Cloning And Function Analysis Of Senescence Associated WRKY Genes In Cotton

WRKY transcription factor is one of the largest gene families in plant, named according to its consereved WRKY domain. WRKY transcription factors wildly participate in regulating plant growth, such as secondary metabolism, abiotic stress, growth and development, immune response, and leaf senescence process and so on. For short season cotton, premature senescence is the most urgent problem needs to be resolved. According to previous study,WRKY transcription factor is the second largest transcrition families involved in cotton leaf senescence process. Moreover, a single transcription factor could regulate multiple genes expression. Therefore, it has significant research values to study senescence associated WRKY genes in cotton. Identify leaf senescence, fiber quality and abiotic stresses associated WRKY genes in cotton which could lay molecular basis for genetic engineering modifications of cotton cultivars with good fiber quality, high resistance, and without premature senescene.This paper studied cotton WRKY genes and which carried out the following works:1. Totally identified 116 GrWRKY genes from the complete geneome sequence of Gossypium raimondii, according to homology-based cloning methods, 102 GhWRKY genes from G.hirsutum cDNA has been cloned, of which 100 GhWRKY genes achieved NCBI accession numbers. Chromosome location analysis of GrWRKY genes on G.raimondii proved that WRKY genes in G.raimondii increased maily becaused of segmental duplication and followed by tandem amplifications. Microarray, expression profiling and qRT-PCR analysis revealed that WRKY genes in G.hirsutum may be involved in the regulation of fiber development, anthers, tissues(roots, stems, leaves and embryos), and also they are involved in the response to stresses. Expression analysis showed that most group II and III GhWRKY genes are highly expressed under diverse stresses, however, group I members are not sensitive to abiotic stresses. Our results indicate that cotton WRKY genes might have evolved by adaptive duplication.2. To comprehensively study group III WRKY genes in cotton, we analyzed the genome sequences of G.hirsutum, G.raimondii and G.arboreum. According to the three genome sequences, 18 group III GhWRKY genes were identified in G.hirsutum, 12 both in G.raimondii and G.arboreum. The ratios of non-synonymous to synonymous of the GhWRKYto GrWRKY or GaWRKY were lower than 1, which indicated that group III WRKY genes in Gossypium species are under purififying selection. Expression analysis indicated that most group III genes could be induced by Salicylic acid, Jasmonic acid, Ethylene, Abscisic acid,mannitol and NaCl both in roots and cotyledons. Moreover, most group III GhWRKY genes expressed with significant difference during fiber development and leaf senescence process,which lay basis for function analysis of group III WRKY genes in cotton species.3. We characterized GhWRKY27 gene sequence and analyzed expression patterns. In short season cotton cultivars CCRI58 and CCRI64 with premature senescence, GhWRKY27 gene expression significantly increased with the leaf age going; however, in cultivars 12 nian pin bi and 29 tiao-41 which without premature senescence, GhWRKY27 expressed almost without significantly changes; GhWRKY27 expressed with larger fold-change in premature senescence cultivars than cultivars without premature senescence. Tissue specific expression analysis proved that GhWRKY27 has higher expression values in stem, leaf and root in CCRI74 premature senescence cultivar than in CCRI28 which without premature senescence.We constructed over expression vector(pBI121-GhWRKY27) and transformed Arabidopsis thaliana, and also construced virus induced gene silence vecotor(pYL-156-GhWRKY27) and infected cotton seedlings. The three over expression lines of A.thaliana showed earlier senescence than wild type, however, the VIGS cotton seedlings showed delay senescence.Above all, GhWRKY27 could promote leaf senescence, and is a positive regulator of cotton leaf senescence.4. Combined chromatin immunoprecipication(ChIP) method and high-throughput sequences(ChIP-Seq) method to analyze target genes of transcription factor GhWRKY27.We totally identifed 98 target genes, of which 56 target gene promoters contain TGAC or TTGACC/T cis element. We used qPCR to validate the sequence result is reliable. According to gene annotation, we concluded that GhWRKY27 involved in redox, proteolysis,photosynthesis, phosphorylation, and transcriptional regulation associated genes to regulate cotton leaf senescence process.