Cloning And Expression Analysis Of Three WRKY Genes From Upland Cotton

WRKY transcription factors are a group of inducible transcription factors which are involved in a variety of stress responses. There is a conserved WRKYGQK amino acids sequence in N-terminal of those proteins. WRKY transcription factors, through binding to (T)(T)TGAC(C/T)sequence, regulate the expression of their target genes which contain the W-box elements in the promoter regions. Therefore, the WRKY transcription factors participate in various defense responses, regulate the growth and development in plant and further paly roles in plant growth, plant metabolism and signal transduction. Although reports on Arabidopsis, tobacco and rice WRKY genes are mounting, the reports on cotton WRKY transcription factors are scarce. We aimed to identify the WRKY genes which are relevant to drought and salt resistance, and consequently lay a solid foundation for cotton breeding.In this study, we isolated3novel WRKY genes from upland cotton by in silico cloning and RT-PCR, and analysed their sequences and expression profile. The key results are as follows:1. Based on74WRKY genes cDNA sequences in Arabidopsis thaliana published at NCBI, we preformed basic local alignment search in the database of cotton EST. We assembled the overlapping and homogenous resulting sequences. The search was repeated until the sequences could not be elongated. ORFs were analyzed by ORF Finder. Then we designed primers using primer5.0for PCR amplification. The results of PCR showed that4pairs of those primers could amplify DNA fragments from Gossypium hirsutumL. shannongSZm. Sequencing analysis showed that one of them was GhWRKY2, which was reported. The other three were the new cotton WRKY genes, named GhWRKY4, GhWRKY5, and GhWRKY6. The GenBank ID of GhWRKY4is JQ081265,787bp in length, it contains a758bp ORF encoding a protein of252amino acids. The GenBank ID of GhWRKY5is JQ081266,865bp in length, it contains a522bp ORF encoding a protein of173amino acids. The GenBank ID of GhWRKY6is JQ081267,681bp in length, it contains a681bp ORF encoding a protein of183amino acids.2. The bioinformation analysis showed that all of the3WRKY proteins contain1WRKY domain, with C2H2-type zinc finger. Consequently they all belong to the group II of WRKY family. Forecast the physicochemical property of the three WRKY proteins by Protparam showed that they were all unstable proteins; Subcellular localization analysis by ProtComp Version showed that they were all located in nucleus; hydrophobic analysis by ProtScale showed that they were hydrophilic proteins. It is predicated by Protfun that the WRKY4protein participates in transcription, transcription regulation, and plant growth, the WRKY5protein participates in transcription and the WRKY6protein participates in transcription, transcription regulation, immune response and plant growth.3. We preformed RT-PCR to analyze the expression profile in different tissues. The results showed that the expression of those three WRKY genes reached the peak in leaf. Simulation salt stress and drought stress by using250mmol/LNaCl and20%PEG6000were proformed to detection the expression of GhWRKY4, GhWRKY5and GhWRKY6. Through real time-qPCR, we explored the expression of those genes under salt stress and drought stress. The result showed that the expression of GhWRKY4was induced by both salt stress and drought stress gene regulation, and GhWRKY5was involved only under drought resistance, while the expression of GhWRKY6was not related to salt stress and drought stress.