过表达OsATG8a基因提高转基因拟南芥对低氮的耐受性和产量
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摘要
自噬(autophagy)是真核生物一种保守的物质降解途径,通过物质的降解和循环实现其再利用。我们鉴定到了水稻(Oryza sativa)自噬核心基因OsATG8a并获得2个独立的过表达OsATG8a转基因拟南芥(Arabidopsisthaliana)纯合株系。研究发现过表达OsATG8a增加了叶片细胞中自噬体的数量和活性,且35S-OsATG8a转基因拟南芥相对于野生型拟南芥(WT)有更好的表型,如氮代谢相关基因表达量增加,正常生长条件下莲座叶增大、产量增多,缺氮条件下转基因株系相对于WT具有更强的抵抗作用。因此,我们认为OsATG8a是一个可以提高产量的优质候选基因。
Autophagy is a conserved degradation pathway of substance in eukaryotes, which degrades and recy-cles cellular components to reuse of substances. We identified an autophagy core gene OsATG8 a in rice(Oryzasativa) and obtained two independent 35 S-OsATG8 a transgenic Arabidopsis thaliana homozygous lines. Wefound that overexpression of OsATG8 a does increase the amount and activity of autophagy in leaf cells, espe-cially under nitrogen-deficiency conditions. 35 S-OsATG8 a transgenic A. thaliana had a better phenotype thanthe wild type(WT), in which the rosette leaves were larger and the yield was higher under normal growth con-ditions. In addition, the transgenic A. thaliana showed stronger resistance to nitrogen starvation and the expres-sion levels of genes related to nitrogen metabolism were higher than those of WT under both nitrogen-sufficientand-deficient conditions. Therefore, we believe that OsATG8 a is a high-quality candidate gene for increasingyield.
Autophagy is a conserved degradation pathway of substance in eukaryotes, which degrades and recy-cles cellular components to reuse of substances. We identified an autophagy core gene OsATG8 a in rice(Oryzasativa) and obtained two independent 35 S-OsATG8 a transgenic Arabidopsis thaliana homozygous lines. Wefound that overexpression of OsATG8 a does increase the amount and activity of autophagy in leaf cells, espe-cially under nitrogen-deficiency conditions. 35 S-OsATG8 a transgenic A. thaliana had a better phenotype thanthe wild type(WT), in which the rosette leaves were larger and the yield was higher under normal growth con-ditions. In addition, the transgenic A. thaliana showed stronger resistance to nitrogen starvation and the expres-sion levels of genes related to nitrogen metabolism were higher than those of WT under both nitrogen-sufficientand-deficient conditions. Therefore, we believe that OsATG8 a is a high-quality candidate gene for increasingyield.
引文
Arnon DI(1949).Copper enzymes in isolated chloroplasts.Polyphenoloxidase in Beta vulgaris.Plant Physiol,24(1):1-15
Boyes DC,Zayed AM,Ascenzi R,et al(2001).Growth stagebased phenotypic analysis of Arabidopsis:a model for high throughput functional genomics in plants.Plant Cell,13(7):1499-1510
Bradford MM(1976).A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem,72(S1-2):248-254
Clough SJ,Bent AF(1998).Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J,16(6):735-743
Contento AL,Xiong Y,Bassham DC(2005).Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein.Plant J,42(4):598-608
Feng Y,He D,Yao Z,et al(2014).The machinery of macroautophagy.Cell Res,24(1):24-41
Good AG,Shrawat AK,Muench DG(2004).Can less yield more?Is reducing nutrient input into the environment compatible with maintaining crop production?Trends Plant Sci,9(12):597-605
Guiboileau A,Yoshimoto K,Soulay F,et al(2012).Autophagy machinery controls nitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis.New Phytol,194(3):732-740
Hanaoka H,Noda T,Shirano Y,et al(2002).Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene.Plant Physiol,129(3):1181-1193
Huang X,Li F(2016).Roles of autophagy in plant programmed cell death.Chin Bull Bot,51(6):859-862(in Chinese with English abstract)[黄晓,李发强(2016).细胞自噬在植物细胞程序性死亡中的作用.植物学报,51(6):859-862]
Izumi M,Hidema J,Wada S,et al(2015).Establishment of monitoring methods for autophagy in rice reveals autophagic recycling of chloroplasts and root plastids during energy limitation.Plant Physiol,167(4):1307-1320
Kichey T,Hirel B,Heumez E,et al(2007).In winter wheat(Triticum aestivum L.),post-anthesis nitrogen uptake and remobilisation to the grain correlates with agronomic traits and nitrogen physiological markers.Field Crop Res,102(1):22-32
Li F,Chung T,Pennington JG,et al(2015).Autophagic recycling plays a central role in maize nitrogen remobilization.Plant Cell,27(5):1389-1408
Li F,Vierstra RD(2012).Autophagy:a multifaceted intracellular system for bulk and selective recycling.Trends Plant Sci,17(9):526-537
Li W,Chen M,Wang E,et al(2016).Genome-wide analysis of autophagy-associated genes in foxtail millet(Setaria italica L.)and characterization of the function of SiAT-G8a in conferring tolerance to nitrogen starvation in rice.BMC Genom,17:797
Masclaux-Daubresse C,Chen Q,HavéM(2017).Regulation of nutrient recycling via autophagy.Curr Opin Plant Biol,39:8-17
Masclaux-Daubresse C,Daniel-Vedele F,Dechorgnat J,et al(2010).Nitrogen uptake,assimilation and remobilization in plants:challenges for sustainable and productive agriculture.Ann Bot,105(7):1141-1157
Masclaux-Daubresse C,Reisdorf-Cren M,Orsel M(2008).Leaf nitrogen remobilization for plant development and grain filling.Plant Biol,10(S1):23-36
Phillips AR,Suttangkakul A,Vierstra RD(2008).The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana.Genetics,178(3):1339-1353
Ren C,Gong Q(2014).Progress on the involvement of plant autophagy in carbon and nitrogen utilization.Chin J Cell Biol,36(4):407-414(in Chinese with English abstract)[任晨霞,龚清秋(2014).细胞自噬在植物碳氮营养中作用的研究进展.中国细胞生物学学报,36(4):407-414]
Rentsch D,Schmidt S,Tegeder M(2007).Transporters for uptake and allocation of organic nitrogen compounds in plants.FEBS Lett,581(12):2281-2289
SlávikováS,Shy G,Yao Y,et al(2005).The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants.J Exp Bot,56(421):2839-2849
Suttangkakul A,Li F,Chung T,et al(2011).The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis.Plant Cell,23(6):3761-3779
Thompson AR,Doelling JH,Suttangkakul A,et al(2005).Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways.Plant Physiol,138(4):2097-2110
Tsukada M,Ohsumi Y(1993).Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae.FEBS Lett,333(1-2):169-174
Wada S,Hayashida Y,Izumi M,et al(2015).Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice.Plant Physiol,168(1):60-73
Wang P,Sun X,Jia X,et al(2016).Characterization of an autophagy-related gene MdATG8i from apple.Front Plant Sci,7:720
Wu B,Cang J,Zhang D,et al(2015).Effects of SA treatment on winter wheat(Triticum aestivum L.)sucrose metabolism in low temperature.Plant Physiol J,51(4):537-545(in Chinese with English abstract)[吴冰,苍晶,张达等(2015).SA处理对低温胁迫下冬小麦(Triticum aestivum L.)蔗糖代谢的影响.植物生理学报,51(4):537-545]
Xia T,Xiao D,Liu D,et al(2012).Heterologous expression of ATG8c from soybean confers tolerance to nitrogen deficiency and increases yield in Arabidopsis.PLoS ONE,7(5):e37217
Xu F,Meng T,Li P,et al(2011).A soybean dual-specificity kinase,GmSARK,and its Arabidopsis homolog,AtSARK,regulate leaf senescence through synergistic actions of auxin and ethylene.Plant Physiol,157(4):2131-2153
Xu G,Fan X,Miller AJ(2012).Plant nitrogen assimilation and use efficiency.Ann Rev Plant Biol,63(1):153-182
Xu W,Liu S,You X,et al(2017).Overexpression of GmAT-G8c gene enhances tolerance to low nitrogen and increases yield in soybean.Plant Physiol J,53(2):241-247(in Chinese with English abstract)[徐伟,刘生,游翔等(2017).过表达GmATG8c基因提高大豆的低氮耐受性和产量.植物生理学报,53(2):241-247]
Yang X,Li Y,Liu Y,et al(2017).Review of selective autophagy in plant cell.Acta Hortic Sin,44(10):2015-2028(in Chinese with English abstract)[杨小龙,李漾漾,刘玉凤等(2017).植物细胞选择性自噬研究进展.园艺学报,44(10):2015-2028]
Yoshimoto K,Hanaoka H,Sato S,et al(2004).Processing of ATG8s,ubiquitin-like proteins,and their deconjugation by ATG4s are essential for plant autophagy.Plant Cell,16(11):2967-2983
Yoshimoto K,Jikumaru Y,Kamiya Y,et al(2009).Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis.Plant Cell,21(9):2914-2927
Yoshimoto K,Takano Y,Sakai Y(2010).Autophagy in plants and phytopathogens.FEBS Lett,584(7):1350-1358
Boyes DC,Zayed AM,Ascenzi R,et al(2001).Growth stagebased phenotypic analysis of Arabidopsis:a model for high throughput functional genomics in plants.Plant Cell,13(7):1499-1510
Bradford MM(1976).A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem,72(S1-2):248-254
Clough SJ,Bent AF(1998).Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J,16(6):735-743
Contento AL,Xiong Y,Bassham DC(2005).Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein.Plant J,42(4):598-608
Feng Y,He D,Yao Z,et al(2014).The machinery of macroautophagy.Cell Res,24(1):24-41
Good AG,Shrawat AK,Muench DG(2004).Can less yield more?Is reducing nutrient input into the environment compatible with maintaining crop production?Trends Plant Sci,9(12):597-605
Guiboileau A,Yoshimoto K,Soulay F,et al(2012).Autophagy machinery controls nitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis.New Phytol,194(3):732-740
Hanaoka H,Noda T,Shirano Y,et al(2002).Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene.Plant Physiol,129(3):1181-1193
Huang X,Li F(2016).Roles of autophagy in plant programmed cell death.Chin Bull Bot,51(6):859-862(in Chinese with English abstract)[黄晓,李发强(2016).细胞自噬在植物细胞程序性死亡中的作用.植物学报,51(6):859-862]
Izumi M,Hidema J,Wada S,et al(2015).Establishment of monitoring methods for autophagy in rice reveals autophagic recycling of chloroplasts and root plastids during energy limitation.Plant Physiol,167(4):1307-1320
Kichey T,Hirel B,Heumez E,et al(2007).In winter wheat(Triticum aestivum L.),post-anthesis nitrogen uptake and remobilisation to the grain correlates with agronomic traits and nitrogen physiological markers.Field Crop Res,102(1):22-32
Li F,Chung T,Pennington JG,et al(2015).Autophagic recycling plays a central role in maize nitrogen remobilization.Plant Cell,27(5):1389-1408
Li F,Vierstra RD(2012).Autophagy:a multifaceted intracellular system for bulk and selective recycling.Trends Plant Sci,17(9):526-537
Li W,Chen M,Wang E,et al(2016).Genome-wide analysis of autophagy-associated genes in foxtail millet(Setaria italica L.)and characterization of the function of SiAT-G8a in conferring tolerance to nitrogen starvation in rice.BMC Genom,17:797
Masclaux-Daubresse C,Chen Q,HavéM(2017).Regulation of nutrient recycling via autophagy.Curr Opin Plant Biol,39:8-17
Masclaux-Daubresse C,Daniel-Vedele F,Dechorgnat J,et al(2010).Nitrogen uptake,assimilation and remobilization in plants:challenges for sustainable and productive agriculture.Ann Bot,105(7):1141-1157
Masclaux-Daubresse C,Reisdorf-Cren M,Orsel M(2008).Leaf nitrogen remobilization for plant development and grain filling.Plant Biol,10(S1):23-36
Phillips AR,Suttangkakul A,Vierstra RD(2008).The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana.Genetics,178(3):1339-1353
Ren C,Gong Q(2014).Progress on the involvement of plant autophagy in carbon and nitrogen utilization.Chin J Cell Biol,36(4):407-414(in Chinese with English abstract)[任晨霞,龚清秋(2014).细胞自噬在植物碳氮营养中作用的研究进展.中国细胞生物学学报,36(4):407-414]
Rentsch D,Schmidt S,Tegeder M(2007).Transporters for uptake and allocation of organic nitrogen compounds in plants.FEBS Lett,581(12):2281-2289
SlávikováS,Shy G,Yao Y,et al(2005).The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants.J Exp Bot,56(421):2839-2849
Suttangkakul A,Li F,Chung T,et al(2011).The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis.Plant Cell,23(6):3761-3779
Thompson AR,Doelling JH,Suttangkakul A,et al(2005).Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways.Plant Physiol,138(4):2097-2110
Tsukada M,Ohsumi Y(1993).Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae.FEBS Lett,333(1-2):169-174
Wada S,Hayashida Y,Izumi M,et al(2015).Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice.Plant Physiol,168(1):60-73
Wang P,Sun X,Jia X,et al(2016).Characterization of an autophagy-related gene MdATG8i from apple.Front Plant Sci,7:720
Wu B,Cang J,Zhang D,et al(2015).Effects of SA treatment on winter wheat(Triticum aestivum L.)sucrose metabolism in low temperature.Plant Physiol J,51(4):537-545(in Chinese with English abstract)[吴冰,苍晶,张达等(2015).SA处理对低温胁迫下冬小麦(Triticum aestivum L.)蔗糖代谢的影响.植物生理学报,51(4):537-545]
Xia T,Xiao D,Liu D,et al(2012).Heterologous expression of ATG8c from soybean confers tolerance to nitrogen deficiency and increases yield in Arabidopsis.PLoS ONE,7(5):e37217
Xu F,Meng T,Li P,et al(2011).A soybean dual-specificity kinase,GmSARK,and its Arabidopsis homolog,AtSARK,regulate leaf senescence through synergistic actions of auxin and ethylene.Plant Physiol,157(4):2131-2153
Xu G,Fan X,Miller AJ(2012).Plant nitrogen assimilation and use efficiency.Ann Rev Plant Biol,63(1):153-182
Xu W,Liu S,You X,et al(2017).Overexpression of GmAT-G8c gene enhances tolerance to low nitrogen and increases yield in soybean.Plant Physiol J,53(2):241-247(in Chinese with English abstract)[徐伟,刘生,游翔等(2017).过表达GmATG8c基因提高大豆的低氮耐受性和产量.植物生理学报,53(2):241-247]
Yang X,Li Y,Liu Y,et al(2017).Review of selective autophagy in plant cell.Acta Hortic Sin,44(10):2015-2028(in Chinese with English abstract)[杨小龙,李漾漾,刘玉凤等(2017).植物细胞选择性自噬研究进展.园艺学报,44(10):2015-2028]
Yoshimoto K,Hanaoka H,Sato S,et al(2004).Processing of ATG8s,ubiquitin-like proteins,and their deconjugation by ATG4s are essential for plant autophagy.Plant Cell,16(11):2967-2983
Yoshimoto K,Jikumaru Y,Kamiya Y,et al(2009).Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis.Plant Cell,21(9):2914-2927
Yoshimoto K,Takano Y,Sakai Y(2010).Autophagy in plants and phytopathogens.FEBS Lett,584(7):1350-1358