of TsGRP7 gene was specific under different stresses and in different tissues. [Conclusion] The TsGRP7 gene involved in responses to low temperature, high salt and drought stress.