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| Cloning and Function Analysis of GhWRKY11 in Cotton (Gossypium hirsutum) |
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Abstract Short season cotton (Gossypium hirsutum) usually accompanies with premature senescence, thus revealing and verifying senescence associated genes in cotton is very important to breed short season cotton cultivars without premature senescence. In this study, a WRKY transcription factor, GhWRKY11 (GenBank accession No. JN604988), was cloned from upland cotton with 1 055 bp cDNA and contained a 1 008 bp open reading frame (ORF), which encoded a putative WRKY group Ⅱd protein with 335 amino acids. Protein sequence phylogenetic analysis showed that GhWRKY11 had high similarity with AtWRKY11. In order to analyze the gene structure, GhWRKY11 was also cloned from genome DNA which contained 1 256 bp (GenBank accession No. JQ522936). Comparison results of GhWRKY11 which were cloned from cDNA and genome DNA revealed that this gene contained 3 exons and 2 introns. Gene sequence of GhWRKY11 was also used to do blast with G. hirsutum genome sequence. The identity of GhWRKY11 with Gh_D07G0023 and Gh_A07G0017 were 100% and 97%, respectively, which indicated that GhWRKY11 was Gh_D07G0023 in genome sequencing work, and Gh_A07G0017 was its homolog. A subcellular localization assay confirmed that GhWRKY11 protein was located only in epidermal cell nucleus of onion (Allium cepa). qRT-PCR for tissue specific expression analysis indicated that GhWRKY11 showed a higher expression level in leaf than that in root, stem and flower. GhWRKY11 had higher expression levels at 2 and 3 week-old cotyledons, and then the expression level decreased with the aging process. At 2 and 3 week-old cotyledons, GhWRKY11 had significantly higher (P＜0.05) expression level in Liao 4086 than that in CCRI10. Also, this gene could be induced by 2 plant senescence inhibitors salicylic acid (SA) and gibberellins (GA3). Compared with the expression level at 0 h, the expression level of GhWRKY11 significantly (P＜0.05) or extremely significantly increased (P＜0.01) after the treatment of GA3 or SA at 2, 4, 8 and 12 h. GhWRKY11 also could be induced by abscisic acid (ABA), jasmonic acid (JA), ethylene (ET) and H2O2 at some specific time points. When the seedlings were treated with JA and ET, expression level of GhWRKY11 significantly increased to the highest expression level at 4 h (P＜0.01), and then the expression level decreased; when the seedlings were treated with H2O2, expression level of GhWRKY11 significantly increased to the highest level at 8 h (P＜0.01). We also detected that GhWRKY11 could be induced by abiotic stresses including low temperature (12 ℃), drought (15% polyethylene glycol (PEG) 6000), salt (200 mmol/L NaCl) and damage. The expression level of GhWRKY11 increased with the time going under the low temperature conditions. Under salt stress, expression level of GhWRKY11 extremely significantly increased (P＜0.01) at 2 and 12 h. GhWRKY11 significantly expressed at 12 h (P＜0.01) when the seedlings were treated with PEG 6000 and it was not sensitive to damage treatment. GhWRKY11 was over-expressed in Arabidopsis thaliana. Compared with wild type Arabidopsis thaliana, the over-expression lines of GhWRKY11 showed delay senescence phenotype after sowing 45 days, which indicated that GhWRKY11 negatively controlled cotton leaf senescence. In this study, cloning and function analysis of GhWRKY11 provided the foundation to create transgenic short season cotton without premature senescent trait.
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Received: 04 December 2015
Published: 01 April 2016
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