Cloning And Expression Analysis Of SSWRKY28 And SSWRKY76 Genes In Sorghum Dochna

The increasing lack of water resources means that drought has become a major restricting factor of plant growth and development.Consequently,Sorghum dochna has garnered attention as a high quality energy plant due to its strong drought resistance,high yield,and rich sugar content.Most studies on Sorghum dochna are on the physiological and biochemical levels,while molecular level studies were few and far between.WRKY?N-terminal contains WRKYGQK highly conserved amino acid sequence?transcription factors are involved in regulating plant growth and development,biological and abiotic stress responses,and many other processes.The present study cloned the Sorghum dochna WRKY gene to study the drought resistance mechanism of Sorghum dochna on the molecular level.Sorghum dochna cultivar Liaotian 1 was used as experimental material,0%,10%and 20% PEG6000 solutions were used to simulate different levels of drought stress.Stress durations were set at 12h,24 h and 48h.RT-PCR was used to clone two Sorghum dochna WRKY genes into full-length cDNA,named SSWRKY28 and SSWRKY76.The results of bioinformatics analysis showed that proteins encoded by the two genes have the core sequence"WRKYGQK"and"C-X4-5-C-X22-23-H-X1-H"zinc finger structure.The total length of SSWRKY28 cDNA sequence is 1179 bp,including a 1029 bp open reading frame that encodes 342 amino acids,the corresponding protein molecular formula is C₁₅₉₂H₂₅₇₉N₄₉₅O₄₉₉S₁₀,with a molecular weight of 36958.61,and the protein's isoelectric point?PI?is 9.38.The full length of the SSWRKY76 cDNA sequence is 1012 bp,including a 984bp open reading frame that encodes 327 amino acids,the corresponding protein molecular formula is C₁₄₇₁H₂₃₉₄N₄₄₂O₅₀₁S₁₃,with a predicted molecular weight of 234704.63,and the protein's isoelectric point?PI?is 6.46.Hydrophobicity tests showed both proteins to be hydrophilic.Sequence homology comparison and phylogenetic tree analysis showed that SSWRKY28 protein was closely related to Sorghum,corn and millet WRKY protein,and SSWRKY76protein was closely related to sorghum,Dichanthelium oligosanthes and corn WRKY protein.Real time-PCR analysis showed that with prolonged drought stress,Sorghum dochna SSWRKY28 and SSWRKY76 genes were down regulated from 0 h to 24 h,and up-regulated after 24 h,indicating their possible roles in drought response.Recombinant SSWRKY28::pHBT-GFP Arabidopsis protoplast expression system was constructed for protein subcellular localization analysis,observations were made using confocal laser microscopy.The results showed that the SSWRKY28 protein is located in the cytoplasm.