Molecular cloning and expression analysis of three ThERF s involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products

Wencai Fan; Ying Yang; Zhiquan Wang; Yunlong Yin; Chaoguang Yu; Qin Shi; Jinbo Guo; Lei Xuan; Jianfeng Hua

doi:10.7717/peerj.4434

Molecular cloning and expression analysis of three ThERF s involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products

PeerJ. 2018 Mar 12:6:e4434. doi: 10.7717/peerj.4434. eCollection 2018.

Authors

Wencai Fan¹, Ying Yang¹, Zhiquan Wang¹, Yunlong Yin¹, Chaoguang Yu¹, Qin Shi¹, Jinbo Guo¹, Lei Xuan¹, Jianfeng Hua¹

Affiliation

¹ Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu, China.

Abstract

As a subfamily of the APETALA 2/ethylene response element binding protein (AP2/EREBP) transcription factor superfamily, the ethylene response factor (ERF) is widely involved in the regulation of growth and response to various abiotic stresses in plants, and has been shown to be the main transcription factor regulating transcription of the genes related to hypoxia and waterlogging stress. In this study, three ThERF genes, with significant differences in expression profile in response to flooding stress, were identified from the transcriptomics data acquired from Taxodium hybrid 'Zhongshanshan 406' (T. mucronatum Tenore × T. distichum (L.) Rich) under waterlogging stress: ThERF15, ThERF39 and ThRAP2.3 (GenBank ID: KY463467, KY463468 and KY463470, respectively).The full-length cDNA of each of the three ERFs was obtained using the RACE (rapid amplification cDNA ends) method, and all three were intron-free. Multiple protein sequence alignments indicated that ThERF15, ThERF39 and ThRAP2.3 proteins all had only one AP2-ERF domain and belonged to the ERF subfamily. A transient gene expression assay demonstrated that ThERF15, ThERF39 and ThRAP2.3 were all localized to the nucleus. Real-time quantitative PCR (qPCR) revealed that the expression of ThERF15, ThERF39 and ThRAP2.3 exhibited significant differences, compared with the control, in response to two levels of flooding treatment (half-flooding or total-submergence) of 'Zhongshanshan 406'. Quantification of ethylene concentration revealed that ethylene was more relevant to the level of expression than the period of flooding treatment. Based on the experimental results above, ThERF15, ThERF39 and ThRAP2.3 were identified as being related to the regulation of downstream flooding- responsive gene expression in 'Zhongshanshan 406'. ThRAP2.3 is most likely to be a key downstream-response ERF gene to respond to the output of the ethylene signal generated by flooding stress.

Keywords: Cloning; Ethylene concentration; Expression analysis; Subcellular localization; Taxodium hybrid ‘Zhongshanshan 406’; ThERF genes.

Grants and funding

This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20150551), National Natural Science Foundation of China (No. 31570593), the Program of Innovation Capacity Construction of Jiangsu Province (No.BM2015019), the Innovation Project of Plant Germplasm Resources of 315 Chinese Academy of Sciences (No.ZSZC-009) and the Natural Science Foundation of Jiangsu Province (No. BK20160601). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.