杨树BAG基因的鉴定及表达模式分析
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摘要
【目的】杨树是我国人工林的重要组成部分,也是公认的模式木本植物。植物BAG蛋白作为保守的分子伴侣,在生长发育和抗逆性中起到关键作用。研究杨树BAG蛋白家族的进化与表达模式,鉴定BAG基因的生物学功能对于林木遗传改良具有重要的指导意义。【方法】以拟南芥7个BAG蛋白为诱饵,搜索毛果杨、水稻和小立碗藓在线数据库(https://phytozome.jgi.doe.gov)获得这3个物种同源的BAG蛋白。利用生物信息学构建毛果杨、拟南芥、水稻和小立碗藓BAG蛋白的系统进化树,分析这些蛋白的生化特性及进化关系。结合杨树芯片(http://bar.utoronto.ca)数据,利用qRT-PCR检测杨树BAG基因的组织和逆境(旱和热胁迫)表达模式。【结果】毛果杨包含14个BAG基因,按照国际命名法(以染色体位置进行排序),将其命名为PtrBAG1—PtrBAG14。系统进化树表明杨树、拟南芥、水稻和小立碗藓BAG蛋白相对保守,大体可以分为2个亚家族。第1个亚家族中,大部分BAG蛋白N端含有UBL结构域,可能作为分子桥梁参与降解某些蛋白;第2个亚家族的大部分BAG蛋白含有IQ结构域,意味着这些蛋白可能与Ca~(2+)信号相关。杨树BAG基因在不同逆境处理(热胁迫和干旱胁迫)条件下有不同的表达模式。热处理诱导所有BAGs基因表达发生变化,其中BAG1、BAG4、BAG6、BAG7和BAG8表达被激活,而另9个BAG基因的表达被抑制;特别是,热诱导BAG4和BAG6表达上调超过90倍。干旱胁迫条件下,大部分BAG表达变化幅度很小。组织表达模式分析表明,大部分杨树BAG基因在根、叶、芽、小苗、雌花序、雄花序和木质部中表达量很低,只有BAG2和BAG12在木质部中大量表达。【结论】杨树BAG家族共有14个成员,大体分为2个亚家族,在进化上与本文选用的陆生植物拟南芥和水稻BAG蛋白有更高的相似性。杨树14个BAG基因可能都参与了热胁迫调控,但不同成员在抗热过程中起不同作用。相比而言,大部分杨树BAG基因可能并不参与抗旱。值得指出的是,杨树BAG4和BAG6可能在热胁迫中作用最明显,而BAG2和BAG12可能参与木材形成。
【Objective】 Poplar(Populus) is widely planted in China and usually used as a model tree. As conserved molecular chaperon, plant BAG proteins play critical roles in growth, development and stress responses. Study on the evolution and biological functions of Populus BAG genes is useful for genetic improvement of tree species.【Method】 Arabidopsis BAG proteins were used as keywords to search online database(https://phytozome.jgi.doe.gov/) in order to identify the homologous BAGs from poplar(Populus trichocarpa), rice(Oryza sativa) and moss(Physcomitrella patens). Protein structures and evolutionary relationship of these BAG proteins were analyzed using the bioinformatic method. Tissue expression patterns and stress(drought and heat) responses of Populus BAG genes were detected based on the microarray(http://bar.utoronto.ca/) and qRT-PCR data.【Result】 P. trichocarpa contains fourteen BAG genes, respectively named as PtrBAG1 to PtrBAG14 according to the international rules for nomenclature(ordered on their positions on the chromosome). BAG proteins are relatively conserved among poplar, Arabidopsis, rice and moss. These Populus BAG proteins are divided into two subfamilies. The members in subfamily Ⅰ contains an UBL domain, suggesting that these proteins might function as bridges to assist the degradation of some proteins. The members in subfamily Ⅱ have an IQ domain, implying the potential roles of these proteins in Ca~(2+) signaling. Poplar BAG genes exhibited different stress(drought and heat) responses. For example, heat treatment resulted in visible changes in BAG expression. Of them, BAG1, BAG4, BAG6, BAG7 and BAG8 were activated whereas others were suppressed by heat stress. By contrast, there were few alterations in the expression of most BAG genes under the treatment of drought. Particularly, heat treatment induced up regulated expression of BAG4 and BAG6 by 90 folds. Tissue expression analysis indicated that most of BAGs had little expression in detected tissues(root, leaves, shoot, seedling, female catkins, male catkins and xylem). However, BAG2 and BAG12 were highly expressed in xylem.【Conclusion】 Poplar BAG family contains 14 members, which are basically divided into two subfamilies. These BAG proteins are closer to BAG homologs from Arabidopsis and rice than those from moss. All poplar BAG genes are responsive to heat stress but not to drought stress. In particular, BAG4 and BAG6 are most highly induced by heat treatment. BAG2 and BAG12 are likely involved in wood formation.
【Objective】 Poplar(Populus) is widely planted in China and usually used as a model tree. As conserved molecular chaperon, plant BAG proteins play critical roles in growth, development and stress responses. Study on the evolution and biological functions of Populus BAG genes is useful for genetic improvement of tree species.【Method】 Arabidopsis BAG proteins were used as keywords to search online database(https://phytozome.jgi.doe.gov/) in order to identify the homologous BAGs from poplar(Populus trichocarpa), rice(Oryza sativa) and moss(Physcomitrella patens). Protein structures and evolutionary relationship of these BAG proteins were analyzed using the bioinformatic method. Tissue expression patterns and stress(drought and heat) responses of Populus BAG genes were detected based on the microarray(http://bar.utoronto.ca/) and qRT-PCR data.【Result】 P. trichocarpa contains fourteen BAG genes, respectively named as PtrBAG1 to PtrBAG14 according to the international rules for nomenclature(ordered on their positions on the chromosome). BAG proteins are relatively conserved among poplar, Arabidopsis, rice and moss. These Populus BAG proteins are divided into two subfamilies. The members in subfamily Ⅰ contains an UBL domain, suggesting that these proteins might function as bridges to assist the degradation of some proteins. The members in subfamily Ⅱ have an IQ domain, implying the potential roles of these proteins in Ca~(2+) signaling. Poplar BAG genes exhibited different stress(drought and heat) responses. For example, heat treatment resulted in visible changes in BAG expression. Of them, BAG1, BAG4, BAG6, BAG7 and BAG8 were activated whereas others were suppressed by heat stress. By contrast, there were few alterations in the expression of most BAG genes under the treatment of drought. Particularly, heat treatment induced up regulated expression of BAG4 and BAG6 by 90 folds. Tissue expression analysis indicated that most of BAGs had little expression in detected tissues(root, leaves, shoot, seedling, female catkins, male catkins and xylem). However, BAG2 and BAG12 were highly expressed in xylem.【Conclusion】 Poplar BAG family contains 14 members, which are basically divided into two subfamilies. These BAG proteins are closer to BAG homologs from Arabidopsis and rice than those from moss. All poplar BAG genes are responsive to heat stress but not to drought stress. In particular, BAG4 and BAG6 are most highly induced by heat treatment. BAG2 and BAG12 are likely involved in wood formation.
引文
尹吴, 孙伟博, 周燕, 等. 2017. 毛果杨Rubisco活化酶基因的克隆与功能分析. 林业科学,53(4): 83-95.(Yin W, Sun W B, Zhou Y, et al. 2017. Cloning and functional analysis of Rubisco activase gene from Populus trichocarpa. Scientia Silvae Sinicae, 53(4): 83-95. [in Chinese])
张进, 李建波, 刘伯斌, 等. 2013. 杨树Hsp90基因家族亚细胞定位及胁迫响应分析. 中国林学会林木遗传育种分会第七届全国林木遗传育种学术大会.(Zhang J, Li J B, Liu B B, et al. 2013. Subcellular localization and stress response analysis of poplar Hsp90 gene family. National Tree Genetics and Breeding Academic Conference, Tree Genetics and Breeding Branch, Chinese Society of Forestry. [in Chinese])
Brive L, Takayama S, Briknarova K, et al. 2001. The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1. Biochemical and Biophysical Research Communications, 289 (5): 1099-1105.
Chai G H, Qi G, Cao Y P, et al. 2014. Poplar PdC3H17 and PdC3H18 are direct targets of PdMYB3 and PdMYB21, and positively regulate secondary wall formation in Arabidopsis and poplar. New Phytologist, 203(2): 520-534.
Cheung J, Smith D F. 2000. Molecular chaperone interactions with steroid receptors: an update. Molecular Endocrinology, 14 (7): 939-946.
Doukhanina E V, Chen S, van der Zalm E, et al. 2006. Identification and functional characterization of the BAG protein family in Arabidopsis thaliana. Journal of Biological Chemistry, 281 (27): 18793-18801.
Frydman J. 2001. Folding of newly translated proteins in vivo: The role of molecular chaperones. Annual Review of Biochemistry, 70: 603-647.
Gonzalez-Arzola K, Moreno-Beltran B, Martinez-Fabregas J, et al. 2015. A common role for cytochrome c in programmed cell death in humans and plants. Protein Science, 24 (1): 259-260.
Kang C H, Jung W Y, Kang Y H, et al. 2006. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants. Cell Death and Differentiation, 13 (1): 84-95.
Kobayashi M, Takato H, Fujita K, et al. 2012. HSG1, a grape Bcl-2-associated athanogene, promotes floral transition by activating CONSTANS expression in transgenic Arabidopsis plant. Molecular Biology Reports, 39 (4): 4367-4374.
Li L, Xing Y, Chang D, et al. 2016a. CaM/BAG5/Hsc70 signaling complex dynamically regulates leaf senescence. Scientific Reports, 6:31889.
Li Y, Kabbage M, Liu W, et al. 2016b. Aspartyl protease-mediated cleavage of BAG6 is necessary for autophagy and fungal resistance in plants. Plant Cell, 28 (1): 233-247.
Li Y R, Williams B, Dickman M. 2017. Arabidopsis B-cell lymphoma2 (Bcl-2)-associated athanogene 7 (BAG7)-mediated heat tolerance requires translocation, sumoylation and binding to WRKY29. New Phytologist, 214 (2): 695-705.
Payapilly A, High S. 2014. BAG6 regulates the quality control of a polytopic ERAD substrate. Journal of Cell Science, 127 (13): 2898-2909.
Schmid M, Davison T S, Henz S R. 2005. A gene expression map of Arabidopsis thaliana development. Nature Genetics, 37 (5): 501-506.
Sturn A, Quackenbush J, Trajanoski Z. 2002. Genesis: cluster analysis of microarray data. Bioinformatics, 18 (1): 207-208.
Tamura K, Peterson D, Peterson N, et al. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28 (10): 2731-2739.
Wilkins O, Nahal H, Foong J, et al. 2008. Expansion and diversification of the Populus R2R3-MYB family of transcription factors. Plant Physiol, 149 (2): 981-993
Wilkins O, Waldron L, Nahal H, et al. 2009. Genotype and time of day shape the Populus drought response. Plant Journal, 60 (4): 703-715.
Williams B, Kabbage M, Britt R, et al. 2010. AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response. Proceedings of the National Academy of Sciences of the United States of America, 107 (13): 6088-6093.
Yan J Q, He C X, Zhang H. 2003. The BAG-family proteins in Arabidopsis thaliana. Plant Science, 165 (1): 1-7.
You Q, Zhai K, Yang D, et al. 2016. An E3 ubiquitin ligase-BAG protein module controls plant innate immunity and broad-spectrum disease resistance. Cell Host Microbe, 20 (6): 758-769.
张进, 李建波, 刘伯斌, 等. 2013. 杨树Hsp90基因家族亚细胞定位及胁迫响应分析. 中国林学会林木遗传育种分会第七届全国林木遗传育种学术大会.(Zhang J, Li J B, Liu B B, et al. 2013. Subcellular localization and stress response analysis of poplar Hsp90 gene family. National Tree Genetics and Breeding Academic Conference, Tree Genetics and Breeding Branch, Chinese Society of Forestry. [in Chinese])
Brive L, Takayama S, Briknarova K, et al. 2001. The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1. Biochemical and Biophysical Research Communications, 289 (5): 1099-1105.
Chai G H, Qi G, Cao Y P, et al. 2014. Poplar PdC3H17 and PdC3H18 are direct targets of PdMYB3 and PdMYB21, and positively regulate secondary wall formation in Arabidopsis and poplar. New Phytologist, 203(2): 520-534.
Cheung J, Smith D F. 2000. Molecular chaperone interactions with steroid receptors: an update. Molecular Endocrinology, 14 (7): 939-946.
Doukhanina E V, Chen S, van der Zalm E, et al. 2006. Identification and functional characterization of the BAG protein family in Arabidopsis thaliana. Journal of Biological Chemistry, 281 (27): 18793-18801.
Frydman J. 2001. Folding of newly translated proteins in vivo: The role of molecular chaperones. Annual Review of Biochemistry, 70: 603-647.
Gonzalez-Arzola K, Moreno-Beltran B, Martinez-Fabregas J, et al. 2015. A common role for cytochrome c in programmed cell death in humans and plants. Protein Science, 24 (1): 259-260.
Kang C H, Jung W Y, Kang Y H, et al. 2006. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants. Cell Death and Differentiation, 13 (1): 84-95.
Kobayashi M, Takato H, Fujita K, et al. 2012. HSG1, a grape Bcl-2-associated athanogene, promotes floral transition by activating CONSTANS expression in transgenic Arabidopsis plant. Molecular Biology Reports, 39 (4): 4367-4374.
Li L, Xing Y, Chang D, et al. 2016a. CaM/BAG5/Hsc70 signaling complex dynamically regulates leaf senescence. Scientific Reports, 6:31889.
Li Y, Kabbage M, Liu W, et al. 2016b. Aspartyl protease-mediated cleavage of BAG6 is necessary for autophagy and fungal resistance in plants. Plant Cell, 28 (1): 233-247.
Li Y R, Williams B, Dickman M. 2017. Arabidopsis B-cell lymphoma2 (Bcl-2)-associated athanogene 7 (BAG7)-mediated heat tolerance requires translocation, sumoylation and binding to WRKY29. New Phytologist, 214 (2): 695-705.
Payapilly A, High S. 2014. BAG6 regulates the quality control of a polytopic ERAD substrate. Journal of Cell Science, 127 (13): 2898-2909.
Schmid M, Davison T S, Henz S R. 2005. A gene expression map of Arabidopsis thaliana development. Nature Genetics, 37 (5): 501-506.
Sturn A, Quackenbush J, Trajanoski Z. 2002. Genesis: cluster analysis of microarray data. Bioinformatics, 18 (1): 207-208.
Tamura K, Peterson D, Peterson N, et al. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28 (10): 2731-2739.
Wilkins O, Nahal H, Foong J, et al. 2008. Expansion and diversification of the Populus R2R3-MYB family of transcription factors. Plant Physiol, 149 (2): 981-993
Wilkins O, Waldron L, Nahal H, et al. 2009. Genotype and time of day shape the Populus drought response. Plant Journal, 60 (4): 703-715.
Williams B, Kabbage M, Britt R, et al. 2010. AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response. Proceedings of the National Academy of Sciences of the United States of America, 107 (13): 6088-6093.
Yan J Q, He C X, Zhang H. 2003. The BAG-family proteins in Arabidopsis thaliana. Plant Science, 165 (1): 1-7.
You Q, Zhai K, Yang D, et al. 2016. An E3 ubiquitin ligase-BAG protein module controls plant innate immunity and broad-spectrum disease resistance. Cell Host Microbe, 20 (6): 758-769.