竹类植物中CYP85A1基因的克隆及表达分析
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摘要
油菜素内酯(BRs)是一种促进植物茎秆伸长的甾类植物激素,CYP85A1基因参与了BRs合成中重要的催化反应步骤。为阐明CYP85A1基因在竹类植物节间伸长中的作用,本研究以20个竹类植物为试验材料,利用生物信息学分析了CYP85A1基因的特点,并利用实时荧光定量PCR技术分析了该基因在孝顺竹及其变种凤尾竹的不同组织和不同发育阶段的相对表达量。结果表明,20个竹种中CYP85A1基因序列在DNA水平上的同源性为92. 23%,cDNA序列的同源性为98. 79%,氨基酸序列的同源性为97. 86%;这些基因均含有9个外显子和8个内含子,编码465～480个氨基酸,蛋白分子量约为54 k Da,理论等电点均约为9. 2。氨基酸序列中均含有细胞色素P450家族保守的血红素结合域、富含脯氨酸和氧的结构域及Glu-X-X-Arg结构域; CYP85A1基因孝顺竹和凤尾竹母竹的叶中表达量均最高,茎中次之,在根中的表达量最低。孝顺竹和凤尾竹幼竹中,该基因在顶部的表达量明显高于第2、第4、第5节间中的表达量; CYP85A1基因在快速伸长阶段的表达量明显高于生长缓慢时期,节间伸长速率越快该基因的表达量越高。综上,CYP85A1基因在竹类植物茎秆及节间伸长发育过程中具有促进作用。本研究结果为阐明CYP85A1在竹类植物中的生物学功能奠定了一定的理论基础。
Brassinosteroids( BRs) is a kind of steroid plant hormones that promote the elongation of plant stem. The enzyme encoded by the CYP85 A1 gene is one of the key enzymes that catalyze the synthesis of brassinosteroids. In order to preliminarily explore the role of CYP85 A1 in the rapid elongation of internode,this study mainly analyzed the sequences of CYP85 A1 homologous genes from twenty different bamboo species by bioinformatics,and detected the relative expression of CYP85 A1 gene in different tissues and different developmental stages of Bambusa multiplex and B.multiplex Fernleaf by real-time PCR. The results showed that the DNA,cDNA and amino acid sequences of 20 orthologous CYP85 A1 genes were 92. 23%,98. 79% and 97. 86% identities,respectively. All analyzed orthologous CYP85 A1 genes contain nine exons and eight introns,encoding 465 to 480 amino acids,with molecular weights of proteins are approximate 54 kDa and the theoretical isoelectric of approximate 9. 2. Their encoded protein sequences contained a conserved heme binding domain of cytochrome P450 family. Tissue specific expression analysis showed that CYP85 A1 gene in in Bambusa multiplex and B. multiplex Fernleaf had the highest expression level in the leaves of mother plants,followed by in the stems,the lowest in the root. The expression level of CYP85 A1 gene in leaves was 6. 3 and2. 6 times higher than that in roots,respectively. During the rapid elongation of newborn plants,the expression of CYP85 A1 gene at the top internode was significantly higher than that at the 2 nd,4 th,5 thinternode,respectively. Analysis of expression variation at different developmental stages showed that the expression level of CYP85 A1 gene was significantly higher during internode rapid growth stage than that during slow growth of bamboo. Taken together,our results demonstrated that CYP85 A1 gene was involved in development of stem and internode elongation of bamboo,provided a strong foundation for further study on functions of CYP85 A1 gene in bamboo.
Brassinosteroids( BRs) is a kind of steroid plant hormones that promote the elongation of plant stem. The enzyme encoded by the CYP85 A1 gene is one of the key enzymes that catalyze the synthesis of brassinosteroids. In order to preliminarily explore the role of CYP85 A1 in the rapid elongation of internode,this study mainly analyzed the sequences of CYP85 A1 homologous genes from twenty different bamboo species by bioinformatics,and detected the relative expression of CYP85 A1 gene in different tissues and different developmental stages of Bambusa multiplex and B.multiplex Fernleaf by real-time PCR. The results showed that the DNA,cDNA and amino acid sequences of 20 orthologous CYP85 A1 genes were 92. 23%,98. 79% and 97. 86% identities,respectively. All analyzed orthologous CYP85 A1 genes contain nine exons and eight introns,encoding 465 to 480 amino acids,with molecular weights of proteins are approximate 54 kDa and the theoretical isoelectric of approximate 9. 2. Their encoded protein sequences contained a conserved heme binding domain of cytochrome P450 family. Tissue specific expression analysis showed that CYP85 A1 gene in in Bambusa multiplex and B. multiplex Fernleaf had the highest expression level in the leaves of mother plants,followed by in the stems,the lowest in the root. The expression level of CYP85 A1 gene in leaves was 6. 3 and2. 6 times higher than that in roots,respectively. During the rapid elongation of newborn plants,the expression of CYP85 A1 gene at the top internode was significantly higher than that at the 2 nd,4 th,5 thinternode,respectively. Analysis of expression variation at different developmental stages showed that the expression level of CYP85 A1 gene was significantly higher during internode rapid growth stage than that during slow growth of bamboo. Taken together,our results demonstrated that CYP85 A1 gene was involved in development of stem and internode elongation of bamboo,provided a strong foundation for further study on functions of CYP85 A1 gene in bamboo.
引文
[1] Yin Y,Vafeados D,Tao Y,Yoshida S,Asami T,Chory J. A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis[J]. Cell,2005,120(2):249-259
[2] Nolan T,Chen J,Yin Y. Cross-talk of brassinosteroid signaling in controlling growth and stress responses[J]. Biochemical Journal,2017,474(16):2641-2661
[3] Cheon J,Park S Y,Schulz B,Choe S. Arabidopsis brassinosteroid biosynthetic mutant dwarf 7-1 exhibits slower rates of cell division and shoot induction[J]. BMC Plant Biology,2010,10(1):270
[4] Choe S. Signal-transduction pathways toward the regulation of brassinosteroid biosynthesis[J]. Journal of Plant Biology,2007,50(3):225-229
[5] Wang X,Chen E,Ge X,Gong Q,Butt H,Zhang C,Yang Z,Li F,Zhang X. Overexpressed BRH1,a RING finger gene,alters rosette leaf shape in Arabidopsis thaliana[J]. Science China Life Sciences,2017,61(1):1-9
[6] Wang X,Zhang J,Yuan M,Ehrhardt D W,Wang Z,Mao T.Arabidopsis microtubule destabilizing protein 40 is involved in brassinosteroid regulation of hypocotyl elongation[J]. The Plant Cell,2012,24(10):4012-4025
[7] Song L,Zhou X Y,Li L,Xue L J,Yang X,Xue H W. Genomewide analysis revealed the complex regulatory network of brassinosteroid effects in photomorphogenesis[J]. Molecular Plant,2009,2(4):755-772
[8] Vogler F,Schmalzl C,Englhart M,Bircheneder M,Sprunck S.Brassinosteroids promote Arabidopsis pollen germination and growth[J]. Plant Reproduction,2014,27(3):153-167
[9] Nomura T, Kushiro T, Yokota T, Kamiya Y, Bishop G J,Yamaguchi S. The last reaction producing brassinolide is catalyzed by cytochrome P-450s,CYP85A3 in tomato and CYP85A2 in Arabidopsis[J]. Journal of Biological Chemistry,2005,280(18):17873-17879
[10] Jager C E,Symons G M,Nomura T,Yamada Y,Smith J J,Yamaguchi S, Kamiya Y, Weller J L, Yokota T, Reid J B.Characterization of two brassinosteroid C-6 oxidase genes in pea[J].Plant Physiology,2007,143(4):1894-1904
[11] KhripachⅤ, ZhabinskiiⅤ, Groot A. Twenty years of brassinosteroids:steroidal plant hormones warrant better crops for the XXI century[J]. Annals of Botany,2000,86(3):441-447
[12] Bancos S,Nomura T,Sato T,Molnár G,Bishop G J,Koncz C,Yokota T,Nagy F,Szekeres M. Regulation of transcript levels of the Arabidopsis cytochrome P450 genes involved in brassinosteroid biosynthesis[J]. Plant Physiology,2002,130(1):504-513
[13] Shimada Y,Fujioka S,Miyauchi N,Kushiro M,Takatsuto S,Nomura T, Yokota T, Kamiya Y, Bishop G J, Yoshida S.Brassinosteroid-6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis[J]. Plant Physiol,2001,126(2):770-779
[14] Mori M,Nomura T,Ooka H,Ishizaka M,Yokota T,Sugimoto K,Okabe K, Kajiwara H, Satoh K, Yamamoto K, Hirochika H,Kikuchi S. Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis[J]. Plant Physiology,2002,130(3):1152-1161
[15] Li X J,Chen X J,Guo X,Yin L L,Jalal A G,Xu C J,Chen K S,Liu C C,Xia X J,Shi K,Zhou J,Zhou Y H,Yu J Q. DWARF overexpression induces alteration in phytohormone homeostasis,development,architecture and carotenoid accumulation in tomato[J]. Plant Biotechnology Journal,2016,14(3):1021-1033
[16]方伟.竹子分类学[M].北京:中国林业出版社,1995
[17]王小利,陈伟,李晚忱,吴佳海,刘晓霞,杨义成.高羊茅春化基因FaVRN1的克隆与分析[J].核农学报,2009,23(5):778-784
[18]马晶晶,刘世男,朱龙飞,戚田田,林新春.雷竹VRN1同源基因克隆及功能分析[J].核农学报,2016,30(9):1699-1705
[19]关鹰,许在恩,郭小勤.毛竹IDD基因家族的生物信息学分析[J].核农学报,2014,28(6):998-1005
[20]龚莺,陈虹君,许在恩,郭小勤.竹类植物Dwarf14(D14)基因的多态性分析[J].核农学报,2018,32(1):48-57
[21] Clouse S D,Daniel M Z,Baker M E,Mcmorris T C. Effect of brassinolide on gene expression in elongating soybean epicotyls[J].Plant Physiology,1992,100(3):1377-1383
[22] Hong Z,Ueguchi-Tanaka M,Shimizu-Sato S,Inukai Y,Fujioka S,Shimada Y,Takatsuto S,Agetsuma M,Yoshida S,Watanabe Y,Uozu S,Kitano H,Ashikari M,Matsuoka M. Loss-of-function of a rice brassinosteroid biosynthetic enzyme,C-6 oxidase,prevents the organized arrangement and polar elongation of cells in the leaves and stem[J]. Plant Journal,2002,32(4):495-508
[23]鲁雪莉,段方猛,万芳源,李宝笃,宋雯雯.过量表达菠菜SoCYP85A1基因增强烟草的耐盐性[J].植物生理学报,2017,53(3):454-460
[24] Wang H,Li W,Qin Y,Pan Y,Wang X,Weng Y,Chen P,Li Y.The cytochrome P450 gene Cs CYP85A1 is a putative candidate for super compact-1(scp-1)plant architecture mutation in cucumber(Cucumis sativus L.)[J]. Frontiers in Plant Science,2017,8,266-279
[25] Duan F,Ding J,Lee D,Lu X,Feng Y,Song W. Overexpression of SoCYP85A1,a spinach cytochrome P450 gene in transgenic tobacco enhances root development and drought stress tolerance[J].Frontiers in Plant Science,2017,8:1909-1923
[26] He J X,Gendron J M,Sun Y,Gampala S S,Gendron N,Sun C Q,Wang Z Y. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses[J]. Science,2005,307(5715):1634-1638
[27] Jin Y L,Tang R J,Wang H H,Jiang C M,Bao Y,Yang Y,Liang M X,Sun Z C,Kong F J,Li B,Zhang H X. Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees[J]. Plant Biotechnology Journal,2017,15(10):1309-1321
[28] Tian X,Li X,Zhou W,Ren Y,Wang Z,Liu Z,Tang J,Tong H,Fang J,Bu Q. Transcription factor Os WRKY53 positively regulates brassinosteroid signaling and plant architecture[J]. Plant Physiology,2017,175(3):1337-1349
[29] Imkampe J,Halter T,Huang S,Schulze S,Mazzotta S,Schmidt N,Manstretta R,Postel S,Wierzba M,Yang Y,van Dongen W M A M,Stahl M,Zipfel C,Goshe M B,Clouse S,de Vries S C,Tax F,Wang X, Kemmerling B. The Arabidopsis leucine-rich repeat receptor kinase BIR3 negatively regulates BAK1 receptor complex formation and stabilizes BAK1[J]. Plant Cell,2017,29(9):2285-2303
[30] Bajguz A,Tretyn A. The chemical characteristic and distribution of brassinosteroids in plants[J]. Phytochemistry,2003,62(7):1027-46
[31] Nomura T,Ueno M,Yamada Y,Takatsuto S,Takeuchi Y,Yokota T. Roles of brassinosteroids and related mRNAs in pea seed growth and germination[J]. Plant Physiology,2007,143(4):1680-1688
[32] Tong H,Liu L,Jin Y,Du L,Yin Y,Qian Q,Zhu L,Chu C.Dwarf and low-tillering acts as a direct downstream target of a gsk3/shaggy-like kinase to mediate brassinosteroid responses in rice[J].Plant Cell,2012,24(6):2562-2577
[2] Nolan T,Chen J,Yin Y. Cross-talk of brassinosteroid signaling in controlling growth and stress responses[J]. Biochemical Journal,2017,474(16):2641-2661
[3] Cheon J,Park S Y,Schulz B,Choe S. Arabidopsis brassinosteroid biosynthetic mutant dwarf 7-1 exhibits slower rates of cell division and shoot induction[J]. BMC Plant Biology,2010,10(1):270
[4] Choe S. Signal-transduction pathways toward the regulation of brassinosteroid biosynthesis[J]. Journal of Plant Biology,2007,50(3):225-229
[5] Wang X,Chen E,Ge X,Gong Q,Butt H,Zhang C,Yang Z,Li F,Zhang X. Overexpressed BRH1,a RING finger gene,alters rosette leaf shape in Arabidopsis thaliana[J]. Science China Life Sciences,2017,61(1):1-9
[6] Wang X,Zhang J,Yuan M,Ehrhardt D W,Wang Z,Mao T.Arabidopsis microtubule destabilizing protein 40 is involved in brassinosteroid regulation of hypocotyl elongation[J]. The Plant Cell,2012,24(10):4012-4025
[7] Song L,Zhou X Y,Li L,Xue L J,Yang X,Xue H W. Genomewide analysis revealed the complex regulatory network of brassinosteroid effects in photomorphogenesis[J]. Molecular Plant,2009,2(4):755-772
[8] Vogler F,Schmalzl C,Englhart M,Bircheneder M,Sprunck S.Brassinosteroids promote Arabidopsis pollen germination and growth[J]. Plant Reproduction,2014,27(3):153-167
[9] Nomura T, Kushiro T, Yokota T, Kamiya Y, Bishop G J,Yamaguchi S. The last reaction producing brassinolide is catalyzed by cytochrome P-450s,CYP85A3 in tomato and CYP85A2 in Arabidopsis[J]. Journal of Biological Chemistry,2005,280(18):17873-17879
[10] Jager C E,Symons G M,Nomura T,Yamada Y,Smith J J,Yamaguchi S, Kamiya Y, Weller J L, Yokota T, Reid J B.Characterization of two brassinosteroid C-6 oxidase genes in pea[J].Plant Physiology,2007,143(4):1894-1904
[11] KhripachⅤ, ZhabinskiiⅤ, Groot A. Twenty years of brassinosteroids:steroidal plant hormones warrant better crops for the XXI century[J]. Annals of Botany,2000,86(3):441-447
[12] Bancos S,Nomura T,Sato T,Molnár G,Bishop G J,Koncz C,Yokota T,Nagy F,Szekeres M. Regulation of transcript levels of the Arabidopsis cytochrome P450 genes involved in brassinosteroid biosynthesis[J]. Plant Physiology,2002,130(1):504-513
[13] Shimada Y,Fujioka S,Miyauchi N,Kushiro M,Takatsuto S,Nomura T, Yokota T, Kamiya Y, Bishop G J, Yoshida S.Brassinosteroid-6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis[J]. Plant Physiol,2001,126(2):770-779
[14] Mori M,Nomura T,Ooka H,Ishizaka M,Yokota T,Sugimoto K,Okabe K, Kajiwara H, Satoh K, Yamamoto K, Hirochika H,Kikuchi S. Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis[J]. Plant Physiology,2002,130(3):1152-1161
[15] Li X J,Chen X J,Guo X,Yin L L,Jalal A G,Xu C J,Chen K S,Liu C C,Xia X J,Shi K,Zhou J,Zhou Y H,Yu J Q. DWARF overexpression induces alteration in phytohormone homeostasis,development,architecture and carotenoid accumulation in tomato[J]. Plant Biotechnology Journal,2016,14(3):1021-1033
[16]方伟.竹子分类学[M].北京:中国林业出版社,1995
[17]王小利,陈伟,李晚忱,吴佳海,刘晓霞,杨义成.高羊茅春化基因FaVRN1的克隆与分析[J].核农学报,2009,23(5):778-784
[18]马晶晶,刘世男,朱龙飞,戚田田,林新春.雷竹VRN1同源基因克隆及功能分析[J].核农学报,2016,30(9):1699-1705
[19]关鹰,许在恩,郭小勤.毛竹IDD基因家族的生物信息学分析[J].核农学报,2014,28(6):998-1005
[20]龚莺,陈虹君,许在恩,郭小勤.竹类植物Dwarf14(D14)基因的多态性分析[J].核农学报,2018,32(1):48-57
[21] Clouse S D,Daniel M Z,Baker M E,Mcmorris T C. Effect of brassinolide on gene expression in elongating soybean epicotyls[J].Plant Physiology,1992,100(3):1377-1383
[22] Hong Z,Ueguchi-Tanaka M,Shimizu-Sato S,Inukai Y,Fujioka S,Shimada Y,Takatsuto S,Agetsuma M,Yoshida S,Watanabe Y,Uozu S,Kitano H,Ashikari M,Matsuoka M. Loss-of-function of a rice brassinosteroid biosynthetic enzyme,C-6 oxidase,prevents the organized arrangement and polar elongation of cells in the leaves and stem[J]. Plant Journal,2002,32(4):495-508
[23]鲁雪莉,段方猛,万芳源,李宝笃,宋雯雯.过量表达菠菜SoCYP85A1基因增强烟草的耐盐性[J].植物生理学报,2017,53(3):454-460
[24] Wang H,Li W,Qin Y,Pan Y,Wang X,Weng Y,Chen P,Li Y.The cytochrome P450 gene Cs CYP85A1 is a putative candidate for super compact-1(scp-1)plant architecture mutation in cucumber(Cucumis sativus L.)[J]. Frontiers in Plant Science,2017,8,266-279
[25] Duan F,Ding J,Lee D,Lu X,Feng Y,Song W. Overexpression of SoCYP85A1,a spinach cytochrome P450 gene in transgenic tobacco enhances root development and drought stress tolerance[J].Frontiers in Plant Science,2017,8:1909-1923
[26] He J X,Gendron J M,Sun Y,Gampala S S,Gendron N,Sun C Q,Wang Z Y. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses[J]. Science,2005,307(5715):1634-1638
[27] Jin Y L,Tang R J,Wang H H,Jiang C M,Bao Y,Yang Y,Liang M X,Sun Z C,Kong F J,Li B,Zhang H X. Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees[J]. Plant Biotechnology Journal,2017,15(10):1309-1321
[28] Tian X,Li X,Zhou W,Ren Y,Wang Z,Liu Z,Tang J,Tong H,Fang J,Bu Q. Transcription factor Os WRKY53 positively regulates brassinosteroid signaling and plant architecture[J]. Plant Physiology,2017,175(3):1337-1349
[29] Imkampe J,Halter T,Huang S,Schulze S,Mazzotta S,Schmidt N,Manstretta R,Postel S,Wierzba M,Yang Y,van Dongen W M A M,Stahl M,Zipfel C,Goshe M B,Clouse S,de Vries S C,Tax F,Wang X, Kemmerling B. The Arabidopsis leucine-rich repeat receptor kinase BIR3 negatively regulates BAK1 receptor complex formation and stabilizes BAK1[J]. Plant Cell,2017,29(9):2285-2303
[30] Bajguz A,Tretyn A. The chemical characteristic and distribution of brassinosteroids in plants[J]. Phytochemistry,2003,62(7):1027-46
[31] Nomura T,Ueno M,Yamada Y,Takatsuto S,Takeuchi Y,Yokota T. Roles of brassinosteroids and related mRNAs in pea seed growth and germination[J]. Plant Physiology,2007,143(4):1680-1688
[32] Tong H,Liu L,Jin Y,Du L,Yin Y,Qian Q,Zhu L,Chu C.Dwarf and low-tillering acts as a direct downstream target of a gsk3/shaggy-like kinase to mediate brassinosteroid responses in rice[J].Plant Cell,2012,24(6):2562-2577
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