| Sunflower(Helianthus annuus L.)is one of the important cash crops and a pioneer crop for improving saline alkali land.As one of the largest transcription factor families in plants,WRKY transcription factor family plays an important biological function in plant abiotic stress response.In this study,sunflower WRKY transcription factor genes HaWRKY9,HaWRKY14,HaWRKY22,HaWRKY30 and HaWRKY79 were cloned and their functions were preliminarily analyzed.The main results are as follows:(1)The salt tolerance of seven sunflower varieties at seedling stage was compared and analyzed by PCA.Among the seven sunflower varieties analyzed,the three varieties least affected by salt stress are Longkuiza 10,Keyang 1 and NK175,and the three varieties most affected by salt stress are 17 hybrid,Neikuiza 4 and Longkuiza 6.Under salt stress,NK175 showed stronger salt stress response ability than Longkuiza 6 and 17 hybrids.(2)The sunflower WRKY transcription factor genes HaWRKY9,HaWRKY14,HaWRKY22,HaWRKY30 and HaWRKY79 were successfully cloned.Fluorescence quantitative PCR analysis showed that HaWRKY9,HaWRKY14,HaWRKY22,HaWRKY30 and HaWRKY79 genes of sunflower could participate in salt stress response.Under salt stress,HaWRKY9,HaWRKY14,HaWRKY22 in sunflower roots play a role in the early stage of stress.Under salt stress,HaWRKY9 and HaWRKY79 in sunflower leaves play a role in the late stage of stress,and HaWRKY30 plays an important role in the early stage of salt stress.(3)Subcellular localization analysis showed that HaWRKY9,HaWRKY14,HaWRKY22,HaWRKY30 and HaWRKY79 were located in the nucleus.Transcriptional activation activity analysis showed that HaWRKY22 and HaWRKY30 had transcriptional activation activity in yeast cells,while HaWRKY9,HaWRKY14 and HaWRKY79 had no transcriptional activation activity.(4)HaWRKY9,HaWRKY14,HaWRKY22 and HaWRKY79 genes were transformed into Arabidopsis by flower dipping method.The overexpression of HaWRKY9,HaWRKY14,HaWRKY22 and HaWRKY79 in Arabidopsis improved the salt tolerance of transgenic Arabidopsis plants.Under salt stress,the overexpression of HaWRKY9 can increase the root length,chlorophyll a and chlorophyll b content of Arabidopsis,the overexpression of HaWRKY14 can increase the root length and fresh weight of Arabidopsis,and the overexpression of HaWRKY22 and HaWRKY79 can promote the root length,fresh weight and the content of chlorophyll a and chlorophyll b.(5)Using yeast two-hybrid technology,it was found that HaWRKY9 could interact with Ha MKS1 in yeast cells,and Ha MKS1 could interact with Ha MAPK11-1,but Ha MAPK11-1 did not interact with HaWRKY9.(6)Overexpression of HaWRKY9 significantly increased the expression of At CAT2 by more than 1.5 times,which may play a role in regulating the balance of reactive oxygen species.Overexpression of HaWRKY14 significantly reduced the expression of At P5CS1 by more than 2 times,the expression of At SOS1 by about 2 times and the expression of At NCED3 by about 2 times,which may be involved in the regulation of osmotic balance,ion balance and ABA signal pathway.Overexpression of HaWRKY22 can significantly increase the expression of At CAT2 by more than 2 times and the expression of At SOS1 by more than 1.5 times,and decrease the expression of At NCED3 by about 2 times.It may play an important role in regulating reactive oxygen species balance,ion balance and ABA signaling pathway.(7)HaWRKY22,HaWRKY30 and HaWRKY79 can bind sequences containing CCAATBOX1 promoter elements.In addition,HaWRKY79 can also bind sequences containing CACTFTPPCA1 elements and "CCCCTCTCGTCCTGGG" sequences that may contain new elements.The salt responsive coexpression genes containing these elements in the promoter may be the candidate target genes of these WRKY transcription factors. |
