Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (14): 2500-2514.doi: 10.3864/j.issn.0578-1752.2019.14.009

• HORTICULTURE • Previous Articles     Next Articles

Genome-Wide Identification and Expression Analysis of SAP Family in Grape

DING Lan,GU Bao,LI PeiYing,SHU Xin,ZHANG JianXia()   

  1. College of Horticulture, Northwest Agriculture and Forestry University/State Key Laboratory of Crop Stress Biology in Arid Areas Yangling 712100, Shaanxi
  • Received:2019-02-25 Accepted:2019-05-07 Online:2019-07-16 Published:2019-07-26
  • Contact: JianXia ZHANG E-mail:zhangjx666@126.com

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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

Table 1

qRT-PCR primers for expression on analysis of VvSAP"

基因Gene 上游引物（5′-3′）Forward primer（5′-3′） 下游引物（5′-3′） Reverse primer（5′-3′）
VvSAP1 TCCACGAAGAAGTCGGAGCA GCAAACGATGCTTCCCACAG
VvSAP2 TGAGCTGCAACAAGAAGGTG TCAGCCTTAACCACCGGATT
VvSAP3 ATTGTCGATGCGGTCACCTT CCTTGACAACAGGGTTGGCT
VvSAP4 CTGCAATTCTCTGCGCCAAT CTTCCTCGATTCCCGAACC
VvSAP5 GCGGCCAATGAAATCACTGTT ACAGAACGTGATCCCACACC
VvSAP6 AGTCCAAGGTCAAGGAGGGA TGCAGCAGTCCGGTAATCAA
VvSAP7 TTCTGCGCTGAGCATCGGTA CAGACTCTCACGATCTTCGCA
VvSAP8 TTGTGCTTCACCAAGCGAGA CGGTCCTGTAGTCGAATGGG
VvSAP9 GGTGTGGGAGTATGTTCT CATAACCTTCCTGGTAACTTC
VvSAP10 TTGTTACCTCCATGACTCAATTATC ACATACTTCCACATCGGCACTT
VvSAP11 CATGAGCTGCAACAAGAAGG AGCCTTAACCACCGGATTAG
VvSAP12 CCGAAACTCTAACCCTATGCG TTCACCTCCCTCCTTCTCACA
VvSAP13 GAGGGAGGTGAACTGGTGCT GTCCTGCCGCCTTGTAATCG
VvSAP14 GATGGAGTGGGACAGAGCAG CTTACAGCAGCTCGGTTGGA
VvSAP15 ACTGCGACCCGTCAAAGAAA AACCGATGCTTGAGGCAGAA

Fig. 1

The chromosome location of the SAP family in Vitis vinifera"

Table 2

Physicochemical property of VvSAP proteins"

基因
Gene		 基因号
Gene ID
 氨基酸数
Amino acid number		 等电点
Theoretical
pI		 分子量
Molecular weight (Da)		 不稳定指数 Instability index 脂溶指数 Aliphatic index 总平均疏水指数
Average of hydropathicity (GRAVY)
VvSAP1 LOC100266326 152 9.10 17119.98 33.30 55.52 -0.600
VvSAP2 LOC100259845 172 8.77 18460.83 46.68 46.05 -0.494
VvSAP3 LOC100267237 172 7.99 18502.95 27.15 56.80 -0.500
VvSAP4 LOC100242410 141 8.28 15277.21 36.57 54.75 -0.509
VvSAP5 LOC100264822 161 8.92 17192.68 49.44 66.21 -0.300
VvSAP6 LOC100243583 172 7.99 18153.57 30.43 68.72 -0.327
VvSAP7 LOC100852428 172 8.95 18359.69 32.98 57.50 -0.486
VvSAP8 LOC100852460 172 8.22 18504.11 46.99 61.40 -0.371
VvSAP9 LOC100265156 153 9.10 17153.98 38.56 50.39 -0.699
VvSAP10 LOC100265151 152 8.77 16850.54 21.92 58.36 -0.558
VvSAP11 LOC100852756 109 9.23 11757.33 54.13 43.85 -0.471
VvSAP12 LOC100251836 126 9.13 14293.29 54.43 53.41 -0.901
VvSAP13 LOC10085429 126 9.68 14575.89 41.04 53.41 -0.904
VvSAP14 LOC100255506 293 8.56 32457.78 40.16 58.84 -0.614
VvSAP15 LOC100245574 189 8.74 21133.11 49.61 47.99 -0.800

Table 3

Subcellular location prediction of SAP family in Vitis vinifera"

基因
Gene		 细胞核
Nucleus		 叶绿体
Chloroplast		 细胞外
Extracellular		 细胞骨架
Cytoskeleton		 线粒体
Mitochondria		 过氧化物酶体
Peroxisome		 细胞质和细胞核
Cyto_nucl		 质膜
Plasma membrane
VvSAP1 6 4 2 1 1 - - -
VvSAP2 8 1 2 1 2 - - -
VvSAP3 9 1 - 4 - - - -
VvSAP4 3 10 - 1 - - - -
VvSAP5 4 3 - 3 4 - - -
VvSAP6 5 4 2 3 - - - -
VvSAP7 4 6 1 1 2 - - -
VvSAP8 8 2 1 3 - - - -
VvSAP9 2.5 4 - 1.5 - 6 2.5 -
VvSAP10 2.5 3 - 2.5 - 6 3 -
VvSAP11 4 2 2 3 3 - - -
VvSAP12 5 6 1 1 1 - - -
VvSAP13 8 2 1 1 1 - - 1
VvSAP14 14 - - - - - - -
VvSAP15 13 - - - - - - 1

Table 4

The secondary structure of VvSAP"

蛋白名称 Protein name α-螺旋 Alpha helix (%) 拓展链结构 Extended strand (%) β-转角 Beta turn (%) 无规则卷曲 Random coil (%)
VvSAP1 30.26 11.18 4.61 53.95
VvSAP2 26.74 11.05 2.91 59.30
VvSAP3 31.40 12.21 3.49 52.91
VvSAP4 29.79 12.77 3.55 53.90
VvSAP5 25.47 10.56 4.35 59.63
VvSAP6 29.65 11.05 3.49 55.81
VvSAP7 23.84 12.21 4.07 59.88
VvSAP8 23.84 12.79 5.23 58.14
VvSAP9 23.53 11.11 5.23 60.13
VvSAP10 34.87 11.84 3.29 50.00
VvSAP11 15.60 23.85 10.09 50.46
VvSAP12 17.46 20.63 3.97 57.94
VvSAP13 27.78 17.46 7.14 47.62
VvSAP14 24.91 10.92 2.05 62.12
VvSAP15 11.11 11.11 4.76 73.02

Fig. 2

SAP family sequence analysis in Vitis vinifera A: SAP family motif analysis in Vitis vinifera; B: LOGO of 8 conserved motif of SAP domain"

Fig. 3

Identify analysis of grape SAP proteins"

Fig. 4

The phylogenetic tree and gene structure of SAP family in Vitis vinifera"

Fig. 5

Phylogenetic tree of SAP gene family in V.vinifera（●）, A. thaliana and O. sativa"

Fig. 6

Relative expression of SAPs under circadian and different treatments CR: Circadian rhythm; ABA: Abscisic acid; SA: Salicylic acid; NaCl: Sodium chloride; LT: Low temperature （Abscissa）CR 1-8: Beijing time 0:00, 3:00, 6:00, 9:00, 12:00, 15:00, 18:00, 21:00;ABA/SA/NaCl/LT 1-8: 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after treatmentError bars represent the standard deviation of three biological replicates. *, **, *** indicate P<0.05, P<0.01and P<0.001, respectively"

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