Functional Analysis Of Three Cotton WRKY Genes And Their Promoters In Gossypiumhirsutum L

Gene expression and regulation is an important part of plant genetic engineering, and the promoter plays a central role of transcriptional regulation, which determining transcription in time and translation in expression intensity. Currently, constitutive promoter is widely used in transgenic crops, and exogenous gene driven by constitutive promoter can express constantly and stably throughout all tissues of plant at every developmental stage, at a high metabolic expense to host plant. So the specific promoter, replacing the constitutive promoter to drive exogenous gene expression in certain time, place and intensity, is the important element of plant genetic engineering.WRKY proteins are a type of proteins which can combine with zinc ions. Most of WRKYs are inducible expression under abiotic and biotic stress. Here, we detected the expression patterns of GhWRKY54, GhWRKY64, and GhWRKY82 in cotton, further isolated their promoters and analyzed their corresponding regulatory elements, respectively. GhWRKY54 promoter includes 15 ROOTMOTIFTAPOX1 and two W-box elements. The transcripts of GhWRKY54 and GhWRKY82 were expressed preferentially in root, while WRKY64 had higher expression levels in roots and leaves. After VD inoculation, drought and salt stress, the transcripts of GhWRKY54 and GhWRKY82 were significantly upregulated post-inoculation, with the highest peak at 144 hours of treatment. GhWRKY64 exhibited higher levels of expression after drought and salt induction, with the highest peak at 10 hours of drought and 2 hours of salt treatments, respectively. The promoters sequences showed that WRKY 64 promoter includes six ROOTMOTIFTAPOX1 cis-acting elements, nine CACTFTPPCA1 cis-acting elements, four W-box, four OSE2ROOTNODULE cis-acting elements, one GT1GMSCAM4 cis-acting elements, and one ABA cis-acting elements. WRKY82 promoter includes 16 ROOTMOTIFTAPOX1 cis-acting elements, five OSE2ROOTNODULE cis-acting elements, two W-box, and six BIHD10S cis-acting elements.Four promoter deletion mutants fused with GUS gene were designed, further four expression vectors were constructed and transformed into tobacco. Expression analysis showed that the promoter of WRKY64 gene specifically express in root of plant, the core promoter of WRKY64 gene was located between-1 and -325bp, which can drive GUS gene specific expression in root. The promoter includes 10 ROOTMOTIFTAPOX1 cis-acting elements and nine CACTFTPPCA1 cis-acting elements, which have highly expression in root and leaf. Further there are four OSE2ROOTNODULE cis-acting elements and two BIHD10S cis-acting elements related to disease response. The WRKY64 promoter was a highly expression in root and leaf which related to disease response. The expression of GUS drived by full-length promoter is higher than other three mutants.