葡萄SAP家族的鉴定与表达分析

doi:10.3864/j.issn.0578-1752.2019.14.009

Abstract

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

DING Lan,GU Bao,LI PeiYing,SHU Xin,ZHANG JianXia. Genome-Wide Identification and Expression Analysis of SAP Family in Grape[J].Scientia Agricultura Sinica, 2019, 52(14): 2500-2514.

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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

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

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

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

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

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