苦豆子赖氨酸脱羧酶基因启动子在拟南芥中的表达分析

doi:10.11686/cyxb2018755

摘要/Abstract

摘要： 赖氨酸脱羧酶(LDC)基因是苦豆子中氧化苦参碱(OMA)生物合成的第一个关键酶基因。在已有苦豆子赖氨酸脱羧酶基因(SaLDC)基础上克隆得到该基因上游1260 bp的启动子序列,GenBank登录号为KY038928,前期在苦豆子愈伤组织中的瞬时表达研究显示该启动子具有启动活性。生物信息学分析发现该启动子区域除了拥有启动子区的基本顺式作用元件TATA-box和CAAT-box外,还具有多个与光信号、逆境应答等相关的顺式作用元件。为进一步研究SaLDC启动子的功能,构建了该启动子与β-葡萄糖苷酸酶(GUS)报告基因融合的植物表达载体并通过农杆菌介导遗传转化拟南芥,同时对光诱导和聚乙二醇(PEG)胁迫的转基因拟南芥进行GUS活性染色和定量分析。结果显示,在T₂代转基因拟南芥幼苗的不同生长阶段和成株的各组织器官中均可检测到GUS酶活性,且随幼苗生长时间的延长,叶片中的表达活性下降;在成株叶片和花萼中的表达活性强于根、茎、花瓣和角果。光和PEG胁迫均能诱导转基因拟南芥中GUS的表达;GUS酶活性定量测定显示,短时间的PEG胁迫(1~2 h)GUS酶活性显著上调(P<0.05),而连续胁迫8 h时GUS酶活性下调至最低(P<0.01),比胁迫前下降了28.2%。以上结果表明SaLDC启动子既有时空表达特异性又有组织表达特异性,光诱导和干旱胁迫对结构基因的表达有重要的调控作用。

关键词: 苦豆子, 赖氨酸脱羧酶, SaLDC启动子, 表达分析

Abstract: Lysine decarboxylase (LDC) gene is the first key enzyme gene in oxymatrine (OMA) biosynthesis in Sophora alopecuroides. The promoter sequence (1260 bp, gene bank accession number: KY038928) upstream of the gene (SaLDC) encoding lysine decarboxylase was cloned from S. alopecuroides genomic DNA. Its promoter activity was evaluated by transient expression in S. alopecuroides callus. A bioinformatics analysis showed that the promoter sequence contains a TATA-box, a CAAT-box, and other cis-acting elements such as drought-responsive, light-responsive, defense-responsive, stress-responsive elements, methyl jasmonate-responsive element, and tissue-specific expression element. To further study the functionality of the SaLDC promoter, a plant expression vector with the SaLDC promoter sequence fused to the GUS reporter gene (encoding β-glucuronidase) was transferred into Arabidopsis thaliana by Agrobacterium. β-glucuronidase activity was quantified by GUS staining and quantitative assays. We detected GUS activity in T₂ transgenic A. thaliana seedlings at different growth stages and in tissues of the adult plants. During seedling growth, GUS activity in the leaves decreased. In adult plants, GUS activity was higher in the leaves and calyxes than in the roots, stems, petals and pods. The expression of GUS was induced by simulated drought (polyethylene glycol, PEG) stress and light in transgenic A. thaliana. Quantitative analyses showed that GUS activity increased significantly in the early stage of PEG stress (1-2 h) (P<0.05) and then decreased to a minimum at 8 h (P<0.01), to around 28.2% of its original level. All these results show that the SaLDC promoter confers tissue-specific and spatio-temporal-specific expression, and that the expression of this structural gene is regulated by light and drought stress.

Key words: Sophra alopecuroides, lysine decarboxylase (LDC), SaLDC promoter, expression analysis

陆姗姗, 洪园淑, 刘萍. 苦豆子赖氨酸脱羧酶基因启动子在拟南芥中的表达分析[J]. 草业学报, 2019, 28(11): 159-167.

LU Shan-shan, HONG Yuan-shu, LIU Ping. Expression analysis of SaLDC promoter from Sophora alopecuroides in Arabidopsis thaliana[J]. Acta Prataculturae Sinica, 2019, 28(11): 159-167.

参考文献

[1] Zhu Y X, Li Y, Zheng X F, et al.Modern molecular biology (Fourth Edition). Beijing: Higher Education Press, 2013.
朱玉贤, 李毅, 郑晓峰, 等. 现代分子生物学(第4版). 北京: 高等教育出版社, 2013.
[2] Wang X C, Yang C D.Biosafe marker genes and their novel approaches. China Biotechnology, 2003, 23(4): 19-22.
王兴春, 杨长登. 转基因植物生物安全标记基因. 中国生物工程杂志, 2003, 23(4): 19-22.
[3] Gu R L, Zhao L, Wang G Y, et al. Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco. Plant Cell Reports, 2006, 25(11): 1157-1165.
[4] Pei L L, Tang Q, Zhang T, et al. Cloning and analysis of promoter of stress-related gene OsPM1 from Oryza sativa. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(11): 2179-2184.
裴柳玲, 唐清, 张涛, 等. 水稻胁迫相关基因OsPM1启动子的克隆与分析. 西北植物学报, 2015, 35(11): 2179-2184.
[5] Li Y H, Wang K, Zhao L, et al. TCT-motif function of photoperiod response element of soybean GmGBP1 gene promoter. Chinese Journal of Oil Crop Sciences, 2018, 40(4): 592-596.
李英华, 王阔, 赵琳, 等. 大豆GmGBP1基因启动子的光周期响应元件TCT-motif功能分析. 中国油料作物学报, 2018, 40(4): 592-596.
[6] Du C, Li J, Tang Y T, et al. Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata. Chinese Journal of Biotechnology, 2014, 30(8): 1299-1307.
杜次, 李菁, 唐云涛, 等. 蛇足石杉赖氨酸脱羧酶基因的克隆、原核表达及其功能分析. 生物工程学报, 2014, 30(8): 1299-1307.
[7] Schoofs G, Teichmann S, Hartmann T, et al. Lysine decarboxylase in plants and its integration in quinolizidine alkaloid biosynthesis. Phytochemistry, 1983, 22(1): 65-69.
[8] Zhang J C, Chen Z Y, Song Y H.Analysis of differential expression of the key enzyme genes involved in huperzine a biosynthesis and metabolic pathways. Chinese Journal of Biochemical Pharmaceutics, 2015, 35(1): 1-5.
张君诚, 陈作毅, 宋育红. 石杉碱甲生物合成与代谢调控关键基因的差异表达分析. 中国生化药物杂志, 2015, 35(1): 1-5.
[9] Waller G R, Nowacki E K.Alkaloid biology and metabolism in plants. Translated by Zhu T P, Tong S H and Ma Z W. Beijing: Science Press, 1984.
Waller G R, Nowacki E K. 生物碱的生物学及其在植物中的代谢作用. 朱太平, 佟绍华, 马忠武, 译. 北京: 科学出版社, 1984.
[10] Bunsupa S, Katayama K, Ikeura E, et al. Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in Leguminosae. The Plant Cell, 2012, 24(3): 1202-1216.
[11] Yang Y, Tian L, Liu P, et al. Relationship between gene expression of lysine decarboxylase and accumulation of matrine and oxymatrine in Sophora alopecuroides. China Journal of Chinese Materia Medica, 2015, 50(10): 846-849.
杨毅, 田蕾, 刘萍, 等. 苦豆子赖氨酸脱羧酶基因表达与苦参碱和氧化苦参碱含量的关系. 中国药学杂志, 2015, 50(10): 846-849.
[12] Yang Y, Lu S S, Liu P, et al. Cloning and expression analysis of a lysine decarboxylase gene in Sophora alopecuroides. Acta Prataculturae Sinica, 2016, 25(8): 128-135.
杨毅, 陆姗姗, 刘萍, 等. 苦豆子赖氨酸脱羧酶基因克隆与表达分析. 草业学报, 2016, 25(8): 128-135.
[13] Lu S S, Meng X S, Liu P, et al. Establishment of genetic transformation system for Sophra alopecuroides and deletion analysis of SaLDC promoter. China Journal of Chinese Materia Medica, 2017, 42(10): 1853-1859.
陆姗姗, 孟祥善, 刘萍, 等. 苦豆子遗传转化体系建立及SaLDC启动子缺失分析. 中国中药杂志, 2017, 42(10): 1853-1859.
[14] Zou Q, Zhao S J, Wang Z, et al.Plant physiology guide. Beijing: China Agricultural Press, 2000.
邹琦, 赵世杰, 王忠, 等. 植物生理学指导. 北京: 中国农业出版社, 2000.
[15] Carninci P, Sandelin A, Lenhard B, et al. Genome-wide analysis of mammalian promoter architecture and evolution. Nature Genetics, 2006, 38(6): 626-635.
[16] Li Y, Lee K K, Walsh S, et al. Establishing glucose and ABA-regulated transcription networks in Arabidopsis by microarray analysis and promoter classification using a relevance vector machine. Genome Research, 2006, 16(3): 414-427.
[17] Liu H, Xue J M, Xu Q Y, et al. Spatio-temporal expression of fatty acid desaturase genes AhFAD2A and AhFAD2B in peanuts. Journal of Zhejiang A&F University, 2019, 36(1): 14-20.
刘华, 薛金嫚, 徐倩玉, 等. 花生油酸脱氢酶基因AhFAD2A和AhFAD2B的时空表达特征. 浙江农林大学学报, 2019, 36(1): 14-20.
[18] Zhang J S, Zhang X L, Lin S F, et al. Cloning and expression analysis of lysine decarboxylase gene NtLDC1 from Nicotiana tabacum. Plant Physiology Journal, 2016, 52(4): 445-453.
张吉顺, 张孝廉, 林世锋, 等. 烟草赖氨酸脱羧酶基因NtLDC1的克隆和表达分析. 植物生理学报, 2016, 52(4): 445-453.
[19] Naoumkina M, Dixon R A.Characterization of the mannan synthase promoter from guar (Cyamopsis tetragonoloba). Plant Cell Reports, 2011, 30: 997-1006.
[20] Jia W S, Xing Y, Lu C M, et al. Signal transduction from water stress perception to ABA accumulation. Acta Botanica Sinica, 2002, 44(10): 1135-1141.
[21] Urao T, Shinozaki K, Urao S, et al. An Arabidopsis MYB homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell, 1993, 5(11): 1529-1539.
[22] Wormit A, Trentmann O, Feifer I, et al. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell, 2006, 18(12): 3476-3490.
[23] Wang Q, Yuan F, Pan Q F, et al. Isolation and functional analysis of the Catharanthus roseus deacetylvindoline-4-O-acetyltransferase gene promoter. Plant Cell Reports, 2010, 29(2): 185-192.
[24] Li X L, Xue C, Li J M, et al. Genome-wide identification, evolution and functional divergence of MYB transcription factors in Chinese white pear (Pyrus bretschneideri). Plant Cell Physiology, 2016, 57(4): 824-847.
[25] Cheng Y S, Chen J Q, Chen D, et al. Cloning and functional analysis of the promoter of PbTMT4 gene related sugar transport in pear. Acta Horticulturae Sinica, 2019, 46(1): 25-36.
程寅胜, 陈健秋, 陈丹, 等. 梨糖转运相关基因PbTMT4启动子克隆及功能分析. 园艺学报, 2019, 46(1): 25-36.
[26] Li X D, Mo B T, Han Y F, et al. Cloning and function analysis of the high temperature inducible promoter pMsMBF1c in alfalfa (Medicago sativa). Acta Prataculturae Sinica, 2019, 28(1): 128-137.
李小冬, 莫本田, 韩永芬, 等. 紫花苜蓿高温诱导启动子pMsMBF1c的克隆与功能分析. 草业学报, 2019, 28(1): 128-137.
[27] Kuznetsov V, Shorina M, Aronova E, et al. NaCl and ethylene-dependent cadaverine accumulation and its possible protective role in the adaptation of the common ice plant to salt stress. Plant Science, 2007, 172: 363-370.
[28] Lu M H, Li X M, Chen J F, et al. Study on chilling tolerance of cucumber during germination and expression of lysine decarboxylase gene. Scientia Agricultura Sinica, 2005, 38(12): 2492-2495.
逯明辉, 李晓明, 陈劲枫, 等. 黄瓜发芽期耐冷性与赖氨酸脱羧酶基因表达. 中国农业科学, 2005, 38(12): 2492-2495.
[29] Miao Y M, Ning Y, Shen J, et al. Cloning of LDC gene and its expression analysis under several adversity stresses from Cucumis sativus L. Journal of Nanjing Agricultural University, 2013, 36(3): 8-14.
苗永美, 宁宇, 沈佳, 等. 黄瓜LDC基因克隆及逆境胁迫下的表达分析. 南京农业大学学报, 2013, 36(3): 8-14.
[30] Lau O S, Deng X W.Plant hormone signaling lightens up: Integrators of light and hormones. Current Opinion in Plant Biology, 2010, 13(5): 571-577.
[31] Li Z X, Chen X B.Research advances on plant inducible promoters and related cis-acting elements. Biotechnology Bulletin, 2015, 31(10): 8-15.
李濯雪, 陈信波. 植物诱导型启动子及相关顺式作用元件研究进展. 生物技术通报, 2015, 31(10): 8-15.
[32] Wang L, Li L, Xu L, et al. Isolation and functional analysis of the poplar RbcS gene promoter. Plant Molecular Biology Reporter, 2013, 31(1): 120-127.