[1]杨 杰,孙 璐,王思瑶,等.3个白桦细胞色素P450基因生物信息学及表达分析[J].南京林业大学学报(自然科学版),2018,42(06):027-34.[doi:10.3969/ j.issn.1000-2006.201803002]
 YANG Jie,SUN Lu,WANG Siyao,et al.Tissue specificity and hormone induced expression of three cytochrome P450 genes from Betula platyphylla Suk[J].Journal of Nanjing Forestry University(Natural Science Edition),2018,42(06):027-34.[doi:10.3969/ j.issn.1000-2006.201803002]





Tissue specificity and hormone induced expression of three cytochrome P450 genes from Betula platyphylla Suk
杨 杰1孙 璐1王思瑶1李 影1翟 睿1林香雨1詹亚光12尹 静1*
(1.东北林业大学生命科学学院,黑龙江 哈尔滨 150040; 2.林木遗传育种与生物技术国家重点实验室,黑龙江 哈尔滨 150040)
YANG Jie1 SUN Lu1 WANG Siyao1 LI Ying1 ZHAI Rui1 LIN Xiangyu1 ZHAN Yaguang12 YIN Jing1*
(1. School of Life Science, Northeast Forestry University, Harbin 150040, China; 2. State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China)
白桦 BpCYP450 表达模式分析 生物信息学分析
Betula platyphylla Suk. BpCYP450 genes expression patterns bioinformatics analysis
Q786; S792.153
10.3969/ j.issn.1000-2006.201803002
【目的】研究白桦细胞色素P450基因表达的组织特异性,以及在茉莉酸甲酯(MeJA)、水杨酸(SA)、赤霉素(GA3)、脱落酸(ABA)、乙烯利和伤害处理下的基因表达模式。【方法】筛选白桦转录组,获得3个BpCYP450 基因,分别命名为BpCYP4、BpCYP5、 BpCYP14,利用生物信息学分析BpCYP450蛋白的分子结构特征及其与其他物种CYP450蛋白的亲缘关系。采用QRT-PCR技术,对白桦BpCYP450组织特异性、激素信号及伤害诱导下的表达特征进行分析。【结果】生物信息学结果表明BpCYP4、BpCYP5、BpCYP14 cDNA序列长度分别为1 569、1 584和1 530 bp,具有完整的开放阅读框(ORF),分别编码522、527和509个氨基酸,均为亲水性跨膜蛋白,主要定位于叶绿体。白桦BpCYP450与百脉根、扁豆及豌豆等豆科植物CYP450蛋白亲缘性较高。组织特异性分析结果显示,3个BpCYP450 基因在叶和根中表达较高,茎中较低。激素信号及伤害诱导结果表明,3个BpCYP450基因不同程度地响应MeJA、SA、GA3、ABA、乙烯利激素信号及伤害诱导。【结论】3个BpCYP450基因均为CYP450基因家族的新成员,具有组织特异性及激素诱导表达特性,可能在白桦生长发育、抵御胁迫及代谢物合成中发挥重要作用。
【Objective】Explore the tissue specificity of the cytochrome P450 genes in the triterpenoids from birch(Betula platyphylla Suk.)and expression pattern under treatments of methyl jasmonate(MeJA), salicylic acid(SA), gibberellin(GA3), abscisic acid(ABA), ethephon and leaf injury.【Method】Three BpCYP450 genes were screened from the transcriptome of B. platyphylla Suk. and named BpCYP4, BpCYP5 and BpCYP14, respectively. The molecular structure of BpCYP450 proteins and relationship with CYP450 proteins of other species of plants were described by bioinformatics analysis. The tissue specificity of BpCYP450 and the expression characteristics under hormone and injury were analyzed by QRT-PCR.【Result】Bioinformatics results showed that the size of BpCYP4, BpCYP5 and BpCYP14 were 1 569, 1 584 and 1 530 bp, respectively, and contained the complete ORF, encoding 522, 527 and 509 amino acids, which were hydrophilic transmembrane proteins and mainly located in the chloroplast. Three BpCYP450 were closely related to leguminous plant CYP450 proteins such as Lotus japonicus, Lens culinaris and Pisum sativum. The ana-lysis of tissue-specific showed that BpCYP450s expression were higher in leaves and in roots,and lower in stems. The results of hormone and injury induction showed that BpCYP450 genes was induced by MeJA, SA, GA3 and ABA, ethephon and injury. 【Conclusion】Three BpCYP450 are new members of the CYP450 genes family, with expression of tissue-specific and hormone induction. These results suggested that three CYP450 genes may play an important role in the growth and development the defense stress and the metabolites synthesis of birch.


[1] NELSON D, WERCK-REICHHART D. A P450-centric view of plant evolution[J]. Plant Journal,2011,66(1):194-211.DOI:10.1111/j.1365-313X.2011.04529.x.
[2] NELSON D R. Progress in tracing the evolutionary paths of cytochrome P450[J]. Biochimica et Biophysica Acta,2011,1814(1):14-18. DOI:10.1016/j.bbapap.2010.08.008.
[3] HAN J Y, HWANG H S, CHOI S W, et al. Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng[J]. Plant and Cell Physiology,2012,53(9):1535-1545. DOI:10.1093/pcp/pcs106.
[4] HAN J Y, KIM H J, KOWN Y S, et al. The cytP450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng[J]. Plant & Cell Physiology,2011,52(12):2062-2073. DOI:10.1093/pcp/pcr150.
[5] PAGE V, SCHWITZGUEBEL J P. Metabolism of sulphonated anthraquinones in rhubarb, maize and celery: the role of cytochromes P450 and peroxidases[J]. Plant Cell Reports, 2009,28(11):1725-1735. DOI:10.1007/s00299-009-0772-5.
[6] BOACHON B, JUNKER R R, MIESCH L, et al. CYP76C1(Cytochrome P450)-mediated linalool metabolism and the formation of volatile and soluble Linalool oxides in Arabidopsis flowers: a strategy for defense against floral antagonists[J]. Plant Cell, 2015,27: 2972-2990. DOI:10.1105/tpc.15.00399.
[7] LI H, PINOT F, SAUVEPLANE V, et al. Cytochrome P450 family member CYP704B2 catalyzes the ω-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice[J]. Plant Cell,2010,22(1):173-190. DOI:10.1105/tpc.109.070326.
[8] YAMAGUCHI S. Gibberellin metabolism and its regulation[J]. Annual Review of Plant Biology, 2008,59:225-251.DOI:10.1146/annurev.arplant.59.032607.092804.
[9] PAK H, YU-LING L I, KIM H, et al. cDNA-ampliifed fragment length polymorphism analysis reveals differential gene expression induced by exogenous MeJA and GA3 in oilseed rape(Brassica napus L.)fowers[J]. Journal of Integrative Agriculture, 2017, 16(1):47-56. DOI:10.1016/S2095-3119(16)61407-7.
[10] LI R, SCHUMAN M C, WANG Y, et al. Jasmonate signaling makes flowers attractive to pollinators and repellant to florivores in nature[J]. Journal of Integrative Plant Biology, 2018, 60(3):190-194. DOI:10.1111/jipb.12607.
[11] MOSES T, POLLIER J, SHEN Q, et al. OSC2 and CYP716A14v2 catalyze the biosynthesis of triterpenoidsfor the cuticle of aerial organs of Artemisia annua[J]. Plant Cell,2015,27(1):286-301. DOI:10.1105/tpc.114.134486.
[12] TAMURA K, FUKAO Y, HATSUGAI N, et al. Nup82 functions redundantly with Nup136 in a salicylic acid-dependent defense response of Arabidopsis thaliana[J]. Nucleus, 2017,8:301-311. DOI:10.1080/19491934.2017.1279774.
[13] MARTINEZMEDINA A, FERNANDEZ I, LOK G B, et al. Shifting from priming of salicylic acid to jasmonic acid-regulated defences by Trichoderma protects tomato against the root knot nematode Meloidogyne incognita[J]. New Phytologist, 2017, 213(3):1363-1377. DOI:10.1111/nph.14251.
[14] DING Y, SUN T, AO K, et al. Opposite roles of salicylic acid receptors NPR1 and NPR3/NPR4 in transcriptional regulation of plant immunity[J]. Cell, 2018, 173(6): 1454-1467. DOI:10.1016/j.cell.2018.03.044.
[15] SUSSMILCH F C, ATALLAH N M, BRODRIBB T J, et al. Abscisic acid(ABA)and key proteins in its perception and signaling pathways are ancient, but their roles have changed through time[J]. Plant Signaling & Behavior, 2017, 12(9):e1365210. DOI:10.1080/15592324.2017.1365210.
[16] VERSLUES P E. Rapid quantification of abscisic acid by GC-MS/MS for studies of abiotic stress response[J]. Methods Mol Biol, 2017, 1631: 325-335. DOI: 10.1007/978-1.4939-7136-7.21.
[17] XI W, LIU C, HOU X, et al.MOTHER OF FT AND TFL1 regulates seed germination through a negative feedback loop modulating ABA signaling in Arabidopsis[J]. Plant Cell, 2010, 22(6):1733-1748. DOI:10.1105/tpc.109.073072.
[18] CHOI Y, LEE Y, HWANG J U. Arabidopsis ROP9 and ROP10 GTPases differentially regulate auxin and ABA responses[J]. Journal of Plant Biology, 2014, 57(4):245-254.DOI:10.1007/s12374-014-0029-x.
[19] KIM S H, LIM S R, HONG S J, et al. Effect of ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris[J]. J Agric Food Chem, 2016, 64(23):4807-4816. DOI:10.1021/acs.jafc.6b00541.
[20] YAO D, HUO X, ZENDA T, et al. Effects of ethephon on DNA methylation and gene expressions associated with shortened internodes in maize[J]. Biotechnology & Biotechnological Equipment, 2017,16:30-40.DOI:10.1080/13102818.2017.1386591.
[21] CHEN B, MA J, XU Z, et al. Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination[J]. Journal of Integrative Plant Biology, 2016, 58(10):859-869.DOI:10.1111/jipb.12479.
[22] 叶银英,何道伟,叶文才,等.23-羟基桦木酸体外和体内抗黑色素瘤作用的研究[J]. 中国肿瘤临床与康复,2000(1):7-9.
YE Y Y, HE D W, YE W C, et al. The study of 23-hydroxyl betulinic acid against melanoma in vivo and in vitro[J].Chinese Journal of Clinical Oncology and Rehabilitation, 2000(1): 7-9.
[23] 李薇,李岩,金雄杰.白桦三萜类物质的抗肿瘤作用及其对免疫功能的增强效应[J].中国免疫学杂志,2001,6(9):485-490.
LI W, LI Y, JIN X J. Antitumor activity and immunoregulatory effect of triterpenes isolated from Betula platyphylla[J]. Chinese Journal of Immunology, 2001,6(9): 485-490.
[24] 李岩,金雄杰,谢湘林,等. 白桦三萜类物质抗黑色素瘤B16、S180肉瘤作用及其机制的实验研究[J].中国药理学通报,2000,16(3):279-281.
LI Y, JIN X J, XIE X L, et al. Antitumor activity and mechanism of tritrepenes isolated form Betula platyphylla on melanoma B16 and sarcoma 180[J]. Chinese Journal of Pharmacology, 2000,16(3): 279-281.
[25] FUJIOKA T, KASHIWADA Y, KILKUSKIE R E, et al. Anti-AIDS agents,11.Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids[J]. Journal of Natural Products, 1994,57(2):243-247.
[26] FULDA S, JEREMIAS I, STEINER H H, et al. Betulinc acid: a new cytotoxic agent against malignant brain-tumor cells Cancer[J].International Journal of Cancer,1999, 82(3):435-441. DOI: 10.1002/(SICI)1097-0215(19990730)82:3<435::AID-IJC18>3.0.CO; 2-1.
[27] FALAMAS A, PINZARU C S, DEHELEAN C A, et al. Betulin and its natural resource as potential anticancer drug candidate seen by FT-Raman and FT-IR spectroscopy[J]. Raman Spectroscopy, 2011, 42(1): 97-107. DOI:10.1002/jrc2658.
[28] 范桂枝,詹亚光.白桦酯醇的研究进展[J].中草药,2008,39(10):1591-1594.
FAN G Z, ZHAN Y G.Advances in studies on betulin[J]. Chinese Herbal Medicine, 2008,39(10): 1591-1594.
[29] 孙华.齐墩果酸类化合物的结构改造及抗癌活性研究[D].沈阳:沈阳药科大学,2007.
SUN H. Studies on synthesis of derivatives of oleanolic acids and their anti-tumor activity[D]. Shenyang: Shenyang Pharmaceutical University, 2007.
[30] 王倩.白桦树皮三萜类物质的分布规律[D].哈尔滨:东北林业大学, 2008.
WANG Q. Study on the distribution of triterpenoids in white birch bark [D]. Harbin: Northeast Forestry University, 2008.
[31] 梁甜.白桦OSC新基因的克隆、RNAi载体构建及遗传转化初步研究[D].哈尔滨:东北林业大学, 2015.
LIANG T. Cloe of OSC new gee, structure of RNAi carrier and study history genetic preliminarilyin Betida platyphylta Suk[D]. Harbin: Northeast Forestry University, 2015.
[32] GHOSH S. Triterpene structural diversification by plant cytochrome P450 enzymes[J]. Frontiers in Plant Science, 2017, 8:1-15. DOI:10.3389/fpls.2017.018886.
[33] ARIMURA G, GARMS S, MAFFEI M, et al. Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling[J]. Planta, 2008, 227(2): 453-464.DOI:10.1007/s00425-007-0631-y.
[34] 牛云云. 三七、西洋参中三萜皂苷合成关键酶基因的克隆及表达模式分析[D].北京:北京协和医学院,2013.
NIU Y Y. Cloning and expression analysis of the key genes involved in triterpene saponin biosynthesis in Panax notoginseng and Panax quinquefolium[D].Beijing: Peking Union Medical College,2013.
[35] CHANG C, KWOK S F, BLEECKER A B, et al. Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators[J]. Science, 1993, 262(5133):539-544.DOI:10.1126/science.8211181.
[36] ZHANG M Y, WANG S Y, YIN J, et al. Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla Suk[J]. Protoplasma,2016, 253,(5):1347-1363.DOI:10.1007/s00709-015-0893-3.
[37] YIN J, LI X, ZHAN Y G, et al. Cloning and expression of BpMYC4 and BpbHLH9 genes and the role of BpbHLH9 in triterpenoid synthesis in birch[J]. BMC Plant Biology, 2017,17(1):214.DOI:10.1186/s12870-071-1150-z.
[38] YU Z X, LI J X, YANG C Q, et al. The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L.[J]. Molecular Plant, 2012, 5(2): 353-365.DOI:10.1093/mp/ssr087.
[39] VODENEEV V, MUDRILOV M, AKINCHITS E, et al. Parameters of electrical signals and photosynthetic responses induced by them in pea seedlings depend on the nature of stimulus[J]. Functional Plant Biology, 2017,45(2):160-170.DOI:10.1071/FP16342.


[1]官民晓,刘雪梅*,张 妍,等.白桦SPL8转录因子基因的分离及转录表达分析[J].南京林业大学学报(自然科学版),2013,37(03):017.[doi:10.3969/j.issn.1000-2006.2013.03.004]
 GUAN Minxiao,LIU Xuemei*,ZHANG Yan,et al.Isolation and transcription expression analysis of SPL8 transcription factors gene of Betula platyphylla[J].Journal of Nanjing Forestry University(Natural Science Edition),2013,37(06):017.[doi:10.3969/j.issn.1000-2006.2013.03.004]
 LI Yuanyuan,YANG Guang,WEI Rui,et al.TabZIP transferred Betula platyphylla generation and salt tolerance analysis[J].Journal of Nanjing Forestry University(Natural Science Edition),2013,37(06):006.[doi:10.3969/j.issn.1000-2006.2013.05.002]
[3]李蕾蕾,孙丰坤,董 恒,等.白桦BpGT14基因表达模式及对非生物 胁迫诱导的响应[J].南京林业大学学报(自然科学版),2016,40(02):041.[doi:10.3969/j.issn.1000-2006.2016.02.007]
 LI Leilei,SUN Fengkun,DONG Heng,et al.The expression patterns of BpGT14 gene in Betula platypylla Suk. and the response to biotic stress[J].Journal of Nanjing Forestry University(Natural Science Edition),2016,40(06):041.[doi:10.3969/j.issn.1000-2006.2016.02.007]
[4]董京祥,任 丽,张 园,等.白桦BpTCPs基因家族生物信息学及时空表达分析[J].南京林业大学学报(自然科学版),2018,42(04):113.[doi:10.3969/j.issn.1000-2006.201709001]
 DONG Jingxiang,REN Li,ZHANG Yuan,et al.Bioinformatics and expression analysis of BpTCPs in Betula platyphylla Suk.[J].Journal of Nanjing Forestry University(Natural Science Edition),2018,42(06):113.[doi:10.3969/j.issn.1000-2006.201709001]
[5]孙 璐,杨 杰,王思瑶,等.白桦BpMYB21基因的克隆及其表达模式分析[J].南京林业大学学报(自然科学版),2018,42(04):119.[doi:10.3969/j.issn.1000-2006.201708017]
 SUN Lu,YANG Jie,WANG Siyao,et al.Cloning and expression pattern of BpMYB21 from Betula platyphylla Suk.[J].Journal of Nanjing Forestry University(Natural Science Edition),2018,42(06):119.[doi:10.3969/j.issn.1000-2006.201708017]


收稿日期:2018-03-01 修回日期:2018-09-11
基金项目:国家自然科学基金项目(31570589); 中央高校基本科研业务费专项资金项目(257-2017DY02); 东北林业大学创新创业训练项目(201710225109)
更新日期/Last Update: 2018-11-30