Genome-wide Identification and Expression Pattern Analysis of the Bcl-2-related Anti-apoptotic Family in Cucumis sativus L. and Cucurbita moschata Duch.

doi:10.13560/j.cnki.biotech.bull.1985.2023-1195

Abstract

Abstract:

【Objective】 In order to provide favorable genes for the analysis of the grafting healing mechanism of Cucumis sativus L. or Cucurbita moschata Duch. grafted seedlings and the molecular breeding of resistance of the cucumber and pumpkin vegetables, we conducted the analysis of the expression patterns of Bcl-2 associated athanogene（BAG）family proteins in the cucumber and pumpkin in response to abiotic stress and in response to light during cucumber or pumpkin grafting healing. 【Method】 Based on the genome information of the cucumber and pumpkin, the BAG gene family in the cucumber and pumpkin was identified through bioinformatics methods, and its physicochemical characteristics, chromosome mapping, gene structure, phylogeny and collinearity were analyzed. The expression patterns of BAG family proteins in the cucumber and pumpkin in response to abiotic stress and in response to light during cucumber or pumpkin grafting healing were analyzed. These data were based on the public database and the transcriptome sequencing data of the cucumber or pumpkin grafted seedlings in the process of grafting and healing. 【Result】 A total of 12 and 18 members of the BAG family were identified in the cucumber and pumpkin, respectively and they were all divided into two subfamilies and the gene members were highly conserved. The BAG of subfamily I was mainly involved in gene regulation and stress response, while the BAG of subfamily II was mainly involved in plant development. The BAG genes of cucumber and pumpkin had multiple linear relationships with Arabidopsis, Oryza sativa L. and Solanum lycopersicum L., respectively, but the CsaV3_1G017210 was relatively conservative and there was no collinearity with the BAG genes in Arabidopsis, rice, tomato and pumpkin. Different BAG genes possessed tissue-specific expression patterns. CsaV3_6G000970 and CmoCh08G008520 belonged to the BAG family molecular chaperone regulator 6. In both cucumber and pumpkin, they were upregulated during the grafting healing process and in response to abiotic stress. 【Conclusion】 BAG family genes respond differently to abiotic stress and light during the grafting healing process of the cucumber or pumpkin. These genes synergistically regulate the growth, development and grafting healing of the cucumber and pumpkin. BAGs play an important role in the process of the cucumber and pumpkin abiotic stress and cucumber or pumpkin grafting healing.

Key words: Cucumis sativus L., Cucurbita moschata Duch., BAG gene family, bioinformatics, grafting healing

HU Yong-bo, LEI Yu-tian, YANG Yong-sen, CHEN Xin, LIN Huang-fang, LIN Bi-ying, LIU Shuang, BI Ge, SHEN Bao-ying. Genome-wide Identification and Expression Pattern Analysis of the Bcl-2-related Anti-apoptotic Family in Cucumis sativus L. and Cucurbita moschata Duch.[J]. Biotechnology Bulletin, 2024, 40(6): 219-237.

Figures/Tables 12

Fig. 1 Chromosome location of the BAG gene family in Cucumis sativus L. (A) and Cucurbita moschata Duch.(B)

Fig. 2 Phylogenetic analysis of the BAG gene family in C. sativus L., C. moschata Duch., Arabidopsis, O. sativa L. and S. lyco-persicum L. : C. sativus L.. : C. moschata Duch.. : Arabidopsis. : O. sativa L.. : S. lycopersicum L

Fig. 3 Gene structure and conserved sequence of the BAG gene family in C. sativus L. (A) and C. moschata Duch. (B)

Fig. 4 Collinear relationship of BAG gene family in C. sativus L., C. moschata Duch., Arabidopsis, O. sativa L. and S. lycop-ersicum L.

Fig. 5 Cis-acting elements in the promoter sequences of BAG gene family in C. sativus L. (A) and C. moschata Duch. (B)

Fig. 6 Expressions of BAG gene family in different tissues and organs of in C. sativus L. (A) and C. moschata Duch. (B)

Fig. 7 Expressions of BAG gene family in C. sativus L. and C. moschata Duch. under abiotic stress A: High temperature stress in C. sativus L. (HT3h, HT6h: High temperature treatment for 3 and 6 h). B: Low temperature stress in C. sativus L. (CS2h, CS6h and CS12h: Low temperature treatment for 2, 6 and 12 h). C: Salt and silicon stress in C. sativus L. (Na: Salt stress treatment; Si: silicon stress treatment; Si+Na: silicon and salt stress treatment). D: High temperature stress in C. moschata Duch. (HT38: 38°C high temperature treatment for 3 h; HD45: 38°C high temperature adaptation for 3 h, recovered at 25°C for 5 h, and then excited at 45°C for 3 h; HT45: high temperature treatment at 45°C for 3 h). E: Low temperature stress in C. moschata Duch. (CS: Low temperature treatment). F: Salt stress in C. moschata Duch. (CT_Leaf mesophyll, CT_Leaf vein, and CT_Root: Control treatment of mesophyll, leaf veins, and roots; Na_Leaf mesophyll, Na_Leaf vein and, Na_Root: salt stress treatment of mesophyll, leaf veins, and roots. CT: Control. The data in the boxes indicate original FPKM value

Fig. 8 Expression patterns of BAG family genes in C. sativus L. (A) and C. moschata Duch. (B) under stress HT: High temperature stress. CS: Low temperature stress. Na: Salt stress. The red color indicates the up-regulation of the expression, the green color indicates the down-regulation of the expression, and the gray color indicates the expression is no significant difference in the expression, the same below

Fig. 9 Expression heat map of BAG gene family in C. sativus L. (A) and C. moschata Duch. (B) in grafting healing CK1, CK2, CK3: Dark control group. T1, T2, T3: Lighting treatment group. The same below

Fig. 10 Co-expression patterns of BAG family genes and differential genes in glucose metabolism pathway in C. sativus L. (A) and C. moschata Duch. (B)

Fig. 11 RT-qPCR validation of BAG gene family in C. sativus L. (A-F) and C. moschata Duch. (G-L) under light and exogenous sugar treatments CK-NO.0d: 0 d dark control group. CK-NO. 2d: 2 d dark control group. T-NO.2d: 2 d lighting treatment group. CK+GLU-NO.2d: 2 d dark + sugar treatment group. T+GLU-NO.2d: 2 d light + sugar treatment group. Different letters indicate significant difference at P<0.05 level

Fig. 12 Expression patterns of BAG family genes in C. sativus L. (A) and C. moschata Duch. (B) during grafting healing Light: Lighting treatment. Sugar: Co-expression of sugar metabolism

References 68

[1]	Thanthrige N, J ain S, Bhowmik SD, et al. Centrality of BAGs in plant PCD, stress responses, and host defense[J]. Trends Plant Sci, 2020, 25(11): 1131-1140. doi: 10.1016/j.tplants.2020.04.012 pmid: 32467063
[2]	Huang SW, Li RQ, Z hang ZH, et al. The genome of the cucumber, Cucumis sativus L.[J]. Nat Genet, 2009, 41(12): 1275-1281.
[3]	Briknarová K, Takayama S, Brive L, et al. Structural analysis of BAG1 cochaperone and its interactions with Hsc70 heat shock protein[J]. Nat Struct Biol, 2001, 8(4): 349-352. pmid: 11276257
[4]	Takayama S, Sato T, Krajewski S, et al. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity[J]. Cell, 1995, 80(2): 279-284. pmid: 7834747
[5]	Doukhanina EV, Chen SR, van der Zalm E, et al. Identification and functional characterization of the BAG protein family in Arabidopsis thaliana[J]. J Biol Chem, 2006, 281(27): 18793-18801. doi: 10.1074/jbc.M511794200 pmid: 16636050
[6]	Poulsen EG, K ampmeyer C, Kriegenburg F, et al. UBL/BAG-domain co-chaperones cause cellular stress upon overexpression through constitutive activation of Hsf1[J]. Cell Stress & Chaperones, 2017, 22(1): 143-154.
[7]	Yang TB, Poovaiah BW. Calcium/calmodulin-mediated signal network in plants[J]. Trends Plant Sci, 2003, 8(10): 505-512. doi: 10.1016/j.tplants.2003.09.004 pmid: 14557048
[8]	Cui BY, Fang SS, Xing YF, et al. Crystallographic analysis of the Arabidopsis thaliana BAG5-calmodulin protein complex[J]. Acta Crystallogr F Struct Biol Commun, 2015, 71(Pt 7): 870-875.
[9]	Chen Y, Wang KK, Di J, et al. Mutation of the BAG-1 domain decreases its protective effect against hypoxia/reoxygenation by regulating HSP70 and the PI3K/AKT signalling pathway in SY-SH5Y cells[J]. Brain Res, 2021, 1751: 147192.
[10]	Brenner CM, Choudhary M, McC ormick MG, et al. BAG3: Nature's quintessential multi-functional protein functions as a ubiquitous intra-cellular glue[J]. Cells, 2023, 12(6): 937.
[11]	Kang CH, Lee JH, Kim YJ, et al. Characterization of AtBAG2 as a novel molecular chaperone[J]. Life-Basel, 2023, 13(3): 687.
[12]	Baeken MW, Behl C. On the origin of BAG(3)and its consequences for an expansion of BAG3's role in protein homeostasis[J]. J Cell Biochem, 2022, 123(1): 102-114.
[13]	Gupta MK, Randhawa PK, Masternak MM. Role of BAG 5 in protein quality control: Double-edged sword?[J]. Front Aging, 2022, 3: 844168.
[14]	Huang HM, Liu CX, Yang C, et al. BAG9 confers thermotolerance by regulating cellular redox homeostasis and the stability of heat shock proteins in Solanum lycopersicum[J]. Antioxidants, 2022, 11(8): 1467.
[15]	杨美玲, 贺丽霞, 于玮玮, 等. 新疆野苹果BAG家族成员分离及BAG7基因的功能分析[J]. 南开大学学报:自然科学版, 2022, 55(6): 1-6.
	Yang ML, He LX, Yu WW, et al. Identification of BAG and functional analysis of BAG7 of malus sieversii[J]. Acta Sci Nat Univ Nankaiensis, 2022, 55(6): 1-6.
[16]	Arif M, Li ZT, Luo Q, et al. The BAG2 and BAG6 genes are involved in multiple abiotic stress tolerances in Arabidopsis thaliana[J]. Int J Mol Sci, 2021, 22(11): 5856.
[17]	Zhang HH, Li YR, Dickman MB, et al. Cytoprotective co-chaperone BcBAG1 is a component for fungal development, virulence, and unfolded protein response(UPR)of Botrytis cinerea[J]. Front Microbiol, 2019, 10: 685.
[18]	Li YR, Williams B, Dickman M. Arabidopsis B-cell lymphoma2(Bcl-2)-associated athanogene 7(BAG7)-mediated heat tolerance requires translocation, sumoylation and binding to WRKY29[J]. New Phytol, 2017, 214(2): 695-705.
[19]	Li LH, Xing YF, Chang D, et al. CaM/BAG5/Hsc70 signaling complex dynamically regulates leaf senescence[J]. Sci Rep, 2016, 6: 31889. doi: 10.1038/srep31889 pmid: 27539741
[20]	Li YR, Dickman M. Processing of AtBAG6 triggers autophagy and fungal resistance[J]. Plant Signal Behav, 2016, 11(6): e1175699.
[21]	Ding HD, Qian L, Jiang HL, et al. Overexpression of a Bcl-2-associated athanogene SlBAG9 negatively regulates high-temperature response in tomato[J]. Int J Biol Macromol, 2022, 194: 695-705.
[22]	Yan JQ, He CX, Zhang H. The BAG-family proteins in Arabidopsis thaliana[J]. Plant Sci, 2003, 165(1): 1-7.
[23]	Zhou H, Li JY, Liu XY, et al. The divergent roles of the rice bcl-2 associated athanogene(BAG)genes in plant development and environmental responses[J]. Plants, 2021, 10(10): 2169.
[24]	Ge SM, Kang Z, Li Y, et al. Cloning and function analysis of BAG family genes in wheat[J]. Funct Plant Biol, 2016, 43(5): 393-402. doi: 10.1071/FP15317 pmid: 32480470
[25]	Yeckel G. Characterization of a soybean BAG gene and its potential role in nematode resistance[D]. Columbia: University of Missouri, 2012.
[26]	Jiang HL, Ji YR, Sheng JR, et al. Genome-wide identification of the Bcl-2 associated athanogene(BAG)gene family in Solanum lycopersicum and the functional role of SlBAG9 in response to osmotic stress[J]. Antioxidants, 2022, 11(3): 598.
[27]	莫心怡, 周海连, 何丹丹, 等. 甘蔗割手密BAG基因家族的鉴定与表达分析[J]. 基因组学与应用生物学, 2023, 42(12): 1364-1378.
	Mo XY, Zhou HL, He DD, et al. Identification and expression analysis of BAG gene family in sugarcane saccharum spontaneum[J]. Genom Appl Biol, 2023, 42(12): 1364-1378.
[28]	张扬, 杜琳, 唐贤丰, 等. 杨树BAG基因的鉴定及表达模式分析[J]. 林业科学, 2019, 55(1): 138-145.
	Zhang Y, Du L, Tang XF, et al. Identification and expression pattern analyses of Populus BAG genes[J]. Sci Silvae Sin, 2019, 55(1): 138-145.
[29]	Dash A, Ghag SB. Genome-wide in silico characterization and stress induced expression analysis of BcL-2 associated athanogene(BAG)family in Musa spp[J]. Sci Rep, 2022, 12(1): 625.
[30]	Lee DW, Kim SJ, Oh YJ, et al. Arabidopsis BAG1 functions as a cofactor in Hsc70-mediated proteasomal degradation of unimported plastid proteins[J]. Mol Plant, 2016, 9(10): 1428-1431.
[31]	You QY, Zhai KR, Yang DL, et al. An E3 ubiquitin ligase-BAG protein module controls plant innate immunity and broad-spectrum disease resistance[J]. Cell Host Microbe, 2016, 20(6): 758-769.
[32]	Locascio A, Marqués MC, García-Martínez G, et al. BCL2-associated athanogene4 regulates the KAT1 potassium channel and controls stomatal movement[J]. Plant Physiol, 2019, 181(3): 1277-1294. doi: 10.1104/pp.19.00224 pmid: 31451552
[33]	He MM, Wang Y, Jahan MS, et al. Characterization of SlBAG genes from Solanum lycopersicum and its function in response to dark-induced leaf senescence[J]. Plants, 2021, 10(5): 947.
[34]	Irfan M, Kumar P, Ahmad I, et al. Unraveling the role of tomato Bcl-2-associated athanogene(BAG)proteins during abiotic stress response and fruit ripening[J]. Sci Rep, 2021, 11(1): 21734.
[35]	Shang KJ, Xiao L, Zhang XP, et al. Tomato chlorosis virus p22 interacts with NbBAG5 to inhibit autophagy and regulate virus infection[J]. Mol Plant Pathol, 2023, 24(5): 425-435.
[36]	Jiang HL, Liu XY, Xiao PX, et al. Functional insights of plant bcl-2-associated ahanogene(BAG)proteins: Multi-taskers in diverse cellular signal transduction pathways[J]. Front Plant Sci, 2023, 14: 1136873.
[37]	张开京, 何帅帅, 贾利, 等. 黄瓜DIR家族基因的全基因组鉴定及其表达分析[J]. 中国农业科学, 2023, 56(4): 711-728. doi: 10.3864/j.issn.0578-1752.2023.04.010
	Zhang KJ, He SS, Jia L, et al. Genome-wide identification and expression analysis of DlR gene family in cucumbers[J]. Sci Agric Sin, 2023, 56(4): 711-728.
[38]	赵振翔, 敖文红, 王新法, 等. 黄瓜DME基因家族的全基因组鉴别及转录分析[J]. 植物生理学报, 2023, 59(1): 209-218.
	Zhao ZX, Ao WH, Wang XF, et al. Genome-wide identification and transcriptional analysis of DME gene family in cucumber[J]. Plant Physiol J, 2023, 59(1): 209-218.
[39]	应奥, 程志华, 张小兰, 等. 黄瓜YABBYs家族及CsYAB1a基因功能初探[J]. 中国农业大学学报, 2022, 27(11): 60-78.
	Ying A, Cheng ZH, Zhang XL, et al. Preliminary study on the function of YABBYs family and CsYAB1a gene in cucumber.(Cucumis sativus L.)[J]. J China Agric Univ, 2022, 27(11): 60-78.
[40]	周国彦, 银珊珊, 高佳鑫, 等. 黄瓜AHP基因家族的鉴定及其非生物胁迫表达分析[J]. 生物技术通报, 2022, 38(6): 112-119. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1338
	Zhou GY, Yin SS, Gao JX, et al. Identification of AHP gene family in cucumis sativus and its expression analysis under abiotic stress[J]. Biotechnol Bull, 2022, 38(6): 112-119.
[41]	Xu MY, Wang YP, Zhang MT, et al. Genome-wide identification of BES1 gene family in six cucurbitaceae species and its expression analysis in Cucurbita moschata[J]. Int J Mol Sci, 2023, 24(3): 2287.
[42]	Davoudi M, Chen JF, Lou QF. Genome-wide identification and expression analysis of heat shock protein 70(HSP70)gene family in pumpkin(Cucurbita moschata)rootstock under drought stress suggested the potential role of these chaperones in stress tolerance[J]. Int J Mol Sci, 2022, 23(3): 1918.
[43]	Hu YP, Zhang TT, Liu Y, et al. Pumpkin(Cucurbita moschata)HSP20 gene family identification and expression under heat stress[J]. Front Genet, 2021, 12: 753953.
[44]	Miao L, Li SZ, Shi AK, et al. Genome-wide analysis of the AINTEGUMENTA-like(AIL)transcription factor gene family in pumpkin(Cucurbita moschata Duch.) and CmoANT1.2 response in graft union healing[J]. Plant Physiol Biochem, 2021, 162: 706-715.
[45]	Li Q, Li HB, Huang W, et al. A chromosome-scale genome assembly of cucumber(Cucumis sativus L.)[J]. Gigascience, 2019, 8(6): giz072.
[46]	Sun HH, Wu S, Zhang GY, et al. Karyotype stability and unbiased fractionation in the paleo-allotetraploid cucurbita genomes[J]. Mol Plant, 2017, 10(10): 1293-1306. doi: S1674-2052(17)30266-6 pmid: 28917590
[47]	Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching[J]. Nucleic Acids Res, 2011, 39(Web Server issue): W29-W37.
[48]	Mistry J, Chuguransky S, Williams L, et al. Pfam: The protein families database in 2021[J]. Nucleic Acids Res, 2021, 49(D1): D412-D419.
[49]	Letunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020[J]. Nucleic Acids Res, 2021, 49(D1): D458-D460.
[50]	Chen CJ, Chen H, Zhang Y, et al. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Mol Plant, 2020, 13(8): 1194-1202. doi: S1674-2052(20)30187-8 pmid: 32585190
[51]	Bailey TL, Williams N, Misleh C, et al. MEME: discovering and analyzing DNA and protein sequence motifs[J]. Nucleic Acids Res, 2006, 34(Web Server issue): W369-W373.
[52]	Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis version 11[J]. Mol Biol Evol, 2021, 38(7): 3022-3027. doi: 10.1093/molbev/msab120 pmid: 33892491
[53]	Lescot M, Déhais P, Thijs G, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Res, 2002, 30(1): 325-327.
[54]	Wang YP, Tang HB, Debarry JD, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity[J]. Nucleic Acids Res, 2012, 40(7): e49.
[55]	Li Z, Zhang ZH, Yan PC, et al. RNA-Seq improves annotation of protein-coding genes in the cucumber genome[J]. BMC Genomics, 2011, 12: 540. doi: 10.1186/1471-2164-12-540 pmid: 22047402
[56]	Chen CH, Chen XQ, Han J, et al. Genome-wide analysis of the WRKY gene family in the cucumber genome and transcriptome-wide identification of WRKY transcription factors that respond to biotic and abiotic stresses[J]. BMC Plant Biol, 2020, 20(1): 443.
[57]	Zhu Y, Yin JL, Liang YF, et al. Transcriptomic dynamics provide an insight into the mechanism for silicon-mediated alleviation of salt stress in cucumber plants[J]. Ecotoxicol Environ Saf, 2019, 174: 245-254.
[58]	Zhou Y, Yang K, Cheng M, et al. Double-faced role of Bcl-2-associated athanogene 7 in plant-Phytophthora interaction[J]. J Exp Bot, 2021, 72(15): 5751-5765. doi: 10.1093/jxb/erab252 pmid: 34195821
[59]	Hu LZ, Chen JT, Guo JJ, et al. Functional divergence and evolutionary dynamics of BAG gene family in maize(Zea mays)[J]. Int J Agric Biol, 2013, 15(2): 200-206.
[60]	UN Food and Agriculture Organization, Corporate Statistical Database(FAOSTAT). Production of cucumbers and gherkins in 2020[EB/OL]. https://www.fao.org/faostat/zh/#data/.2020-03-30/2023-12-18.
[61]	Jain M, Tyagi AK, Khurana JP. Genome-wide analysis, evolutionary expansion, and expression of early auxin-responsive SAUR gene family in rice(Oryza sativa)[J]. Genomics, 2006, 88(3): 360-371.
[62]	Zhang SB, C hen C, Li L, et al. Evolutionary expansion, gene structure, and expression of the rice wall-associated kinase gene family[J]. Plant Physiol, 2005, 139(3): 1107-1124. pmid: 16286450
[63]	Nollen EA, Brunsting JF, Song J, et al. Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity[J]. Mol Cell Biol, 2000, 20(3): 1083-1088. doi: 10.1128/MCB.20.3.1083-1088.2000 pmid: 10629065
[64]	Takayama S, Xie Z, Reed JC. An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators[J]. J Biol Chem, 1999, 274(2): 781-786. doi: 10.1074/jbc.274.2.781 pmid: 9873016
[65]	Takayama S, Bimston DN, Matsuzawa S, et al. BAG-1 modulates the chaperone activity of Hsp70/Hsc70[J]. EMBO J, 1997, 16(16): 4887-4896. doi: 10.1093/emboj/16.16.4887 pmid: 9305631
[66]	Nawkar GM, Maibam P, Park JH, et al. In silico study on Arabidopsis BAG gene expression in response to environmental stresses[J]. Protoplasma, 2017, 254(1): 409-421.
[67]	Wang J, Nan N, Li N, et al. A DNA methylation reader-chaperone regulator-transcription factor complex activates OsHKT1;5 expression during salinity stress[J]. Plant Cell, 2020, 32(11): 3535-3558.
[68]	Wang JY, Yeckel G, Kandoth PK, et al. Targeted suppression of soybean BAG6-induced cell death in yeast by soybean cyst nematode effectors[J]. Mol Plant Pathol, 2020, 21(9): 1227-1239. doi: 10.1111/mpp.12970 pmid: 32686295