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| Cloning and Expression Analysis of LsMYB5 Gene in Lycoris sprengeri |
| HOU Shuo, GAO Yan-Hui*, TONG Zai-Kang |
| State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin' an 311300, China |
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Abstract Lycoris sprengeri is perennial herbaceous plants of Amaryllidaceae, Lycoris with the red and blue multicolor flower, which is a good material for cut flower, potted flower and landscape gardening, so L. sprengeri has the very high commercial value. To enrich the germplasm resources and cultivate varieties in L. sprengeri, a R2R3-MYB transcription factor gene LsMYB5 cDNA sequence (GenBank No. MK779710) was successfully screened by using reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The LsMYB5 gene length was 871 bp, and the open reading frame (ORF) was 681 bp, which encoded 226 amino acids. The c-terminal of LsMYB5 protein contained conservative EAR inhibition domain (pdLNLD/ELxiG/s) and zinc finger domain (CX-(1-2), CX-(7-12), CX-(1-2)C), which may have transcriptional inhibition function. LsMYB5 gene mainly expressed in leaves and the fading period of petals, and there were significant differences in the expression of LsMYB5 in clones with different colors (P<0.05). To further analyze LsMYB5 gene function, prokaryotic expression vector of pET-30(a)-LsMYB5 was constructed and transformed into Escherichia coli BL21 (DE3), the expression of recombinant proteins was induced under the induction of isopropyl glucosinolates galactose glucoside (IPTG), and the His label protein was purified. The results will provide the theory basis for screening structural genes regulated by LsMYB5 transcription factor. which related to flower color formation in Lycoris sprengeri.
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Received: 15 April 2019
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Corresponding Authors:
* gaoyanhui408@126.com
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[1] 陈清, 汤浩茹, 董晓莉, 等. 2009. 植物Myb转录因子的研究进展[J]. 基因组学与应用生物学, 28(02): 365-372,(Chen Q, Tang H R, Dong X L, et al. 2009. Progress in the study of plant myb transcription factors[J]. Genomics and Applied Biology, 28(02): 365-372.)
[2] 戴智慧, 龙骏, 俞雷民, 等. 2016. 宁波地区野生换锦花鳞茎的主要成分含量测定[J]. 天然产物研究与开发, 28(08): 1238-1243+1250,(Dai Z H, Long J, Yu L M, et al. 2016. Determination of main components in bulbs of wild Lycoris sprengeri from Ningbo[J]. Natural Product Research and Development, 28(08): 1238-1243+1250.)
[3] 杜照奎, 李钧敏. 2015. 花生AhRRF基因的克隆与原核表达[J]. 核农学报, 29(10): 1867-1875.
(Du Z K, Li J M.2015. Peanut AhRRF cloning and prokaryotic expression of genes[J]. Journal of Nuclear Agricultural Sciences, 29(10): 1867-1875.)
[4] 高燕会, 黄春红, 朱玉球, 等. 2012. 植物花青素苷生物合成及调控的研究进展[J], 中国生物工程杂志, 32(08): 94-99.
(Gao Y H, Huang C H, Zhu Y Q, et al.2012. Advances in studies on biosynthesis and regulation of plant anthocyanins[J]. China Biotechnology, 32(08): 94-99.)
[5] 葛翠莲, 黄春辉, 徐小彪. 2012. 果实花青素生物合成研究进展[J], 园艺学报, 39(09): 1655-1664.
(Ge C L, Huang C H, Xu X B.2012. Research on anthocyanins biosynthesis in fruit[J]. Acta Horticulturae Sinica, 39(09): 1655-1664.)
[6] 宫硖, 薛静, 张晓东. 2011. 植物花青素合成途径中的调控基因研究进展[J], 生物技术进展, 01(6): 381-390.
(Gong X, Xue J, Zhang X D.2011. Regulation genes in plant anthocyanin synthesis pathway[J]. Current Biotechnology, 01(6): 381-390.)
[7] 官丽莉, 张雪, 韩怡来, 等. 2015. 红花转录因子CtMYB1基因的克隆及原核表达[J].中草药, 46(17): 2603-2609.
(Guan L L, Zhang X, Han Y L, et al.2015. Cloning and prokaryotic expression of transcription factor CtMYB1 gene in Carthamus tinctorius[J]. Chinese Traditional and Herbal Drugs, 46(17): 2603-2609.)
[8] 胡可, 韩科厅, 戴思兰. 2010. 环境因子调控植物花青素苷合成呈色的机理[J]. 植物学报, 45(03): 307-317.
(Hu K, Han K T, Dai S L.2010. Regulation of plant anthocyaninsynthesis and pigmentation by environmental factors[J]. Bulletin of Botany, 45: 307-317.)
[9] 贾赵东, 马佩勇, 边小峰, 等. 2014. 植物花青素合成代谢途径及其分子调控[J]. 西北植物学报, 34(07): 1496-1506.
(Jiao Z D, Ma P Y, Bian X F, et al.2014. Biosynthesis metabolic pathway and molecular regulation of plants anthocyanin[J]. Acta Botanica Boreali-Occidentalia Sinica, 34(07): 1496-1506.)
[10] 蒋婷婷, 高燕会, 童再康. 2015. 石蒜属植物实时荧光定量PCR内参基因的选择[J]. 园艺学报, 42(06): 1129-1138.
(Jiang T T, Gao Y H, Tong Z K.2015. Selection of reference genes for quantitative real-time PCR in Lycoris[J]. Acta Horticulturae Sinica, 42(06): 1129-1138.)
[11] 刘欣, 李云. 2006. 转录因子与植物抗逆性研究进展[J]. 中国农学通报, 22(4): 61-61.
(Liu X, Li Y.2006. Transcription factors re1ated to plant stress-to1erance[J]. Chinese Agricultural Science Bulletin, 22(4): 61-61.)
[12] 孟祥春, 张玉进, 王小菁. 2001. 矮牵牛花瓣发育过程中花色素苷、还原糖及蛋白质含量的变化[J]. 华南师范大学学报(自然科学版), (02): 96-99.
(Meng X C, Zhang Y J, Wang X J.2001. Anthocyanin and reducing sugar and the protein content change during petal development of Petunia hybrida[J]. Ournal of South China Normal University (Natural Science Edition), (02):96-99.)
[13] 庞红霞, 祝长青, 覃建兵. 2010. 植物花青素生物合成相关基因研究进展[J]. 种子, 29(03): 60-64.
(Pang H X, Zhu C Q, Qin J B, 2010. Advancement of plant genesrelated with anthocyanins synthetic biology[J]. Seed, 29(3): 60-64.)
[14] 乔玲玲, 马雪蕾, 张昂, 等. 2016. 不同因素对赤霞珠果实理化性质及果皮花色苷含量的影响[J]. 西北农林科技大学学报(自然科学版), 44(02):129-136.
(Qiao L L, Ma X L, Zhang A, et al.2016. Effects of different factors on physicochemical properties and the content of anthocyanin in the skin of cabernet sauvignon[J]. Journal of Northwest A&F University(Natural Science Edition), 44(02): 129-136. )
[15] 佘琳芳, 陈菁珏, 常乐, 等. 2014. 两种石蒜属植物花器官愈伤组织诱导及其亚显微结构观察[J]. 核农学报, 28(10):1798-1805.
(Yu L F, Chen J Y, Chang L, et al.2014. Callus induction of flower organs in two Lycoris and its submicroscopic structure observation[J]. Journal of Nuclear Agricultural Sciences, 28(10): 1798-1805. )
[16] 时剑, 童再康, 刘志高, 等. 2011. 换锦花种胚和叶片的组织培养研究[J]. 江西农业大学学报, 33(4): 665-669.
(Shi J, Tong Z K, Liu Z G, et al.2011. Tissue culture of leaves and embryo in Lycoris sprengeri[J]. Acta Agriculturae Universitatis Jiangxiensis, 33(4): 665-669.)
[17] 石艳, 童再康, 高燕会. 2018. 换锦花EST-SSR标记开发及遗传多样性分析[J]. 核农学报, 32(06): 1089-1096.
(Shi Y, Tong Z K, Gao Y H.2018, Development of EST-SSR markers and analysis of genetic diversity in Lycoris sprengeri[J]. Journal of Nuclear Agricultural Sciences, 32(06): 1089-1096.)
[18] 舒庆艳, 朱瑾, 门思琦, 等. 2018. 基于牡丹类黄酮糖基转移酶基因建立VIGS技术体系[J]. 园艺学报, 45(01): 168-176.
(Shu Q Y, Zhu J, Men S Q, et al.2018. Establishing virus induced gene silencing (VIGS) system in tree peony using PsUFGT gene[J]. Acta Horticulturae Sinica, 45(01): 168-176.)
[19] 宿红艳, 王磊, 王仲礼, 等. 2010. 杨树R2R3 MYB基因PeMYBL1的克隆及表达[J]. 林业科学, 46(01): 142-146.
(Su H Y, Wang L, Wang Z L, et al.2010. Cloning and characterization of PeMYBL1, an R2R3 Gene from poplar[J]. Scientia Silvea Snicae, 46(01): 142-146.)
[20] 王芳, 董美玲, 董乐, 等. 2019. 蓖麻延伸因子基因的克隆与表达分析[J]. 核农学报, 33(03): 464-472.
(Wang F, Dong M L, Dong L, et al.2019. Cloning and expression analysis of ramie elongation factor gene[J]. Journal of Nuclear Agricultural Sciences, 33(03): 464-472.)
[21] 王玲, 汤浩茹, 王小蓉, 等. 2017. 利用VIGS技术研究草莓FaMYB5的功能[J]. 园艺学报, 44(01): 33-42.
(Wang L, Tang H R, Wang X R, et al.2017. Virus-induced gene silencing as a tool for FaMYB5 gene functional studies in strawberry[J]. Acta Horticulturae Sinica, 44(01): 33-42.)
[22] 汪仁, 徐晟, 蒋明敏, 等. 2014. 换锦花和中国石蒜对干旱胁迫的生理响应[J]. 西北植物学报, 34(10): 2041-2048.
(Wang R, Xu S, Jiang M M, et al.2014. Physiological response of Lycoris sprengeri and Lycoris chinensis to drought stress[J]. Acta Botanica Boreali-Occidentalia Sinica, 34(10): 2041-2048.)
[23] 许振渊, 高燕会, 周芬静, 等. 2014. 换锦花LsMYB4基因的克隆与表达分析[J]. 园艺学报, 41(11):2281-2290.
(Xu Z Y, Gao Y H, Zhou F J, et al.2014. Cloning and expression analysis of LsMYB4 gene in Lycoris sprengeri[J]. Acta Horticulturae Sinica, 41(11): 2281-2290.)
[24] 杨雪, 雒军, 杨彩霞, 等. 2018. 当归MYB4转录因子基因的克隆及表达分析[J]. 河南农业科学, 47(12): 48-56.
(Yang X, Luo J, Yang C X, et al.2018. Cloning and expression analysis of transcription factor MYB4 Gene from Angelica sinensis[J]. Journal of Henan Agricultural Sciences, 47(12): 48-56.)
[25] 朱重胜, 谢树禄. 2008. 石蒜植物的种质资源、药用开发及其快速繁育[J]. 江西林业科技, (05): 41-42.
(Zhu C S, Xie S L.2008. Germplasm resources of Lycoris plants, medicinal development and rapid breeding[J]. South China Forestry Science,(05): 41-42.).
[26] Ai T N, Naing A H, Arun M, et al.2017. Expression of RsMYB1 in Petunia enhances anthocyanin production in vegetative and floral tissues[J]. Scientia Horticulturae, 214: 58-65.
[27] An J P, Li R, Qu F J, et al.2018. R2R3-MYB transcription factor MdMYB23 is in involved in the cold tolerance and proanthocyanidin accumlation in apple[J]. Plant Journal, 96(3): 562-577
[28] Cedroni M L, Cronn R C, Adams K L, et al.2003. Evolution and expression of MYB genes in diploid and polyploid cotton[J]. Plant Molecular Biology, 51(3): 313-325
[29] Chen K L, Liu H L, Lou Q, et al.2017. Ectopic expression of the grape hyacinth (Muscari armeniacum) R2R3-MYB transcription factor gene, MaAN2, induces anthocyanin accumulation in tobacco[J]. Frontiers in Plant Science, 8(29)1722.
[30] Chen Y H, Yang X Y, He K, et al.2006. The MYB transcription factor superfamily of Arabidopsis: Expression analysis and phylogeneticcomparison with the rice MYB family[J]. Plant Molecular Biology, 60(1): 107-124
[31] Dubos C, Gourrierec J L, Baudry A, et al.2008. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana[J]. The Plant Journal, 55(6): 940-953.
[32] Espley R V, Hellens R P, Putterill J, et al.2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10[J]. Plant Journal, 49(3): 414-427.
[33] Feng K, Xu Z S, Que F, et al.2017. An R2R3-MYB transcription factor, OjMYB1, functions in anthocyanin biosynthesis in Oenanthe javanic[J]. Planta, 247(4): 1-15.
[34] Gonzalez, Zhao, M, Leavitt, et al.2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings[J]. The Plant Journal, 53(5): 814-827.
[35] Kazan K.2006. Negative regulation of defence and stress genes by EAR-motif-containing repressors[J]. Trends in Plant Science, 11(3): 0-112.
[36] Liu Y F, Zhou B, Qi Y W, et al.2017. Expression differences of pigment structural genes and transcription factors explain flesh coloration in three contrasting kiwifruit cultivars[J]. Frontiers in Plant Science, 8: 1507.
[37] Petroni K, Tonelli C.2011. Recent advances on the regulation of anthocyanin synthesis in reproductive organs[J]. Plant Science, 181(3): 219-229.
[38] Preston J, Wheeler J, Heazlewood J.2005. AtMYB32 is required for normal pollen development in Arabidopsis thaliana[J]. The Plant Journal, 40(6): 979-995.
[39] Shen X, Zhao K, Liu L, et al.2014. A Role for PacMYBA in ABA-Regulated anthocyanin biosynthesis in red-colored sweet cherry cv. Hong Deng (Prunus avium L.)[J]. Plant and Cell Physiology, 55(5): 862-880.
[40] Stracke R, Ishihara H, Huep G, et al.2007. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling[J]. The Plant Journal, 50(4): 660-677.
[41] Wu Q, Wu J, Li S S, et al.2016. Transcriptome sequencing and metabolite analysis for revealing the blue flower formation in waterlily[J]. BMC Genomics, 17(1): 897-910.
[42] Zhou H, Peng Q, Zhao J B, et al.2016. Multiple R2R3-MYB transcription factors involved in the regulation of anthocyanin accumulation in peach flower[J]. Frontiers in Plant Science, 7(28): 1557.
[43] Zuluaga D L, Gonzali S, Loreti E, et al.2008. Arabidopsis thaliana MYB75/PAP1 transcription factor induces anthocyanin production in transgenic tomato plants[J]. Functional Plant Biology, 35: 606-618. |
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