Cloning And Preliminary Function Analysis Of HaWRKY18/29/59/82 Genes In Helianthus Annuus

Sunflower(Helianthus annuus L.)has a strong tolerance to salt and alkali and is one of the most important economic crops in the world.Transcription factors can reconstruct ion balance,osmotic balance and reactive oxygen species balance in plants through regulating Na~+/K~+homeostasis,the synthesis of osmotic protective substances and the activity of antioxidant enzymes,which finally makes plants adapt to salt stress.Salt stress-responsive WRKY transcription factors have been widely studied in many plants,but there is little research on WRKY genes in response to salt stress in sunflower.Based on the bioinformatics analysis and literature data,the sunflower Ha WRKY18,Ha WRKY29,Ha WRKY59 and Ha WRKY82 genes were screened and cloned,and their functions were preliminarily analyzed in this study.The main results are as follows:(1)The Ha WRKY18,Ha WRKY29,Ha WRKY59 and Ha WRKY82 genes were successfully cloned from sunflower.Fluorescence quantitative PCR analysis showed that under 140 m M Na Cl treatment,the expression levels of Ha WRKY18,Ha WRKY29,Ha WRKY59 and Ha WRKY82 genes in sunflower leaves were higher than the initial level at all time points of 0 h,3 h,6 h,12 h and 24 h,besides this,the expression levels of Ha WRKY18,Ha WRKY29,Ha WRKY59 and Ha WRKY82 genes in sunflower roots were higher than the initial level at 2-4 time points,and only a few time points were lower than the initial level.At the early stage of stress,the expressions of Ha WRKY18,Ha WRKY29 and Ha WRKY59 genes in sunflower roots and leaves were in the process of rapid accumulation.The results showed that the four WRKY genes responded to salt stress,and Ha WRKY18,Ha WRKY29 as well as Ha WRKY59 may play an important role in the early salt response process.(2)Subcellular localization analysis showed that Ha WRKY18,Ha WRKY29,Ha WRKY59 and Ha WRKY82 were located in the nucleus.The transcriptional activity analysis showed that Ha WRKY29 and Ha WRKY59 had transcriptional activation activities in yeast cells,while Ha WRKY18 and Ha WRKY82 had no transcriptional activation activities in yeast cells.(3)Agrobacterium with p BI121-Ha WRKY18 recombinant plasmid was transformed into sunflower HA89,and the PCR results showed that Ha WRKY18 was successfully transformed into sunflower and Ha WRKY18 overexpression sunflower was obtained.(4)Ha WRKY18,Ha WRKY29,Ha WRKY59 and Ha WRKY82 were transformed into Arabidopsis by Agrobacterium-mediated method for preliminary functional analysis.Under salt stress,overexpression of Ha WRKY18,Ha WRKY29,Ha WRKY59 and Ha WRKY82 reduced the damage caused by salt stress.Compared with WT,the transgenic plants grew better and the leaves were more stretched.The overexpression of Ha WRKY29 and Ha WRKY59could increase the fresh weight of Arabidopsis;overexpression of Ha WRKY29,Ha WRKY59 and Ha WRKY82 could increase the root length of Arabidopsis;overexpression of four WRKY genes resulted in higher chlorophyll content in Arabidopsis under salt stress.(5)Yeast two-hybrid analysis showed that,in yeast cells,Ha WRKY59interacted with Ha MKS1,Ha MKS1 interacted with Ha MAPK11-1 and Ha WRKY59 did not interact with Ha MAPK11-1.Ha WRKY59,Ha MKS1 and Ha MAPK11-1 might form ternary complexes.(6)Fluorescence quantitative PCR analysis showed that compared with WT,the expression levels of salt stress response marker genes At SOS1,At P5CS1,At CAT2 and At NCED3 in OE-Ha WRKY18 were significantly up-regulated by more than twofold,indicating that Ha WRKY18 may play an important role in maintaining ion balance,osmotic balance,ROS balance and the regulation of ABA signal pathway;the expression level of At NCED3 gene in OE-Ha WRKY29 was significantly up-regulated by more than 2.6 times,indicating that Ha WRKY29 may be involved in ABA signal transduction pathway;the expression levels of At P5CS1 and At NCED3 genes in OE-Ha WRKY59 were significantly up-regulated by more than 4 times,this suggests that Ha WRKY59 may be involved in the synthesis of osmotic protective substances and ABA signal transduction pathway;the expression levels of At P5CS1 and At NCED3 in OE-Ha WRKY82 were significantly down-regulated to less than 0.3 times of WT.This suggests that Ha WRKY82may be involved in the negative regulation of the synthesis of osmotic protective substances and ABA signal transduction pathway.(7)The possible binding DNA sequences of Ha WRKY29 were screened by yeast single hybridization,and"CCCAATCTCCAATGG"contained CCAATBOX1 element;"CCCAATCTCCAATGGG"contains GTGANTG10elements;"CCCTGACTGTTTTGG"contains W-BOX element.