Identification of GAE Gene Family in Antirrhinum majus and Mining of Resistance Genes to Sclerotinia sclerotiorum
XIA Wen-Nian, YANG Dong-Mei, SONG Jia-Yi, YANG Jie, WANG Zhong-Yi, LIN Hai-Di, HU Hui-Zhen*
College of Landscape Architecture and Horticulture Sciences, Southwest Forestry University/Yunnan Province Engineering Research Center for Functional Flower Resources and Industrialization, Kunming 650224, China
Abstract：Pectin, a crucial constituent of plant cell walls, assumes a significant role in both plant development and cell wall fortification. The enzymatic catalyst responsible for pectin polymerization is glucuronate isomerase, also known as UDP-D-glucuronate-4-epimerase (GAE). This enzymatic process involves the utilization of UDP-α-D-galacturonic acid (UDP-GalA) as the monosaccharide donor to form the pectin skeleton. To investigate the cell wall resistance mediated by GAE of Antirrhinum majus, 8 AmGAEs genes were identified at the whole genome level. Bioinformatics analysis of their physical and chemical properties, subcellular localization, phylogeny, conserved motifs, gene structure, chromosome localization and cis-acting elements showed that all the members of the AmGAE family were alkaline hydrophilic proteins located in Golgi apparatus. The genes had resistance-related response elements such as defense and stress, methyljasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) in the promoter region. Furthermore, RNA-seq and qRT-PCR were used to analyse and reverify the resistant and susceptible materials to S. sclerotiorum in snapdragon. The results showed that the expression of AmGAE1a, AmGAE3, AmGAE5 and AmGAE2 genes were significantly induced in the resistant material Am6 (R), while the expression of AmGAE7 and AmGAE4 genes was significantly induced in the susceptible material Am1 (S). Among them, the expression patterns of AmGAE1a, AmGAE3, AmGAE7 and AmGAE4 genes in qRT-PCR were consistent with differentially expressed genes (DEGs) by RNA-seq, and thus finally identified as candidate genes. The results provide a theoretical basis and new genetic resources of AmGAE family genes involved in the resistance to S. sclerotiorum for further study.
夏文念, 杨冬梅, 宋佳怡, 杨洁, 汪仲毅, 蔺海娣, 胡慧贞. 金鱼草GAE基因家族鉴定及核盘菌抗性基因挖掘[J]. 农业生物技术学报, 2024, 32(9): 2049-2059.
XIA Wen-Nian, YANG Dong-Mei, SONG Jia-Yi, YANG Jie, WANG Zhong-Yi, LIN Hai-Di, HU Hui-Zhen. Identification of GAE Gene Family in Antirrhinum majus and Mining of Resistance Genes to Sclerotinia sclerotiorum. 农业生物技术学报, 2024, 32(9): 2049-2059.
[1] 曹舸洋. 2018. 调节金鱼草叶序发育AmPIN1a基因克隆和转基因分析[D]. 硕士学位论文, 安徽农业大学, 导师: 王冬良, pp.19-50. (Cao G Y.2018. Cloning and transgenic analysis of AmPIN1a regulating phyllotaxic development in Antirrhinum majus[J]. Thesis for M.S., Anhui Agricultural University, Supervisor: Wang D L, pp. 19-50.) [2] 陈宇华, 陈剑锋, 钟声远, 等. 2022. 20份金鱼草种质资源花色性状鉴定与分析[J]. 福建农业科技, 53(7): 1-7. (Chen Y H, Chen J F, Zhong S Y, et al.2022. Identification and analysis of flower color traits of 20 germplasm resources of Antirrhinum majus[J]. Fujian Agricultural Science and Technology, 53(7): 1-7. [3] 葛廷, 黄雪, 谢让金. 2019. 柑橘CitPG34的克隆、定位与表达分析[J]. 中国农业科学, 52(19): 3404-3416. (Ge T, Hang X, Xie R J.2019. Cloning, subcellular localization and expression analysis of CitPG34 in citrus[J]. Scientia Agricultura Sinica, 52(19): 3404-3416.) [4] 李秀丽, 高智谋. 2013. 核盘菌(Seleroinia slerotiorum)致病分子机理研究进展[J]. 安徽农业大学学报, 40(2): 266-272. (Li X L, Gao Z M.2013. Research progress on molecular pathogenic mechanisms of Sclerotinia sclerotiorum (Lib.) de Bary[J]. Journal of Anhui Agricultural University, 40(2): 266-272. [5] 刘晓慧. 2017. 不同光质对金鱼草花香合成与释放的影响[D]. 硕士学位论文, 北京农学院, 导师: 冷平生, pp. 8-19. (Liu X H.2017. Effect of different light qualities on the synthesis and emission of floral scent from snapdragon[D]. Thesis for M.S., Beijing University of Agriculture, Supervisor: Leng P S, pp. 8-19.) [6] 罗维平, 陈少萍, 龚衍熙. 2008. 金鱼草的繁殖与病虫害防治[J]. 中国花卉园艺, (08): 23-25. (Luo W P, Chen S P, Gong Y X. 2008. Breeding and pest control in Antirrhinum majus[J]. China Flowers ＆ Horticulture, (08): 23-25.) [7] 师莹莹, 李大勇. 张慧娟. 2011. 植物细胞壁介导的抗病性及其分子机制[J]. 植物生理学报, 47(07): 661-668. (Shi Y Y, Li D Y, Zhang H J.2011. Cell wall-mediated disease resistance and its molecular mechanism in plants[J]. Plant Physiology Journal, 47(07), 661-668. [8] 童超, 张兴国, 鄢巧灵, 等. 2008. AtUGAE4基因反义表达载体构建及对烟草 (N. tabacum L.)的转化[J]. 南方农业, 2(3): 12-14. (Tong C, Zhang X G, Yan Q L, et al.2008. Construction of the AtUGAE4 antisense gene expression vector and its transformation into tobacco (N. tabacum L.)[J]. South China Agriculture, 2(3): 12-14. [9] 鄢巧灵. 2007. AtUGAE4反义基因对番茄、拟南芥和烟草离体培养细胞粘连性的影响[D]. 硕士学位论文, 西南大学, 导师: 张兴国, pp.19-29. (Yan Q L.2007. Effects of AtUGAE4 anticense gene on the inter-cellular adhesion in vitro of tomato Arabidopsis and tobacco[D]. Thesis for M.S., Southwest University, Supervisor: Zhang X G, pp. 19-29.) [10] 张松雨, 王敬敬, 刘正文, 等. 2019. 四倍体棉花GAE基因家族的鉴定及其在棉纤维发育中的表达分析[J]. 棉花学报, 31(03): 169-181. (Zhang S Y, Wang J J, Liu Z W, et al.2019. Genome-wide identification and expression analysis of GAE gene family in fiber developmental stages of tetraploid cotton[J]. Cotton Science, 31(03): 169-181. [11] Ahmed R I, Ren A, Yang D, et al.2020. Identification and characterization of pectin related gene NbGAE6 through virus-induced gene silencing in Nicotiana benthamiana[J]. Gene, 741:144522. [12] Amselem J, Cuomo C A, van Kan J A, et al.2011. Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea[J]. Plos Genetics, 7: e1002230. [13] Bari R, Jones J D.2009. Role of plant hormones in plant defence responses[J]. Plant Molecular Biology, 69(4): 473-488. [14] Bacete L, Mélida H, Miedes E, et al.2018. Plant cell wall-mediated immunity: Cell wall changes trigger disease resistance responses[J]. Plant Journal, 93(4): 614-636. [15] Barber C, Rosti J, Rawat A, et al.2006. Distinct properties of the five UDP-D-glucose/ UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana[J]. The Journal of Biological Chemistry, 281(25): 17276-17285. [16] Bethke G, Thao A, Xiong G, et al.2016. Pectin biosynthesis is critical for cell wall integrity and immunity in Arabidopsis thaliana[J]. Plant Cell, 28(2): 537-556. [17] Burget E G, Verma R, Molhoj M, et al.2003. The biosynthesis of L-arabinose in plants: Molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis[J]. Plant Cell, 15(2): 523-531. [18] Cosgrove D J.2005. Growth of the plant cell wall[J]. Nature Reviews Molecular Cell Biology, 6(11): 850-861. [19] Dodds P N, Rathjen J P.2010. Plant immunity: Towards an integrated view of plant-pathogen interactions[J]. Nature Reviews Genetics, 11(8): 539-548. [20] Du H, Zhang L, Liu L, et al.2009, Biochemical and molecular characterization of plant MYB transcription factor family[J]. Biochemistry (Moscow), 74(1): 1-11. [21] Guo X M, Stotz H U.2007. Defense against Sclerotinia sclerotiorum in Arabidopsis is dependent on jasmonic acid, salicylic acid, and ethylene signaling[J]. Molecular Plant Microbe Interactions, 20: 1384-1395. [22] Hardham A R, Jones D A, Takemoto D.2007. Cytoskeleton and cell wall function in penetration resistance[J]. Current Opinion In Plant Biology, 10(40): 342-348. [23] Hegedus D D, Li R, Buchwaldt L, et al.2008. Brassica napus possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to Sclerotinia sclerotiorum infection, wounding and defense hormone treatment[J]. Planta, 228(2): 241-253. [24] Hibbett D S, Binder M, Bischoff J F, et al.2007. A higher-level phylogenetic classification of the fungi[J]. Mycological Research, 111(5): 509-547. [25] Hu H Z, Tang Y W, Wu J, et al.2021. Brassica napus mediator subunit16 induces BnMED25- and BnWRKY33-activated defense signaling to confer Sclerotinia sclerotiorum resistance[J]. Frontiers in Plant Science, 12: 663536. [26] Jornvall H, Persson B, Krook M, et al.1995. Short-chain dehydrogenases/reductases (SDR)[J]. Advances in Experimental Medicine & Biology, 372(18): 383. [27] Kabbage M, Yarden O, Dickman M B.2015. Pathogenic attrib-utes of Sclerotinia sclerotiorum: Switching from a biotro-phic to necrotrophic lifestyle[J]. Plant Science, 233: 53-60. [28] Li M, Zhang D, Gao Q, et al.2019. Genome structure and evolution of Antirrhinum majus L.[J]. Nature Plants, 5(2): 174-183. [29] Molhoj M, Verma R, Reiter W D.2004. The biosynthesis of D-Galacturonate in plants. Functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis[J]. Plant Physiology, 135(3): 1221-1230. [30] Riou C, Freyssinet G, Fevre M.1991. Production of cell wall-degrading enzymes by the phytopathogenic fungus Sclerotinia sclerotiorum[J]. Applied and Environmental Microbiology, 57: 1478-1484. [31] Ren Y J, Zou W H, Feng J F, et al.2021. Characterization of the sugarcane MYC gene family and the negative regulatory role of ShMYC4 in response to pathogen Stress[J]. Industrial Crops and Products, 176(2022): 114292. [32] Sun Q, Lin L, Liu D, et al.2018. CRISPR/cas9-mediated multiplex genome editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L[J]. International Journal of Molecular Sciences, 19(9): 2716. [33] Thoden J B, Hegeman A D, Wesenberg G, et al.1997. Structural analysis of UDP-sugar binding to UDP-galactose 4-epimerase from Escherichia coli[J]. Biochemistry, 36(21): 6294-6304. [34] Usadel B, Schlüter U, Molhoj M, et al.2004. Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis[J]. FEBS Letters, 569(1-3): 327-331. [35] Wierenga R K, Terpstra P, Hol W G.1986. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint[J]. Journal of Molecular Biology, 187(1): 101-107. [36] Wu J, Zhao Q, Yang Q Y, et al.2016. Comparative transcriptomic analysis uncovers the complex genetic network for resistance to Sclerotinia cclerotiorum in Brassica napus[J]. Scientific Reports, 6: 19007. [37] Xu G, Yuan M, Ai C, et al.2017. ORF-mediated translation allows engineered plant disease resistance without fitness costs[J]. Nature, 545(7655): 491-494.
