Analysis Of Cold Resistance Function Of VaSAP15 Gene In Vitis Amurensis

The northwest of China is one of the main grape production areas,and the severe cold in winter is an important environmental factor restricting the development of grape production.China has rich wild grapes resources,among which Vitis amurensis is one of the species with the strongest cold resistance.It is of great significance to explore the cold resistance genes for the improvement of grape quality and yield.SAP15 was selected from the cold stress transcriptome database of Vitis amurensis‘Shuangyou’and Vitis vinifera‘Red Globe’which was obviously up-regulated expressed.Then we started the work from two aspects:（1）Exploring SAPs from grapes’genome,analyzing its structure,physical and chemical properties,and the expression pattern under adverse circumstance;（2）VaSAP15 was cloned from Vitis amurensis‘Shuangyou’,and its cold resistance function was analyzed by subcellular localization,tissue and organ analysis and low temperature treatment in transgenic plants.The main results were as follows:1.A total of 15 members of SAP family were identified from grape genome,distributing on 9 chromosomes of grape.According to their positions on chromosomes and conserved structural domains,they were named Vv SAP1-Vv SAP15 respectively.Phylogenetic tree analysis showed that Vv SAPs could be divided into three subfamilies with similar conserved domains.The expression patterns of Vv SAPs under biological rhythm and stress were analyzed,and the results showed that except Vv SAP1 and Vv SAP9 were pseudogenes with no expression,Vv SAPs all responded to ABA treatment,and Vv SAP15showed the most significant up-regulated expression in response to low temperature.2.The CDS sequence of VaSAP15 was cloned by q RT-PCR from‘Shuangyou’（Vitis amurensis）,with a length of 567 bp,encoding 189 amino acids and two AN1 conserved domains.Sequence comparison analysis showed that VaSAP15 was 97.4%similar to the homology sequence in‘Pinot Noir’.The subcellular localization analysis showed that VaSAP15 was located in the nucleus and cell membrane.Tissue analysis showed that SAP15 had different expression specificity in different varieties:it was highly expressed in the root of‘Shuangyou’and in the stem of‘Red Globe’.In order to verify the cold resistance function of VaSAP15,we transformed the agrobacterium with VaSAP15overexpression vector into‘Thompson Seedless’.Under low temperature stress,leaves of wild-type‘Thompson Seedless’wilted earlier than transgenic lines,and the content of electrical conductivity and malondialdehyde were significantly higher than transgenic lines,but the activity of three antioxidant enzymes SOD,POD and CAT was lower.Real-time quantitative PCR analysis of cold-related genes showed that the overexpression of VaSAP15significantly up-regulated the expressions of MYB14,MYB306 and U-Box54,while the expressions of Los1,DREB1E and ERF025 were significantly down-regulated.3.SAP15 promoters were cloned from the genomic DNA of Vitis vinifera‘Red Globe’and Vitis amurensis‘Shuangyou’:P_{Vv SAP15} and P_VaSAP15.Cis-elements in the promoters related to adversity were noticed,like ABRE,MBS,etc.And there were difference in the cis-elements between two promoters.Accordingly,fragments of promoters were cloned,and along with the whole promoters,had been constructed to GUS fusion vector.By employing the agrobacterium GV3101,they were instantaneously transformed into Nicotiana benthamiana’s leaf.After 4℃treatment,quantitative analysis of GUS protein activity was carried out.Results showed that P_{Vv SAP15} and P_VaSAP15 were both induced by 4℃low temperature.By comparision,the GUS expression of P_VaSAP15 was stronger,and the expression of the first fragment was stronger than the total promoter,so it was predicted that there were some inhibitions in this missing parts.Moreover,the difference in the activity and cis-elements between P_{Vv SAP15} and P_VaSAP15 may be one of the important factors contributing to the difference in expression under low temperature stress.