Cloning of Cold Tolerance-related Genes RsMYBS3 and RsRCI2B of Rhododendron sphaeroblastum var. wumengense and Their Expression Analysis
PENG Wan-Ming, TAN Yi, ZHANG Xiao-Li, LUO Liang, LI Xin-Yi, HUANG Hai-Quan*, HUANG Mei-Juan*
College of Landscape Architecture and Horticulture Sciences, Southwest Research Center for Engineering Technology of Landscape Architecture (State Forestry and Grassland Administration)/Yunnan Engineering Research Center for Functional Flower Resources and Industrialization/Research and Development Center of Landscape Plants and Horticulture Flowers, Southwest Forestry University, Kunming 650224, China
Abstract：Rhododendron sphaeroblastum var. wumengense is a high-altitude and cold-tolerant plant. MYBS3 and rare cold inducible 2 (RCI2B) genes were found to play a significant role in enhancing cold stress tolerance in plants based on the pre-screening from R. sphaeroblastum transcriptome. In this study, the key transcription factors RsMYBS3 (GenBank No. OR178946) and RsRCI2B (GenBank No. OR178945) genes that regulated cold tolerance of R. sphaeroblastum were cloned, whose cDNA lengths were 795 and 165 bp, encoding 264 and 54 aa, respectively. Bioinformatics analysis showed that RsMYBS3 was a hydrophilic unstable protein with a typical SANT conserved structural domain of the SANT superfamilies; RsRCI2B was a hydrophobic stable protein with a highly conservative structural domain typical PMP3. The results of gene homology comparison showed that both RsMYBS3 and RsRCI2B proteins were highly homologous to MYBS3 and RCI2B proteins in red horse psyllium (Rhododendron vialii). The similarities were 93.73% and 94.44%, respectively. Phylogenetic analysis showed that RsMYBS3 and RsRCI2B were clustered in a clade with RvMYBS3 and RvRCI2B from R. vialii, which suggested that 4 genes were orthologous. qPCR analysis showed that both genes were significantly expressed in R. sphaeroblastum under the low-temperature stress, but had different expression patterns under different stress temperatures and times. The RsMYBS3 gene had the highest expression level at -6 ℃ treatment on 1 d, and then gradually decreased on 4 d with the prolonging of stress time, and expression gradually up-regulated on 7 d. The expression level gradually increased and reached a peak from 1 d to 4 d, and gradually decreased on 7 d under the -12 ℃ treatment. While the expression level of RsRCI2B gene had gradually increased from 1 d to the 7 day at -6 ℃ treatment, and at -12 ℃ treatment, except for the slight decrease expression on 1 d, the expression level gradually increased and reached a peak from 4 d to 7 d as the stress time prolonged. The above mentioned results suggested that both RsMYBS3 and RsRCI2B genes might be involved in the cold-tolerance-response physiological process of R. sphaeroblastum. This study provides some certain basic data and theoretical basis for exploring their cold tolerance molecular mechanism in the future.
[1] 窦同心. 2016. 香蕉抗寒、抗病相关基因的遗传转化验证[D]. 博士学位论文, 华南农业大学, 导师: 易干军; 彭新湘, pp. 18-69. (Dou T X.2016. Genetic transformation verification of cold-tolerance or disease-resistance related genes in Banana[D.]. Thesis for M.S., South China Agricultural University, Supervisor: Yi G J; Peng X X, pp. 18-69.) [2] 丁小玲, 张宁波, 焦淑珍, 等. 2017. 山葡萄COR413家族基因克隆及其参与低温胁迫的表达分析[J]. 农业生物技术学报, 25(03): 366-377. (Ding X L, Zhang N B, Jiao S Z, et al.2017. Cloning and expression analysis of COR413 family genes from Vitisamurensis in response to cold stress[J]. Journal of Agricultural Biotechnology, 25(03): 366-377.) [3] 高洁. 2017. 大蕉与香牙蕉抗寒性差异的分子机理解析[D]. 博士学位论文, 华南农业大学, 导师: 易干军; 彭新湘. pp. 25-26. (Go J.2017. Analysis of molecular mechanism of the different cold tolerance between da jiao and Cavendish banana[D]. Thesis for Ph.D., South China Agricultural University, Supervisor: Yi G J; Peng X X, pp. 25-26.) [4] 何盈, 雷云生, 张劲, 等. 2023. 杜鹃bHLH转录因子RsMYC2的克隆及在非生物胁迫下的表达分析[J]. 分子植物育种, 21(04): 1103-1110. (He Y, Lei Y S, Zhang J, et al.2023. Cloning and expression analysis of Rhododendron bHLH transcription factor RsMYC2 under abiotic Stress[J]. Molecular Plant Breeding, 21(04): 1103-1110.) [5] 金锋, 丁莲鑫, 骆骏, 等. 2023. 水稻MYB转录因子的研究进展[J]. 植物遗传资源学报, 24(04): 917-926. (Jin F, Ding L X, Luo J, et al.2023. Research progress of MYB transcription factors in rice[J]. Journal of Plant Genetic Resources, 24(04): 917-926.) [6] 蔺海娇, 梁雨晨, 李玲, 等. 2023. 薰衣草CBF途径相关耐寒基因挖掘与调控网络分析[J]. 园艺学报, 50(01): 131-144. (Lin H J, Liang Y C, Li L, et al.2023. Exploration and regulation network analysis of CBF pathway related cold tolerance genes in Lavandula angustifolia[J]. Acta Horticulturae Sinica, 50(01): 131-144.) [7] 刘楚楚, 吴玉香. 2021. 棉花RCI2基因家族的鉴定及表达分析[J]. 山西农业大学学报(自然科学版), 41(4): 41-49. (Liu C C, Wu Y X.2021. Identification and expression analysis of RCI2 gene family in cotton[J]. Journal of Shanxi Agricultural University (Natural Science Edition), 41(4): 41-49.) [8] 饶席兵, 钱禛锋, 张蓉琼, 等. 2022. 蔗茅EfMYB1基因的克隆与表达分析[J]. 西北植物学报, 42(09): 1487-1494. (Rao X B, Qian Z F, Zhang R Q, et al.2022. Molecular cloning and expression analysis of EfMYB1 in Erianthus fulvus[J]. Acta Botanica Boreali-Occidentalia Sinica, 42(09): 1487-1494.) [9] 任鸿雁, 魏强. 2015. 水稻磷胁迫响应基因OsRCI2-9的克隆及功能鉴定[J]. 中国农业科学, 48(05): 831-840. (Ren H Y, Wei Q.2015. Isolation and functional analysis of phosphate-responsive gene OsRCI2-9 in Oryza sativa[J]. Scientia Agricultura Sinica, 48(05): 831-840.) [10] 童海青. 2013. 玉米全基因组RCI2基因特征及表达分析[D]. 硕士学位论文, 安徽农业大学, 导师: 朱苏文, pp. 16-22. (Tong H Q.2013. Genome-wide survey and characterization of the RC12 gene family in Zea mays[D.]. Thesis for M.S., Anhui Agricultural University, Supervisor: Zhu S W, pp. 16-22.) [11] 战力峰, 李淳馨, 何凯, 等. 2021. MsRCI2A、MsRCI2B、MsRCI2C基因超量表达对紫花苜蓿耐冷的影响[J]. 北方园艺, (18): 67-74. (Zhan L F, Li C X, He K, et al. 2021. Effects of overexpression of MsRCI2A, MsRCI2B and MsRCI2C genes on cold tolerance of Alfalfa[J]. Northern Horticulture, (18): 67-74.) [12] 朱志鹏, 刘慧玲, 吴可鑫, 等. 2022. 秋葵AeMYB1R1转录因子基因克隆及对胁迫的响应[J]. 西北植物学报, 42(06): 909-919. (Zhu Z P, Liu H L, Wu K X, et al.2022. Cloning transcription factor AeMYB1R1 gene of Abelmoschus esculentus and analysis of its response to abiotic stress[J]. Acta Botanica Boreali-Occidentalia Sinica, 42(06): 909-919.) [13] 张蓉琼, 钱禛锋, 谷书杰, 等. 2022. 基于转录组的蔗茅EfPHD-finger家族基因鉴定及冷胁迫表达分析[J]. 农业生物技术学报, 30(11): 2128-2140. (Zhang R Q, Qian Z F, Gu S J, et al.2022. Transcriptome-wide identification and cold stress expression analysis of EfPHD-finger family genes in Erianthus fulvus[J]. Journal of Agricultural Biotechnology, 30(11): 2128-2140.) [14] Chen S, Hang H A, Chen J H, et al.2020. SgRVE6, a LHY-CCA1-like transcription factor from fine-stem stylo, Upregulates NB-LRR gene expression and enhances cold tolerance in tobacco[J]. Frontiers in Plant Science, 11: 1 276. [15] Dou T X, Hu C H, Sun X X, et al.2016. MpMYBS3 as a crucial transcription factor of cold signaling confersthe cold tolerance of banana[J]. Plant Cell, Tissue and Organ Culture, 125(1): 93-106. [16] Feng D R, Liu B, Li W Y, et al.2009. Over-expression of a cold-induced plasma membrane protein gene (MpRCI) from plantain enhances low temperature-resistance in transgenic tobacco[J]. Environmental and Experimental Botany, 65(2-3): 395-402. [17] Capel J A, Jarillo J, Salinas, et al. 1997. Two Homologous Low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins[J]. Oxford University Press (oup), 115(2): 569-576. [18] Joaquín M, Luisa M B, Julio S.2007. Phylogenetic and functional analysis of Arabidopsis RCI2 genes[J]. Journal of experimental botany, 58(15-16): 4333-4346. [19] Kim H S, Lee J E, Jang H Y, et al.2016. CsRCI2A and CsRCI2E genes show opposite salt sensitivity reaction due to membrane potential control[J]. Acta Physiologiae Plantarum, 38(2): 1-13. [20] Kim H S, Park W, Lim H G, et al.2019. NaCl-induced CsRCI2E and CsRCI2F interact with aquaporin CsPIP2; 1 to reduce water transport in Camelina sativa L[J]. Biochemical and Biophysical Research Communications, 513(1): 213. [21] Kim H S, Park W, Lee H, et al.2021. Subcellular journey of rare cold inducible 2 protein in plant under stressful condition[J]. Frontiers in Plant Science, 11: 2201. [22] Li H L, Guo D, Peng S Q.2014. Molecular characterization of the Jatropha curcas JcR1MYB1 gene encoding a putative R1-MYB transcription factor[J]. Genetics and Molecular Biology, 37(3): 549-555. [23] Lu C A, Ho T H, Ho S L, et al.2002. Three novel MYB proteins with one DNA binding repeat mediate sugar and hormone regulation of alpha-amylase gene expression[J]. The Plant Cell, 14(8): 1963-1980. [24] Liu J, Shi Y, Yang S, et al.2018. Insights into the regulation of C-repeat binding factors in plant cold signaling[J]. Journal of Integrative Plant Biology, 60(9): 780-795. [25] Medina J, Catala R, Salinas J, et al.2001. Developmental and stress regulation of RCI2A and RCI2B, two cold-inducible genes of Arabidopsis encoding highly conserved hydrophobic proteins[J]. Plant Physiology, 125(4): 1655-1687. [26] Navarre C.2000. Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane[J]. Springer Science and Business Media LLC, 19(11): 2515-2524. [27] Pedro S. C. F. Rocha.2016. Erratum to: Plant abiotic stress-related RCI2/PMP3s: Multigenes for multiple roles[J]. Planta, 244: 287. [28] Sun W Q, Li M D, Wang J B, 2022. Genome-wide identification and characterization of the RCl2 gene family in allotetraploid Brassica napus compared with lts diploid progenitors[J]. International Journal of Molecular Sciences, 23(2): 614. [29] Su C F, Wang Y C, Hsieh T H, et al.2010. A novel MYBS3-dependent pathway confers cold tolerance in rice[J]. Plant Physiololy, 153(1): 145-158. [30] Yang A, Dai X Y, Zhang W H.2012. A R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice[J]. Journal of Experimental Botany, 63(7): 2541-2556. [31] Yang L C, Li P, Qiu L A, et al.2022. Identification and comparative analysis of the rosaceae RCI2 gene family and characterization of the cold stress response in Prunus mume[J]. Horticulturae Pub Date, 8(11): 997. [32] Zhou Y, Ge L L, Li G H, et al.2019. In silico identification and expression analysis of rare cold inducible 2 (RCI2) gene family in cucumber[J]. Journal of Plant Biochemistry and Biotechnology, 29(1): 56-66.
