多腺毛烟草K326品种基因编辑材料的创制与分析
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摘要
烟草腺毛与烟株抗性和烟叶品质密切相关。为了定向改良烟草腺毛密度、创制多腺毛烟草材料,以烟草品种K326为材料,利用CRISPR-Cas9技术对腺毛发生负向调控基因NtCycB2进行敲除,创制了多腺毛K326定向改良材料HK326(Hairy K326),并分析了NtCycB2基因敲除对烤烟农艺性状和化学成分的影响。结果表明:NtCycB2基因敲除后烟株发育正常,生长旺盛,农艺性状与对照相比无明显变化;烟草叶面腺毛密度显著提高,分泌型腺毛发育完善,物质合成和分泌旺盛,西柏烷二萜化合物含量显著提高;烤后烟叶中性香气成分总量增加,其中,茄酮及新植二烯类成分增幅最为明显。因此,NtCycB2基因的敲除,不仅能够提高烟草的腺毛密度,而且对烟株生长发育和重要香气物质的积累也有促进作用,在烟草品种改良中具有较大的应用潜力。
Tobacco trichomes are closely related to disease resistance and leaf quality. To improve tobacco trichome density and create a high trichome density variety, CRISPR-Cas9 technique was used to knock out NtCycB2, which negatively regulated trichome formation, in cv. K326. A high trichome density line was obtained as a result, named as HK326(Hairy K326). Furthermore, the effects of NtCycB2 on the agronomic traits and chemical composition of flue-cured tobacco were studied. The results showed that HK326 grew and developed normally, and its agronomic trait was no different to the controls. The trichome density on the surface of HK326 leaves increased. Glandular trichomes of HK326 had strongly biosynthetic and secretory functions, and cembranoids content significantly raised. The total content of neutral aroma components in fluecured leaves of HK326 was increased, especially solanone and neophytadiene. Therefore, the trichome density,growing, development and main aroma component accumulation of K326 was promoted by knocking out NtCycB2, which suggested that HK326 had promising application potential in tobacco variety improvement.
Tobacco trichomes are closely related to disease resistance and leaf quality. To improve tobacco trichome density and create a high trichome density variety, CRISPR-Cas9 technique was used to knock out NtCycB2, which negatively regulated trichome formation, in cv. K326. A high trichome density line was obtained as a result, named as HK326(Hairy K326). Furthermore, the effects of NtCycB2 on the agronomic traits and chemical composition of flue-cured tobacco were studied. The results showed that HK326 grew and developed normally, and its agronomic trait was no different to the controls. The trichome density on the surface of HK326 leaves increased. Glandular trichomes of HK326 had strongly biosynthetic and secretory functions, and cembranoids content significantly raised. The total content of neutral aroma components in fluecured leaves of HK326 was increased, especially solanone and neophytadiene. Therefore, the trichome density,growing, development and main aroma component accumulation of K326 was promoted by knocking out NtCycB2, which suggested that HK326 had promising application potential in tobacco variety improvement.
引文
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[3]Wang E M,Wang R,DeParasis J,et al.Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance[J].Nature Biotechnology,2001,19(4):371-374.
[4]Huchelmann A,Boutry M,Hachez C.Plant glandular trichomes:natural cell factories of high biotechnological interest[J].Plant Physiology,2017,175(1):6-22.
[5]翁梦苓,梁志敏,崔红.遮荫对烟草中部叶片腺毛密度及其分泌物含量的影响[J].河南农业科学,2009,38(4):52-55.WENG Mengling,LIANG Zhimin,CUI Hong.Effects of shaded treatment on density of central trichome and content of glandular secretions in tobacco[J].Journal of Henan Agricultural Sciences,2009,38(4):52-55.
[6]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):4550-4551,4553.WEI Jianyu,JIN Yabo,QU Ran,et al.Effect of different ecological conditions on the quality of tobacco[J].Journal of Anhui Agricultural Sciences,2008,36(11):4550-4551,4553.
[7]张华,崔红,冀浩,等.水分胁迫对烤烟腺毛形态发育及基因表达的影响[J].郑州牧业工程高等专科学校学报,2012,32(1):4-8.ZHANG Hua,CUI Hong,JI Hao,et al.Effects of drought stress on the development and gene expression of tobacco trichomes[J].Journal of Zhengzhou College of Animal Husbandry Engineering,2012,32(1):4-8.
[8]朱显灵,潘文杰,陈懿,等.不同气候条件下烟叶腺毛分泌物主要成分的差异分析[J].烟草科技,2011(1):60-65.ZHU Xianling,PAN Wenjie,CHEN Yi,et al.Comparison of major components in leaf trichome exudate of flue-cured tobacco grown under different climatic conditions[J].Tobacco Science&Technology,2011(1):60-65.
[9]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 type on glandular trichome density of oriental tobacco lamina[J].Tobacco Science&Technology,2007(7):55-57.
[10]李鹏飞,周冀衡,张建平,等.氮、磷、钾、镁亏缺对烤烟生长和叶片腺毛发育的影响[J].烟草科技,2009(12):49-54.LI Pengfei,ZHOU Jiheng,ZHANG Jianping,et al.Effects of N,P,K,Mg deficiency on plant growth and leaf glandular trichome development of flue-cured tobacco[J].Tobacco Science&Technology,2009(12):49-54.
[11]梁志敏,冀浩,张华,等.施肥对烟草腺毛叶绿体形态结构的影响[J].西北植物学报,2009,29(2):291-295.LIANG Zhimin,JI Hao,ZHANG Hua,et al.Morphology and structure of chloroplast in flue-cured tobacco trichomes after applying fertilizer[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(2):291-295.
[12]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,2014,20(5):38-42.YANG Yongxia,WEI Yuewei,FENG Qi,et al.Comparative research of the leaf trichome development and exudates of tobacco growing in typical ecological regions[J].Acta Tabacaria Sinica,2014,20(5):38-42.
[13]Huang M Y,Zhang H Y,Wang Z J,et al.Comparative studies of leaf surface chemical biosynthesis in different tobacco cultivars[J].Acta Physiologiae Plantarum,2018,40(4):67.
[14]刘彩云,常志隆,常爱霞,等.普通烟草品种生长发育过程中叶表腺毛密度及其形态的比较[J].烟草科技,2011(5):71-73,84.LIU Caiyun,CHANG Zhilong,CHANG Aixia,et al.Comparison of density and morphology of leaf glandular trichome at different growth stages of Nicotiana tabacum L.[J].Tobacco Science&Technology,2011(5):71-73,84.
[15]李艳华,张洪映,魏跃伟,等.典型烤烟品种腺毛形态及分泌特性比较分析[J].中国烟草学报,2017,23(2):84-91.LI Yanhua,ZHANG Hongying,WEI Yuewei,et al.Comparison analysis of trichome morphology and secretion of typical flue-cured tobacco varieties[J].Chinese Journal of Tobacco,2017,23(2):84-91.
[16]杨铁钊,李伟,李钦奎,等.烤烟叶面腺毛密度及其分泌物变化动态的相关分析[J].中国烟草科学,2005,26(1):43-46.YANG Tiezhao,LI Wei,LI Qinkui,et al.Analysis of the varying trends and correlation of the density of glandular trichome and leaf surface exudate of fluecured tobacco[J].Chinese Tobacco Science,2005,26(1):43-46.
[17]时向东,杨会丽,高致明,等.烤烟叶片腺毛发育过程的扫描电镜观察[J].河南农业大学学报,2005,39(2):155-157.SHI Xiangdong,YANG Huili,GAO Zhiming,et al.Observation of trichomes on developing flue-cured tobacco leaf by scan electronic microscope[J].Journal of Henan Agricultural University,2005,39(2):155-157.
[18]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.
[19]Schnittger A,Sch?binger U,Stierhof Y D,et al.Ectopic b-type cyclin expression induces mitotic cycles in endoreduplicating arabidopsis trichomes[J].Current Biology,2002,12(5):415-420.
[20]Yang C X,Li H X,Zhang J H,et al.A regulatory gene induces trichome formation and embryo lethality in tomato[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(29):11836-11841.
[21]Yang C X,Gao Y N,Gao S H,et al.Transcriptome profile analysis of cell proliferation molecular processes during multicellular trichome formation induced by tomato Wovgene in tobacco[J].BMC Genomics,2015,16:868.
[22]Gao S H,Gao Y N,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.
[23]Lee J,Das A,Yamaguchi M,et al.Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclindependent kinase[J].Plant Journal for Cell&Molecular Biology,2010,34(4):417-425.
[24]Guo J,Wang F,Song J,et al.The expression of Orysa;CycB1;1 is essential for endosperm formation and causes embryo enlargement in rice[J].Planta,2009,231:293.
[25]连文力,张松涛,李艳华,等.AtPAP1基因在烟草中的表达[J].烟草科技,2016,49(7):1-7.LIAN Wenli,ZHANG Songtao,LI Yanhua,et al.Expression of AtPAP1 gene in tobacco[J].Tobacco Science&Technology,2016,49(7):1-7.
[26]YC/T 143-1998烟草育苗基本技术规程[S].YC/T 143-1998 Basic rules for cultivate seedling technology of tobacco[S].
[27]GB/T 23221-2008烤烟栽培技术规程[S].GB/T 23221-2008 Rules for cultivation of flue-cured tobacco[S].
[28]YC/T 142-2010烟草农艺性状调查测量方法[S].YC/T 142-2010 Investigating and measuring methods of agronomical character of tobacco[S].
[29]娄亚楠,王召军,杨欣玲,等.茉莉酸甲酯对烟草分泌型和非分泌型腺毛形态发生的影响[J].中国烟草学报,2018,24(2):28-29.LOU Yanan,WANG Zhaojun,YANG Xinling,et al.Effects of methyl jasmonic acid on morphogenesis of tobacco glandular and non-glandular trichomes[J].Acta Tabacaria Sinica,2018,24(2):28-29.
[30]王霄龙,杨永霞,张松涛,等.中间香型烤烟叶面腺毛形态及分泌特性研究[J].中国烟草学报,2013(3):45-48.WANG Xiaolong,YANG Yongxia,ZHANG Songtao,et al.Trichome morphology and secreting feature in neutral flavor type flue-cured tobacco leaf[J].Acta Tabacaria Sinica,2013(3):45-48.
[31]邸慧慧,史宏志,马永建,等.烤烟叶片中性香气成分含量的分布[J].中国烟草科学,2010,31(6):5-8.DI Huihui,SHI Hongzhi,MA Yongjian,et al.Distribution of neutral aroma components in flue-cured tobacco leaves[J].Chinese Tobacco Science,2010,31(6):5-8.
[32]冯琦.烟草腺毛发育及类萜代谢研究[D].郑州:河南农业大学,2013.FENG Qi.Studies on development and terpenoid metabolism of tobacco trichome[D].Zhengzhou:Henan Agricultural University,2013.
[2]Johnson A,Severson R,Hudson J,et al.Tobacco leaf trichomes and their exudates[J].Tobacco Science,1985,29:67-72.
[3]Wang E M,Wang R,DeParasis J,et al.Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance[J].Nature Biotechnology,2001,19(4):371-374.
[4]Huchelmann A,Boutry M,Hachez C.Plant glandular trichomes:natural cell factories of high biotechnological interest[J].Plant Physiology,2017,175(1):6-22.
[5]翁梦苓,梁志敏,崔红.遮荫对烟草中部叶片腺毛密度及其分泌物含量的影响[J].河南农业科学,2009,38(4):52-55.WENG Mengling,LIANG Zhimin,CUI Hong.Effects of shaded treatment on density of central trichome and content of glandular secretions in tobacco[J].Journal of Henan Agricultural Sciences,2009,38(4):52-55.
[6]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):4550-4551,4553.WEI Jianyu,JIN Yabo,QU Ran,et al.Effect of different ecological conditions on the quality of tobacco[J].Journal of Anhui Agricultural Sciences,2008,36(11):4550-4551,4553.
[7]张华,崔红,冀浩,等.水分胁迫对烤烟腺毛形态发育及基因表达的影响[J].郑州牧业工程高等专科学校学报,2012,32(1):4-8.ZHANG Hua,CUI Hong,JI Hao,et al.Effects of drought stress on the development and gene expression of tobacco trichomes[J].Journal of Zhengzhou College of Animal Husbandry Engineering,2012,32(1):4-8.
[8]朱显灵,潘文杰,陈懿,等.不同气候条件下烟叶腺毛分泌物主要成分的差异分析[J].烟草科技,2011(1):60-65.ZHU Xianling,PAN Wenjie,CHEN Yi,et al.Comparison of major components in leaf trichome exudate of flue-cured tobacco grown under different climatic conditions[J].Tobacco Science&Technology,2011(1):60-65.
[9]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 type on glandular trichome density of oriental tobacco lamina[J].Tobacco Science&Technology,2007(7):55-57.
[10]李鹏飞,周冀衡,张建平,等.氮、磷、钾、镁亏缺对烤烟生长和叶片腺毛发育的影响[J].烟草科技,2009(12):49-54.LI Pengfei,ZHOU Jiheng,ZHANG Jianping,et al.Effects of N,P,K,Mg deficiency on plant growth and leaf glandular trichome development of flue-cured tobacco[J].Tobacco Science&Technology,2009(12):49-54.
[11]梁志敏,冀浩,张华,等.施肥对烟草腺毛叶绿体形态结构的影响[J].西北植物学报,2009,29(2):291-295.LIANG Zhimin,JI Hao,ZHANG Hua,et al.Morphology and structure of chloroplast in flue-cured tobacco trichomes after applying fertilizer[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(2):291-295.
[12]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,2014,20(5):38-42.YANG Yongxia,WEI Yuewei,FENG Qi,et al.Comparative research of the leaf trichome development and exudates of tobacco growing in typical ecological regions[J].Acta Tabacaria Sinica,2014,20(5):38-42.
[13]Huang M Y,Zhang H Y,Wang Z J,et al.Comparative studies of leaf surface chemical biosynthesis in different tobacco cultivars[J].Acta Physiologiae Plantarum,2018,40(4):67.
[14]刘彩云,常志隆,常爱霞,等.普通烟草品种生长发育过程中叶表腺毛密度及其形态的比较[J].烟草科技,2011(5):71-73,84.LIU Caiyun,CHANG Zhilong,CHANG Aixia,et al.Comparison of density and morphology of leaf glandular trichome at different growth stages of Nicotiana tabacum L.[J].Tobacco Science&Technology,2011(5):71-73,84.
[15]李艳华,张洪映,魏跃伟,等.典型烤烟品种腺毛形态及分泌特性比较分析[J].中国烟草学报,2017,23(2):84-91.LI Yanhua,ZHANG Hongying,WEI Yuewei,et al.Comparison analysis of trichome morphology and secretion of typical flue-cured tobacco varieties[J].Chinese Journal of Tobacco,2017,23(2):84-91.
[16]杨铁钊,李伟,李钦奎,等.烤烟叶面腺毛密度及其分泌物变化动态的相关分析[J].中国烟草科学,2005,26(1):43-46.YANG Tiezhao,LI Wei,LI Qinkui,et al.Analysis of the varying trends and correlation of the density of glandular trichome and leaf surface exudate of fluecured tobacco[J].Chinese Tobacco Science,2005,26(1):43-46.
[17]时向东,杨会丽,高致明,等.烤烟叶片腺毛发育过程的扫描电镜观察[J].河南农业大学学报,2005,39(2):155-157.SHI Xiangdong,YANG Huili,GAO Zhiming,et al.Observation of trichomes on developing flue-cured tobacco leaf by scan electronic microscope[J].Journal of Henan Agricultural University,2005,39(2):155-157.
[18]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.
[19]Schnittger A,Sch?binger U,Stierhof Y D,et al.Ectopic b-type cyclin expression induces mitotic cycles in endoreduplicating arabidopsis trichomes[J].Current Biology,2002,12(5):415-420.
[20]Yang C X,Li H X,Zhang J H,et al.A regulatory gene induces trichome formation and embryo lethality in tomato[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(29):11836-11841.
[21]Yang C X,Gao Y N,Gao S H,et al.Transcriptome profile analysis of cell proliferation molecular processes during multicellular trichome formation induced by tomato Wovgene in tobacco[J].BMC Genomics,2015,16:868.
[22]Gao S H,Gao Y N,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.
[23]Lee J,Das A,Yamaguchi M,et al.Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclindependent kinase[J].Plant Journal for Cell&Molecular Biology,2010,34(4):417-425.
[24]Guo J,Wang F,Song J,et al.The expression of Orysa;CycB1;1 is essential for endosperm formation and causes embryo enlargement in rice[J].Planta,2009,231:293.
[25]连文力,张松涛,李艳华,等.AtPAP1基因在烟草中的表达[J].烟草科技,2016,49(7):1-7.LIAN Wenli,ZHANG Songtao,LI Yanhua,et al.Expression of AtPAP1 gene in tobacco[J].Tobacco Science&Technology,2016,49(7):1-7.
[26]YC/T 143-1998烟草育苗基本技术规程[S].YC/T 143-1998 Basic rules for cultivate seedling technology of tobacco[S].
[27]GB/T 23221-2008烤烟栽培技术规程[S].GB/T 23221-2008 Rules for cultivation of flue-cured tobacco[S].
[28]YC/T 142-2010烟草农艺性状调查测量方法[S].YC/T 142-2010 Investigating and measuring methods of agronomical character of tobacco[S].
[29]娄亚楠,王召军,杨欣玲,等.茉莉酸甲酯对烟草分泌型和非分泌型腺毛形态发生的影响[J].中国烟草学报,2018,24(2):28-29.LOU Yanan,WANG Zhaojun,YANG Xinling,et al.Effects of methyl jasmonic acid on morphogenesis of tobacco glandular and non-glandular trichomes[J].Acta Tabacaria Sinica,2018,24(2):28-29.
[30]王霄龙,杨永霞,张松涛,等.中间香型烤烟叶面腺毛形态及分泌特性研究[J].中国烟草学报,2013(3):45-48.WANG Xiaolong,YANG Yongxia,ZHANG Songtao,et al.Trichome morphology and secreting feature in neutral flavor type flue-cured tobacco leaf[J].Acta Tabacaria Sinica,2013(3):45-48.
[31]邸慧慧,史宏志,马永建,等.烤烟叶片中性香气成分含量的分布[J].中国烟草科学,2010,31(6):5-8.DI Huihui,SHI Hongzhi,MA Yongjian,et al.Distribution of neutral aroma components in flue-cured tobacco leaves[J].Chinese Tobacco Science,2010,31(6):5-8.
[32]冯琦.烟草腺毛发育及类萜代谢研究[D].郑州:河南农业大学,2013.FENG Qi.Studies on development and terpenoid metabolism of tobacco trichome[D].Zhengzhou:Henan Agricultural University,2013.