Analysis On Thermotolerance Mechanism Of Lily Regulated By LlWRKY39 Transcription Factor

Lily（Lilium spp.）originates from China and likes cool and shade environment.Lily has a higher ability to withstand low temperature,but less ability to undertake the high temperature.Lily often grows weaken and shortness under temperature above 30℃,which seriously affects the yield and quality of cut flowers.Therefore,it is important to analyze the response mechanism of lily varieties under high temperature,which may contribute to improving the thermotolerance of lily.At present,the studies on thermotolerance of transcription factor（TF）in lily mainly focus on heat stress transcription factors（HSFs）,and research on the response mechanism involved in WRKY TF under high temperature is relatively lacking.In this experiment,’White Heaven’（Longiflorum hybrids）was used as the material to study heat-stress response（HSR）mechanism of lily WRKY TF.The LlWRKY39 was isolated from leaves of ’White Heaven’ and its expression pattern and roles in the regulatory network of HSR were analyzed.The main results are as follows:（1）LlWRKY39 was isolated from the leaves of the lily ’White Heaven’.The open reading frame（ORF）was 858 bp which encoded 285 amino acids.Phylogenetic tree analysis with Arabidopsis thaliana WRKY family revealed that LlWRKY39 was closer to AtWRKY39 and it belonged to WRKY-IId family.Phylogenetic tree analysis with WRKY39 of different monocotyledonous,such as wheat,rice,brachypodium,date,oil palm,pineapple,apple and tomato showed that LlWRKY39 had the highest homology and closest relationship with pineapple AcWRKY39.（2）The LlWRKY39 was localized to the nucleus and had no transcription activation activity in yeast cells.The expression of LlWRKY39 in leaves showed a rising trend after continuous heat stress treatment with 37℃,which indicated that the expression of LlWRKY39 was induced by high temperature.Using the hiTAIL-PCR method,the 737 bp promoter of LlWRKY39 was isolated.Luciferase reporter assay was performed to test the promoter activity of LlWRKY39.The results showed that the activity of the promoter of LlWRKY39 could be activated by high temperature.（3）In order to further verify the biological function of LlWRKY39 under HS,heterologous transformation of Arabidopsis thaliana was performed by inflorescence infiltration.Three transgenic lines（OE-2,OE-4,OE-5）were used for basal thermotolerance（BT）and acquired thermotolerance（AT）treatments.The results showed that the survival rate of the three transgenic lines（OE-2,OE-4,OE-5）was significantly higher than WT in BT and AT,which were about 41.4%,42.6%and 49%higher than WT in BT and 52%,33.9%and 35.1%higher than WT in AT.The expression levels of HS induced genes AtHSFAl,AtHSFA2,AtMBF1c,AtGolSl and AtDREB2C in wild-type and transgenic plants were detected by qRT-PCR.The results showed that the expression of these genes was significantly increased in transgenic lines.Agrobacterium-mediated transient transformation method was used to transform lily.It was found that transient overexpression of LIWRKY39 in lily could significantly reduce its ion leakage under HS to improve the thermotolerance of lily.（4）Transient transformation of Agrobacterium-mediated was used to transform ’White heaven’ to detect the regulation of LlWRKY39 on LIMBF1c in lily.The qRT-PCR results showed that overexpression of LlWRKY39 in lily could activate the expression of LlMBF1c;We isolated the LlMBF1c promoter sequence,which contains W-box elements,then yeast one-hybrid assay and the effector-reporter assay were used to identify the relationship between LlWRKY39 and LlMBF1c.The results exhibited that LlWRKY39 could bind the W-box element on its promoter in yeast cells and LlWRKY39 could significantly activate LlMBF1c promoter activity in tobacco leaves,suggesing that LlWRKY39 may be upstream regulator of LlMBF1c.（5）Based on previous study on the interaction between WRKY-Ⅱd and CaM,LlCaM3 was selected as the candidate protein of LlWRKY39 for interaction detection.Bimolecular fluorescence complementation and the luciferase complementation image assay were used to detect whether LlWRKY39 could interact with LlCaM3.The results showed that an interaction between them exsited.Then,the dual luciferase reporter assay was used to detect the effect of LlCaM3 on the activation of LlMBFlc activated by LlWRKY39.The results show that LlWRKY39-LlCaM3 interaction can inhibit activity of LlMBF1c promoter.