Activity And Salt Response Regulatory Mechanism Analysis Of Transcription Factor HcSCL13 Promoter From Halostachys Caspica

As a sessile organism,plants face a variety of environmental stresses throughout their life cycles.Among them,salt is one of the adverse environmental factors affecting plant growth and yield.As a plant-specific transcription factor,GRAS plays an important role in plant growth and abiotic stress.When plants are exposed to high concentration of salt stress,the relevant receptors on the plasma membrane sense the signal,and regulate the transcription factors such as GRAS at the promoter level through a series of intracellular signal transduction,thereby regulating the expression of downstream genes and making positive effects on salt stress.Previous studies have found that the Halostachys caspica transcription factor HcSCL13 which belonged to the PAT1 branch of the GRAS family was regulate the growth and salt tolerance of transgenic Arabidopsis.In order to clarify the transcriptional regulation mechanism of HcSCL13 transcription factor,histochemical staining and enzyme activity assay of GUS were used for HcSCL13 gene promoter P_HcSCL13cSCL13 in different developmental stages,different abiotic stresses?salt,cold,drought,damage and light?and the key areas of the activity and salt response of the truncated deletion promoters of different lengths were analyzed.The specific results were as follows:?1?The activity of HcSCL13 promoter in Arabidopsis young seedlings?germination on 1d,3 d,5 d,10 d,12 d and 15 d?and adult seedlings?55 d?were analyzed by GUS histochemical staining.The transgenic Arabidopsis thaliana showed strong GUS activity in both young seedlings and adult seedlings,among which the colour of seedling cotyledon and radical was the strongest,in addition,it showed some activities in various tissues and organs such as roots,stems,leaves,flowers and pods in adult seedings.The above results indicated that HcSCL13 promoter of Halostachys caspica could regulate the expression of downstream genes in the whole growth stage of plants,especially the expression of cotyledons and radicals in young seedlings.?2?Bioinformatics analysis showed that the HcSCL13 promoter has some cis-acting elements related to salt,cold,drought and light stress.Therefore,we performed histochemical staining and enzyme activity analysis of GUS under the above-mentioned stress conditions in Arabidopsis thaliana with the GUS reporter gene coupled to the HcSCL13 promoter.The results showed that the GUS activity of HcSCL13 promoter Arabidopsis thaliana was significantly higher than that of the control group after 3 days of 300 mM NaCl stress.However,the activity of HcSCL13 promoter was not significantly change after treatment with cold and drought stress.And the GUS activity was significantly increased in leaves,petioles and stems after mechanical damage.Under the net light treatment,the Arabidopsis hypocotyls on different germination days had no GUS activity,while under the net darkness?except germination 1 d?and 16 h light/8 h dark photoperiod treatment,the hypocotyls showed a certain GUS activity.The GUS activity was the strongest in the hypocotyls under the 16 h light/8 h dark photoperiod;The Arabidopsis seedlings germinated for 5 d in the dark condition were transferred to the net light for 2 d,the hypocotyls without GUS activity exhibited a certain GUS activity;The cotyledons have a similar light response regulation pattern as the hypocotyls.The radicle of 1 d seedling had the strongest GUS activity under the16 h light/8 h dark photoperiod,and the weakest under the net dark condition;On 3 d,5 d and10 d after germination,the whole root under net light had strong expression of GUS gene.The expression of GUS gene in root crown under net dark condition was significantly stronger than that in meristematic,elongation and mature zone,and GUS gene expression in whole roots was more uniform under 16 h light/8 h dark photoperiod condition.Noticebly,the expression of GUS gene in the whole root increased significantly on the 10th day after seed germination.This result indicated that the P_HcSCL13cSCL13 was responded to salt and damage stress and regulated by photoperiod.?3?To explore the key regions affecting promoter activity and salt response,based on the bioinformatics analysis of the promoter of P_HcSCL13,the HcSCL13 promoter was truncated and deleted into five fragments of different lengths:SP1?-1730 bp?truncated deleted fragment containing anaerobic inductioncis cis-elements?ARE?and associated with defense and emergency response element?TC-rich repeats?;SP2?-1450 bp?truncated deleted fragment containing cis-acting elements associated with anaerobic induction?ARE?and defense and emergency response?TC-rich repeats?;SP3?-1120 bp?truncated deleted fragment containing a salt response-related element?CACG?;SP4?-720 bp?truncated deleted fragment containing a fungal elicitor element?Box-W1?;SP5?-239 bp?truncated deleted fragment containing a salt-responsive correlation?GAAAAA?with a defense and stress response cis-elements?TC-rich repeats?.The truncated deletion promoter sequence was constructed on the plant expression vector pBI121 containing the GUS gene,and the Nicotiana benthamiana leaves were transiently injected and dark treatment for 12 h,then treated plants with 300 mM NaCl for 24 h,then GUS histochemical staining and GUS enzyme activity analysis were performed.The results showed that the promoter fragments of different lengths under normal conditions could initiate the expression of GUS gene,but the expression levels of GUS gene in different lengths were significantly different.The full-length promoter SP was consistent with the staining degree of the truncated deletion SP1 and SP2 promoter,and there was no significant difference in GUS enzyme activity,while the activity of GUS enzyme in truncated deletion promoter fragments SP3 and SP4 were significantly decreased,but the activity of GUS enzyme in SP5 fragment was significantly enhanced.It was speculated that the truncated deletion SP2 and SP3 promoter may contain the key cis-elements that dominated promoter activity,an enhancer element may exist between SP4 and SP5,SP3 and SP5 truncated deleted sequences contain elements that affect promoter activity.After 300 mM NaCl stress treatment,the GUS activity of the full-length promoter SP was significantly increased,and it was able to respond positively to salt stress.However,SP1,SP2,SP3 and SP4 deleted promoter had no significant effect on GUS activity after salt stress.The activity of GUS in SP5 was significantly decreased after salt stress,These results suggested that the HcSCL13 promoter of Halostachys caspica can respond positively to salt stress,and the fragment deleted by SP5?-239 bp?contains salt-related negative regulatory elements.In summary,the HcSCL13 promoter of Halostachys caspica can significantly regulated the growth and development of plant,especially to cotyledon and radical of seedling.In the mean time,the HcSCL13 promoter was regulated by photoperiod and can respond to abiotic stresses such as damage and salt.