Functional Analysis Of The E3 Ubiquitin Ligase HOS1 Gene And Promoter In Vitis Vinifera Under Low Temperature Stress

With the rapid development of the wine industry,the main variety planted is the Vitis vinifera,which has good quality but poor resistance to adversity.Especially for the northern cold viticulture areas,winter buried soil,spring vines will cause damage to the vine body,and seriously affect the quality of grapes.Therefore,it is of great significance to breed cold-resistant grape varieties for cold region viticulture.RING type E3 ubiquitin ligase HOS1 gene is a negative regulator of cold response in Arabidopsis thaliana and can interact with key factors in the cold signaling pathway to regulate cold resistance metabolism.However,the way HOS1 gene participates in cold regulation network in Vitis vinifera is still unclear.In this study,HOS1 gene and its promoter from Vitis vinifera were cloned,and the expression pattern and subcellular localization of VvHOS1 gene involved in low temperature stress,as well as the expression pattern of VvHOS1 promoter involved in low temperature stress response were analyzed,and the regulatory network of the key of VvHOS1 promoter was analyzed by DNA pull down-MS test.The main results are as follows:1.The homology of HOS1 protein was up to 98.77%in Vitis vinifera and Vitis amurensis.HOS1 gene was constitutively expressed in different tissues of both Vitis vinifera and Vitis amurensis.After the two germplasms were subjected to low temperature stress,the expression level of HOS1 gene in Vitis vinifera was downregulated except for 3 h,and the expression level of Vitis amurensis tended to be silent.At room temperature,VvHOS 1 protein was localized in both cytoplasm and nucleus.After 4℃ treatment,the localization of green fluorescent protein VvHOS 1 was observed to migrate from cytoplasm to nucleus.Western blot results showed that the expression of VvHOS1 cytoplasm protein decreased after low temperature treatment,while the expression of VvHOS 1 in nucleus increased.It was proved that VvHOS 1 migrated from cytoplasm to nucleus after cold treatment.In addition,VvHOS1 gene responded to ABA and ethephon exogenous hormone signals.2.Cloning and analysis of HOS1 promoter in Vitis vinifera and Vitis amurensis indicated that 313 bp of HOS1 promoter was missing in Vitis amurensis.Col-0 Arabidopsis thaliana was infected with constructed PVvHOS1,PVaHOS1 and PVvHOSIDE313 fusion vectors by Agrobacteria-mediated flocculant transformation system.After obtaining T3 generation of plants,GUS staining of tissues and organs in different growth stages and analysis of expression patterns under different abiotic stresses were conducted.PVvHOS1 transgenic Arabidopsis thaliana can stain blue throughout the growth cycle,and the stain is the deepest in leaves and the lightest in roots.However,PVaHOS1 did not stain blue before seedling,and the stain is the deepest in leaves and the lightest in roots after seedling.PVvHOS1 staining is always stronger than PVaHOS1 throughout the growth cycle.Moreover,the HOS1 promoter 313 bp deletion fragment in Vitis vinifera did not stain blue in different tissues and under different stress treatments.3.Cloning and cis-acting element analysis of the 313 bp fragment of VvHOS1 promoter revealed that the region contained 2 PREA,1 WBOX,1 MYB cis-acting element and 2 transcription start sites.This region was transferred into Agrobacterium GV3101 receptive state and infected with Col-0 Arabidopsis thaliana.After the T3 generation of plants was obtained,GUS activity analysis was conducted after low temperature stress.The activity of different tissue parts showed no significant staining changes after low temperature treatment,but the staining of leaves and stomata was enhanced after low temperature treatment.GUS activity was analyzed in the whole transgenic Arabidopsis thaliana plant at 3 weeks of age.It was found that GUS expression increased rapidly after 1 h and 6 h of low temperature treatment.The nucleoprotein of ’Chardonnay’ was extracted and verified to be normal by Western blot.At the same time,biotin probe was prepared by 313 bp,and DNA pull down technology was used.Biotin labeled probe was used as the test group and non-biotin labeled probe as the control group.The obtained differential bands were sequenced by MS,and the results showed that there were 341 differential proteins in the biotin labeled probe group,which were mainly divided into 15 categories.There were 3 chromatin/chromatin binding or regulatory proteins and 7 gene transcriptional regulatory factors.