Cloning And Characterization Of Wrky Transcription Factors From Reaumuria Trigyna

Reaumuria trigyna is an endangered small shrub endemic to the Eastern Alxa-Western Ordos area in Inner Mongolia. This recretohalophyte has developed unique morphological characteristics and adaption strategies that allow it to cope with the stress imposed by semi-desert saline soil. Due to its superior tolerance to salinity, R. trigyna is considered to be excellent candidates to investigate salt-tolerance mechanisms and to identify effective salt-response genes in the plant. As an important transcription factor family, WRKY transcription factors play a vital role in plant responding to abiotic stresses. In this study, we focused on the members of WRKY transcription factors which screened from transcription library of R. trigyna. Meanwhile, we analyzed its expression pattern under artificial adverse stresses. Finally, Two WRKY transcription factors were isolated from R. trigyna, and function analysis of the two genes was carried out. The main results are as follows:1.67unigene sequences of WRKY transcription factors were obtained from transcription library of R. trigyna. Among them, there are23unigenes that share highly similarities with the homologous genes from Populus tomentosa.2.10genes were selected from all67WRKY fragments. Their expression pattern was analyzed under salt, cold, drought and ABA stress. The results suggested that10genes were induced by salt and cold stress,8and7genes were upregulated by drought and ABA treatment.3. Two novel WRKY genes named RtWRKYl and RtWRKY23were isolated from R. trigyna by rapid amplification of cDNA ends (RACE) cloning. Sequence analysis revealed that they belonged to the class I and class Ⅱ WRKY transcription factor, respectively. RtWRKY1shared69%similarities with Arabidopsis thaliana AtWRKY1, and RtWRKY23was40%identities with AtWRKY23. A phylogenetic tree of the deduced amino acid sequences of WRKY genes from different plant species was constructed by MEGA4.0software. The result showed that RtWRKYl was related more closely to A. thaliana, Brassica campestris and P. tomentosa, while RtWRKY23was closer to WRKY proteins from Solanum tuberosum, Capsicum annuum and Glycine max.4. RtWRKYl and RtWRKY23transcripts were analyzed in three organs including the root, shoot, and leaves. Research showed that expression of these two genes varied between organs. RtWRKY1was expressed only in shoot. RtWRKY23was induced in all organs that we tested, but less transcripts were generated in root and leaves.5. Transient expression of GFP/RtWRKY1or GFP/RtWRKY23in onion epidermal cells showed that two RtWRKY proteins were localized in the nucleus, which suggested that RtWRKY1and RtWRKY23might regulate the expression of downstream genes in the nucleus by specific binding with them.6. Two eukaryotic expression vectors pMETa-RtWRKY1and pMETa-RtWRKY23were constructed, which lay the foundation for the heterologous expression and functional identification in yeast.