Cloning And Functional Analysis Of Salt And Drought Resistance Of The LpWRKY49 Gene In Lilium Pumilum

Abiotic environmental factors have increasingly affected plant growth in recent years,and salinity and drought stress are the two of the most serious environmental factors that hinder normal plant growth and development,and WRKY transcription factors play an important regulatory role in plant response to abiotic stresses.Lilium pumilum has strong resistance to disease,heat,cold and salinity,and has both edible,medicinal and ornamental functions,making it an excellent germplasm resource of the Lilium.In this study,a WRKY transcription factor family gene,named LpWRKY49（Gen Bank ID:OQ623131）,was screened from the salt stress transcriptome of L.pumilum,and cloning of LpWRKY49 gene by homologous recombination method and bioinformatics analysis of it;its promoter sequence was cloned by FPNI-PCR technology and analyzed;the LpWRKY49 overexpression vector was constructed and transformed into tobacco by Agrobacterium-mediated leaf disc method.The transgenic tobacco was analyzed for salt and drought tolerance to further elaborate the mechanism of LpWRKY49 in plant response to salt and drought stress.This research provides a theoretical basis for breeding new resistant lily varieties and improves the reference for further research on the role of WRKY transcription factors in plant response to abiotic stresses.The main findings of this experiment are as follows:1.The cloned LpWRKY49 gene belongs to the WRKY transcription factor family.It has an open reading frame（ORF）length of 990 bp,encoding 329 amino acids..Its protein is hydrophilic,lacking transmembrane structures and signal peptides,but contains multiple phosphorylation sites.lt is localized in the cell nucleus..The highset homology of the LpWRKY49 gene was 55.85%with Elaeis guineensis,and it had the colsest evolutionary relationship with a homologous gene in Asparagus officinalis.Its promoter contains phytohormones,light-responsive and stress-responsive elements in addition to transcriptional core elements.2.Overexpression of LpWRKY49 gene enhanced the salt tolerance of transgenic tobacco.Under salt stress,the seed germination of transgenic tobacco was higher than that of the control.The net photosynthetic rate（Pn）,stomatal conductance（Gs）,intercellular CO₂concentration（Ci）,transpiration rate（Tr）,chlorophyll content,maximum photochemical quantum yield of PS II（Fv/Fm）,and antioxidant enzyme activities（SOD,POD,CAT）and proline（Pro）content were higher in the transgenic tobacco than in wild-type tobacco（WT）and turning empty carrier tobacco（EV）.However,the malondialdehyde（MDA）content,relative conductivity,hydrogen peroxide（H₂O₂）and superoxide anion（O₂·^-）content were lower than those of WT and EV.DAB and NBT chemical tissue staining results indicated that tobacco overexpressing LpWRKY49 gene accumulated less H₂O₂and O₂·^-.Under salt stress,the relative expression of salt tolerance-related genes,including Nt SOD,Nt POD,Nt CAT,Nt NHX1,and Nt HKT1,were up-regulated.3.Overexpression of LpWRKY49 gene enhanced the drought tolerance of transgenic tobacco.Under drought stress,the seed germination rate of transgenic tobacco was higher than that of the control,and its seedlings showed higher Pn,Gs,Ci,Tr,chlorophyll content,Fv/Fm,and SOD,POD,CAT activity and Pro content than those of WT and EV,but lower MDA content,relative conductivity,H₂O₂and O₂·^-content than those of WT and EV.DAB and NBT chemical tissue staining showed that tobacco overexpressing the LpWRKY49 gene accumulated less H₂O₂and O₂·^-under drought stress.The relative expressions of antioxidant enzyme genes,including Nt SOD,Nt POD,Nt CAT,as well as Nt Sn RK2.2 and Nt NCED3,were up-regulated under drought stress.