关键词: 葡萄, SAP家族, 生物节律, 激素, 非生物胁迫, 表达分析

Abstract:

【Objective】The aim of this paper was to identify the Stress Associated Protein (SAP) family genes of grape (Vitis vinifera L.) genome and to know the profile of SAP family regarding the number and structure of genes. The biological rhythm of SAP family and its expression pattern under different hormones and abiotic stress were analyzed by qRT-PCR. The study would provide theoretical basis for further exploring the roles of SAP family genes in grapevine. 【Method】SAP15, a gene selected from the transcriptome database of Vitis amurensis cv. ‘Shuangyou’ and Vitis vinifera cv. ‘Red Globe’, had a significantly up-regulated expression under the cold treatment. According to the conserved structural domains, the SAPs in grape genome-wide were identified by BLAST of NCBI and grape genome database. DNAMAN5.0, MEME, GSDS2.0, ExPASy and MEGA6 software were used for various bioinformatics analysis of VvSAPs, including the study of nucleotide sequences, gene structure, protein structure, physical and chemical properties, chromosome localization and phylogeny. Quantitative real-time PCR (qRT-PCR) was employed to detect the circadian rhythm of VvSAPs and their relative expression levels under hormone and abiotic stress.【Result】In total, 15 SAPs with conservative domains of AN1 were identified from grape genome and most of them contained A20 conservative domain. They could be classified into Class Ⅰ, Class Ⅱ and Class Ⅲ based on their conservative structure domain and the location on chromosome in grape. Analysis of physical and chemical properties of VvSAP family showed that the numbers of amino acids were between 109 and 293, and the theoretical equivalence points were between 7.99 and 9.68. Localization of genes on the chromosome revealed that 15 VvSAPs were distributed on 9 chromosomes of the grape, and there were 3 SAPs on chromosome 8. By the sub-cellular location analysis, we noticed that VvSAPs were mainly expressed in nucleus, chloroplast and cytoskeleton in grape. The secondary structure of VvSAP were mainly irregular crimp and α-helix. The gene structures were highly conserved, and VvSAP1-VvSAP12 had no intron, while VvSAP13-VvSAP15 contained one intron. The results of qRT-PCR analysis showed that VvSAP1 and VvSAP9 had extremely low expression or no expression under hormone and abiotic treatment, so they were preliminarily identified as pseudogenes. While VvSAP10 was proved to be down-regulated under 400 mmol·L ^-1NaCl treatment, the remaining genes were up-regulated under 50 μmol·L ^-1 ABA, 100 μmol·L ^-1SA and 400 mmol·L ^-1NaCl treatment, respectively. VvSAP10-VvSAP14 were highly responsive to 50 μmol·L ^-1ABA treatment, and their relative expressions were improved by 37.19, 36.63, 21.69, 58.34 and 267.35 times, respectively, after 24 hours treatment. After 4 hours under NaCl treatment, VvSAP8 and VvSAP11 had the highest relative expression level, which were 13.16 and 12.42 times higher than those without NaCl treatment, respectively. The VvSAP15 had the highest response towards 4℃ treatment, and its relative expression level was 35.90 times after 8 hours treatment comparing to the 0 hour.【Conclusion】15 VvSAPs family were identified from the grape genome, of which VvSAP1 and VvSAP9 were preliminarily identified as pseudogenes. These members were localized across 9 chromosomes of the grape, and the evolution of VvSAP was highly conservative. All members of VvSAP family responded to adversity and had circadian rhythm, However, the expression pattern of VvSAPs was varied under different stress.

Key words: grape, SAP family, circadian rhythm, hormone, abiotic stress, expression analysis