NtCycB2基因表达对烟草腺毛发生的影响
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摘要
烟草腺毛是烟草表皮细胞的特化结构,对烟株抗性及烟叶品质具有重要影响。为探索烟草腺毛密度的分子调控途径,本文对烟草B型细胞周期蛋白基因NtCycB2进行了同源克隆和生物信息学分析,并对其表达特性和生物学功能进行研究。结果表明该基因包含333bp的编码区,编码110个氨基酸,该序列在栽培烟草不同类型、不同品种间高度保守,但与SlCycB2和AtCycB2同源性分别为71.05%和52.38%。RT-PCR分析表明NtCycB2在叶面腺毛和花中的表达水平较高,并受到盐、温度、MeJA等逆境信号的诱导;分别利用基因过表达和基因编辑技术获得了NtCycB2过量表达和敲除株系;植株腺毛形态学观察发现NtCycB2过表达植株腺毛密度显著减少,基因敲除植株分泌型腺毛密度显著增加,而非分泌型腺毛变化不大,该研究表明NtCycB2对烟草分泌型腺毛发生具有明显的负调控作用,在烟草腺毛密度分子调控和叶面化学定向改良中具有较大潜力。
Tobacco trichome, as a specialized structure of tobacco epidermal cells, has an important influence on plant resistance and leaf quality. In order to explore the molecular regulation pathway for the density of tobacco trichome, t homologous cloning and bioinformatics analysis of tobacco B-type cyclin gene NtCycB2 were carried out, and the expression characteristics and biological functions were studied. Results showed that NtCycB2 contained a 333 bp coding region and encoded 110 amino acids. NtCycB2 sequence was highly conserved among cultivated tobacco varieties and has 71.05% and 52.38% homology with SlCycB2 and AtCycB2, respecitvely. RT-PCR analysis showed that NtCycB2 was highly expressed in leaf trichome and flowers, and was induced by stress signals such as NaCl, temperature and MeJA. The NtCycB2 overexpression line and NtCycB2 knock-out line were obtained using transgenic technology and CRISPR-Cas9. Morphological observation of trichome showed that density of trichome was reduced significantly in overexpression lines, while density of glandular trichome was increased significantly in knockout lines and non-glandular trichome showed a little change, which indicated that NtCycB2 acted as a negative regulator for morphogenesis of glandular trichome and played an important role in tobacco leaf chemical orientation improvement.
Tobacco trichome, as a specialized structure of tobacco epidermal cells, has an important influence on plant resistance and leaf quality. In order to explore the molecular regulation pathway for the density of tobacco trichome, t homologous cloning and bioinformatics analysis of tobacco B-type cyclin gene NtCycB2 were carried out, and the expression characteristics and biological functions were studied. Results showed that NtCycB2 contained a 333 bp coding region and encoded 110 amino acids. NtCycB2 sequence was highly conserved among cultivated tobacco varieties and has 71.05% and 52.38% homology with SlCycB2 and AtCycB2, respecitvely. RT-PCR analysis showed that NtCycB2 was highly expressed in leaf trichome and flowers, and was induced by stress signals such as NaCl, temperature and MeJA. The NtCycB2 overexpression line and NtCycB2 knock-out line were obtained using transgenic technology and CRISPR-Cas9. Morphological observation of trichome showed that density of trichome was reduced significantly in overexpression lines, while density of glandular trichome was increased significantly in knockout lines and non-glandular trichome showed a little change, which indicated that NtCycB2 acted as a negative regulator for morphogenesis of glandular trichome and played an important role in tobacco leaf chemical orientation improvement.
引文
[1]Tooker J, Peiffer M, Luthe D S, et al. Trichomes as sensors[J].Plant Signaling&Behavior, 2010, 5, 1:73-75.
[2]Stratmann J W, Bequette C J. Hairless but no longer clueless:understanding glandular trichome development[J]. Journal of Experimental Botany, 2016, 67, 18:5285-5287.
[3]Wagner G J, Wang E, Shepherd R W. New approaches for studying and exploiting an old protuberance, the plant trichome[J]. Annals of Botany, 2004, 93(1):3-11.
[4]Shepherd R W, Bass W T, Houtz R L, et al. Phylloplanins of tobacco are defensive proteinsdeployed on aerial surfaces by short glandular trichomes[J]. The Plant Cell, 2005, 17(6):1851.
[5]Loughner R, Goldman K, Loeb G, et al. Influence of leaf trichomes on predatory mite(Typhlodromus pyri)abundance in grape varieties[J]. Experimental&Applied Acarology, 2008, 45(3-4):111-122.
[6]Payne T,ClementJ, ArnoldD,etal.Heterologousmyb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum[J]. Development, 1999,126(4):671-682.
[7]Serna L, Martin C. Trichomes:different regulatory networks lead to convergent structures[J]. Trends in Plant Science, 2006, 11(6):274-280.
[8]Yang C, Ye Z. Trichomes as models for studying plant cell differentiation[J]. Cellular and Molecular Life Sciences, 2013,70(11):1937-1948.
[9]Pines J. Cyclins and cyclin-dependent kinases:theme and variations[J]. Advances in Cancer Research, 1995, 66(6):181-212.
[10]Menges M, Jager S M D, Gruissem W, et al. Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes,reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control[J]. Plant Journal, 2010, 41(4):546-566.
[11]Weingartner M; Pelayo H R; Binarova P, et al. A plant cyclin B2 is degraded early in mitosis and its ectopic expression shortens G2-phase and alleviates the DNA-damage checkpoint[J]. Journal of Cell Science, 2003, 116(3):487-498.
[12]Lee J, Das A, Yamaguchi M, et al. Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase[J].Plant Journal, 2010, 34(4):0417-425.
[13]Sabelli P A, Dante R A, Nguyen H N, et al. Expression, regulation and activity of a B2-type cyclin in mitotic and endoreduplicating maize endosperm[J]. Frontiers in Plant Science, 2014, 5(561):561-574.
[14]Schnittger A, Schobinger U, Bouyer D, et al. Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes[J]. Proceedings of the National Academy of Sciences, 2002, 99(9):6410-6415.
[15]Yang C, Li H, Zhang J, et al. Fine-mapping of the woolly gene controlling multicellular trichome formation and embryonic development in tomato[J]. Theoretical&Applied Genetics, 2011,123(4):625-633.
[16]Gao S, Gao Y, Xiong C, et al. The tomato B-type cyclin gene,SlCycB2, plays key roles in reproductive organ development,trichome initiation, terpenoids biosynthesis and prodenia litura defense[J]. Plant Science, 2017, 262:103-114.
[17]孔光辉,徐照丽,王伟,等.不同肥料对红花大金元中部叶片腺毛及分泌物积累的影响[J].中国烟草学报, 2007, 13(4):41-44.KONG Guanghui, XU Zhaoli, WANG Wei, et al. Effect of fertilizer on glandular trichome density and exudates accumulation in middle leaf of “Honhuadajinyuan”[J].Acta Tabacaria Sinica, 2007,13(4):41-44.
[18]周世民,韩延,符云鹏,等.肥料类型对香料烟叶片腺毛密度的影响[J].烟草科技, 2007(7):55-57.ZHOU Shimin, HAN Yan, FU Yunpeng, et al. Effects of fertilizer typeon glandular trichome density of oriental tobacco lamina[J].Tobacco Science&Technology, 2007(7):55-57
[19]翁梦苓,梁志敏,崔红,等.遮荫对烟草叶片腺毛形态结构及分泌物含量的影响[J].河南农业大学学报, 2008, 42(6):613-616.WENG Mengling, LIANG Zhimin, CUI Hong, et al.Effects of shading on leaf trichome morphology and structure and secretions content of flue-cured tobacco[J]. Journal of Henan Agricultural University, 2008, 42(6):613-616.
[20]梁志敏,翁梦苓,崔红.施用有机肥对烟草腺毛形态结构及分泌物的影响[J].厦门大学学报(自然版), 2008, 47(s2):149-152.LIANG Zhimin, WENG Mengling, CUI Hong, et al. Effects of applying organic fertilizer on morphology and structure of chloroplast and exudates of flue-cured tobacco trichomes[J]. Journal of Xiamen University(Natural Science), 2008, 47(s2):149-152.
[21]张华,赵百东,冀浩,等.水分胁迫对烤烟腺毛超微结构的影响[J].中国烟草学报, 2008, 14(5):45-47.ZHANG Hua, ZHAO Baidong, JI Hao, et al. Effects of water stress on ultrastructure of tobacco leaf trichome[J]. Acta Tabacaria Sinica, 2008, 14(5):45-47.
[22]齐永杰,徐锦锦,梁伟.干旱胁迫对烟草腺毛密度及叶面分泌物的影响[J].广东农业科学, 2008(6):39-41.QI Yongjie, XU Jinjin, LIANG Wei. The influence of arid coercion to tobacco glandular hair density and leaf surface secretion[J].Guangdong Agricultural Sciences, 2008(6):39-41.
[23]武东玲,冯琦,王霄龙,等.光照度对烟草腺毛发育及其物质代谢的影响[J].河南农业大学学报, 2013, 47(6):659-662.WU Dongling, FENG Qi, WANG Xiaolong, et al. Effects of light intensity on trichome development and secretion metabolism of tobacco[J].Journal of Henan Agricultural University, 2013, 47(6):659-662.
[24]薛小平,潘文杰,陈伟,等.不同生态条件对烟叶腺毛密度的影响[J].西南农业学报, 2010, 23(6):2185-2188.XUE Xiaoping,PAN Wenjie,CHEN Wei, et al. Effect of different ecological condition on trichome density of flue-cured tobacco leaves[J]. Southwest China Journal of Agricultural Sciences, 2010,23(6):2185-2188.
[25]杨永霞,魏跃伟,冯琦,等.典型生态区烟叶腺毛发育及其分泌物比较研究[J].中国烟草学报, 2014(5):38-42.YANG Yongxia, WEI Yuewei, FENG Qi, et al. Comparative study in leaf trichome development and exudates in typical ecological regions[J]. Acta Tabacaria Sinica, 2014(5):38-42.
[26]韦建玉,金亚波,屈冉,等.不同生态条件对烟叶品质的影响[J].安徽农业科学, 2008, 36(11):4550-4551.WEI Jianyu, JIN Yabo, QU Ran, et al.Effect of different ecological conditionsonthequalityoftobacco[J].Journalof Anhui Agricultural Sciences, 2008, 36(11):4550-4551.
[27]张纪利.贵州烟区气候和土壤对烤烟生长、腺毛和质体色素的影响[D].安徽农业大学, 2010.ZHANG Jili. Effects of climate and soil on flue-cured tobacco growth, trichomes and plastid pigments in Guizhou tobacco growing areas[D]. Anhui Agricultural University, 2010.
[28]SchmidtGW, DelaneySK.Stable internal reference genes for normalization of real-time RT-PCR in tobacco(Nicotiana tabacum)during development and abioticstress.[J]. Molecular genetics&genomics, 2010, 283(3):233-241.
[29]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J]. Methods, 2001, 25:402-408.
[30]王关林,方宏均.植物基因工程技术原理与技术[M].科学出版社, 1998, 229-609.WANG Guanlin, FANG Hongjun. Principles and techniques of plant genetic engineering technology[M]. Science Press, 1998,229-609.
[31]Lin Y, Wagner G J. Rapid and simple method for estimation of sugar esters.[J]. Journal of Agricultural&Food Chemistry, 1994,42(8):1709-1712.
[32]Valverde PL, Fornoni J, Núez-Farfán J. Defensive role of leaf trichomes in resistance to herbivorous insects in Datura stramonium. Journal of Evolutionary Biology. 2001, 14:424-432.
[33]Nadakuduti S S, Pollard M, Kosma D K, et al. Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[J]. Plant Physiology. 2012, 159(3):945-960.
[34]Zhang J L. Influence of Ecological and cultivation factors on density and secretions of glandular trichome in tobacco leaves[J].Journal of Anhui Agricultural Sciences. 2010.
[35]Hülskamp M. Plant trichomes:a model for cell differentiation.Nature Reviews Molecular Cell Biology. 2004,5:471-480.
[36]Kang J H, Liu G, Shi F, et al. The tomato odorless-2 mutant is defective in trichome-based production of diverse specialized metabolites and broad-spectrum resistance to insect herbivores[J].Plant Physiology. 2010, 154(1):262-272.
[37]高升华. SlCycB2调控植物多细胞表皮毛形成和花器发育的机制研究.[D].华中农业大学, 2017.GAO Shenghua. Mechanism ofSlCycB2 regulating multicellular trichome formation and flower organ development in plant[D].Huazhong Agricultural University, 2017.
[2]Stratmann J W, Bequette C J. Hairless but no longer clueless:understanding glandular trichome development[J]. Journal of Experimental Botany, 2016, 67, 18:5285-5287.
[3]Wagner G J, Wang E, Shepherd R W. New approaches for studying and exploiting an old protuberance, the plant trichome[J]. Annals of Botany, 2004, 93(1):3-11.
[4]Shepherd R W, Bass W T, Houtz R L, et al. Phylloplanins of tobacco are defensive proteinsdeployed on aerial surfaces by short glandular trichomes[J]. The Plant Cell, 2005, 17(6):1851.
[5]Loughner R, Goldman K, Loeb G, et al. Influence of leaf trichomes on predatory mite(Typhlodromus pyri)abundance in grape varieties[J]. Experimental&Applied Acarology, 2008, 45(3-4):111-122.
[6]Payne T,ClementJ, ArnoldD,etal.Heterologousmyb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum[J]. Development, 1999,126(4):671-682.
[7]Serna L, Martin C. Trichomes:different regulatory networks lead to convergent structures[J]. Trends in Plant Science, 2006, 11(6):274-280.
[8]Yang C, Ye Z. Trichomes as models for studying plant cell differentiation[J]. Cellular and Molecular Life Sciences, 2013,70(11):1937-1948.
[9]Pines J. Cyclins and cyclin-dependent kinases:theme and variations[J]. Advances in Cancer Research, 1995, 66(6):181-212.
[10]Menges M, Jager S M D, Gruissem W, et al. Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes,reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control[J]. Plant Journal, 2010, 41(4):546-566.
[11]Weingartner M; Pelayo H R; Binarova P, et al. A plant cyclin B2 is degraded early in mitosis and its ectopic expression shortens G2-phase and alleviates the DNA-damage checkpoint[J]. Journal of Cell Science, 2003, 116(3):487-498.
[12]Lee J, Das A, Yamaguchi M, et al. Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase[J].Plant Journal, 2010, 34(4):0417-425.
[13]Sabelli P A, Dante R A, Nguyen H N, et al. Expression, regulation and activity of a B2-type cyclin in mitotic and endoreduplicating maize endosperm[J]. Frontiers in Plant Science, 2014, 5(561):561-574.
[14]Schnittger A, Schobinger U, Bouyer D, et al. Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes[J]. Proceedings of the National Academy of Sciences, 2002, 99(9):6410-6415.
[15]Yang C, Li H, Zhang J, et al. Fine-mapping of the woolly gene controlling multicellular trichome formation and embryonic development in tomato[J]. Theoretical&Applied Genetics, 2011,123(4):625-633.
[16]Gao S, Gao Y, Xiong C, et al. The tomato B-type cyclin gene,SlCycB2, plays key roles in reproductive organ development,trichome initiation, terpenoids biosynthesis and prodenia litura defense[J]. Plant Science, 2017, 262:103-114.
[17]孔光辉,徐照丽,王伟,等.不同肥料对红花大金元中部叶片腺毛及分泌物积累的影响[J].中国烟草学报, 2007, 13(4):41-44.KONG Guanghui, XU Zhaoli, WANG Wei, et al. Effect of fertilizer on glandular trichome density and exudates accumulation in middle leaf of “Honhuadajinyuan”[J].Acta Tabacaria Sinica, 2007,13(4):41-44.
[18]周世民,韩延,符云鹏,等.肥料类型对香料烟叶片腺毛密度的影响[J].烟草科技, 2007(7):55-57.ZHOU Shimin, HAN Yan, FU Yunpeng, et al. Effects of fertilizer typeon glandular trichome density of oriental tobacco lamina[J].Tobacco Science&Technology, 2007(7):55-57
[19]翁梦苓,梁志敏,崔红,等.遮荫对烟草叶片腺毛形态结构及分泌物含量的影响[J].河南农业大学学报, 2008, 42(6):613-616.WENG Mengling, LIANG Zhimin, CUI Hong, et al.Effects of shading on leaf trichome morphology and structure and secretions content of flue-cured tobacco[J]. Journal of Henan Agricultural University, 2008, 42(6):613-616.
[20]梁志敏,翁梦苓,崔红.施用有机肥对烟草腺毛形态结构及分泌物的影响[J].厦门大学学报(自然版), 2008, 47(s2):149-152.LIANG Zhimin, WENG Mengling, CUI Hong, et al. Effects of applying organic fertilizer on morphology and structure of chloroplast and exudates of flue-cured tobacco trichomes[J]. Journal of Xiamen University(Natural Science), 2008, 47(s2):149-152.
[21]张华,赵百东,冀浩,等.水分胁迫对烤烟腺毛超微结构的影响[J].中国烟草学报, 2008, 14(5):45-47.ZHANG Hua, ZHAO Baidong, JI Hao, et al. Effects of water stress on ultrastructure of tobacco leaf trichome[J]. Acta Tabacaria Sinica, 2008, 14(5):45-47.
[22]齐永杰,徐锦锦,梁伟.干旱胁迫对烟草腺毛密度及叶面分泌物的影响[J].广东农业科学, 2008(6):39-41.QI Yongjie, XU Jinjin, LIANG Wei. The influence of arid coercion to tobacco glandular hair density and leaf surface secretion[J].Guangdong Agricultural Sciences, 2008(6):39-41.
[23]武东玲,冯琦,王霄龙,等.光照度对烟草腺毛发育及其物质代谢的影响[J].河南农业大学学报, 2013, 47(6):659-662.WU Dongling, FENG Qi, WANG Xiaolong, et al. Effects of light intensity on trichome development and secretion metabolism of tobacco[J].Journal of Henan Agricultural University, 2013, 47(6):659-662.
[24]薛小平,潘文杰,陈伟,等.不同生态条件对烟叶腺毛密度的影响[J].西南农业学报, 2010, 23(6):2185-2188.XUE Xiaoping,PAN Wenjie,CHEN Wei, et al. Effect of different ecological condition on trichome density of flue-cured tobacco leaves[J]. Southwest China Journal of Agricultural Sciences, 2010,23(6):2185-2188.
[25]杨永霞,魏跃伟,冯琦,等.典型生态区烟叶腺毛发育及其分泌物比较研究[J].中国烟草学报, 2014(5):38-42.YANG Yongxia, WEI Yuewei, FENG Qi, et al. Comparative study in leaf trichome development and exudates in typical ecological regions[J]. Acta Tabacaria Sinica, 2014(5):38-42.
[26]韦建玉,金亚波,屈冉,等.不同生态条件对烟叶品质的影响[J].安徽农业科学, 2008, 36(11):4550-4551.WEI Jianyu, JIN Yabo, QU Ran, et al.Effect of different ecological conditionsonthequalityoftobacco[J].Journalof Anhui Agricultural Sciences, 2008, 36(11):4550-4551.
[27]张纪利.贵州烟区气候和土壤对烤烟生长、腺毛和质体色素的影响[D].安徽农业大学, 2010.ZHANG Jili. Effects of climate and soil on flue-cured tobacco growth, trichomes and plastid pigments in Guizhou tobacco growing areas[D]. Anhui Agricultural University, 2010.
[28]SchmidtGW, DelaneySK.Stable internal reference genes for normalization of real-time RT-PCR in tobacco(Nicotiana tabacum)during development and abioticstress.[J]. Molecular genetics&genomics, 2010, 283(3):233-241.
[29]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J]. Methods, 2001, 25:402-408.
[30]王关林,方宏均.植物基因工程技术原理与技术[M].科学出版社, 1998, 229-609.WANG Guanlin, FANG Hongjun. Principles and techniques of plant genetic engineering technology[M]. Science Press, 1998,229-609.
[31]Lin Y, Wagner G J. Rapid and simple method for estimation of sugar esters.[J]. Journal of Agricultural&Food Chemistry, 1994,42(8):1709-1712.
[32]Valverde PL, Fornoni J, Núez-Farfán J. Defensive role of leaf trichomes in resistance to herbivorous insects in Datura stramonium. Journal of Evolutionary Biology. 2001, 14:424-432.
[33]Nadakuduti S S, Pollard M, Kosma D K, et al. Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato[J]. Plant Physiology. 2012, 159(3):945-960.
[34]Zhang J L. Influence of Ecological and cultivation factors on density and secretions of glandular trichome in tobacco leaves[J].Journal of Anhui Agricultural Sciences. 2010.
[35]Hülskamp M. Plant trichomes:a model for cell differentiation.Nature Reviews Molecular Cell Biology. 2004,5:471-480.
[36]Kang J H, Liu G, Shi F, et al. The tomato odorless-2 mutant is defective in trichome-based production of diverse specialized metabolites and broad-spectrum resistance to insect herbivores[J].Plant Physiology. 2010, 154(1):262-272.
[37]高升华. SlCycB2调控植物多细胞表皮毛形成和花器发育的机制研究.[D].华中农业大学, 2017.GAO Shenghua. Mechanism ofSlCycB2 regulating multicellular trichome formation and flower organ development in plant[D].Huazhong Agricultural University, 2017.
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