Identification of grapevine AQP family and prediction for transcriptional regulatory network under drought stress

Abstract:【Objective】Grape is one of the oldest fruit crops in the world. Most of the grapes in the world are cultivated in arid and semi-arid areas. Facing with the increasingly severe environment on the earth, the grape industry will face a serious impact in the future, drought will become one of the key fac- tors restricting grape production. The aim of this study was to identify members of the AQPs gene fami- ly in the whole grape genome. In addition, the transcriptional regulatory network of VvAQPs under drought stress was explored to provide theoretical basis for the subsequent research on the function and the transcriptional regulation mechanism of drought resistance genes in grape.【Methods】Through BLAST Arabidopsis thaliana AQP protein sequences and the HMM file (PF00230), 37 VvAQPs were identified based on the grape genome database. The phylogenetic analysis, gene structure and protein se- quence analysis, chromosome location and series replication analysis of grape AQP genes family were performed by using bioinformatics method, interspecific collinear analysis, targeted VvAQPs transcrip- ion factor regulatory network analysis. Through drought stress and ABA treatment to further identify the VvAQPs involved in drought, and explore possible transcriptional regulatory relationships.【Results】Thirty- seven VvAQPs members were identified from the grape genome. The sequence lengths of VvAQPs varied from 65 to 354, the molecular weights of VvAQPs varied from 6.79 to 37.33 kDa, and the isoelectric points of VvAQPs varied from 4.33 to 9.79. Most of the VvAQPs protein had 6 trans- membrane domains. Phylogenetic analysis showed that the family was divided into PIPs, TIPs, NIPs and SIPs. These VvAQPs were distributed among 17 chromosomes, one VvAQP was distributed on 4, 11, 12, 18_random chromosome, and two on 2, 5, 9, 10, 16, 7_random, Un chromosome. There were 3VvAQPs on 3, 6, 13, 14, and 15, 4VvAQPs on the chr8 chromosomes. VvAQPs were accompanied with 5 pairs of segmental copies and 2 pairs of tandem copies. There were 27 AQP homologous genes be- tween grape and Arabidopsis thaliana, 31 AQP homologous genes in tomato, 36 AQP homologous genes in peach, 55 AQP homologous genes in poplar and 44 AQP homologous genes in kiwifruit, re- spectively. In addition, the number of homologous genes on the 6, 8 and 13 chromosomes of grape were large, 5 VvAQPs were direct homologous genes with other 6 species, and 7 VvAQPs were homologous with other 5 dicotyledons. Gene structure and protein sequence analysis showed that there was a conserved domain characteristic (MIP) of plant aquaporin in each grape AQP protein sequence and genes from the same subgroup had similar structures. The prediction of transcriptional regulatory network re- vealed that the transcriptional regulatory factors targeting VvAQPs were mainly divided into 16 types (HD-ZIP, MYB, ERF, bZIP etc.). Combined with transcriptome data, most VvAQPs were significantly down-regulated in roots and leaves under drought stress, and VvAQPs in most roots or leaves were not differentially expressed during the T1 period of drought stress. In roots, VvPIP3-1, VvNIP3-1, VvTIP1-2and VvTIP2-1 were significantly down-regulated, while VvPIP2-3 showed the opposite trend and was significantly up-regulated. VvNIP2-3, VvPIP1-3, VvNIP1-1 and VvTIP4-1 were significantly down-regu- lated in the later stage of drought stress, and VvTIP2-2 was only significantly down-regulated in the M4 drought-tolerant genotype. In leaves, VvTIP1-4, VvTIP2-1 and VvPIP3-2 were significantly down-regu- lated, VvTIP1-2 and VvNIP3-1 were up-regulated first and then down-regulated, and most genes of grape PIP subfamily members were significantly down- regulated in later stages, such as VvPIP1- 3,VvPIP1-5, VvPIP2-1, VvPIP2-2, VvPIP2-4and VvPIP2-5. VvAQPs were induced by ABA in different tissues. In roots, 4 VvAQPs were significantly up- regulated after being induced by ABA, such asVvPIP3-1, VvPIP1-6, VvPIP2-2 and VvTIP3-1, and VvNIP4-2 was down-regulated after ABA treatment. In leaves, VvNIP1-1, VvPIP3-1 and VvTIP1-3 were induced to be up-regulated, while VvPIP2-2 was in- duced to be down-regulated. Combined transcriptome expression data with transcriptional regulatory network prediction results, 15 differentially expressed transcription factors may regulate 13 VvAQPs un- der drought stress. Under drought stress, HD-ZIP (VIT_16s0098g01170, VIT_04s0023g01330 and VIT_ 15s0048g02870) showed an up- regulation trend, and their target genes VvPIP2- 1, VvPIP2- 4 andVvTIP1- 1 basically showed a trend of up- regulation first and then down- regulation. bZIP (VIT_ 01s0010g00930, VIT_18s0001g10450 and VIT_15s0046g01440) was all up- regulated. Their target genes VvTIP1-4, VvNIP1-1, VvPIP1-5, VvPIP1-4, VvPIP2-5, VvTIP1-3 and VvTIP1-1 basically showed a downward-regulation trend in roots and leaves under drought stress. VvTIP1-2 was first up-regulated and then down-regulated in the leaves, while it was continuously down-regulated in roots. NAC (VIT_ 01s0026g02710 and VIT_19s0014g03290) all showed an up-regulation trend, and they jointly targeted the VvTIP2-1, which was continuously down-regulated in roots and leaves under drought stress. In addi- tion, VIT_11s0016g02410 (MYB_related) and target gene VvNIP3-1 continued to be down-regulated in roots under drought stress, VIT_16s0013g01000 (ERF) and target gene VvPIP1-7 were continuously down- regulated in roots under drought stress, and VIT_07s0197g00060 (G2- like) and target geneVvTIP2-1 showed opposite trends in roots and leaves under drought stress.【Conclusion】The basic infor- mation about grape AQPs family members was identified and provided, and the VvAQPs involved in drought stress and the predicted transcriptional regulatory networks were further identified, which pro- vided a theoretical basis for further study on grape AQPs involved in drought stress and transcriptional regulation mechanism.