Molecular Cloning And Function Analysis Of GhWRKY11Gene In Cotton (Gossypium Hirsutum L.)

Leaf senescence, constituting the terminal stage of leaf development, is a type of programmed celldeath (PCD).Leaf senescence is regulated by both intrinsic and environmental signals. Expressionprofiling revealed that senescence is accompanied by extensive changes in gene expression with12-16%of the Arabidopsis genes up-or down-regulated. Massive changes in the transcriptome implyan important role of transcription factors. NAC and WRKY transcription factors constitute the twolargest groups of transcription factors in the senescence transcriptome, and members of these twotranscription factor families have been shown to play central roles in regulating senescence in wheat andArabidopsis.WRKY transcription factors are one of the largest families of transcriptional regulators inplants. The defining feature of WRKY transcription factors is their DNA binding domain. This is calledthe WRKY domain after the almost invariant WRKY amino acid sequence at the N-terminus. TheWRKY domain is about60residues in length, as well as containing the WRKY signature it also has anatypical zinc-finger structure at the C-terminus.Most members of WRKY family have been implicatedin transcriptional reprogramming linked with the plant immune responses where they serve as centralcomponents of the innate immune system. Additionally, specific WRKY proteins have beendemonstrated to be involved in cold, heat, drought and salt tolerance thereby indicating that the WRKYweb of signaling encompasses both biotic stress and abiotic stress responses. Further, WRKYtranscription factors are also involved in regulating the complex hormonal crosstalk during senescencein different plants. In the present study, a WRKY gene GhWRKY11, was cloned from cotton(Gossypium hirsutum L.) and encoding a putative group IIc WRKY protein. Subcellular localizationassay was used to confirm where it is targeted in. Quantitative RT-PCR was used to confirm itsexpression. Ectopic over-expression in Arabidopsis was used to confirm its functions.1、 Obtaining A WRKY transcription factor EST from the natural SAGs in cotton, in silico cloningand PCR was performed to get the full length of the gene；2、 Based on the primary amino acid sequence of the single WRKY domain that GhWRKY11possesses,it has been assigned to group IId of WRKY transcription factors. The putative WRKY clone fromcotton exhibits high sequence similarity to Arabidopsis thaliana AtWRKY11, so the novel cottonWRKY gene was designated as GhWRKY11；3、 Three predicted NLS sequence were uncovered using the PSORT program and nuclear localizationof GhWRKY11has been established by making a GhWRKY11::GFP fusion protein anddemonstrating it was localized to the nucleus. These results proved that GhWRKY11mightfunction in the nucleus;4、 Tissue-specific expression indicated that GhWRKY11gene had different expression patterns atdifferent development stages. During the seedling stage，whatmore，GhWRKY11might be involvedin complex signaling pathways and play an important role in plant defense responses mediated byphytohormones and the GhWRKY11might be involved in regulating plant responses to drought andlow temperature stress conditions;5、 Cloning2000bp promoter sequence,the1800-bp promoter fragment was transcriptionally fused to the promoterless reporter gene-glucuronidase (GUS)(P GhWRKY11::GUS) and GhWRKY11promoter can activate the expression of GUS6、 Ectopic over-expression of GhWRKY11in Arabidopsis results in delay leaf senescence...