Cloning And Functional Analysis Of GhWRKY27/42/91 From Gossypium Hirsutum L

Cotton is an important economic crop in China,which plays an important role in the development of national economy and society.Premature senescence and abiotic stress are important factors that limit the yield and quality of cotton.WRKY transcription factors play an important role in plant leaf senescence and abiotic stress responses,but little is known about the WRKY transcription factors involved in regulating cotton leaf senescence and abiotic stress.Previous studies have reported the whole genome analysis of the WRKY gene family in cotton.Based on the published expression profile data of leaf senescence and various stress treatments,we identified that the expression of GhWRKY27/42/91 genes was up-regulated during leaf senescence and abiotic stress.The purpose of this study was to clone GhWRKY27/42/91 genes from cotton and analyze their function roles.The main contents and conclusions are as follows:1.GhWRKY27,an aging up-regulated gene,promoted leaf senescence in transgenic Arabidopsis,as determined by reduced chlorophyll content and elevated expression of senescence-associated genes(SAGs).Transcriptional activation assays indicated that GhWRKY27 has no transcriptional activation activity in yeast cells.Yeast two-hybrid(Y2H)library screening identified 67 potential interaction proteins of GhWRKY27.Y2 H and bimolecular fluorescence complementation(BiFC)assays showed that GhWRKY27 interacted with the potential interaction protein MYB transcription factor GhTT2.Yeast one-hybrid(Y1H)assay and electrophoretic mobility shift assay(EMSA)revealed that GhWRKY27 binds directly to the promoters of cytochrome P450 94C1(GhCYP94C1)and ripening-related protein 2(GhRipen2-2).Transient dual-luciferase reporter assay indicated that GhWRKY27 could activate the expression of GhCYP94C1 and GhRipen2-2.In addition,the expression patterns of GhTT2,GhCYP94C1 and GhRipen2-2 were examined during leaf senescence.2.The GhWRKY42 gene was isolated from cotton variety CCRI10.Multiple sequence alignment and evolutionary analysis indicated that GhWRKY42 belongs to group IId subfamily.Quantitative real-time PCR(qRT-PCR)analysis showed that GhWRKY42 was predominantly expressed in roots,stems and leaves;it was up-regulated during leaf senescence;it was induced after exposure to abscisic acid(ABA),methyl jasmonate(MeJA),PEG6000 and NaCl stresses.A transactivation assay in yeast demonstrated that GhWRKY42 had no transcriptional activation activity.A GUS activity assay revealed that the promoter of GhWRKY42 showed fragment deletion activity in Nicotiana tabacum.In addition,GUS expression was mainly expressed in the roots,stems and leaves,and was induced by mannitol and MeJA treatments in ProGhWRKY42::GUS transgenic Arabidopsis plants.Constitutive expression of GhWRKY42 in Arabidopsis led to a premature aging phenotype,which was correlated with an increased number of senescent leaves,reduced chlorophyll content and elevated expression of SAGs.In addition,virus-induced gene silencing(VIGS)was used to silence the endogenous GhWRKY42 gene in cotton,and this silencing reduced plant height.3.The GhWRKY91 gene was cloned from cotton variety CCRI10.The gene structure,amino acid sequence and evolutionary tree were studied.qRT-PCR analysis showed that GhWRKY91 was predominantly expressed in leaves,pistils,petals and sepals;it was induced by leaf senescence and highly expressed in early-aging cotton variety than non-early-aging cotton variety;it was down-regulated after exposure to ABA and PEG6000 stresses.Transcriptional activation assays indicate that GhWRKY91 had transcriptional activation activity.GUS activity driven by the GhWRKY91 promoter in transgenic Arabidopsis was expressed in roots,stems and leaves and reduced upon exposure to ABA and drought treatments.Constitutive expression of GhWRKY91 in Arabidopsis caused small type plants and delayed natural leaf senescence.The GhWRKY91 transgenic plants exhibited increased drought tolerance and presented delayed drought-induced leaf senescence,which were accompanied by the reinforced expression of stress-related genes and the attenuated expression of SAGs.The VIGS plants of GhWRKY91 reduced drought tolerance.Y1 H and EMSA assays revealed that GhWRKY91 directly targets the promoter of GhWRKY17 and activates the expression of GhWRKY17 in the transient dual-luciferase reporter system.