光皮桦MYB基因的克隆及表达和调控分析
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摘要
【目的】MYB转录因子是植物中一类重要的转录因子,在调控次生壁纤维素和木质素等的合成中具有重要作用。为了挖掘参与光皮桦木材形成的MYB基因,本研究通过克隆MYB基因,分析其序列特征、表达模式和下游调控基因,以期为后续功能深入解析和分子辅助育种提供理论依据和基因资源。【方法】利用RACE技术分离到4个光皮桦BlMYB基因的cDNA片段。利用生物信息学工具对这些基因的序列特征进行分析,利用实时荧光定量PCR分析BlMYB及预测的下游基因在不同器官(雌花序、雄花序、嫩芽、嫩叶、成熟叶、茎)、组织(内外层木质部、韧皮部、形成层)中,以及应拉木诱导早期阶段的表达差异。以2个木质部特异表达的BlMYB为指导基因,采用相互排名法和Cytoscape软件构建共表达网;使用Plant CARE在线查询共表达的下游基因启动子区元件。【结果】分离到4个BlMYB的全长cDNA序列,分别命名为BlMYB1、BlMYB2、BlMYB3和BlMYB4,它们编码的蛋白分别由395、252、258、320个氨基酸残基组成,且在靠近N端都有R2R3结构域。4个BlMYB氨基酸序列间一致性27%～37%,而与它们同源的拟南芥AtMYB氨基酸序列间的一致性为39%～55%。4个BlMYB基因都含有1～2个内含子。进化树构建分析发现4个BlMYB分属于4个不同的分支,其中BlMYB2、BlMYB4分别与细胞次生壁形成相关的MYB聚在一起。BlMYB1、BlMYB3在成熟叶片中优势表达,且随叶片成熟表达水平呈递增趋势,可能与叶片的发育有关。BlMYB2在木质化茎段的木质部强烈表达,而在叶片、韧皮部等组织中的表达相对较弱;在应拉木诱导形成的早期阶段,应拉木中BlMYB2呈下调表达,尤其是拉弯处理48 h和7天时的相应数值均显著低于直立木。BlMYB4在茎的木质部、成熟雄花序中优势表达,在根、嫩叶中的表达水平较低;同时,在应拉木诱导形成早期阶段,BlMYB4在应拉木和对应木中分别呈现上调和下调表达。另外,基于共表达分析和特异性AC元件筛选,推测FRK、COMT、HCT、CesA3、CesA4、4CL、CCoAOMT 7个纤维素、木质素合成酶基因可能受BlMYB2和BlMYB4所调控。【结论】获得的4个光皮桦BlMYB基因属于R2R3-MYB家族,具不同的基因结构。4个基因呈现不同的表达模式暗示它们可能参与调控不同代谢途径。结合光皮桦应拉木特征和共表达分析结果,可初步推测BlMYB2和BlMYB4可能参与光皮桦木材形成过程的调控。
【Objective】 MYB protein is one of the important transcription factors, which play critical roles in regulating the syntheses of cellulose and lignin. In order to identify those MYB genes involved in wood formation of Betula luminifera, four BlMYB genes were cloned and their sequence characteristics, expression patterns and downstream regulatory genes were also analyzed. These results would provide a theoretical basis and genetic resources for further gene function analysis and molecular assisted breeding.【Method】 cDNA fragments of four BlMYB genes of B. luminifera were isolated by using RACE technology. Their sequence features were analyzed by bioinformatic tools, and then, the relative expression levels of these BlMYB genes and seven downstream regulatory genes in different organs/tissues(female inflorescence, male inflorescence, bud, young leaf, mature leaf, stem, phloem, cambium, inner and outer xylem) and early stage of tension wood(TW) were analyzed by real-time quantitative PCR. Furthermore, two xylem specific expressed BlMYB were used as the guide genes to construct a co-expression network by mutual ranking method and Cytoscape software. Cis-elements in the promoter regions of the putative downstream genes were analyzed by using the Plant CARE website.【Result】 The full-length cDNA of four BlMYB genes were isolated and were named BlMYB1, BlMYB2, BlMYB3 and BlMYB4, respectively. These four BlMYB genes encode 395, 252, 258, 320 amino acid residues, respectively, and all these proteins contain R2 R3 domains near the N-terminus. The four BlMYB protein sequences show 27%-37% identity with each other, but exhibit higher identity(39%-55%) with their homologs in Arabidopsis thaliana. There are one or two introns in these BlMYB genes. The phylogenetic analysis revealed that the four BlMYB belonged to different branches, and BlMYB2 and BlMYB4 belonged to two branches which contained MYB genes involved in the formation of secondary cell wall. BlMYB1 and BlMYB3 dominantly expressed in mature leaf, and their expression levels increased with the maturation of leaf, which suggested that they may be associated with development of leaf. BlMYB2 strongly expressed in the xylem of lignified stem, while weakly expressed in leaves, phloem and other tissues. During the early stages of TW formation, the expression of BlMYB2 in TW was down-regulated, especially after 48 hours and 7 days bending treatment. BlMYB4 predominantly expressed in the xylem and male inflorescence, and its expression levels were relatively low in root and leaves. And, its expression was up-regulated in TW and down-regulated in OW(Opposite Wood). In addition, according to co-expression analysis and specific AC element screening, seven Unigenes annotated as cellulose and lignin biosynthesis enzyme genes(FRK, COMT, HCT, CesA3, CesA4, 4 CL and CCoAOT) might be regulated by BlMYB2 and BlMYB4.【Conclusion】 Four BlMYB genes belonging to the R2 R3-MYB family were cloned from B.luminifera and showed different gene structures. The expression patterns of BlMYB indicated that they might participate in different metabolic pathways. In consideration of tension wood feature and the result of co-expression analysis, it could be speculated that the BlMYB2 and BlMYB4 might regulate the wood formation of B. luminifera.
【Objective】 MYB protein is one of the important transcription factors, which play critical roles in regulating the syntheses of cellulose and lignin. In order to identify those MYB genes involved in wood formation of Betula luminifera, four BlMYB genes were cloned and their sequence characteristics, expression patterns and downstream regulatory genes were also analyzed. These results would provide a theoretical basis and genetic resources for further gene function analysis and molecular assisted breeding.【Method】 cDNA fragments of four BlMYB genes of B. luminifera were isolated by using RACE technology. Their sequence features were analyzed by bioinformatic tools, and then, the relative expression levels of these BlMYB genes and seven downstream regulatory genes in different organs/tissues(female inflorescence, male inflorescence, bud, young leaf, mature leaf, stem, phloem, cambium, inner and outer xylem) and early stage of tension wood(TW) were analyzed by real-time quantitative PCR. Furthermore, two xylem specific expressed BlMYB were used as the guide genes to construct a co-expression network by mutual ranking method and Cytoscape software. Cis-elements in the promoter regions of the putative downstream genes were analyzed by using the Plant CARE website.【Result】 The full-length cDNA of four BlMYB genes were isolated and were named BlMYB1, BlMYB2, BlMYB3 and BlMYB4, respectively. These four BlMYB genes encode 395, 252, 258, 320 amino acid residues, respectively, and all these proteins contain R2 R3 domains near the N-terminus. The four BlMYB protein sequences show 27%-37% identity with each other, but exhibit higher identity(39%-55%) with their homologs in Arabidopsis thaliana. There are one or two introns in these BlMYB genes. The phylogenetic analysis revealed that the four BlMYB belonged to different branches, and BlMYB2 and BlMYB4 belonged to two branches which contained MYB genes involved in the formation of secondary cell wall. BlMYB1 and BlMYB3 dominantly expressed in mature leaf, and their expression levels increased with the maturation of leaf, which suggested that they may be associated with development of leaf. BlMYB2 strongly expressed in the xylem of lignified stem, while weakly expressed in leaves, phloem and other tissues. During the early stages of TW formation, the expression of BlMYB2 in TW was down-regulated, especially after 48 hours and 7 days bending treatment. BlMYB4 predominantly expressed in the xylem and male inflorescence, and its expression levels were relatively low in root and leaves. And, its expression was up-regulated in TW and down-regulated in OW(Opposite Wood). In addition, according to co-expression analysis and specific AC element screening, seven Unigenes annotated as cellulose and lignin biosynthesis enzyme genes(FRK, COMT, HCT, CesA3, CesA4, 4 CL and CCoAOT) might be regulated by BlMYB2 and BlMYB4.【Conclusion】 Four BlMYB genes belonging to the R2 R3-MYB family were cloned from B.luminifera and showed different gene structures. The expression patterns of BlMYB indicated that they might participate in different metabolic pathways. In consideration of tension wood feature and the result of co-expression analysis, it could be speculated that the BlMYB2 and BlMYB4 might regulate the wood formation of B. luminifera.
引文
程龙军, 童再康, 黄华宏, 等. 2010. 光皮桦中4-香豆酸辅酶 A 连接酶基因 Bl4CL 的克隆和表达分析. 植物生理学通讯, (1): 5-10.(Cheng L J, Tong Z K, Huang H H, et al. 2010. Cloning and expression analysis of 4-coumarate: CoA ligase gene Bl4CL in Betula luminifera H. Winkl. Plant Physiology Communications, (1): 5-10. [in Chinese])
何辉, 楼雄珍, 林二培, 等. 2016. 光皮桦应拉木的显微特征及其形成早期内源激素分布. 林业科学, 52(10): 38-44.(He H, Lou X Z, Lin E P, et al. 2016. Xylem characteristics of tension wood and endogenous hormones distributions during its early formation period in Betula luminifera. Scientia Silvae Sinicae, 52(10): 38-44. [in Chinese])
黄华宏. 2012. 基于转录组测序的光皮桦应拉木形成分子机制研究. 杭州: 浙江大学博士学位论文.(Huang H H. 2012. Studies on molecular mechanism of tension wood formation in Betula luminifera using RNA-Seq. Hangzhou: PhD thesis of Zhejiang University. [in Chinese])
江成. 2014. 光皮桦BlOFPs基因的克隆及其功能研究. 杭州: 浙江农林大学硕士学位论文.(Jiang C. 2014. Isolation and functional analysis of BlOFPs genes in Betula luminifera. Hangzhou: MS thesis of Zhejiang Agricultural and Forestry University. [in Chinese])
刘雪梅. 2005. 白桦木质素生物合成酶基因分离及遗传转化的研究. 哈尔滨: 东北林业大学博士学位论文.(Liu X M. 2005. Genes isolation and genetic transformation of biosynthetic enzymes in Betula platyphylla. Harbin: PhD thesis of Northeast Forestry University. [in Chinese])
Bomal C, Bedon F, Caron S, et al. 2008. Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. Journal of Experimental Botany, 59(14): 3925-3939.
Campanella J J, Bitincka L, Smalley J. 2003. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics, 4(1): 29.
Chai G, Wang Z, Tang X, et al. 2014. R2R3-MYB gene pairs in Populus: evolution and contribution to secondary wall formation and flowering time. Journal of Experimental Botany, 65(15): 4255-4269.
Demura T, Fukuda H. 2006. Transcriptional regulation in wood formation. Trends in Plant Science, 12(2): 64-70.
Goicoechea M, Lacombe E, Legay S, et al. 2005. EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant Journal, 43(4):553-567.
Groover A T. 2005. What genes make a tree a tree? Trends in Plant Science, 10(5): 210-214.
Groover A, Robischon M. 2006. Developmental mechanisms regulating secondary growth in woody plants. Current Opinion in Plant Biology, 9(1): 55-58.
Huang H, Jiang C, Tong Z, et al. 2014. Eight distinct cellulose synthase catalytic subunit genes from Betula luminifera are associated with primary and secondary cell wall biosynthesis. Cellulose, 21(4): 2183-2198.
Kalluri U C, Joshi C P. 2004. Differential expression patterns of two cellulose synthase genes are associated with primary and secondary cell wall development in aspen trees. Planta, 220(1): 47-55.
Karpinska B, Karlesson M, Srivastava M, et al. 2004. MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen. Plant Molecular Biology, 56(2): 255-270.
Krauskopf E, Harris P J, Putterill J. 2005. The cellulose synthase gene PrCESA10 is involved in cellulose biosynthesis in developing tracheids of the gymnosperm Pinus radiata. Gene, 350(2):107-116.
Larson P R. 2012. The vascular cambium: development and structure. Springer Science & Business Media.
Li C, Wang X, Lu W, et al. 2014. A poplar R2R3-MYB transcription factor, PtrMYB152, is involved in regulation of lignin biosynthesis during secondary cell wall formation. Plant Cell, Tissue and Organ Culture, 119(3): 553-563.
Li C, Wang X, Ran L, et al. 2015. PtoMYB92 is a transcriptional activator of the lignin biosynthetic pathway during secondary cell wall formation in Populus tomentosa. Plant and Cell Physiology, 56(12): 2436-2446.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25(4): 402-408.
McCarthy R L, Zhong R, Fowler S, et al. 2010. The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis. Plant and Cell Physiology, 51(6): 1084-1090.
Nairn C J, Haselkorn T. 2005. Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms. New Phytologist, 166(3): 907-915.
Obayashi T, Kinoshita K, Nakai K, et al. 2007. ATTED-II: a database of co-expressed genes and cis elements for identifying co-regulated gene groups in Arabidopsis. Nucleic Acids Research, 35(suppl 1): D863-D869.
Öhman D, Demedts B, Kumar M, et al. 2013. MYB103 is required for FERULATE-5-HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems. The Plant Journal, 73(1): 63-76.
Patzlaff A, Mclnnis S, Courtenay A, et al. 2003. Characterisation of a pine MYB that regulates lignification. The Plant Journal, 36(6):743-754.
Vélez-Bermúdez I C, Salazar-Henao J E, Fornalé S, et al. 2015. A MYB/ZML complex regulates wound-induced lignin genes in maize. The Plant Cell, 27(11): 3245-3259.
Wu L, Joshi C P, Chiang V L.2000. A xylem-specific cellulose synthase gene from aspen (Popolus tremuloides) is responsive to mechanical stress. The Plant Journal, 22(6):495-502.
Zhong R, Lee C, Zhou J, et al. 2008. A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. The Plant Cell, 20(10): 2763-2782.
Zhong R, McCarthy R L, Lee C, et al. 2011. Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar. Plant Physiology, 157(3): 1452-1468.
Zhong R, Mccarthy R L, Haghighat M, et al. 2013. The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation. PLoS One, 8(7): e69219.
何辉, 楼雄珍, 林二培, 等. 2016. 光皮桦应拉木的显微特征及其形成早期内源激素分布. 林业科学, 52(10): 38-44.(He H, Lou X Z, Lin E P, et al. 2016. Xylem characteristics of tension wood and endogenous hormones distributions during its early formation period in Betula luminifera. Scientia Silvae Sinicae, 52(10): 38-44. [in Chinese])
黄华宏. 2012. 基于转录组测序的光皮桦应拉木形成分子机制研究. 杭州: 浙江大学博士学位论文.(Huang H H. 2012. Studies on molecular mechanism of tension wood formation in Betula luminifera using RNA-Seq. Hangzhou: PhD thesis of Zhejiang University. [in Chinese])
江成. 2014. 光皮桦BlOFPs基因的克隆及其功能研究. 杭州: 浙江农林大学硕士学位论文.(Jiang C. 2014. Isolation and functional analysis of BlOFPs genes in Betula luminifera. Hangzhou: MS thesis of Zhejiang Agricultural and Forestry University. [in Chinese])
刘雪梅. 2005. 白桦木质素生物合成酶基因分离及遗传转化的研究. 哈尔滨: 东北林业大学博士学位论文.(Liu X M. 2005. Genes isolation and genetic transformation of biosynthetic enzymes in Betula platyphylla. Harbin: PhD thesis of Northeast Forestry University. [in Chinese])
Bomal C, Bedon F, Caron S, et al. 2008. Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. Journal of Experimental Botany, 59(14): 3925-3939.
Campanella J J, Bitincka L, Smalley J. 2003. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics, 4(1): 29.
Chai G, Wang Z, Tang X, et al. 2014. R2R3-MYB gene pairs in Populus: evolution and contribution to secondary wall formation and flowering time. Journal of Experimental Botany, 65(15): 4255-4269.
Demura T, Fukuda H. 2006. Transcriptional regulation in wood formation. Trends in Plant Science, 12(2): 64-70.
Goicoechea M, Lacombe E, Legay S, et al. 2005. EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant Journal, 43(4):553-567.
Groover A T. 2005. What genes make a tree a tree? Trends in Plant Science, 10(5): 210-214.
Groover A, Robischon M. 2006. Developmental mechanisms regulating secondary growth in woody plants. Current Opinion in Plant Biology, 9(1): 55-58.
Huang H, Jiang C, Tong Z, et al. 2014. Eight distinct cellulose synthase catalytic subunit genes from Betula luminifera are associated with primary and secondary cell wall biosynthesis. Cellulose, 21(4): 2183-2198.
Kalluri U C, Joshi C P. 2004. Differential expression patterns of two cellulose synthase genes are associated with primary and secondary cell wall development in aspen trees. Planta, 220(1): 47-55.
Karpinska B, Karlesson M, Srivastava M, et al. 2004. MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen. Plant Molecular Biology, 56(2): 255-270.
Krauskopf E, Harris P J, Putterill J. 2005. The cellulose synthase gene PrCESA10 is involved in cellulose biosynthesis in developing tracheids of the gymnosperm Pinus radiata. Gene, 350(2):107-116.
Larson P R. 2012. The vascular cambium: development and structure. Springer Science & Business Media.
Li C, Wang X, Lu W, et al. 2014. A poplar R2R3-MYB transcription factor, PtrMYB152, is involved in regulation of lignin biosynthesis during secondary cell wall formation. Plant Cell, Tissue and Organ Culture, 119(3): 553-563.
Li C, Wang X, Ran L, et al. 2015. PtoMYB92 is a transcriptional activator of the lignin biosynthetic pathway during secondary cell wall formation in Populus tomentosa. Plant and Cell Physiology, 56(12): 2436-2446.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25(4): 402-408.
McCarthy R L, Zhong R, Fowler S, et al. 2010. The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis. Plant and Cell Physiology, 51(6): 1084-1090.
Nairn C J, Haselkorn T. 2005. Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms. New Phytologist, 166(3): 907-915.
Obayashi T, Kinoshita K, Nakai K, et al. 2007. ATTED-II: a database of co-expressed genes and cis elements for identifying co-regulated gene groups in Arabidopsis. Nucleic Acids Research, 35(suppl 1): D863-D869.
Öhman D, Demedts B, Kumar M, et al. 2013. MYB103 is required for FERULATE-5-HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems. The Plant Journal, 73(1): 63-76.
Patzlaff A, Mclnnis S, Courtenay A, et al. 2003. Characterisation of a pine MYB that regulates lignification. The Plant Journal, 36(6):743-754.
Vélez-Bermúdez I C, Salazar-Henao J E, Fornalé S, et al. 2015. A MYB/ZML complex regulates wound-induced lignin genes in maize. The Plant Cell, 27(11): 3245-3259.
Wu L, Joshi C P, Chiang V L.2000. A xylem-specific cellulose synthase gene from aspen (Popolus tremuloides) is responsive to mechanical stress. The Plant Journal, 22(6):495-502.
Zhong R, Lee C, Zhou J, et al. 2008. A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. The Plant Cell, 20(10): 2763-2782.
Zhong R, McCarthy R L, Lee C, et al. 2011. Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar. Plant Physiology, 157(3): 1452-1468.
Zhong R, Mccarthy R L, Haghighat M, et al. 2013. The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation. PLoS One, 8(7): e69219.