毛竹PeCPD基因克隆与表达分析
|
摘要
[目的]通过对毛竹(Phyllostachys edulis(Carri.)H. deLehaie)CPD基因的分子特征和表达模式进行研究分析,为揭示CPD在参与毛竹笋生长调控、光诱导以及响应胁迫过程中的作用提供参考依据。[方法]在毛竹基因组数据库(BambooGDB)中查找CPD的同源序列,设计引物并克隆PeCPD。通过生物信息学的方法分析PeCPD的基因结构、顺式调控元件、其编码蛋白的基本理化性质、保守结构域、进化关系以及该基因在不同组织中的表达模式等,利用实时定量PCR方法分析该基因在不同高度笋、昼夜节律光照条件下以及干旱、低温胁迫处理下叶片和根中的表达模式。[结果]获得了毛竹CPD同源基因PeCPD(PH01003419G0030),cDNA全长为1 584 bp,包含5′和3′端非编码区分别为110 bp和64 bp,编码区为1 410 bp,对应的基因组长度为2 796 bp,外显子和内含子数量分别为6个和5个。克隆获得了PeCPD编码区,与BambooGDB中PH01003419G0030序列完全一致,编码一个470 aa的蛋白,分子量约为52.2 kDa,理论等电点为9.063。同时获得了PeCPD上游序列(1 999 bp),与数据库中序列完全一致,分析发现,其中除了启动子基本元件外,还含有多种与环境相关的作用元件,如参与低温应答的LTR、干旱响应的MBS和光响应元件AE-box、TCT-motif等。基于CPD氨基酸序列的系统进化分析表明,毛竹与水稻、玉米、谷子和二穗短柄草等单子叶植物聚类到一个大的分支,其中与二穗短柄草的亲缘关系最近。基于转录组数据的基因表达热图分析发现,PeCPD在毛竹7个不同组织中的表达存在明显差异,其中在20 cm笋中的表达量最高,根中表达量最低。实时定量PCR分析表明,随着笋的增高,PeCPD表达量呈上升趋势;在昼夜节律光照条件下,PeCPD表达量随着光照时间延长而上升,随着黑暗时间延长而下降;干旱和低温胁迫条件下,叶片和根中PeCPD的表达量均呈先上升后下降的趋势。[结论]从毛竹中获取得到了CPD同源基因PeCPD,该基因为组成型表达,且随着笋的增高其表达量呈上升趋势,可能通过参与BRs的生物合成对竹笋的生长起调控作用;PeCPD在叶片中的表达呈现昼夜节律变化,表明该基因可能会参与毛竹的光形态建成;干旱和低温胁迫条件下,PeCPD表达量的变化有助于提高毛竹适应逆境胁迫的能力。
[Objective] To study the molecular characteristics and expression pattern of constitutive photomorphogenesis and dwarf of Phyllostachys edulis(PeCPD), a kind of the key rate-limiting enzymes in biosynthesis of brasinosteroids, aiming at revealing the role of PeCPD in regulating the rapid growth of bamboo shoots and the response to light induction and stresses. [Method] Primers were designed based on the CPD homologous sequence of PH01003419 G0030 in the Bamboo Genome Database(BambooGDB) and used for PeCPD cloning. The bioinformatics method was used for further analyses, including the gene structure, the cis-elements, the basic physicochemical properties and the conserved domains of the protein encoded by PeCPD, the evolutionary relationships, and the gene expression patterns in different tissues. Quantitative real-time PCR(qRT-PCR) method was used to analyze the gene expression in different height shoots and that in leaves and roots under the circadian rhythm light conditions and stresses of drought and cold. [Result] PeCPD, a homologous gene of CPD in Ph. edulis was obtained, which cDNA was 1 584 bp in full-length including 5' untranslated region(UTR) 110 bp, 3' UTR 64 bp and coding sequence(CDS) 1 410 bp. The corresponding genomic sequence to the CDS was 2 796 bp containing 6 exons and 5 introns. PeCPD encoded a 470 aa protein with a molecular weight of approximately 52.2 kDa and the theoretical isoelectric point of 9.063. At the same time, the upstream sequence of PeCPD(1 999 bp) was obtained, which was completely consistent with the sequence in the database. Besides the basic elements of the promoter, the upstream sequence of PeCPD also contained a variety of environmentally relevant action elements, such as LTR involved in low temperature response, MBS in response to drought, and light responsive elements(AE-box and TCT-motif). Phylogenetic analysis based on the CPD amino acid sequences showed that Ph. edulis was clustered together with the monocotyledon plants such as Oryza sativa, Zea mays, Setaria italica and Brachypodium distachyon, which was closed to B. distachyon. Expression pattern analysis based on the transcriptome data demonstrated that PeCPD expressed obviously different in the seven tissues of Ph. edulis, with the highest level in 20 cm shoot and the lowest in root. The result of qRT-PCR showed that the expression level of PeCPD in shoots increased with the increasing height of bamboo shoots, those in leaves under the circadian rhythm light conditions demonstrated an increasing trend in the daytime and a decreasing trend at night(darkness). Under both drought and cold stresses, the expression of PeCPD in leaves and roots all showed similar trends of rising at first and falling then. [Conclusion] CPD homologous gene(PeCPD) is obtained from Ph. edulis. The PeCPD is constitutively expressed in Ph. edulis. Moreover, its expression level in shoots increases with the increasing height of bamboo shoots, which suggests that PeCPD may regulate the growth of bamboo shoots by participating in the biosynthesis process of brasinosteroids. The expression of PeCPD in leaves shows circadian rhythm changes, indicating that it may be involved in the photomorphogenesis of Ph. edulis. The expression changes of PeCPD under drought and low temperature stresses indicate that PeCPD is helpful to improve the ability of bamboo to adapt to stresses.
[Objective] To study the molecular characteristics and expression pattern of constitutive photomorphogenesis and dwarf of Phyllostachys edulis(PeCPD), a kind of the key rate-limiting enzymes in biosynthesis of brasinosteroids, aiming at revealing the role of PeCPD in regulating the rapid growth of bamboo shoots and the response to light induction and stresses. [Method] Primers were designed based on the CPD homologous sequence of PH01003419 G0030 in the Bamboo Genome Database(BambooGDB) and used for PeCPD cloning. The bioinformatics method was used for further analyses, including the gene structure, the cis-elements, the basic physicochemical properties and the conserved domains of the protein encoded by PeCPD, the evolutionary relationships, and the gene expression patterns in different tissues. Quantitative real-time PCR(qRT-PCR) method was used to analyze the gene expression in different height shoots and that in leaves and roots under the circadian rhythm light conditions and stresses of drought and cold. [Result] PeCPD, a homologous gene of CPD in Ph. edulis was obtained, which cDNA was 1 584 bp in full-length including 5' untranslated region(UTR) 110 bp, 3' UTR 64 bp and coding sequence(CDS) 1 410 bp. The corresponding genomic sequence to the CDS was 2 796 bp containing 6 exons and 5 introns. PeCPD encoded a 470 aa protein with a molecular weight of approximately 52.2 kDa and the theoretical isoelectric point of 9.063. At the same time, the upstream sequence of PeCPD(1 999 bp) was obtained, which was completely consistent with the sequence in the database. Besides the basic elements of the promoter, the upstream sequence of PeCPD also contained a variety of environmentally relevant action elements, such as LTR involved in low temperature response, MBS in response to drought, and light responsive elements(AE-box and TCT-motif). Phylogenetic analysis based on the CPD amino acid sequences showed that Ph. edulis was clustered together with the monocotyledon plants such as Oryza sativa, Zea mays, Setaria italica and Brachypodium distachyon, which was closed to B. distachyon. Expression pattern analysis based on the transcriptome data demonstrated that PeCPD expressed obviously different in the seven tissues of Ph. edulis, with the highest level in 20 cm shoot and the lowest in root. The result of qRT-PCR showed that the expression level of PeCPD in shoots increased with the increasing height of bamboo shoots, those in leaves under the circadian rhythm light conditions demonstrated an increasing trend in the daytime and a decreasing trend at night(darkness). Under both drought and cold stresses, the expression of PeCPD in leaves and roots all showed similar trends of rising at first and falling then. [Conclusion] CPD homologous gene(PeCPD) is obtained from Ph. edulis. The PeCPD is constitutively expressed in Ph. edulis. Moreover, its expression level in shoots increases with the increasing height of bamboo shoots, which suggests that PeCPD may regulate the growth of bamboo shoots by participating in the biosynthesis process of brasinosteroids. The expression of PeCPD in leaves shows circadian rhythm changes, indicating that it may be involved in the photomorphogenesis of Ph. edulis. The expression changes of PeCPD under drought and low temperature stresses indicate that PeCPD is helpful to improve the ability of bamboo to adapt to stresses.
引文
[1] Fujioka S,Yokota T.Biosynthesis and metabolism of brassinosteroids[J].Physiologia Plantarum,2010,100(3):710-750.
[2] Bajguz A.Metabolism of brassinosteroids in plants[J].Plant Physiology Biochemistry,2007,45(2):95-107.
[3] Mandava N B.Plant growth-promoting brassinosteroids [J].Annual Review of Plant Physiology and Plant Molecular Biology,1988,39(1):23-52.
[4] Szekeres M,Nemeth K,Konczkalman Z,et al.Brassinosteroids rescue the deficiency of CYP90,a cytochrome P450,controlling cell elongation and de-etiolation in Arabidopsis [J].Cell,1996,85(2):171-182.
[5] Krishna P.Brassinosteroid-mediated stress responses[J].Journal of Plant Growth Regulation,2003,22(4):289-297.
[6] Divi U K,Rahman T,Krishna P,et al.Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid,ethylene and salicylic acid pathways[J].BMC Plant Biology,2010,10(1):151-160.
[7] Nehert D W,Gonzalez F J.P450 genes:Structure,evolution,and regulation[J].Annual Review of Biochemistry,1987,56(1):945-993.
[8] Mathur J,Molnár G,Fujioka S,et al.Transcription of the Arabidopsis CPD gene,encoding a steroidogenic cytochrome P450,is negatively controlled by brassinosteroids[J].The Plant Journal,1998,14(5):593-602.
[9] Ohnishi T,Godza B,Watanabe B,et al.CYP90A1/CPD,a brassinosteroid biosynthetic cytochrome P450 of Arabidopsis,catalyzes C-3 oxidation[J].Journal of Biological Chemistry,2012,287(37):31551-31560.
[10] Sakamoto T,Matsuoka M.Characterization of constitutive photomorphogenesis and dwarfism homologs in rice (Oryza sativa L.)[J].Journal of Plant Growth Regulation,2006,25(3):245-251.
[11] Koka C V,Cerny R E,Gardner R G,et al.A putative role for the tomato genes DUMPY and CURL-3 in brassinosteroid biosynthesis and response[J].Plant Physiology,2000,122(1):85-98.
[12] Bancos S,Szatmári A,Castle J,et al.Diurnal regulation of the brassinosteroid-biosynthetic CPD gene in Arabidopsis[J].Plant Physiology,2006,141(1):299-309.
[13] 江泽慧.世界竹藤[M].沈阳:辽宁科学技术出版社,2002:3-10.
[14] 徐丽君,包刚,刘清平,等.曙箣竹自然降温过程中生理特性的变化[J].世界竹藤通讯,2015,13(4):5-9.
[15] 裴晶晶,施拥军.高温干旱下毛竹林受灾状况和自恢复能力研究[J].世界竹藤通讯,2017,15(3):31-36.
[16] 胡尚连,卢学琴,陈其兵,等.四川雅安地区2种大型丛生竹耐低温能力 [J].竹子研究汇刊,2011,30(2):43-47.
[17] 刘济明,李鹏,廖小锋,等.干旱胁迫对小蓬竹生理化特性的影响[J].西北农业学报,2013,22(9):153-157.
[18] 应叶青,郭璟,魏建芬,等.干旱胁迫对毛竹幼苗生理特性的影响 [J].生态学杂志,2011,30(2):262-266.
[19] Gao Z M,Li X P,Li L B,et al.An effective method for total RNA isolation from bamboo[J].Chinese Forestry Science and Technology,2006,5(3):52-54.
[20] 高志民,范少辉,高健,等.基于CTAB法提取毛竹基因组DNA的探讨[J].林业科学研究,2006,19(6):725-728.
[21] Zhao H,Peng Z,Fei B,et al.BambooGDB:a bamboo genome database with functional annotation and an analysis platform[J].Database(Oxford),2014,2014(10):bau006.
[22] Peng Z H,Lu Y,Li L B,et al.The draft genome of the fast growing non-timber forest species mosobamboo (Phyllostachys heterocycla)[J].Nature Genetics,2013,45(4):456-461.
[23] Fan C,Ma J,Guo Q,et al.Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis)[J].PLoS One,2013,8(2):e56573.
[24] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod[J].Methods,2001,25(4):402-408.
[25] Moore M J,Query C C,Sharp P A.Splicing of precursors to mRNA by the spliceosome[M]// Gesteland R F,Atkins J F.The RNA World.New York:Cold Spring Harbor Laboratory Press,1993:303-357.
[26] 崔凯,何彩云,张建国,等.毛竹茎秆组织速生的时空发育特征[J].林业科学研究,2012,25(4):425-431.
[2] Bajguz A.Metabolism of brassinosteroids in plants[J].Plant Physiology Biochemistry,2007,45(2):95-107.
[3] Mandava N B.Plant growth-promoting brassinosteroids [J].Annual Review of Plant Physiology and Plant Molecular Biology,1988,39(1):23-52.
[4] Szekeres M,Nemeth K,Konczkalman Z,et al.Brassinosteroids rescue the deficiency of CYP90,a cytochrome P450,controlling cell elongation and de-etiolation in Arabidopsis [J].Cell,1996,85(2):171-182.
[5] Krishna P.Brassinosteroid-mediated stress responses[J].Journal of Plant Growth Regulation,2003,22(4):289-297.
[6] Divi U K,Rahman T,Krishna P,et al.Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid,ethylene and salicylic acid pathways[J].BMC Plant Biology,2010,10(1):151-160.
[7] Nehert D W,Gonzalez F J.P450 genes:Structure,evolution,and regulation[J].Annual Review of Biochemistry,1987,56(1):945-993.
[8] Mathur J,Molnár G,Fujioka S,et al.Transcription of the Arabidopsis CPD gene,encoding a steroidogenic cytochrome P450,is negatively controlled by brassinosteroids[J].The Plant Journal,1998,14(5):593-602.
[9] Ohnishi T,Godza B,Watanabe B,et al.CYP90A1/CPD,a brassinosteroid biosynthetic cytochrome P450 of Arabidopsis,catalyzes C-3 oxidation[J].Journal of Biological Chemistry,2012,287(37):31551-31560.
[10] Sakamoto T,Matsuoka M.Characterization of constitutive photomorphogenesis and dwarfism homologs in rice (Oryza sativa L.)[J].Journal of Plant Growth Regulation,2006,25(3):245-251.
[11] Koka C V,Cerny R E,Gardner R G,et al.A putative role for the tomato genes DUMPY and CURL-3 in brassinosteroid biosynthesis and response[J].Plant Physiology,2000,122(1):85-98.
[12] Bancos S,Szatmári A,Castle J,et al.Diurnal regulation of the brassinosteroid-biosynthetic CPD gene in Arabidopsis[J].Plant Physiology,2006,141(1):299-309.
[13] 江泽慧.世界竹藤[M].沈阳:辽宁科学技术出版社,2002:3-10.
[14] 徐丽君,包刚,刘清平,等.曙箣竹自然降温过程中生理特性的变化[J].世界竹藤通讯,2015,13(4):5-9.
[15] 裴晶晶,施拥军.高温干旱下毛竹林受灾状况和自恢复能力研究[J].世界竹藤通讯,2017,15(3):31-36.
[16] 胡尚连,卢学琴,陈其兵,等.四川雅安地区2种大型丛生竹耐低温能力 [J].竹子研究汇刊,2011,30(2):43-47.
[17] 刘济明,李鹏,廖小锋,等.干旱胁迫对小蓬竹生理化特性的影响[J].西北农业学报,2013,22(9):153-157.
[18] 应叶青,郭璟,魏建芬,等.干旱胁迫对毛竹幼苗生理特性的影响 [J].生态学杂志,2011,30(2):262-266.
[19] Gao Z M,Li X P,Li L B,et al.An effective method for total RNA isolation from bamboo[J].Chinese Forestry Science and Technology,2006,5(3):52-54.
[20] 高志民,范少辉,高健,等.基于CTAB法提取毛竹基因组DNA的探讨[J].林业科学研究,2006,19(6):725-728.
[21] Zhao H,Peng Z,Fei B,et al.BambooGDB:a bamboo genome database with functional annotation and an analysis platform[J].Database(Oxford),2014,2014(10):bau006.
[22] Peng Z H,Lu Y,Li L B,et al.The draft genome of the fast growing non-timber forest species mosobamboo (Phyllostachys heterocycla)[J].Nature Genetics,2013,45(4):456-461.
[23] Fan C,Ma J,Guo Q,et al.Selection of reference genes for quantitative real-time PCR in bamboo (Phyllostachys edulis)[J].PLoS One,2013,8(2):e56573.
[24] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod[J].Methods,2001,25(4):402-408.
[25] Moore M J,Query C C,Sharp P A.Splicing of precursors to mRNA by the spliceosome[M]// Gesteland R F,Atkins J F.The RNA World.New York:Cold Spring Harbor Laboratory Press,1993:303-357.
[26] 崔凯,何彩云,张建国,等.毛竹茎秆组织速生的时空发育特征[J].林业科学研究,2012,25(4):425-431.