拟南芥CPK6在钙离子信号转导过程中的生理功能研究
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摘要
为了研究拟南芥CPK6基因在钙离子信号转导过程中的生理功能,进一步明确植物适应缺钙环境的分子机制,首先对CPK6基因缺失突变体(cpk6)进行纯合鉴定,获得了2种纯合突变体:cpk6-2和cpk6-3。通过分析野生型拟南芥Col-0和cpk6突变体在Ca~(2+)、Fe~(2+)、Mg~(2+)、Zn~(2+)、Mn~(2+)二价阳离子缺失培养基上的生长表型,结果发现,拟南芥cpk6突变体在缺Ca~(2+)条件下,相对野生型Col-0,表现出生长受抑制的表型。构建含有CPK6启动子驱动β-葡萄糖苷酸酶基因(GUS)的转基因材料,通过GUS组织化学染色法,发现CPK6启动子在叶片生长点部位和根上有活性,并且在缺Ca~(2+)条件下,CPK6启动子在叶片中的活性明显增强。通过原子吸收光谱法,发现拟南芥野生型Col-0和cpk6突变体之间,在对照和缺Ca~(2+)条件下,总钙含量没有显著差别,表明CPK6基因不影响植物对钙的吸收和积累。利用组织化学染色法和荧光探针,结果发现,缺Ca~(2+)条件下,cpk6突变体根和叶中H2O2的积累量明显高于Col-0,表明CPK6是介导缺Ca~(2+)诱导植物积累H2O2的负调控因子。
The objective was to research the physiological function of CPK6 in Ca~(2+)signal transduction and to reveal the mechanism of plants to adapt to Ca~(2+)deficient environment. By homozygous identification of the CPK6 deletion mutant( cpk6),two homozygous mutants,cpk6-2 and cpk6-3 were obtained. And the growth phenotype of wild-type Arabidopsis Col-0 and cpk6 mutants in the deficiency of Ca~(2+),Fe~(2+),Mg~(2+),Zn~(2+)and Mn~(2+)were analyzed.The results showed that the mutants showed a growth-inhibiting phenotype relative to wild-type Col-0 in the deficiency of Ca~(2+). The Arabidopsis transgenic material expressing CPK6 promoter-driven β-glucuronidase were obtained. Using GUS histochemical staining,it was found that CPK6 promoter had activity in the roots and the growth point of the leaves. And the activity of CPK6 promoter in leaves was visibly enhanced under Ca~(2+)deficient condition. The total Ca content of Col-0 and cpk6 was determined by atomic absorption spectrometry( AAS). It was found that there was no significant difference in Ca content between Col-0 and cpk6 under control and Ca~(2+)deficiency conditions. The results indicated that CPK6 gene did not participate in the absorption and accumulation of Ca in Arabidopsis. Using histochemical staining and fluorescent probes methods,it was found that the accumulation of H2 O2 in the leaves and roots of cpk6 was visibly higher than that in Col-0 under Ca~(2+)deficient condition. The results showed that CPK6 was a negative regulator of the accumulation of H2 O2 induced by Ca~(2+)deficiency in plants.
The objective was to research the physiological function of CPK6 in Ca~(2+)signal transduction and to reveal the mechanism of plants to adapt to Ca~(2+)deficient environment. By homozygous identification of the CPK6 deletion mutant( cpk6),two homozygous mutants,cpk6-2 and cpk6-3 were obtained. And the growth phenotype of wild-type Arabidopsis Col-0 and cpk6 mutants in the deficiency of Ca~(2+),Fe~(2+),Mg~(2+),Zn~(2+)and Mn~(2+)were analyzed.The results showed that the mutants showed a growth-inhibiting phenotype relative to wild-type Col-0 in the deficiency of Ca~(2+). The Arabidopsis transgenic material expressing CPK6 promoter-driven β-glucuronidase were obtained. Using GUS histochemical staining,it was found that CPK6 promoter had activity in the roots and the growth point of the leaves. And the activity of CPK6 promoter in leaves was visibly enhanced under Ca~(2+)deficient condition. The total Ca content of Col-0 and cpk6 was determined by atomic absorption spectrometry( AAS). It was found that there was no significant difference in Ca content between Col-0 and cpk6 under control and Ca~(2+)deficiency conditions. The results indicated that CPK6 gene did not participate in the absorption and accumulation of Ca in Arabidopsis. Using histochemical staining and fluorescent probes methods,it was found that the accumulation of H2 O2 in the leaves and roots of cpk6 was visibly higher than that in Col-0 under Ca~(2+)deficient condition. The results showed that CPK6 was a negative regulator of the accumulation of H2 O2 induced by Ca~(2+)deficiency in plants.
引文
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[24]张小莉,王鹏程,宋纯鹏.植物细胞过氧化氢的测定方法[J].植物学报,2009,44(1):103-106.
[25] Munemasa S,Hossain M A,Nakamura Y,et al. The Arabidopsis calcium-dependent protein kinase,CPK6,functions as a positive regulator of methyl jasmonate signaling in guard cells[J]. Plant Physiology,2011,155(1):553-561.
[2] Aldon D,Mbengue M,Mazars C,et al. Calcium signalling in plant biotic interactions[J]. International Journal of Molecular Sciences,2018,19(3):1-19.
[3] Harmon A C,Gribskov M,Harper J F. CDPKs-a kinase for every Ca2+signal[J]. Trends in Plant Science,2000,5(4):154-159.
[4] Boudsocq M,Sheen J. CDPKs in immune and stress signaling[J]. Trends in Plant Science,2013,18(1):30-40.
[5]王娇娇,韩胜芳,李小娟,等.钙依赖蛋白激酶(CDPKs)介导植物信号转导的分子基础[J].草业学报,2009,18(3):241-250.
[6] Laanemets K,Brandt B,Li J,et al. Calcium-dependent and independent stomatal signaling network and compensatory feedback control of stomatal opening via Ca2+sensitivity priming[J]. Plant Physiology,2013,163(2):504-513.
[7] Mori I C,Murata Y,Yang Y,et al. CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion-and Ca2+-permeable channels and stomatal closure[J]. Plo S Biology,2006,4(10):1749-1762.
[8] Poovaiah B W,Du L,Wang H,et al. Recent advances in calcium/calmodulin-mediated signaling with an emphasis on plant-microbe interactions[J]. Plant Physiology,2013,163(2):531-542.
[9] Van Loon L C. The intelligent behavior of plants[J].Trends in Plant Science,2015,21(4):286-294.
[10] Kissoudis C,Wiel C V D,Visser R G,et al. Futureproof crops:challenges and strategies for climate resilience improvement[J]. Current Opinion in Plant Biology,2016,30:47-56.
[11] Boudsocq M,Sheen J. CDPKs in immune and stress signaling[J]. Trends in Plant Science,2013,18(1):30-40.
[12] Poovaiah B W,Du L,Wang H,et al. Recent advances in calcium/calmodulin-mediated signaling with an emphasis on plant-microbe interactions[J]. Plant Physiology,2013,163(2):531-542.
[13] Schulz P,Herde M,Romeis T. Calcium-dependent protein kinases:hubs in plant stress signaling and development[J]. Plant Physiology,2013,163(2):523-530.
[14] Zipfel C,Oldroyd G E. Plant signalling in symbiosis and immunity[J]. Nature,2017,543(7645):328-336.
[15]潘孝武,黎用朝,李小湘,等.非生物胁迫条件下植物H2O2的代谢及信号转导[J].中国农业科技导报,2010,12(2):38-43.
[16] Keller T,Damude H G,Werner D,et al. A plant homolog of the neutrophil NADPH oxidase subunit gene encodes a plasma membrane protein with Ca2+binding motifs[J]. Plant Cell,1998,10(2):255-266.
[17] Harding S A,Oh S,Roberts D M. Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species[J]. Embo Journal,2014,16(6):1137-1144.
[18] Yang T,Poovaiah B W. Hydrogen peroxide homeostasis:activation of plant catalase by calcium/calmodulin[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(6):4097-4102.
[19] Stael S,Kmiecik P,Willems P,et al. Plant innate immunity-sunny side up[J]. Trends in Plant Science,2015,20(1):3-11.
[20]李敏,杨双,阮燕晔,等.拟南芥T-DNA插入突变体atsuc3的PCR鉴定[J].植物生理学通讯,2006,42(1):91-94.
[21]周卫,汪洪.植物钙吸收、转运及代谢的生理和分子机制[J].植物学报,2007,24(6):762-778.
[22]高天,铁英,王妍,等.拟南芥低钙诱导c DNA文库的构建[J].北方农业学报,2016,44(5):8-14.
[23]李师翁,薛林贵,冯虎元,等.植物中的H2O2信号及其功能[J].中国生物化学与分子生物学报,2007(10):804-810.
[24]张小莉,王鹏程,宋纯鹏.植物细胞过氧化氢的测定方法[J].植物学报,2009,44(1):103-106.
[25] Munemasa S,Hossain M A,Nakamura Y,et al. The Arabidopsis calcium-dependent protein kinase,CPK6,functions as a positive regulator of methyl jasmonate signaling in guard cells[J]. Plant Physiology,2011,155(1):553-561.