Study On The Role Of Cotton(Gossypium Hirsutum)Transcription Factors GhWRKY33 And GhERF108

As the important source of natural fibers in the textile industry,cotton fiber quality and yield are often restricted to abiotic stresses,especiallly drought conditions because more than half of cotton plants in the world grow in the regions with water shortage.WRKY transcription factors regulate multiple plant physiological processes,including drought stress response.However,little is known of how the WRKY genes respond to drought stress in cotton.Our previous study revealed that GhWRKY33 is leaf-specific and induced by drought stress and ABA treatment,and based on this clue,we studied how GhWRKY33 participates in drought stress and ABA signal transduction.Cotton fiber which has important economic value in modern industrial and agricultural fields develops from ovule epidermal cells.As the model cells that grow fastest and synthesis the most cellulose in higher plants,fiber cells develop through five independent and overlapping period,including the initiation of cotton fiber(0-3 DPA),cotton fiber elongation(3-16 DPA),the primary wall to the secondary wall transition(16-20 DPA),secondary wall thickening(20-40 DPA)and maturation(40-50 DPA)five stages.In addition,this paper preliminarily identified a transcription factor,GhERF108 from ERF family,that may play a role in secondary wall thickening.The main results are as follows:1.GhWRKY33 is a typical transcription factorWe transferred the 35S:GhWRKY33:GFP construct into Arabidopsis to generate the transgenic plants,and we observed GFP signal in hypocotyls cell nucleus of transgenic seedlings,suggesting that GhWRKY33 is a nuclear-localized protein.We also verified that GhWRKY33 recognize and bind W-box element by yeast one-hybrid system.Both suggest that GhWRKY33 is a typical transcription factor.2.Overexpression of GhWRKY33 enhances transgenic plant sensitivity to drought stressTo further investigate the function of GhWRKY33,GhWRKY33 was introduced to ectopically express in Arabidopsis thaliana.Statistical analysis indicated that seed germination rate of transgenic Arabidopsis was lower than wild type in the presence of mannitol.Additionally,the root elongation assay revealed that the roots of transgenic lines were shorter that wild type in the presence of mannitol.GhWRKY33 overexpression transgenic plants appeared to be yellowing and more withered than wild type controls which were still green when kept away from water.The experimental results revealed that total chlorophyll content and proline accumulation in leaves of the transgenic lines were as same high as wild type under normal conditions,but much lower than wild type under drought stress.Stomatal movement assay and water loss rate assay also implied that overexpression of GhWRKY33 enhances transgenic Arabidopsis sensitivity to drought stress.3.Overexpression of GhWRKY33 enhances transgenic plant tolerance to ABATo investigate whether GhWRKY33 involve in ABA signaling pathway,transgenic Arabidopsis and wild type were grown on MS medium with different concentration of ABA.Statistical analysis indicated that seed germination rate of transgenic Arabidopsis was higher than wild type in the presence of ABA.Additionally,the root elongation assay revealed that the roots of transgenic lines were longer than wild type in the presence of ABA.Stomatal movement assay revealed that the ratio of stomatal length to width in the transgenic lines was lower than that in wild type.All these results suggest that overexpression of GhWRKY33 enhances transgenic plant tolerance to ABA.4.GhWRKY33 might regulate stress-related genes in transgenic ArabidopsisWe analyzed expressions of several drought-related and ABA-responsive genes(such as RD29A,DREB2A,ERD15,SOS2,ABIl and RAB18)in GhWRKY33 overexpression transgenic plants through qRT-PCR.The results indicated that there were significant difference of the expression levels of these stress-related genes.It suggested that GhWRKY33 might regulate the expression of these marker genes to involve in drought stress response and ABA signaling pathway.5.Identification of GhERF108 and tissue expression level analysisWe identified GhERF108 of AP2/ERF transcript factors from cotton cDNA library.The GhERF108 gene contains a complete ORF of 879bp that encodes a protein with 292 amino acids.With only one AP2 conserved domain whose 14th and 19th amino acid were Glycine(A)and Aspartic acid(D)and the results of further homology analysis,GhERF108 was classified into ERF family.Tissue expression level analysis reveals that GhERF108 has high expression level in fibers and has the highest expression in 25DPA fiber.Therefore,we speculate that GhERF108 may play an important role in secondary wall thickening phase.6.GhERF108 functions as a typical transcription factorWe transferred the 35S:GhERF108:GFP construct into Arabidopsis to generate the transgenic plants,and we observed GFP signal in hypocotyls cell nucleus of transgenic seedlings,suggesting that GhERF108 is a nuclear-localized protein.Yeast transcriptional self-activation experiments revealed that GhERF108 could activate the expression of LacZ,Ade and His.Both suggest that GhERF108 is a typical transcription factor.7.Overexpression of GhERF108 Promotes secondary cell wall synthesis in transgenic ArabidopsisWhen paraffin sections of the stems of 40d's wild type and GhERF108 overexpressing transgenic Arabidopsis were observed under UV channel,it was found that GhERF108 overexpressing transgenic Arabidopsis slices had stronger fluorescence than the wild type in the epidermis,and this might be due to ectopic deposition of lignin in the transgenic Arabidaopsis.The stained slices were observed,and it was found that the interfascicular fiber cell wall of the over-expression line was deeper than wild type,and the cell wall thickness was thicker than wild type.Further statistical analysis confirmed the observations.8.Phenotypic analysis of GhERF108 RNAi transgenic cotton.We transferred the 35S:GhWRKY33 and 35S:GhERF108RNAi constructs into cotton respectively to generate the transgenic plants by Agrobacterium mediated transformation method.Then we extracted gDNA from cotton leaves to identify positive plants through Polymerase Chain Reaction(PCR).The stained paraffin sections of wild-type and GhERF108 RNAi transgenic cotton 25DPA fibers were observed,it was found that 25DPA fiber cell wall of GhERF108 RNAi transgenic cotton was thinner and the length of the mature fibers of GhERF108 RNAi transgenic cotton was shorter than wild type cotton.The expression of Marker gene related to ethylene synthesis in GhERF108 RNAi transgenic cotton and wild type was detected.The expression of AC03,AC04 and ACO5 was significantly higher than that in the wild type.