Cloning and Expression Analysis of MYB62 Transcription Factors from Kohlrabi

doi:10.7668/hbnxb.20194583

Abstract

Abstract:

In order to clarify the sequence characteristics of the MYB62 transcription factor,expression changes after stress,and further explore the biological function of the MYB62 transcription factor,kohlrabis were used as plant materials.MYB62 transcription factors were obtained by homologous cloning method and bioinformatics was analyzed.Spatiotemporal expression analysis of MYB62 and its expression after stress were performed by Real-time PCR.Results of gene cloning showed that gDNA length of BocMYB62 gene was 1 353 bp,the length of CDS was 837 bp,which contained four exons and three introns and encoded 278 amino acids.The sequence structure analysis showed that BocMYB62 was a hydrophilic protein with two SANT-MYB domains,belonging to the R2R3-MYB type in the MYB transcription factor family.The spatial structure prediction revealed a typical α-helix structure.Phylogenetic analysis indicated that BocMYB62 was closely related to MYB62 in Brassica napus.The spatiotemporal expression results showed that BocMYB62 was consistently higher in green kohlrabi than that in purple with clear tissue specificity.The expression of BocMYB62 was significantly increased during drought stress with the highest expression under 12 h.The BocMYB62 expression was significantly lower than control and the lowest at 4 ℃ cold stress.These results inferred that BocMYB62 may be involved in the regulation of anthocyanin biosynthesis and the regulation of stress.It provides a theoretical basis for the subsequent functional identification of MYB62 transcription factors.

Key words: Kohlrabi, MYB62, Sequence analysis, Conversed motifs, Relationship, Real-time PCR

WANG Xinmiao, ZHAO Mengliang, SHAO Dengkui, GA Sang, REN Yanjing. Cloning and Expression Analysis of MYB62 Transcription Factors from Kohlrabi[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 62-70. doi: 10.7668/hbnxb.20194583.

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Figures/Tables 10

Fig.1 Amplification of BocMYB62 gene M.DL2000 DNA Marker;1.Green kohlrabi;2.Purple kohlrabi.The gDNA amplification bands were shown on the left and the cDNA amplification bands were shown on the right.

Fig.2 BocMYB62 gene structure of Brassica oleracea L.var.caulorapa

Fig.3 Nucleotide sequence of BocMYB62 gene and its encoded amino acid sequences of Brassica oleracea L.var.caulorapa ^*.Termination codon;—.Intron;Uppercase letter.Amino acid sequence.

Fig.4 Multiple alignment analysis of BocMYB62 protein Wild cabbage BooMYB108-like.XP_013596475.1;Brassica napus BnMYB62-like.XP_013659722.1; Brassica rapa BrMYB62.XP_009115302.1;Wild cabbage BooMYB24.XP_013584285.1.

Fig.5 Prediction of secondary structure of BocMYB62 protein in Brassica oleracea L.var.caulorapa Blue curve.α-helix;Green curve.β-angle;Purple curve.Irregular coil;Red curve.Extended strand.

Fig.6 BocMYB62 protein structure prediction analysis

Fig.7 Conserved motif analysis of the BocMYB62 protein of Brassica oleracea L.var.caulorapa

Fig.8 Phylogenetic relationship of BocMYB62 in Brassica oleracea L.var.caulorapa and anthocyanin-related MYB transcription factors in other species.

Fig.9 Expression levels of BocMYB62 in different tissues of green and purple kohlrabi Different lowercase letters indicate significant difference at the P<0.05 level.The same as Fig.10.

Fig.10 Expression level changes of BocMYB62 under stress A.Expression level changes of BocMYB62 under drought stress in green kohlrabi;B.Expression level of BocMYB62 under cold stress in purple kohlrabi.

References 41

[1]	隋好林, 于尧, 张志芬. 紫苤蓝引种栽培试验[J]. 山东农业科学, 1996, 28(4):24-25.
	Sui H L, Yu Y, Zhang Z. Introduction and cultivation experiment of purple kohlrabi[J]. Shandong Agricultural Sciences, 1996, 28(4):24-25.
[2]	林骁. 特菜新品种——紫苤蓝栽培技术[J]. 农村实用技术, 2005(1):12.
	Lin X. Cultivation techniques of a new special vegetable variety-purple kohlrabi[J]. Nongcun Shiyong Jishu, 2005(1):12.
[3]	夏明艳, 金静华. 苤蓝的特征特性及高产栽培技术[J]. 上海蔬菜, 2020(5):41,43.doi:10.3969/j.issn.1002-1469.2020.05.017.
	Xia M Y, Jin J H. Characteristics and high-yield cultivation techniques of kohlrabi[J]. Shanghai Vegetables, 2020(5):41,43.
[4]	袁菱婧, 罗盼, 蒋明, 李温平, 何建婷. 紫苤蓝花青素合成酶基因BocANS的克隆与分析[J]. 浙江农业科学, 2012, 53(7):1060-1062,1066.doi:10.3969/j.issn.0528-9017.2012.07.047.
	Yuan L J, Luo P, Jiang M, Li W P, He J T. Cloning and analysis of anthocyanin synthase gene BocANS from Echinacea purpurea[J]. Journal of Zhejiang Agricultural Sciences, 2012, 53(7):1060-1062,1066.
[5]	张金鹏, 王利波, 陈莹, 张爽爽, 惠长敏. 紫苤蓝北方寒地秋冬季温室栽培技术[J]. 吉林蔬菜, 2019(1):14-15.doi:10.16627/j.cnki.cn22-1215/s.2019.01.010.
	Zhang J P, Wang L B, Chen Y, Zhang S S, Hui C M. Cultivation techniques of purple kohlrabi in greenhouse in autumn and winter in northern cold region[J]. Jilin Vegetable, 2019(1):14-15.
[6]	牛义岭, 姜秀明, 许向阳. 植物转录因子MYB基因家族的研究进展[J]. 分子植物育种, 2016, 14(8):2050-2059.doi:10.13271/j.mpb.014.002050.
	Niu Y L, Jiang X M, Xu X Y. Research advance on transcription factor MYB gene family in plant[J]. Molecular Plant Breeding, 2016, 14(8):2050-2059.
[7]	艾佳琦, 庞鸿涛, 胡天华, 包崇来, 魏庆镇. 茄果类蔬菜MYB转录因子研究进展[J]. 中国蔬菜, 2023(6):23-33.doi:10.19928/j.cnki.1000-6346.2023.2029.
	Ai J Q, Pang H T, Hu T H, Bao C L, Wei Q Z. Research progress on MYB transcription factors of solanaceous fruit vegetable[J]. China Vegetables, 2023(6):23-33.
[8]	郭凯, 侯留迪, 张莹莹, 周开明, 张丙林, 邹华文. 植物MYB基因家族研究进展[J]. 长江大学学报(自然科学版), 2020, 17(6):93-98.doi:10.3969/j.issn.1673-1409.2020.06.016.
	Guo K, Hou L D, Zhang Y Y, Zhou K M, Zhang B L, Zou H W. Research progress of MYB gene family in plants[J]. Journal of Yangtze University (Natural Science Edition), 2020, 17(6):93-98.
[9]	Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T, Kanai H, Sarai A, Ishii S, Nishimura Y. Solution structure of a specific DNA complex of the Myb DNA-binding do-main with cooperative recognition helices[J]. Cell, 1994, 79(4):639-648.doi:10.1016/0092-8674(94)90549-5. pmid: 7954830
[10]	靳亚忠, 齐娟, 何淑平, 吴兴彪, 张天一, 王雪娇, 胡文曲, 卢朝晖, 张鹏. R2R3-MYB转录因子调控果蔬品质形成的研究进展[J]. 农业生物技术学报, 2021, 29(2):364-374.doi:10.3969/j.issn.1674-7968.2021.02.016.
	Jin Y Z, Qi J, He S P, Wu X B, Zhang T Y, Wang X J, Hu W Q, Lu Z H, Zhang P. Recent advance of R2R3-MYB transcription factors regulating the formation of fruit and vegetable qualities[J]. Journal of Agricultural Biotechnology, 2021, 29(2):364-374.
[11]	关淑艳, 焦鹏, 蒋振忠, 齐拙, 夏海丰, 曲静, 马义勇. MYB转录因子在植物非生物胁迫中的研究进展[J]. 吉林农业大学学报, 2019, 41(3):253-260.doi:10.13327/j.jjlau.2019.5023.
	Guan S Y, Jiao P, Jiang Z Z, Qi Z, Xia H F, Qu J, Ma Y Y. Research progress of MYB transcription factors in plant abiotic stress[J]. Journal of Jilin Agricultural University, 2019, 41(3):253-260.
[12]	张磊, 曹德美, 胡建军. 植物叶色形成调控机制研究进展[J]. 植物遗传资源学报, 2021, 22(2):293-303.doi:10.13430/j.cnki.jpgr.20200805001.
	Zhang L, Cao D M, Hu J J. Advance of the regulation mechanism of leaf color formation in plants[J]. Journal of Plant Genetic Resources, 2021, 22(2):293-303.
[13]	张驰, 王艳芳, 陈静, 王义, 张美萍. 植物MYB转录因子调控次生代谢的研究进展[J]. 基因组学与应用生物学, 2020, 39(9):4171-4177.doi:10.13417/j.gab.039.004171.
	Zhang C, Wang Y F, Chen J, Wang Y, Zhang M P. Research advances on the regulation of secondary metabolism by plant MYB transcription factors[J]. Genomics and Applied Biology, 2020, 39(9):4171-4177.
[14]	谷佳, 王童欣, 饶英, 潘庆龙, 李霆格, 赵莹, 周扬, 彭婷, 王健. 基于三色堇全长转录组的MYB基因家族鉴定[J]. 江苏农业科学, 2022, 50(22):11-19.doi:10.15889/j.issn.1002-1302.2022.22.002.
	Gu J, Wang T X, Rao Y, Pan Q L, Li T G, Zhao Y, Zhou Y, Peng T, Wang J. Identification of MYB gene family based on full-length transcriptome of Corydalis tricolor[J]. Jiangsu Agricultural Sciences, 2022, 50(22):11-19.
[15]	Wang X, Niu Y, Zheng Y. Multiple functions of MYB transcription factors in abiotic stress responses[J]. International Journal of Molecular Sciences, 2021, 22(11):6125.doi:10.3390/ijms22116125. URL
[16]	Bogs J, JafféF W, Takos A M, Walker A R, Robinson S P. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development[J]. Plant Physiology, 2007, 143(3):1347-1361.doi:10.1104/pp.106.093203. pmid: 17208963
[17]	Walker A R, Lee E, Bogs J, McDavid D A J, Thomas M R, Robinson S P. White grapes arose through the mutation of two similar and adjacent regulatory genes[J]. The Plant Journal, 2007, 49(5):772-785.doi:10.1111/j.1365-313x.2006.02997.x.
[18]	Spelt C, Quattrocchio F, Mol J N, Koes R. anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes[J]. The Plant Cell, 2000, 12(9):1619-1632.doi:10.1105/tpc.12.9.1619. URL
[19]	Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis[J]. The EMBO Journal, 2000, 19(22):6150-6161.doi:10.1093/emboj/19.22.6150. URL
[20]	陈江, 唐小慧, 任超翔, 陈骁, 何雯, 张思源, 吴清华, 裴瑾. 红花MYB转录因子基因克隆及表达分析[J]. 药学学报, 2018, 53(1):141-146.doi:10.16438/j.0513-4870.2017-0614.
	Chen J, Tang X H, Ren C X, Chen X, He W, Zhang S Y, Wu Q H, Pei J. Cloning and expression analysis of MYB transcription factor genes in safflower[J]. Acta Pharmaceutica Sinica, 2018, 53(1):141-146.
[21]	李军, 赵爱春, 刘长英, 吕蕊花, 刘晓清, 余茂德. 桑树花青素合成相关MYB类转录因子的鉴定与功能分析[J]. 西北植物学报, 2016, 36(6):1110-1116.doi:10.7606/j.issn.1000-4025.2016.06.1110.
	Li J, Zhao A C, Liu C Y, Lü R H, Liu X Q, Yu M D. Identification and function analysis of anthocyanin biosynthesis related MYB genes in mulberry[J]. Acta Botanica Boreali-Occidentalia Sinica, 2016, 36(6):1110-1116.
[22]	Devaiah B N, Madhuvanthi R, Karthikeyan A S, Raghothama K G. Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis[J]. Molecular Plant, 2009, 2(1):43-58.doi:10.1093/mp/ssn081. URL
[23]	Chen Y H, Yang X Y, He K, Liu M H, Li J G, Gao Z F, Lin Z Q, Zhang Y F, Wang X X, Qiu X M, Shen Y P, Zhang L, Deng X H, Luo J C, Deng X W, Chen Z L, Gu H Y, Qu L J. The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family[J]. Plant Molecular Biology, 2006, 60(1):107-124.doi:10.1007/s11103-005-2910-y. URL
[24]	Qi X, Tang W S, Li W W, He Z, Xu W Y, Fan Z J, Zhou Y B, Wang C X, Xu Z S, Chen J, Gao S Q, Ma Y Z, Chen M. Arabidopsis G-protein β subunit AGB1 negatively regulates DNA binding of MYB62,a suppressor in the gibberellin pathway[J]. International Journal of Molecular Sciences, 2021, 22(15):8270.doi:10.3390/ijms22158270. URL
[25]	Wei B, Hou K, Zhang H H, Wang X Y, Wu W. Integrating transcriptomics and metabolomics to studies key metabolism,pathways and candidate genes associated with drought-tolerance in Carthamus tinctorius L.under drought stress[J]. Industrial Crops and Products, 2020,151:112465.doi:10.1016/j.indcrop.2020.112465.
[26]	丁霄, 李朋波, 雷梦林, 温思钰, 杨六六, 罗晓莉, 秦丽霞, 潘转霞, 李换丽, 吴翠翠, 朱永红, 夏芝, 曹彩荣. 一种改良CTAB法提取棉花基因组DNA:CN107699561A[P].2018-02-16.
	Ding X, Li P B, Lei M L, Wen S Y, Yang L L, Luo X L, Qin L X, Pan Z X, Li H L, Wu C C, Zhu Y H, Xia Z, Cao C R. An improved CTAB method for extracting cotton genomic DNA:CN107699561A[P].2018-02-16.
[27]	赵文菊, 王紫莹, 王鑫淼, 巴桑措姆, 李江, 任延靖. 球茎甘蓝MYB115转录因子的克隆与表达分析[J]. 青海大学学报, 2023, 41(1):21-27.doi:10.13901/j.cnki.qhwxxbzk.2023.01.004.
	Zhao W J, Wang Z Y, Wang X M, Basangcuomu, Li J, Ren Y J. Cloning and expression analysis of MYB115 transcription factor from Brassica oleracea L.var.caulorapa[J]. Journal of Qinghai University, 2023, 41(1):21-27.
[28]	杨雪, 雒军, 王引权. 调控植物黄酮类化合物生物合成的MYB转录因子研究进展[J]. 甘肃中医药大学学报, 2018, 35(6):77-81.doi:10.16841/j.issn1003-8450.2018.06.17.
	Yang X, Luo J, Wang Y Q. MYB transcription factors involved in regulation of flavonoid biosynthesis of medicinal plants[J]. Journal of Gansu University of Chinese Medicine, 2018, 35(6):77-81.
[29]	Zhou H P, He J X, Zhang Y Y, Zhao H Y, Sun X, Chen X, Liu X R, Zheng Y, Lin H H. RHA2b-mediated MYB30 degradation facilitates MYB75-regulated,sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings[J]. Plant Communications, 2024, 5(3):100744.doi:10.1016/j.xplc.2023.100744. URL
[30]	He Q, Wu J Q, Xue Y H, Zhao W B, Li R, Zhang L G. The novel gene BrMYB2,located on chromosome A07,with a short intron 1 controls the purple-head trait of Chinese cabbage(Brassica rapa L.)[J]. Horticulture Research, 2020,7:97.doi:10.1038/s41438-020-0319-z.
[31]	Yang J F, Song H D, Chen Y Z, Chen B W, Kim M, Kim P, Kawabata S, Li Y H, Wang Y. A single amino acid substitution in the R2R3 conserved domain of the BrPAP1a transcription factor impairs anthocyanin production in turnip(Brassica rapa subsp.Rapa)[J]. Plant Physiology and Biochemistry, 2021, 162:124-136.doi:10.1016/j.plaphy.2021.02.011. URL
[32]	An G H, Chen J J. Frequent gain-and loss-of-function mutations of the BjMYB113 gene accounted for leaf color variation in Brassica juncea[J]. BMC Plant Biology, 2021, 21(1):301.doi:10.1186/s12870-021-03084-5.
[33]	Lim S H, Kim D H, Lee J Y. Molecular mechanism controlling anthocyanin composition and content in radish plants with different root colors[J]. Plant Physiology and Biochemistry, 2023,204:108091.doi:10.1016/j.plaphy.2023.108091.
[34]	Xie T, Zan X Y, Chen X, Zhu H T, Rong H, Wang Y P, Jiang J J. An R3-MYB repressor,BnCPC forms a feedback regulation with MBW complex to modulate anthocyanin biosynthesis in Brassica napus[J]. Biotechnology for Biofuels and Bioproducts, 2022, 15(1):133.doi:10.1186/s13068-022-02227-6.
[35]	Xu J, Fan Y R, Han X J, Pan H H, Dai J H, Wei Y, Zhuo R Y, Liu J. Integrated transcriptomic and metabolomic analysis reveal the underlying mechanism of anthocyanin biosynthesis in toona sinensis leaves[J]. International Journal of Molecular Sciences, 2023, 24(20):15459.doi:10.3390/ijms242015459. URL
[36]	秦子璐, 何威, 王雨芳, 吴敏. 毛竹PheMYB2R-4的克隆与功能分析[J]. 林业科学研究, 2023, 36(5):100-110.doi:10.12403/j.1001-1498.20230026.
	Qin Z L, He W, Wang Y F, Wu M. Cloning and functional analysis of PheMYB2R-4 in Phyllostachys edulis[J]. Forest Research, 2023, 36(5):100-110.
[37]	刘保国, 张艳文, 姚珂心, 史志坤, 李菲, 李永华, 张开明. 四季秋海棠BsMYB62的抗旱性功能研究[J]. 西北农林科技大学学报(自然科学版), 2024(4):1-10.doi:10.13207/j.cnki.jnwafu.2024.04.011.
	Liu B G, Zhang Y W, Yao K X, Shi Z K, Li F, Li Y H, Zhang K M. A study on the drought resistance function of four seasons autumn Begonia BsMYB62 [J]Journal of Northwest A&F University(Natural Science Edition), 2024(4):1-10.
[38]	刘壮斌. 低温胁迫下番茄MYB113基因的功能分析[D]. 泰安: 山东农业大学, 2019.doi: 10.7666/d.D01699056.
	Liu Z B. Functional analysis of tomato MYB113 gene under low temperature stress[D]. Taian: Shandong Agricultural University,2019.
[39]	刁鹏飞. SlMYB41基因的克隆及其在番茄低温胁迫响应中的功能分析[D]. 泰安: 山东农业大学, 2020.doi:10.27277/d.cnki.gsdnu.2020.000955.
	Diao P F. Cloning of SlMYB41 gene and its functional analysis in tomato response to low temperature stress[D]. Taian: Shandong Agricultural University,2020.
[40]	冯盼盼. 番茄SlMYB71基因在干旱和盐胁迫响应中的功能研究[D]. 重庆: 重庆大学, 2021.doi:10.27670/d.cnki.gcqdu.2021.001401.
	Feng P P. Study on the function of tomato SlMYB71 gene in response to drought and salt stress[D]. Chongqing: Chongqing University,2021.
[41]	Wang M L, Hao J, Chen X H, Zhang X C. SlMYB102 expression enhances low-temperature stress resistance in tomato plants[J]. PeerJ, 2020,8:e10059.doi:10.7717/peerj.10059.

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