毛竹SLAC家族基因鉴定及PheSLAC1功能分析

doi:10.16420/j.issn.0513-353x.2023-0098

摘要/Abstract

摘要：

为了研究S型阴离子通道蛋白（slow type anion channel，SLAC）基因家族在毛竹（Phyllostachys edulis）响应干旱胁迫中的作用，利用生物信息方法以拟南芥和水稻中的SLAC/SLAH家族蛋白序列为模板对毛竹中SLAC家族成员进行鉴定，对其基因结构、进化关系和组织表达等进行分析，并对PheSLAC1的保守功能位点以及在干旱胁迫中的功能进行初步研究。结果表明：毛竹中包含15个SLAC家族基因，其中PheSLAC1.1（PH02Gene01933.t1）和PheSLAC1.2（PH02Gene00592.t1）在叶片中表达量最高，基因编码区（coding sequence，CDS）序列长度分别为1 716和1 677 bp，分别编码572和559个氨基酸，且包含保守的关键功能位点。亚细胞定位结果表明，PheSLAC1.1和PheSLAC1.2均定位于细胞质膜中。PheSLAC1.1和PheSLAC1.2在拟南芥slac1-3突变体中的表达结果表明，其能够部分减轻突变体的干旱敏感表型，表明两者均能够通过调控气孔关闭在毛竹响应干旱胁迫中发挥重要作用。

关键词: 毛竹, SLAC基因家族, 干旱胁迫, 功能鉴定

Abstract:

To investigate the function of slow anion channel（SLAC）protein of Phyllostachys edulis in response to drought stress，genes encoding slow anion channel proteins in moso bamboo genome were identified by TBtools software based on SLAC/SLAH genes from Arabidopsis thaliana and Oryza sativa，respectively. Phylogenetic relationship，conserved region and motif，and gene expression pattern were investigated. Moreover，the function of PheSLAC1 in response to drought stress was verified by transgenic method. There were 15 members in SLAC family in bamboo，and the expression of PheSLAC1.1（PH02Gene01933.t1）and PheSLAC1.2（PH02Gene00592.t1）were the highest in leaves. The coding sequence（CDS）of PheSLAC1.1 and PheSLAC1.2 were 1 716 and 1 677 bp in length，encoding 572 and 559 amino acids，respectively. Subcellular localization exhibited that PheSLAC1.1 and PheSLAC1.2 were mainly located in cell membrane. Transgenic plants expressing PheSLAC1.1 and PheSLAC1.2 in slac1-3 mutants showed that PheSLAC1.1 and PheSLAC1.2 were able to restore the drought-sensitive phenotype of slac1-3. These results indicated that both PheSLAC1.1 and PheSLAC1.2 played important roles in response to drought stress by regulating stomatal closure.

Key words: Phyllostachys edulis, SLAC gene family, drought stress, functional identification

刘晋红, 王峥, 于昊, 辛依睿, 亓果宁, 柳参奎, 任慧敏. 毛竹SLAC家族基因鉴定及PheSLAC1功能分析[J]. 园艺学报, 2024, 51(3): 545-559.

LIU Jinhong, WANG Zheng, YU Hao, XIN Yirui, QI Guoning, LIU Shenkui, REN Huimin. Identification of SLAC Gene Family in Phyllostachys edulis and Characterization of PheSLAC1[J]. Acta Horticulturae Sinica, 2024, 51(3): 545-559.

图/表 11

参考文献 33

[34]	Sun S J, Qi G N, Gao Q F, Wang H Q, Yao F Y, Hussain J, Wang Y F. 2016. Protein kinase OsSAPK 8 functions as an essential activator of S-type anion channel OsSLAC1,which is nitrate-selective in rice. Planta, 243 (2):489-500.
[35]	Vahisalu T, Kollist H, Wang Y F, Nishimura N, Chan W Y, Valerio G, Lamminmaki A, Brosche M, Moldau H, Desikan R, Schroeder J I, Kangasjarvi J. 2008. SLAC 1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature, 452 (7186):487-491.
[36]	Vahisalu T, Puzorjova I, Brosche M, Valk E, Lepiku M, Moldau H, Pechter P, Wang Y S, Lindgren O, Salojarvi J, Loog M, Kangasjarvi J, Kollist H. 2010. Ozone-triggered rapid stomatal response involves the production of reactive oxygen species,and is controlled by SLAC1 and OST1. Plant J, 62 (3):442-453.
[37]	Wasilewska A, Vlad F, Sirichandra C, Redko Y, Jammes F, Valon C, Frei dit Frey N, Leung J. 2008. An update on abscisic acid signaling in plants and more. Mol Plant, 1 (2):198-217. doi: 10.1093/mp/ssm022 pmid: 19825533
[38]	Yamamoto Y, Negi J, Wang C, Isogai Y, Schroeder J I, Iba K. 2016. The transmembrane region of guard cell SLAC 1 channels perceives CO₂ signals via an ABA-independent pathway in Arabidopsis. Plant Cell, 28 (2):557-567.
[1]	Bailey T L, Boden M, Buske F A, Frith M, Grant C E, Clementi L, Li W W, Noble W S. 2009. MEME SUITE:tools for motif discovery and searching. Nucleic Acids Res,37 (Web Server issue):W202-W208.
[2]	Brandt B, Brodsky D E, Xue S, Negi J, Iba K, Kangasjarvi J, Ghassemian M, Stephan A B, Hu H, Schroeder J I. 2012. Reconstitution of abscisic acid activation of SLAC 1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proc Natl Acad Sci U S A, 109 (26):10593-10598.
[3]	Chen C J, Chen H, Zhang Y, Thomas H R, Frank M H, He Y H, Xia R. 2020. TBtools:an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13 (8):1194-1202.
[4]	Chen G D, Shi Y Y, Shen X, Zhang Y A, Lu X Y, Li Y, Jin C, Wang J Z, Wu J Y. 2022. Guard cell anion channel PbrSLAC1 regulates stomatal closure through PbrSnRK2.3 protein kinases. Plant Science,325:111487.
[5]	Chen Y H, Hu L, Punta M, Bruni R, Hillerich B, Kloss B, Rost B, Love J, Siegelbaum S A, Hendrickson W A. 2010. Homologue structure of the SLAC 1 anion channel for closing stomata in leaves. Nature, 467 (7319):1074-1080.
[6]	Cheng Mingsheng, Zou Huanhuan, Huang Xiaofeng, Chen Huijing, Zou Na, Yang Qingpei. 2021. Effects of drought and salt stress on growth and physiology of Phyllostachys edulis seedlings. Acta Agriculturae Universitatis Jiangxiensis, 43 (4):842-852. (in Chinese)
	程明圣, 邹欢欢, 黄孝风, 陈慧晶, 邹娜, 杨清培. 2021. 干旱与盐胁迫对毛竹幼苗生长及生理的影响. 江西农业大学学报, 43 (4):842-852.
[7]	Dreyer I, Gomez-Porras J L, Riano-Pachon D M, Hedrich R, Geiger D. 2012. Molecular evolution of slow and quick anion channels(SLACs and QUACs/ALMTs). Front Plant Sci,3:263.
[8]	Fan C J, Ma J M, Guo Q R, Li X T, Wang H, Lu M Z. 2013. Selection of reference genes for quantitative real-time PCR in bamboo(Phyllostachys edulis). PLoS ONE, 8 (2):e56573.
[9]	Geiger D, Scherzer S, Mumm P, Marten I, Ache P, Matschi S, Liese A, Wellmann C, Al-Rasheid K A, Grill E, Romeis T, Hedrich R. 2010. Guard cell anion channel SLAC 1 is regulated by CDPK protein kinases with distinct Ca²⁺ affinities. Proc Natl Acad Sci U S A, 107 (17):8023-8028.
[10]	Geiger D, Scherzer S, Mumm P, Stange A, Marten I, Bauer H, Ache P, Matschi S, Liese A, Al-Rasheid K A, Romeis T, Hedrich R. 2009. Activity of guard cell anion channel SLAC 1 is controlled by drought-stress signaling kinase-phosphatase pair. Proc Natl Acad Sci U S A, 106 (50):21425-21430.
[11]	Guzel Deger A, Scherzer S, Nuhkat M, Kedzierska J, Kollist H, Brosche M, Unyayar S, Boudsocq M, Hedrich R, Roelfsema M R. 2015. Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal closure. New Phytol, 208 (1):162-173. doi: 10.1111/nph.13435 pmid: 25932909
[12]	Hedrich R, Geiger D. 2017. Biology of SLAC1-type anion channels - from nutrient uptake to stomatal closure. New Phytol, 216 (1):46-61. doi: 10.1111/nph.14685 pmid: 28722226
[13]	Hinckley T, Duhme F, Hinckley A, Richter H. 1980. Water relations of drought hardy shrubs:osmotic potential and stomatal reactivity. Plant,Cell & Environment,3:131-140.
[14]	Keller B U, Hedrich R, Raschke K. 1989. Voltage-dependent anion channels in the plasma membrane of guard cells. Nature, 341 (6241):450-453.
[15]	Kim T H, Bohmer M, Hu H, Nishimura N, Schroeder J I. 2010. Guard cell signal transduction network:advances in understanding abscisic acid,CO₂,and Ca²⁺ signaling. Annu Rev Plant Biol,61:561-591.
[16]	Kumar S, Stecher G, Tamura K. 2016. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 33 (7):1870-1874.
[17]	Kusumi K, Hirotsuka S, Kumamaru T, Iba K. 2012. Increased leaf photosynthesis caused by elevated stomatal conductance in a rice mutant deficient in SLAC1,a guard cell anion channel protein. J Exp Bot, 63 (15):5635-5644.
[18]	Lee S C, Lan W, Buchanan B B, Luan S. 2009. A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells. Proc Natl Acad Sci U S A, 106 (50):21419-21424.
[19]	Li J L, Han L, Su Y H, Guo H E, Zhang H C. 2019. Functional identification of Ammopiptanthus mongolicus anion channel AmSLAC 1 involved in drought induced stomata closure. Plant Physiol Biochem,143:340-350.
[20]	Lind C, Dreyer I, Lopez-Sanjurjo E J, von Meyer K, Ishizaki K, Kohchi T, Lang D, Zhao Y, Kreuzer I, Al-Rasheid K A, Ronne H, Reski R, Zhu J K, Geiger D, Hedrich R. 2015. Stomatal guard cells co-opted an ancient ABA-dependent desiccation survival system to regulate stomatal closure. Curr Biol, 25 (7):928-935. doi: 10.1016/j.cub.2015.01.067 pmid: 25802151
[21]	Liu C G, Wang Y J, Jin Y Q, Pan K W, Zhou X M, Li N. 2017. Photoprotection regulated by phosphorus application can improve photosynthetic performance and alleviate oxidative damage in dwarf bamboo subjected to water stress. Plant Physiol Biochem,118:88-97.
[22]	Ma J F, Shen R F, Zhao Z Q, Wissuwa M, Takeuchi Y, Ebitani T, Yano M. 2002. Response of rice to Al stress and identification of quantitative trait loci for Al tolerance. Plant and Cell Physiology, 43 (6):652-659. doi: 10.1093/pcp/pcf081 pmid: 12091719
[23]	Maierhofer T, Diekmann M, Offenborn J N, Lind C, Bauer H, Hashimoto K, S Al-Rasheid KA, Luan S, Kudla J, Geiger D, Hedrich R. 2014. Site- and kinase-specific phosphorylation-mediated activation of SLAC1,a guard cell anion channel stimulated by abscisic acid. Sci Signal, 7 (342):ra86.
[24]	Muller H M, Schafer N, Bauer H, Geiger D, Lautner S, Fromm J, Riederer M, Bueno A, Nussbaumer T, Mayer K, Alquraishi S A, Alfarhan A H, Neher E, Al-Rasheid K A S, Ache P, Hedrich R. 2017. The desert plant Phoenix dactylifera closes stomata via nitrate-regulated SLAC 1 anion channel. New Phytol, 216 (1):150-162.
[25]	Negi J, Matsuda O, Nagasawa T, Oba Y, Takahashi H, Kawai-Yamada M, Uchimiya H, Hashimoto M, Iba K. 2008. CO₂ regulator SLAC1 and its homologues are essential for anion homeostasis in plant cells. Nature, 452 (7186):483-486.
[39]	Ying Yeqing, Guo Jing, Wei Jianfen, Jiang Qin, Xie Nannan. 2011. Effects of drought stress on physiological characteristics of Phyllostachys edulis seedlings. Chinese Journal of Ecology, 30 (2):262-266. (in Chinese)
	应叶青, 郭璟, 魏建芬, 姜琴, 解楠楠. 2011. 干旱胁迫对毛竹幼苗生理特性的影响. 生态学杂志, 30 (2):262-266.
[40]	Zhao H S, Peng Z H, Fei B H, Li L B, Hu T, Gao Z M, Jiang Z H. 2014. BambooGDB:a bamboo genome database with functional annotation and an analysis platform. Database (Oxford):bau006.
[26]	Pardo J M. 2010. Biotechnology of water and salinity stress tolerance. Curr Opin Biotechnol, 21 (2):185-196.
[27]	Park S Y, Fung P, Nishimura N, Jensen D R, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow T F, Alfred S E, Bonetta D, Finkelstein R, Provart N J, Desveaux D, Rodriguez P L, McCourt P, Zhu J K, Schroeder J I, Volkman B F, Cutler S R. 2009. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science, 324 (5930):1068-1071.
[28]	Qi G N, Yao F Y, Ren H M, Sun S J, Tan Y Q, Zhang Z C, Qiu B S, Wang Y F. 2018. The S-type anion channel ZmSLAC 1 plays essential roles in stomatal closure by mediating nitrate efflux in maize. Plant Cell Physiol, 59 (3):614-623.
[29]	Ren H M, Su Q S, Hussain J, Tang S W, Song W, Sun Y Q, Liu H F, Qi G N. 2021. Slow anion channel GhSLAC1 is essential for stomatal closure in response to drought stress in cotton. J Plant Physiol,258-259:153360.
[30]	Roelfsema M R, Hedrich R. 2005. In the light of stomatal opening:new insights into‘the Watergate’. New Phytol, 167 (3):665-691. pmid: 16101906
[31]	Schmidt C, Schroeder J I. 1994. Anion selectivity of slow anion channels in the plasma membrane of guard cells(large nitrate permeability). Plant Physiol, 106 (1):383-391. pmid: 12232336
[32]	Shi K, Li X, Zhang H, Zhang G Q, Liu Y, Zhou Y H, Xia X J, Chen Z X, Yu J Q. 2015. Guard cell hydrogen peroxide and nitric oxide mediate elevated CO₂ -induced stomatal movement in tomato. New Phytol, 208 (2):342-353.
[33]	Song Q N, Lu H, Liu J, Yang J, Yang G Y, Yang Q P. 2017. Accessing the impacts of bamboo expansion on NPP and N cycling in evergreen broadleaved forest in subtropical China. Sci Rep,7:40383.

引物用途 Primer use	引物名称 Primer name	引物序列（5′-3′） Primer sequence
实时荧光定量引物 qRT-PCR primer	NTB	F：TCTTGTTTGACACCGAAGAGGAG；	R：AATAGCTGTCCCTGGAGGAGTTT
	PheSLAC1.1	F：FTTCTTCATGGCGCTCTTCTT；	R：GACATGAGGGAGAGGCTCAG
	PheSLAC1.2	F：GCGCGGACATTATTCTTCAT；	R：GACATGAGGGAGAGGCTCAG
	PH02Gene35134	F：ATCTACGGCCAGTGGTTCAC；	R：TGACGAACAGCACGAGGTAG
	PH02Gene08796	F：CATGAACTACCTCTTCGCGC；	R：GAACCACTGGCCGTAGATCT
	PH02Gene45054	F：ACTACAGCGTGCTGTTCGTG；	R：GAACCCACGGAAGAAGTTGA
	PH02Gene22907	F：TGTTCGTGACGCTCTACCAG；	R：TGAACCCCCTGAAAAAGTTG
	PH02Gene22131	F：CCAGAGTCCAACGCTTCTTC；	R：ATCTTGAGCGTGATGTGCAG
	PH02Gene10869	F：AGCTAAGGTCGGAGGAGGAG；	R：ATCTTGAGCGTGATGTGCAG
	PH02Gene20948	F：ATCTACGGGCAGTGGATGTC；	R：TGGTACAGCGTGACGAAGAG
	PH02Gene48421	F：GCCTCCCTGAATGAGCATAA；	R：ATGCTGAGCGAAAGGGAGTA
	PH02Gene25481	F：GTGGATGAACAGGGGAAAGA；	R：GGACCTCGAGTTTTGTCTCG
	PH02Gene41494	F：GGAAGGACGTCGTGATTAGC；	R：GGACCTCGAGTTTTGTCTCG
	PH02Gene06334	F：CTGTCCGCCGAGAGTTTTAC；	R：GAGCTCAAGGCAGAAGATGG
	PH02Gene06335	F：GGCTTCGTCTCGGTGATCTA；	R：CATCCACTGCCCGTAGATCT
	PH02Gene20553	F：ATCTACGGGCAGTGGATGTC；	R：TGACGAACAGCACGAGGTAG
	AtACTIN2	F：GGTAACATTGTGCTCAGTGGTGG；	R：AACGACCTTAATCTTCATGCTGC
转基因和亚细胞定位 Transgene and subcellular localization	PheSLAC1.1-1304	F： GAAGATCTTATGGCAGCCGAGCCATCGTC R： CGGACTAGTGTCTGTTTTCTCCTCTTCGTCCTT
	PheSLAC1.2-1304	F： GAAGATCTTATGGCAGCCGAGCCATCGTC R： CGGACTAGTTTCTGTTTCCTCCTCTTCATCCTTG
	PheSLAC1.1-GFP	F： TCCCCCCGGGATGGCAGCCGAGCCATCGTCT R： CTAGTCTAGAGTCTGTTTTCTCCTCTTCGTCCTT
	PheSLAC1.2-GFP	F： TCCCCCCGGGATGGCAGCCGAGCCATCGTC R： CTAGTCT AGATTCTGTTTCCTCCTCTTCATCCT

引物用途 Primer use	引物名称 Primer name	引物序列（5′-3′） Primer sequence
实时荧光定量引物 qRT-PCR primer	NTB	F：TCTTGTTTGACACCGAAGAGGAG；	R：AATAGCTGTCCCTGGAGGAGTTT
	PheSLAC1.1	F：FTTCTTCATGGCGCTCTTCTT；	R：GACATGAGGGAGAGGCTCAG
	PheSLAC1.2	F：GCGCGGACATTATTCTTCAT；	R：GACATGAGGGAGAGGCTCAG
	PH02Gene35134	F：ATCTACGGCCAGTGGTTCAC；	R：TGACGAACAGCACGAGGTAG
	PH02Gene08796	F：CATGAACTACCTCTTCGCGC；	R：GAACCACTGGCCGTAGATCT
	PH02Gene45054	F：ACTACAGCGTGCTGTTCGTG；	R：GAACCCACGGAAGAAGTTGA
	PH02Gene22907	F：TGTTCGTGACGCTCTACCAG；	R：TGAACCCCCTGAAAAAGTTG
	PH02Gene22131	F：CCAGAGTCCAACGCTTCTTC；	R：ATCTTGAGCGTGATGTGCAG
	PH02Gene10869	F：AGCTAAGGTCGGAGGAGGAG；	R：ATCTTGAGCGTGATGTGCAG
	PH02Gene20948	F：ATCTACGGGCAGTGGATGTC；	R：TGGTACAGCGTGACGAAGAG
	PH02Gene48421	F：GCCTCCCTGAATGAGCATAA；	R：ATGCTGAGCGAAAGGGAGTA
	PH02Gene25481	F：GTGGATGAACAGGGGAAAGA；	R：GGACCTCGAGTTTTGTCTCG
	PH02Gene41494	F：GGAAGGACGTCGTGATTAGC；	R：GGACCTCGAGTTTTGTCTCG
	PH02Gene06334	F：CTGTCCGCCGAGAGTTTTAC；	R：GAGCTCAAGGCAGAAGATGG
	PH02Gene06335	F：GGCTTCGTCTCGGTGATCTA；	R：CATCCACTGCCCGTAGATCT
	PH02Gene20553	F：ATCTACGGGCAGTGGATGTC；	R：TGACGAACAGCACGAGGTAG
	AtACTIN2	F：GGTAACATTGTGCTCAGTGGTGG；	R：AACGACCTTAATCTTCATGCTGC
转基因和亚细胞定位 Transgene and subcellular localization	PheSLAC1.1-1304	F： GAAGATCTTATGGCAGCCGAGCCATCGTC R： CGGACTAGTGTCTGTTTTCTCCTCTTCGTCCTT
	PheSLAC1.2-1304	F： GAAGATCTTATGGCAGCCGAGCCATCGTC R： CGGACTAGTTTCTGTTTCCTCCTCTTCATCCTTG
	PheSLAC1.1-GFP	F： TCCCCCCGGGATGGCAGCCGAGCCATCGTCT R： CTAGTCTAGAGTCTGTTTTCTCCTCTTCGTCCTT
	PheSLAC1.2-GFP	F： TCCCCCCGGGATGGCAGCCGAGCCATCGTC R： CTAGTCT AGATTCTGTTTCCTCCTCTTCATCCT

种类 Species	蒸腾速率/（mmol · m^-2 · s^-1） Transpiration rate	气孔导度/（mmol · m^-2 · s^-1） Stomatal conductance
毛竹 Phyllostachys edulis	0.938	0.058
菲黄竹 Pleioblastus viridistriatus	1.198	0.073
绿槽罗汉竹 Phyllostachys aureosulcata	0.592	0.037
唐竹 Sinobambusa tootsik	0.482	0.029
紫竹 Phyllostachys nigra	0.641	0.040
橄榄竹 Indosasa gigantea	0.655	0.048
凤尾竹 Bambusa multiplex	1.562	0.101

种类 Species	蒸腾速率/（mmol · m^-2 · s^-1） Transpiration rate	气孔导度/（mmol · m^-2 · s^-1） Stomatal conductance
毛竹 Phyllostachys edulis	0.938	0.058
菲黄竹 Pleioblastus viridistriatus	1.198	0.073
绿槽罗汉竹 Phyllostachys aureosulcata	0.592	0.037
唐竹 Sinobambusa tootsik	0.482	0.029
紫竹 Phyllostachys nigra	0.641	0.040
橄榄竹 Indosasa gigantea	0.655	0.048
凤尾竹 Bambusa multiplex	1.562	0.101

基因登录号 ID	染色体位置 Chromosome location	等电点 pI	相对分子量/kD MW	长度/aa Length	跨膜区数量 No. of transmembrane domain
PH02Gene00592.t1	hic_scaffold_24	9.63	61 635.52	558	9
PH02Gene01933.t1	hic_scaffold_23	9.94	62 657.88	571	10
PH02Gene35134.t1	hic_scaffold_16	10.18	33 127.26	294	9
PH02Gene08796.t1	hic_scaffold_6	9.87	42 370.12	384	10
PH02Gene45054.t2	hic_scaffold_9	9.13	64 409.35	586	10
PH02Gene22907.t1	hic_scaffold_18	8.52	67 869.30	621	10
PH02Gene22131.t1	hic_scaffold_16	9.26	64 301.36	583	10
PH02Gene10869.t1	hic_scaffold_14	9.18	67 749.68	609	10
PH02Gene20948.t2	hic_scaffold_16	8.82	72 183.69	651	10
PH02Gene48421.t1	hic_scaffold_14	8.70	68 667.45	613	10
PH02Gene25481.t1	hic_scaffold_342	8.02	73 583.66	663	9
PH02Gene41494.t1	hic_scaffold_9	7.71	69 005.90	623	9
PH02Gene06334.t1	hic_scaffold_7	7.03	70 670.36	640	8
PH02Gene06335.t1	hic_scaffold_7	7.30	70 425.26	640	10
PH02Gene20553.t1	hic_scaffold_13	7.09	77 827.50	708	9