多基因瞬时操纵体系的建立及其在草莓ABA信号产生中的应用研究
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摘要
植物激素脱落酸(abscisic acid, ABA)具有广泛的功能,其中ABA与植物抗逆、果实发育及成熟调控的关系国内外的一个研究热点。无论对于抗逆还是果实发育及成熟调控,ABA作用的根本基础在于在特定的条件下ABA水平大幅度提高。因此,ABA信号的产生是操纵ABA功能的根本基础。近年来,虽然国内外围绕ABA的生理功能及其作用机理,也就是ABA信号转导的机理开展了大量研究,以往的研究表明,ABA信号产生可能是多基因协同操纵的,但对ABA信号产生的机理研究较少。对ABA信号产生研究匮乏的原因之一是缺乏高效的基因操纵体系,特别是多基因操纵体系。草莓不仅是重要的果树品种之一,而且日益成为研究果树分子机理的模式材料。近年来的研究表明,ABA在草莓果实发育及成熟调控中起着重要作用。为此,本论文首先建立多基因瞬时操纵体系,在此基础上对ABA信号产生的机理进行了深入研究。主要结果和结论如下：
首先以草莓为试材,确定了草莓原生质体游离的优化条件,成功建立了草莓叶片和果实的原生质体游离体系,但是,未能成功建立草莓原生质体多基因转化体系。随后,探索了玉米叶片原生质体的多基因瞬时操纵体系,并证明该体系适用于草莓ABA信号产生研究。分析了草莓的ABA合成关键酶基因FαNCED1、FαNCED2和FαNCED3。克隆NCED2启动子序列,生物信息学分析表明,NCED2启动子序列含有许多响应植物激素、生物和非生物胁迫响应的顺式作用元件。试验证明,外源ABA、甘露醇、蔗糖都能明显的诱导FαNCED2启动子的表达,表明FαNCED2在ABA及逆境应答的ABA信号产生中起着关键作用。
基于以前的研究及生物信息学分析,确定了一系列与ABA信号产生相关的信号蛋白。这些信号蛋白从属于三个信号蛋白家族：G蛋白信号蛋白家族、酪氨酸蛋白磷酸酶(Protein Tyrosine Phosphatases, PTPs)家族、促分裂原活化蛋白激酶家族(Mitogen-activated protein kinases, MAPKs)。本文利用生物信息学的方法筛选得到了草莓G蛋白相关基因,通过半定量分析基因在草莓果实发育不同时期的表达情况,农杆菌瞬时侵染果实未检测到G蛋白相关基因在果实成熟发育中的作用,在草莓中筛选得到了19个PTPs成员以及12个MAPKs。依据FaPTPs家族中DSP1、 DSP3和DSP4,以及FaMAPKs家族中MPK2、MPK4、MPK6和MPK12基因详细的时空表达分析,推定这些信号蛋白与ABA信号产生可能有着密切的关系。
利用所建立的原生质体瞬时转化体系,以FαNCED2启动子驱动GUS做报告基因,以CaMV35s强启动子驱动LUC做内参基因,对候选基因在ABA信号产生中的功能进行了深入研究。结果表明,MPK4、MPK6、MPK12的过量表达可以抑制报告基因的表达；相反,DSP1、DSP3、DSP4的过量表达可激活报告基因的表达。有趣的是,DSP1、DSP3和DSP4三个基因中,两个基因及三个基因的共表达极大地促进了报告基因的表达,说明FaPTPs对ABA信号产生的控制存在着协同效应。综合以上结果,本研究揭示了操纵ABA信号产生的六个信号蛋白成员,即DSP1、 DSP3、DSP4, MPK4、MPK6和MPK12。其中,FaPTPs对ABA信号产生的控制存在着协同效应。
As a plant hormone, Abscisic acid (ABA) plays an important role in a wide range, especially the connection between ABA and plants defense as well as fruits development and maturity regulation. The researches about those have drawn people's attentions, and it is a hotspot around the world. The fundamental role of ABA is that its level can greatly improve under a certain condition, no matter in plant defense or fruits development and maturity regulation. In other words, only the level of ABA accumulates to a certain level, ABA can effectively activate its downstream signal transduction. Therefore, the generation of ABA signaling is the foundation to manipulate the functions of ABA. In recent years, the researches about the physiological function and mechanism of ABA, which is the mechanism ABA signal transduction have been carried out. The previous researches have indicated that the origin of ABA may be synergistically manipulated by multiple genes, but there is no substantive understanding about the mechanism of ABA's emergency. One of the reasons for the lack of researches about the origin of ABA signaling is the lack of an efficient gene manipulation system, especially the polygenic manipulation system. Strawberry is not only one of the important fruit trees, but also becoming a model material for the study of molecular mechanism in fruit trees. In recent years, research has shown that ABA plays an important role in development and ripening of strawberry fruits. In our study, we established the polygenic transient expression system of protoplast. Bases on the system, we carried out a thorough research on the mechanism of the generation of ABA signaling. The main results and conclusions as follows:
First, we identified the optimal conditions of strawberry protoplasts dissociation and successfully established strawberry protoplasts dissociation system in leaves and fruits. However, we failed to establish the multiple genes transformation system of strawberry protoplasts. Then, we explored the corn leaves protoplasts polygenic instantaneous transient expression system, and proved that the system was suitable for the study of ABA signaling generation in strawberry. We cloned three key enzyme genes of ABA synthesis in strawberry, FaNCEDl, FaNCED2and FaNCED3. Bioinformatics analysis showed that NCED2promoter sequences contain many cis-elements which respond to plant hormones, biotic and abiotic stress responses. Experiments have proved that exogenous ABA, mannitol, sucrose can significantly induce the expression of FaNCED2promoter. This shows that FaNCED2plays a key role in ABA and ABA signaling generation in stress responses.
Based on the previous researches and bioinformatics analysis, we determined a series of signaling proteins related to the generation of ABA signaling. These signaling proteins belong to three families, G protein genes, Protein Tyrosine Phosphatases (PTPs) family and Mitogen Activated Protein kinases (MAPKs) family. In this paper, we got many strawberry G protein genes used the method of bioinformatics, and through a quantitative analysis of gene expression in different period in strawberry fruit development, provided the evidence for the role of G protein genes in fruit mature development,We got19PTPs members and12MAPKs in strawberry. According to the genes expression analysis of DSP1, DSP3, DSP4in PTPs family and MPK2, MPK4, MPK6and MPK12in MAPKs family, we presumed that these proteins had a close relation to the generation of ABA signaling.
Using the established protoplasts transient transformation system, we conducted some researches about the candidate genes in the generation of ABA signaling, with FaNCED2promoter-driven GUS as a reporter gene and CaMV35s strong promoter-driven LUC as an internal control gene. The results showed that overexpression of MPK4, MPK6, MPK12can inhibit the reporter gene expression. On the contrary, overexpression of DSP1, DSP3, DSP4can activate the reporter gene expression. Interestingly, among DSP1, DSP3and DSP4, two or three genes co-expression can greatly promote the reporter gene expression, which showed that the FaPTPs have a synergistic effect on the generation of ABA signaling. Synthesizing the above results, our study reveals six signaling proteins, DSP1, DSP3, DSP4, MPK4, MPK6and MPK12, which can manipulate the generation of ABA signaling. And the FaPTPs have a synergistic effect on the origin of ABA signaling.
首先以草莓为试材,确定了草莓原生质体游离的优化条件,成功建立了草莓叶片和果实的原生质体游离体系,但是,未能成功建立草莓原生质体多基因转化体系。随后,探索了玉米叶片原生质体的多基因瞬时操纵体系,并证明该体系适用于草莓ABA信号产生研究。分析了草莓的ABA合成关键酶基因FαNCED1、FαNCED2和FαNCED3。克隆NCED2启动子序列,生物信息学分析表明,NCED2启动子序列含有许多响应植物激素、生物和非生物胁迫响应的顺式作用元件。试验证明,外源ABA、甘露醇、蔗糖都能明显的诱导FαNCED2启动子的表达,表明FαNCED2在ABA及逆境应答的ABA信号产生中起着关键作用。
基于以前的研究及生物信息学分析,确定了一系列与ABA信号产生相关的信号蛋白。这些信号蛋白从属于三个信号蛋白家族：G蛋白信号蛋白家族、酪氨酸蛋白磷酸酶(Protein Tyrosine Phosphatases, PTPs)家族、促分裂原活化蛋白激酶家族(Mitogen-activated protein kinases, MAPKs)。本文利用生物信息学的方法筛选得到了草莓G蛋白相关基因,通过半定量分析基因在草莓果实发育不同时期的表达情况,农杆菌瞬时侵染果实未检测到G蛋白相关基因在果实成熟发育中的作用,在草莓中筛选得到了19个PTPs成员以及12个MAPKs。依据FaPTPs家族中DSP1、 DSP3和DSP4,以及FaMAPKs家族中MPK2、MPK4、MPK6和MPK12基因详细的时空表达分析,推定这些信号蛋白与ABA信号产生可能有着密切的关系。
利用所建立的原生质体瞬时转化体系,以FαNCED2启动子驱动GUS做报告基因,以CaMV35s强启动子驱动LUC做内参基因,对候选基因在ABA信号产生中的功能进行了深入研究。结果表明,MPK4、MPK6、MPK12的过量表达可以抑制报告基因的表达；相反,DSP1、DSP3、DSP4的过量表达可激活报告基因的表达。有趣的是,DSP1、DSP3和DSP4三个基因中,两个基因及三个基因的共表达极大地促进了报告基因的表达,说明FaPTPs对ABA信号产生的控制存在着协同效应。综合以上结果,本研究揭示了操纵ABA信号产生的六个信号蛋白成员,即DSP1、 DSP3、DSP4, MPK4、MPK6和MPK12。其中,FaPTPs对ABA信号产生的控制存在着协同效应。
As a plant hormone, Abscisic acid (ABA) plays an important role in a wide range, especially the connection between ABA and plants defense as well as fruits development and maturity regulation. The researches about those have drawn people's attentions, and it is a hotspot around the world. The fundamental role of ABA is that its level can greatly improve under a certain condition, no matter in plant defense or fruits development and maturity regulation. In other words, only the level of ABA accumulates to a certain level, ABA can effectively activate its downstream signal transduction. Therefore, the generation of ABA signaling is the foundation to manipulate the functions of ABA. In recent years, the researches about the physiological function and mechanism of ABA, which is the mechanism ABA signal transduction have been carried out. The previous researches have indicated that the origin of ABA may be synergistically manipulated by multiple genes, but there is no substantive understanding about the mechanism of ABA's emergency. One of the reasons for the lack of researches about the origin of ABA signaling is the lack of an efficient gene manipulation system, especially the polygenic manipulation system. Strawberry is not only one of the important fruit trees, but also becoming a model material for the study of molecular mechanism in fruit trees. In recent years, research has shown that ABA plays an important role in development and ripening of strawberry fruits. In our study, we established the polygenic transient expression system of protoplast. Bases on the system, we carried out a thorough research on the mechanism of the generation of ABA signaling. The main results and conclusions as follows:
First, we identified the optimal conditions of strawberry protoplasts dissociation and successfully established strawberry protoplasts dissociation system in leaves and fruits. However, we failed to establish the multiple genes transformation system of strawberry protoplasts. Then, we explored the corn leaves protoplasts polygenic instantaneous transient expression system, and proved that the system was suitable for the study of ABA signaling generation in strawberry. We cloned three key enzyme genes of ABA synthesis in strawberry, FaNCEDl, FaNCED2and FaNCED3. Bioinformatics analysis showed that NCED2promoter sequences contain many cis-elements which respond to plant hormones, biotic and abiotic stress responses. Experiments have proved that exogenous ABA, mannitol, sucrose can significantly induce the expression of FaNCED2promoter. This shows that FaNCED2plays a key role in ABA and ABA signaling generation in stress responses.
Based on the previous researches and bioinformatics analysis, we determined a series of signaling proteins related to the generation of ABA signaling. These signaling proteins belong to three families, G protein genes, Protein Tyrosine Phosphatases (PTPs) family and Mitogen Activated Protein kinases (MAPKs) family. In this paper, we got many strawberry G protein genes used the method of bioinformatics, and through a quantitative analysis of gene expression in different period in strawberry fruit development, provided the evidence for the role of G protein genes in fruit mature development,We got19PTPs members and12MAPKs in strawberry. According to the genes expression analysis of DSP1, DSP3, DSP4in PTPs family and MPK2, MPK4, MPK6and MPK12in MAPKs family, we presumed that these proteins had a close relation to the generation of ABA signaling.
Using the established protoplasts transient transformation system, we conducted some researches about the candidate genes in the generation of ABA signaling, with FaNCED2promoter-driven GUS as a reporter gene and CaMV35s strong promoter-driven LUC as an internal control gene. The results showed that overexpression of MPK4, MPK6, MPK12can inhibit the reporter gene expression. On the contrary, overexpression of DSP1, DSP3, DSP4can activate the reporter gene expression. Interestingly, among DSP1, DSP3and DSP4, two or three genes co-expression can greatly promote the reporter gene expression, which showed that the FaPTPs have a synergistic effect on the generation of ABA signaling. Synthesizing the above results, our study reveals six signaling proteins, DSP1, DSP3, DSP4, MPK4, MPK6and MPK12, which can manipulate the generation of ABA signaling. And the FaPTPs have a synergistic effect on the origin of ABA signaling.
引文
何飞龙,周嫦.玉帘的组织和原生质体培养.武汉大学学报(自然科学版),1995,41(2)：213-217.
何晓明,王鸣,王之.辣椒子叶原生质体分离条件的研究.西北植物学报,1997,17(1)：112-117.
胡家金,熊金耀,张秋明,等.美味猕猴桃原生质体培养及植株再生技术研究.湖南农业大学学报,1998,24(1)：184-190.
金万梅,董静,尹淑萍,等.冷诱导转录因子CBF1转化草莓及其抗寒性鉴定.西北植物学报,2007(02)：223-227.
李杰,黄敏仁,王明庥,等.植物原生质体培养和体细胞融合技术研究进展.仲恺农业技术学院学报,2003(04)：64-71.
李绍清,周小梅.茴香原生质体培养研究.山西大学学报(自然科学版),1996,19(3)：328-330.
李新锋,赵淑清.转基因植物中报道基因GUS的活性检测及其应用.生命的化学,2004(01)：71-74.
李彦舫,程肖蕊,张亚兰,等.野大麦幼穗原生质体的分离和培养.植物学通报,1999,16(1)：67-71.
林顺权,陈振光.山梨醇对枇杷原生质体分离和培养的效应.福建农业大学学报,1997,26(4)：401-406.
马锋旺,李嘉瑞.抗氧化剂对杏和中国李原生质体培养的影响.西北农业大学学报,1998,26(6)：10-13.
马利加P,克莱森D F,卡什莫尔A R.植物分子生物学实验指南[M].刘进元,吴庆余等译.北京：科学出版社,2000：37-38.
潘增光,邓秀新.苹果原生质体分离培养及植株再生.园艺学报,2000,27(2)：95-101.
史永忠,万蜀渊,薛光荣.草莓花药愈伤组织原生质体培养再生多细胞团.华中农业大学学报,1995,14(2)：181-189.
孙勇如,李向辉,孙宝林,等.新疆甜瓜子叶原生质体培养和植株再生.植物学报,1989,31(12)：916-922.
王关林,方宏筠.植物基因工程[M].北京：科学出版社,2009.
肖尊安,韩碧文.猕猴桃属种间体细胞杂种.植物学报,1997(39)：1110-1117.
邢宇,王幼群,张蜀秋,等.酪氨酸蛋白磷酸酶可能影响ABA的积累和参与植物细胞水分胁迫信号传递.科学通报,2003(04)：369-374.
徐子勤,贾敬芬,胡之德,苜蓿根癌农杆菌转化系原生质体培养研究.武汉植物学研究,1997,15(3)：283-285.
许智宏,卫志明.植物原生质体培养和遗传操作.上海：科学技术出版社,1997.
张菊梅,吴清平,周小燕,等.荧光素酶研究进展.微生物学通报,2001(05)：98-101.
赵华,梁婉琪,杨永华,等.绿色荧光蛋白及其在植物分子生物学研究中的应用.植物生理学通讯,2003(02)：171-178.
周延清,苑保军,张根发,等.决明原生质体的分离与培养研究.华北农学报,1998,13(3)：107-111.
Abel S, Theologis A. Transient transformation of Arabidopsis leaf protoplasts:a versatile experimental system to study gene expression. Plant J,1994,5(3):421-427.
Aflalo C. Biologically localized firefly luciferase:a tool to study cellular processes. Int Rev Cytol, 1991,130:269-323.
Alexander L, Grierson D. Ethylene biosynthesis and action in tomato:a model for climacteric fruit ripening. J Exp Bot,2002,53(377):2039-2055.
Anderson J D, Lieberman M, Stewart R N. Ethylene production by apple protoplasts. Plant Physiol,1979,63(5):931-935.
Asao H, Arai S, Nishizawa Y, et al. Environmental risk evaluation of transgenic strawberry expressing a rice chitinase gene. J Biosci Bioeng,2003,81 (2):57-63.
Bartels S, Gonzalez B M, Lang D, et al. Emerging functions for plant MAP kinase phosphatases. Trends Plant Sci,2010,15(6):322-329.
Bates G W, Rabussay D, Piastuch W. Transient and stable expression of foreign DNA introduced into plant protoplasts by electroporation. Methods Mol Biol,1990,6:309-322.
Bogre L, Meskiene I, Heberle-Bors E, et al. Stressing the role of MAP kinases in mitogenic stimulation. Plant Mol Biol,2000,43(5-6):705-718.
Burbidge A, Grieve T M, Jackson A, et al. Characterization of the ABA-deficient tomato mutant notabilis and its relationship with maize Vpl4. Plant J,1999,17(4):427-431.
Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases:a gene family for control of MAP kinase function. FASEB J,2000,14(1l):6-16.
Chai Y M, Jia H F, Li C L, et al. FaPYR1 is involved in strawberry fruit ripening. J Exp Bot,2011, 62(14):5079-5089.
Chen S, Tao L, Zeng L, et al. A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol,2006,7(5):417-427.
Chen Y, Ji F, Xie H, et al. The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination. Plant Physiol,2006,140(1):302-310.
Chernys J T, Zeevaart J A. Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado. Plant Physiol,2000,124(1):343-353.
Cocking E C. A method for the isolation of plant protoplasts and vacuoles. Nature,1960, 187:927-929.
Colcombet J, Hirt H. Arabidopsis MAPKs:a complex signalling network involved in multiple biological processes. Biochem J,2008,413(2):217-226.
Craig W, Gargano D, Scotti N, et al. Direct gene transfer in potato:a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts. Plant Cell Rep, 2005,24(10):603-611.
Denu J M, Stuckey J A, Saper M A, et al. Form and function in protein dephosphorylation. Cell, 1996,87(3):361-364.
Diaz I,Moreno P Power J B.Plant regeneration from protoplasts of Capsicumannuum.Plant Cell Rep,1988,13:397-400.
Ding A P, Wang H F. Plant regeneration from cotyledon and cell suspension protoplasts of apple(Malusexdomestia,cv.starkrimson).Plant Cell Tissue and Organ Culture.1995,40:145-149.
Ding L, Pandey S, Assmann S M. Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis. Plant J,2008,53(2):248-263.
Dunphy W G. The decision to enter mitosis. Trends Cell Biol,1994,4(6):202-207.
Fait A, Hanhineva K, Beleggia R, et al. Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development. Plant Physiol,2008,148(2):730-750.
Fujii H, Chinnusamy V, Rodrigues A, et al. In vitro reconstitution of an abscisic acid signalling pathway. Nature,2009,462(7273):660-664.
Fujisawa Y, Kato H, Iwasaki Y. Structure and function of heterotrimeric G proteins in plants. Plant Cell Physiol,2001,42(8):789-794.
Gao Y, Zeng Q, Guo J, et al. Genetic characterization reveals no role for the reported ABA receptor, GCR2, in ABA control of seed germination and early seedling development in Arabidopsis. Plant J,2007,52(6):1001-1013.
Gelvin S B. The introduction and expression of transgenes in plants. Curr Opin Biotechnol, 1998,9(2):227-232.
Gilroy S, Jones R L. Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts. Proc Natl Acad Sci USA,1992,89(8):3591-3595.
Gookin T E, Kim J, Assmann S M. Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar:computational prediction and in-vivo protein coupling. Genome Biol,2008,9(7):R120.
Green A A, Mcelroy W D. Crystalline firefly luciferase. Biochim Biophys Acta, 1956,20(1):170-176.
Griesser M, Hoffmann T, Bellido M L, et al. Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit. Plant Physiol, 2008,146(4):1528-1539.
Guan K L. The mitogen activated protein kinase signal transduction pathway:from the cell surface to the nucleus. Cell Signal,1994,6(6):581-589.
Guo J, Morrell-Falvey J L, Labbe J L, et al. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS One,2012,7(9):e44908.
Gupta R, Luan S. Redox control of protein tyrosine phosphatases and mitogen-activated protein kinases in plants. Plant Physiol,2003,132(3):1149-1152.
Harkins K R, Jefferson R A, Kavanagh T A, et al. Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. Proc Natl Acad Sci USA,1990,87(2):816-820.
Hassanein A, Hamama L, Loridon K, et al. Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium xhortorum, Tanache Sud1. Plant Cell Rep,2009,28(10):1521-1530.
Higo K, Ugawa Y, Iwamoto M, et al. Plant cis-acting regulatory DNA elements (PLACE) database:1999. Nucleic Acids Res,1999,27(1):297-300.
Hoffmann T, Kalinowski G, Schwab W. RNAi-induced silencing of gene expression in strawberry fruit(Fragaria ×ananassa) by agroinfiltration:a rapid assay for gene function analysis. The Plant Journal,2006,48:818-826.
Honda C, Moriguchi T. High GUS expression in protoplasts isolated from immature peach fruits. Sci Hort,2006,109:244-247.
Hoyos B, Imam A, Chua R, et al. The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors. J Exp Med,2000,192(6):835-845.
Huang Y, Li H, Gupta R, et al. ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEKl through threonine phosphorylation. Plant Physiol, 2000,122(4):1301-1310.
Ichimura K, Shinozaki K, Tena G, et al. Mitogen-activated protein kinase cascades in plants:a new nomenclature. Trends Plant Sci,2002,7(7):301-308.
Iuchi S, Kobayashi M, Taji T, et al. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J, 2001,27(4):325-333.
Iuchi S, Kobayashi M, Yamaguchi-Shinozaki K, et al. A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea. Plant Physiol,2000,123(2):553-562.
James D J, Passey AJ, and Barbera DJ. Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria xananassa Duch) using disarmed binary vectors. Plant Science,1990,69:79-94.
Jammes F, Song C, Shin D, et al. MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling. Proc Natl Acad Sci USA, 2009,106(48):20520-20525.
Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J,1987,6(13):3901-3907.
Jefferson R A. The GUS reporter gene system. Nature,1989,342(6251):837-838.
Jefferson RA.Assaying chimeric genes in plants:the GUS gene fusion system. Plant Mol Biol Rep.1987,5:387-405.
Ji K, Chen P, Sun L, et al. Non-climacteric ripening in strawberry fruit is linked to ABA, FaNCED2 and FaCYP707A1. Functional Plant Biology,2012,39:351-357.
Jia H F, Chai Y M, Li C L, et al. Abscisic acid plays an important role in the regulation of strawberry fruit ripening. Plant Physiol,2011,157(1):188-199.
Jonak C, Okresz L, Bogre L, et al. Complexity, cross talk and integration of plant MAP kinase signalling. Curr Opin Plant Biol,2002,5(5):415-424. Josefsson L G, Rask L. Cloning of a putative G-protein-coupled receptor from Arabidopsis thaliana. Eur J Biochem,1997,249(2):415-420.
Kerk D, Bulgrien J, Smith D W, et al. The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis. Plant Physiol,2002,129(2):908-925.
Kim J, Somers D E. Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts. Plant Physiol,2010,154(2):611-621.
Kim M J, Shin R, Schachtman D P. A nuclear factor regulates abscisic acid responses in Arabidopsis. Plant Physiol,2009,151(3):1433-1445.
Knetsch M, Wang M, Snaar-Jagalska B E, et al. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts. Plant Cell,1996,8(6):1061-1067.
Knight M R, Campbell A K, Smith S M, et al. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature,1991,352(6335):524-526.
Krens F A,Molendijk L,Wullems G L.In vitro transformation of plant protoplasts with Ti-plasmid DNA.Nature,1982,296:72-74.
Lee J S, Ellis B E. Arabidopsis MAPK phosphatase 2 (MKP2) positively regulates oxidative stress tolerance and inactivates the MPK3 and MPK6 MAPKs. J Biol Chem,2007,282(34):25020-25029.
Lee J S, Wang S, Sritubtim S, et al. Arabidopsis mitogen-activated protein kinase MPK12 interacts with the MAPK phosphatase IBR5 and regulates auxin signaling. Plant J,2009,57(6):975-985.
Lee Y R, Assmann S M. Arabidopsis thaliana 'extra-large GTP-binding protein' (AtXLGl):a new class of G-protein. Plant Mol Biol,1999,40(1):55-64.
Lescot M, Dehais P, Thijs G, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 2002,30(1):325-327.
Lessard P, Kreis M, Thomas M. Protein phosphatases and protein kinases in higher plants. C R Acad Sci Ⅲ,1997,320(9):675-688.
Li H, Lin Y, Heath R M, et al. Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell,1999,11(9):1731-1742.
Li H, Wu G, Ware D, et al. Arabidopsis Rho-related GTPases:differential gene expression in pollen and polar localization in fission yeast. Plant Physiol,1998,118(2):407-417.
Li Q, Ji K, Sun Y, et al. The role of FaBG3 in fruit ripening and B. cinerea fungal infection of strawberry. Plant J,2013,76(1):24-35.
Ligterink W, Hirt H. Mitogen-activated protein (MAP) kinase pathways in plants:versatile signaling tools. Int Rev Cytol,2001,201:209-275.
Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001,25(4):402-408.
Lu C, Han M H, Guevara-Garcia A, et al. Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid. Proc Natl Acad Sci USA, 2002,99(24):15812-15817.
Luan S. Protein phosphatases in plants. Annu Rev Plant Biol,2003,54:63-92.
Luan S. Tyrosine phosphorylation in plant cell signaling. Proc Natl Acad Sci USA,2002, 99(18):11567-11569.
Luehrsen K R, de Wet J R, Walbot V. Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol,1992,216:397-414.
Ma H, Yanofsky M F, Meyerowitz E M. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc Natl Acad Sci USA, 1990,87(10):3821-3825.
Ma Y, Szostkiewicz I, Korte A, et al. Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science,2009,324(5930):1064-1068.
MacRobbie E A. Evidence for a role for protein tyrosine phosphatase in the control of ion release from the guard cell vacuole in stomatal closure. Proc Natl Acad Sci USA,2002,99(18):11963-11968.
Maehama T, Taylor G S, Dixon J E. PTEN and myotubularin:novel phosphoinositide phosphatases. Annu Rev Biochem,2001,70:247-279.
Mason M G, Botella J R. Completing the heterotrimer:isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA. Proc Natl Acad Sci USA, 2000,97(26):14784-14788.
Mason M G, Botella J R. Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with Gbeta. Biochim Biophys Acta,2001,1520(2):147-153.
Mizoguchi T, Ichimura K, Shinozaki K. Environmental stress response in plants:the role of mitogen-activated protein kinases. Trends Biotechnol,1997,15(1):15-19.
Mizoguchi T, Irie K, Hirayama T, et al. A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc Natl Acad Sci USA, 1996,93(2):765-769.
Monroe-Augustus M, Zolman B K, Bartel B. IBR5, a dual-specificity phosphatase-like protein modulating auxin and abscisic acid responsiveness in Arabidopsis. Plant Cell,2003,15(12):2979-2991.
Nagata T, Takebe I. Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta,1970,92(4):301-308.
Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol, 2005,56:165-185.
Neel B G, Tonks N K. Protein tyrosine phosphatases in signal transduction. Curr Opin Cell Biol, 1997,9(2):193-204.
Nehra N S, Chibbar R N, Kartha K K, et al. Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants.Plant Cell Rep,1990a,9(1):10-13.
Nehra N S, Chibbar R N, Kartha K K, et al. Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Rep,1990b,9(6):293-298.
Nuhse T S, Peck S C, Hirt H, et al. Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK6. J Biol Chem,2000,275(11):7521-7526.
Ochatt S J, Caso O J. Shoot regeneration from leaf mesophyll protoplasts of vild pear.Plant Physiology.1986,122:243-249.
Ortiz-Masia D, Perez-Amador M A, Carbonell J, et al. Diverse stress signals activate the Cl subgroup MAP kinases of Arabidopsis. FEBS Lett,2007,581(9):1834-1840.
Ou-Lee T M, Turgeon R, Wu R. Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat, and sorghum. Proc Natl Acad Sci USA,1986,83(18):6815-6819.
Owens C L, Iezzoni A F, Hancock J F, et al. CBF1 orthologs in sour cherry and strawberry and the heterologous expression of CBF1 in strawberry. J Amer Soc Hort Sci,2002,127(4):489-494.
Owens C L, Iezzoni A F, Hancock J F. Enhancement of freezing tolerance of strawberry by heterologous expression of CBF1. Acta Hort.2003,626:93-100.
Pandey S, Nelson D C, Assmann S M. Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell,2009,136(1):136-148.
Parihar D S,maheshwari S C.Influence of heat shock and UV irradiation on PEG-mediated DNA uptake and transient expression of NPT Ⅱgene in protoplasts of Brassicanapas.Indian J of Expbio,1998, 36(10):1002-1006.
Park S Y, Fung P, Nishimura N, et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science,2009,324(5930):1068-1071.
Parry A D, Babiano M J, Horgan R. The role of cis-carotenoids in abscisic acid biosynthesis. Planta, 1990,182(1):118-128.
Petersen M, Brodersen P, Naested H, et al. Arabidopsis map kinase 4 negatively regulates systemic acquired resistance. Cell,2000,103(7):1111-1120.
Qin X, Zeevaart J A. The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean. Proc Natl Acad Sci USA,1999,96(26):15354-15361.
Quettier A L, Bertrand C, Habricot Y, et al. The phs1-3 mutation in a putative dual-specificity protein tyrosine phosphatase gene provokes hypersensitive responses to abscisic acid in Arabidopsis thaliana. Plant J,2006,47(5):711-719.
Rakotondrafara A M, Jackson J R, Kneller E P, et al. Preparation and electroporation of oat protoplasts from cell suspension culture. Curr Protoc Microbiol,2007,Chapter 16:13D-16D.
Rasmussen J O,Rasmussen O S.PEG mediated DNA uptake and transient GUS expression in carrot,rapeseed and soybean protoplasts.Plant Sci,1993(89):199-207.
Rodrigo M J, Alquezar B, Zacarias L. Cloning and characterization of two 9-cis-epoxycarotenoid dioxygenase genes, differentially regulated during fruit maturation and under stress conditions, from orange (Citrus sinensis L. Osbeck). J Exp Bot,2006,57(3):633-643.
Rodrigo M J, Marcos J F, Alferez F, et al. Characterization of Pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content. J Exp Bot,2003,54(383):727-738.
Rudnicki R. Biosynthesis and metabolism of abscisic acid in plants. Postepy Biochem,1971, 17(1):43-55.
Sakamoto S, Fujikawa Y, Esaka M. Analysis of ascorbic acid biosynthesis using a simple transient gene expression system in tomato fruit protoplasts. Biosci Biotechnol Biochem,2013,77(3):673-675.
Santiago J, Rodrigues A, Saez A, et al. Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. Plant J,2009,60(4):575-588.
Schaffer R, Landgraf J, Accerbi M, et al. Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. Plant Cell,2001,13(1):113-123.
Schaffner A R, Sheen J. Maize rbcS promoter activity depends on sequence elements not found in dicot rbcS promoters. Plant Cell,1991,3(9):997-1012.
Schoenbeck M A, Samac D A, Fedorova M, et al. The alfalfa (Medicago sativa) TDY1 gene encodes a mitogen-activated protein kinase homolog. Mol Plant Microbe Interact, 1999,12(10):882-893.
Sheen J. Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell,1991,3(3):225-245.
Sheen J. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol, 2001,127(4):1466-1475.
Sheen, J.2002. A transient expression assay using maize mesophyll protoplasts.
Shimomura O, Johnson F H, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol, 1962,59:223-239.
Shinozaki K, Yamaguchi-Shinozaki K, Seki M. Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol,2003,6(5):410-417.
Spolaore S, Trainotti L, Casadoro G. A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium. Journal of Experimental Botany,2001,52:845-850.
Stone R L, Dixon J E. Protein-tyrosine phosphatases. J Biol Chem,1994,269(50):31323-31326.
Tan B C, Joseph L M, Deng W T, et al. Molecular characterization of the Arabidopsis 9-cis-epoxycarotenoid dioxygenase gene family. Plant J,2003,35(1):44-56.
Tan B C, Schwartz S H, Zeevaart J A, et al. Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA,1997,94(22):12235-12240.
Tanoue T, Adachi M, Moriguchi T, et al. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat Cell Biol,2000,2(2):110-116.
Tena G, Asai T, Chiu W L, et al. Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol,2001,4(5):392-400.
Thompson A J, Jackson A C, Symonds R C, et al. Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J, 2000,23(3):363-374.
Tonks N K, Neel B G. Combinatorial control of the specificity of protein tyrosine phosphatases. Curr Opin Cell Biol,2001,13(2):182-195.
Tonks N K. Protein tyrosine phosphatases:from genes, to function, to disease. Nat Rev Mol Cell Biol,2006,7(11):833-846.
Ulm R, Ichimura K, Mizoguchi T, et al. Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1. EMBO J,2002,21(23):6483-6493.
Van Vactor D,0,Reilly A M,Neel BG.Genetic analysis of Protein tyrosine phosphatase.Curr Opin Genet Develop,1998,8:112-126.
Wang J L, Ge H B, Peng S Q, et al. Transformation of strawberry (Fragariax ananassa Duch.) with late embryogenesis abundant protein gene. J Hort Sci Biotech,2004,79(5):735-738.
Wang X Q, Ullah H, Jones A M, et al. G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science,2001,292(5524):2070-2072.
Weiss C A, Garnaat C W, Mukai K, et al. Isolation of cDNAs encoding guanine nucleotide-binding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc Natl Acad Sci USA, 1994,91(20):9554-9558.
Weiss C A, Huang H, Ma H. Immunolocalization of the G protein alpha subunit encoded by the GPA1 gene in Arabidopsis. Plant Cell,1993,5(11):1513-1528.
Wenzler H C, Mignery G A, Fisher L M, et al. Analysis of a chimeric class-I patatin-GUS gene in transgenic potato plants:High-level expression in tubers and sucrose-inducible expression in cultured leaf and stem explants. Plant Mol Biol,1989,12(1):41-50.
Winge P, Brembu T, Bones A M. Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana. Plant Mol Biol,1997,35(4):483-495.
Winge P, Brembu T, Kristensen R, et al. Genetic structure and evolution of RAC-GTPases in Arabidopsis thaliana. Genetics,2000,156(4):1959-1971.
Xiong L, Schumaker K S, Zhu J K. Cell signaling during cold, drought, and salt stress. Plant Cell, 2002,14 Suppl:S165-S183.
Xiong L, Yang Y. Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell,2003,15(3):745-759.
Xu Q, Fu H H, Gupta R, et al. Molecular characterization of a tyrosine-specific protein phosphatase encoded by a stress-responsive gene in Arabidopsis. Plant Cell,1998,10(5):849-857.
Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol,2006,57:781-803.
Yang Z. Small GTPases:versatile signaling switches in plants. Plant Cell,2002,14 Suppl:S375-S388.
Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis. Nat Protoc,2007,2(7):1565-1572.
Yoshida R, Umezawa T, Mizoguchi T, et al. The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABIl and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J Biol Chem,2006,281(8):5310-5318.
Yuasa T, Ichimura K, Mizoguchi T, et al. Oxidative stress activates ATMPK6, an Arabidopsis homologue of MAP kinase. Plant Cell Physiol,2001,42(9):1012-1016.
Zhai Z, Sooksa-nguan T, Vatamaniuk O K. Establishing RNA interference as a reverse-genetic approach for gene functional analysis in protoplasts. Plant Physiol,2009,149(2):642-652.
Zhang H M, Yang H, Rech E L, et al. Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Rep,1988,7(6):379-384.
Zhang M, Yuan B, Leng P. The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit. J Exp Bot,2009,60(6):1579-1588.
Zhang S, Klessig D F. MAPK cascades in plant defense signaling. Trends Plant Sci, 2001,6(11):520-527.
Zheng Z L, Nafisi M, Tam A, et al. Plasma membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis. Plant Cell, 2002,14(11):2787-2797.
Zheng Z L, Yang Z. The Rop GTPase:an emerging signaling switch in plants. Plant Mol Biol, 2000,44(1):1-9.
Zhu J K. Salt and drought stress signal transduction in plants. Annu Rev Plant Biol, 2002,53:247-273.
何晓明,王鸣,王之.辣椒子叶原生质体分离条件的研究.西北植物学报,1997,17(1)：112-117.
胡家金,熊金耀,张秋明,等.美味猕猴桃原生质体培养及植株再生技术研究.湖南农业大学学报,1998,24(1)：184-190.
金万梅,董静,尹淑萍,等.冷诱导转录因子CBF1转化草莓及其抗寒性鉴定.西北植物学报,2007(02)：223-227.
李杰,黄敏仁,王明庥,等.植物原生质体培养和体细胞融合技术研究进展.仲恺农业技术学院学报,2003(04)：64-71.
李绍清,周小梅.茴香原生质体培养研究.山西大学学报(自然科学版),1996,19(3)：328-330.
李新锋,赵淑清.转基因植物中报道基因GUS的活性检测及其应用.生命的化学,2004(01)：71-74.
李彦舫,程肖蕊,张亚兰,等.野大麦幼穗原生质体的分离和培养.植物学通报,1999,16(1)：67-71.
林顺权,陈振光.山梨醇对枇杷原生质体分离和培养的效应.福建农业大学学报,1997,26(4)：401-406.
马锋旺,李嘉瑞.抗氧化剂对杏和中国李原生质体培养的影响.西北农业大学学报,1998,26(6)：10-13.
马利加P,克莱森D F,卡什莫尔A R.植物分子生物学实验指南[M].刘进元,吴庆余等译.北京：科学出版社,2000：37-38.
潘增光,邓秀新.苹果原生质体分离培养及植株再生.园艺学报,2000,27(2)：95-101.
史永忠,万蜀渊,薛光荣.草莓花药愈伤组织原生质体培养再生多细胞团.华中农业大学学报,1995,14(2)：181-189.
孙勇如,李向辉,孙宝林,等.新疆甜瓜子叶原生质体培养和植株再生.植物学报,1989,31(12)：916-922.
王关林,方宏筠.植物基因工程[M].北京：科学出版社,2009.
肖尊安,韩碧文.猕猴桃属种间体细胞杂种.植物学报,1997(39)：1110-1117.
邢宇,王幼群,张蜀秋,等.酪氨酸蛋白磷酸酶可能影响ABA的积累和参与植物细胞水分胁迫信号传递.科学通报,2003(04)：369-374.
徐子勤,贾敬芬,胡之德,苜蓿根癌农杆菌转化系原生质体培养研究.武汉植物学研究,1997,15(3)：283-285.
许智宏,卫志明.植物原生质体培养和遗传操作.上海：科学技术出版社,1997.
张菊梅,吴清平,周小燕,等.荧光素酶研究进展.微生物学通报,2001(05)：98-101.
赵华,梁婉琪,杨永华,等.绿色荧光蛋白及其在植物分子生物学研究中的应用.植物生理学通讯,2003(02)：171-178.
周延清,苑保军,张根发,等.决明原生质体的分离与培养研究.华北农学报,1998,13(3)：107-111.
Abel S, Theologis A. Transient transformation of Arabidopsis leaf protoplasts:a versatile experimental system to study gene expression. Plant J,1994,5(3):421-427.
Aflalo C. Biologically localized firefly luciferase:a tool to study cellular processes. Int Rev Cytol, 1991,130:269-323.
Alexander L, Grierson D. Ethylene biosynthesis and action in tomato:a model for climacteric fruit ripening. J Exp Bot,2002,53(377):2039-2055.
Anderson J D, Lieberman M, Stewart R N. Ethylene production by apple protoplasts. Plant Physiol,1979,63(5):931-935.
Asao H, Arai S, Nishizawa Y, et al. Environmental risk evaluation of transgenic strawberry expressing a rice chitinase gene. J Biosci Bioeng,2003,81 (2):57-63.
Bartels S, Gonzalez B M, Lang D, et al. Emerging functions for plant MAP kinase phosphatases. Trends Plant Sci,2010,15(6):322-329.
Bates G W, Rabussay D, Piastuch W. Transient and stable expression of foreign DNA introduced into plant protoplasts by electroporation. Methods Mol Biol,1990,6:309-322.
Bogre L, Meskiene I, Heberle-Bors E, et al. Stressing the role of MAP kinases in mitogenic stimulation. Plant Mol Biol,2000,43(5-6):705-718.
Burbidge A, Grieve T M, Jackson A, et al. Characterization of the ABA-deficient tomato mutant notabilis and its relationship with maize Vpl4. Plant J,1999,17(4):427-431.
Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases:a gene family for control of MAP kinase function. FASEB J,2000,14(1l):6-16.
Chai Y M, Jia H F, Li C L, et al. FaPYR1 is involved in strawberry fruit ripening. J Exp Bot,2011, 62(14):5079-5089.
Chen S, Tao L, Zeng L, et al. A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol,2006,7(5):417-427.
Chen Y, Ji F, Xie H, et al. The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination. Plant Physiol,2006,140(1):302-310.
Chernys J T, Zeevaart J A. Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado. Plant Physiol,2000,124(1):343-353.
Cocking E C. A method for the isolation of plant protoplasts and vacuoles. Nature,1960, 187:927-929.
Colcombet J, Hirt H. Arabidopsis MAPKs:a complex signalling network involved in multiple biological processes. Biochem J,2008,413(2):217-226.
Craig W, Gargano D, Scotti N, et al. Direct gene transfer in potato:a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts. Plant Cell Rep, 2005,24(10):603-611.
Denu J M, Stuckey J A, Saper M A, et al. Form and function in protein dephosphorylation. Cell, 1996,87(3):361-364.
Diaz I,Moreno P Power J B.Plant regeneration from protoplasts of Capsicumannuum.Plant Cell Rep,1988,13:397-400.
Ding A P, Wang H F. Plant regeneration from cotyledon and cell suspension protoplasts of apple(Malusexdomestia,cv.starkrimson).Plant Cell Tissue and Organ Culture.1995,40:145-149.
Ding L, Pandey S, Assmann S M. Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis. Plant J,2008,53(2):248-263.
Dunphy W G. The decision to enter mitosis. Trends Cell Biol,1994,4(6):202-207.
Fait A, Hanhineva K, Beleggia R, et al. Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development. Plant Physiol,2008,148(2):730-750.
Fujii H, Chinnusamy V, Rodrigues A, et al. In vitro reconstitution of an abscisic acid signalling pathway. Nature,2009,462(7273):660-664.
Fujisawa Y, Kato H, Iwasaki Y. Structure and function of heterotrimeric G proteins in plants. Plant Cell Physiol,2001,42(8):789-794.
Gao Y, Zeng Q, Guo J, et al. Genetic characterization reveals no role for the reported ABA receptor, GCR2, in ABA control of seed germination and early seedling development in Arabidopsis. Plant J,2007,52(6):1001-1013.
Gelvin S B. The introduction and expression of transgenes in plants. Curr Opin Biotechnol, 1998,9(2):227-232.
Gilroy S, Jones R L. Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts. Proc Natl Acad Sci USA,1992,89(8):3591-3595.
Gookin T E, Kim J, Assmann S M. Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar:computational prediction and in-vivo protein coupling. Genome Biol,2008,9(7):R120.
Green A A, Mcelroy W D. Crystalline firefly luciferase. Biochim Biophys Acta, 1956,20(1):170-176.
Griesser M, Hoffmann T, Bellido M L, et al. Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit. Plant Physiol, 2008,146(4):1528-1539.
Guan K L. The mitogen activated protein kinase signal transduction pathway:from the cell surface to the nucleus. Cell Signal,1994,6(6):581-589.
Guo J, Morrell-Falvey J L, Labbe J L, et al. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS One,2012,7(9):e44908.
Gupta R, Luan S. Redox control of protein tyrosine phosphatases and mitogen-activated protein kinases in plants. Plant Physiol,2003,132(3):1149-1152.
Harkins K R, Jefferson R A, Kavanagh T A, et al. Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. Proc Natl Acad Sci USA,1990,87(2):816-820.
Hassanein A, Hamama L, Loridon K, et al. Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium xhortorum, Tanache Sud1. Plant Cell Rep,2009,28(10):1521-1530.
Higo K, Ugawa Y, Iwamoto M, et al. Plant cis-acting regulatory DNA elements (PLACE) database:1999. Nucleic Acids Res,1999,27(1):297-300.
Hoffmann T, Kalinowski G, Schwab W. RNAi-induced silencing of gene expression in strawberry fruit(Fragaria ×ananassa) by agroinfiltration:a rapid assay for gene function analysis. The Plant Journal,2006,48:818-826.
Honda C, Moriguchi T. High GUS expression in protoplasts isolated from immature peach fruits. Sci Hort,2006,109:244-247.
Hoyos B, Imam A, Chua R, et al. The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors. J Exp Med,2000,192(6):835-845.
Huang Y, Li H, Gupta R, et al. ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEKl through threonine phosphorylation. Plant Physiol, 2000,122(4):1301-1310.
Ichimura K, Shinozaki K, Tena G, et al. Mitogen-activated protein kinase cascades in plants:a new nomenclature. Trends Plant Sci,2002,7(7):301-308.
Iuchi S, Kobayashi M, Taji T, et al. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J, 2001,27(4):325-333.
Iuchi S, Kobayashi M, Yamaguchi-Shinozaki K, et al. A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea. Plant Physiol,2000,123(2):553-562.
James D J, Passey AJ, and Barbera DJ. Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria xananassa Duch) using disarmed binary vectors. Plant Science,1990,69:79-94.
Jammes F, Song C, Shin D, et al. MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling. Proc Natl Acad Sci USA, 2009,106(48):20520-20525.
Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J,1987,6(13):3901-3907.
Jefferson R A. The GUS reporter gene system. Nature,1989,342(6251):837-838.
Jefferson RA.Assaying chimeric genes in plants:the GUS gene fusion system. Plant Mol Biol Rep.1987,5:387-405.
Ji K, Chen P, Sun L, et al. Non-climacteric ripening in strawberry fruit is linked to ABA, FaNCED2 and FaCYP707A1. Functional Plant Biology,2012,39:351-357.
Jia H F, Chai Y M, Li C L, et al. Abscisic acid plays an important role in the regulation of strawberry fruit ripening. Plant Physiol,2011,157(1):188-199.
Jonak C, Okresz L, Bogre L, et al. Complexity, cross talk and integration of plant MAP kinase signalling. Curr Opin Plant Biol,2002,5(5):415-424. Josefsson L G, Rask L. Cloning of a putative G-protein-coupled receptor from Arabidopsis thaliana. Eur J Biochem,1997,249(2):415-420.
Kerk D, Bulgrien J, Smith D W, et al. The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis. Plant Physiol,2002,129(2):908-925.
Kim J, Somers D E. Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts. Plant Physiol,2010,154(2):611-621.
Kim M J, Shin R, Schachtman D P. A nuclear factor regulates abscisic acid responses in Arabidopsis. Plant Physiol,2009,151(3):1433-1445.
Knetsch M, Wang M, Snaar-Jagalska B E, et al. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts. Plant Cell,1996,8(6):1061-1067.
Knight M R, Campbell A K, Smith S M, et al. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature,1991,352(6335):524-526.
Krens F A,Molendijk L,Wullems G L.In vitro transformation of plant protoplasts with Ti-plasmid DNA.Nature,1982,296:72-74.
Lee J S, Ellis B E. Arabidopsis MAPK phosphatase 2 (MKP2) positively regulates oxidative stress tolerance and inactivates the MPK3 and MPK6 MAPKs. J Biol Chem,2007,282(34):25020-25029.
Lee J S, Wang S, Sritubtim S, et al. Arabidopsis mitogen-activated protein kinase MPK12 interacts with the MAPK phosphatase IBR5 and regulates auxin signaling. Plant J,2009,57(6):975-985.
Lee Y R, Assmann S M. Arabidopsis thaliana 'extra-large GTP-binding protein' (AtXLGl):a new class of G-protein. Plant Mol Biol,1999,40(1):55-64.
Lescot M, Dehais P, Thijs G, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 2002,30(1):325-327.
Lessard P, Kreis M, Thomas M. Protein phosphatases and protein kinases in higher plants. C R Acad Sci Ⅲ,1997,320(9):675-688.
Li H, Lin Y, Heath R M, et al. Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell,1999,11(9):1731-1742.
Li H, Wu G, Ware D, et al. Arabidopsis Rho-related GTPases:differential gene expression in pollen and polar localization in fission yeast. Plant Physiol,1998,118(2):407-417.
Li Q, Ji K, Sun Y, et al. The role of FaBG3 in fruit ripening and B. cinerea fungal infection of strawberry. Plant J,2013,76(1):24-35.
Ligterink W, Hirt H. Mitogen-activated protein (MAP) kinase pathways in plants:versatile signaling tools. Int Rev Cytol,2001,201:209-275.
Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods,2001,25(4):402-408.
Lu C, Han M H, Guevara-Garcia A, et al. Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid. Proc Natl Acad Sci USA, 2002,99(24):15812-15817.
Luan S. Protein phosphatases in plants. Annu Rev Plant Biol,2003,54:63-92.
Luan S. Tyrosine phosphorylation in plant cell signaling. Proc Natl Acad Sci USA,2002, 99(18):11567-11569.
Luehrsen K R, de Wet J R, Walbot V. Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol,1992,216:397-414.
Ma H, Yanofsky M F, Meyerowitz E M. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc Natl Acad Sci USA, 1990,87(10):3821-3825.
Ma Y, Szostkiewicz I, Korte A, et al. Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science,2009,324(5930):1064-1068.
MacRobbie E A. Evidence for a role for protein tyrosine phosphatase in the control of ion release from the guard cell vacuole in stomatal closure. Proc Natl Acad Sci USA,2002,99(18):11963-11968.
Maehama T, Taylor G S, Dixon J E. PTEN and myotubularin:novel phosphoinositide phosphatases. Annu Rev Biochem,2001,70:247-279.
Mason M G, Botella J R. Completing the heterotrimer:isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA. Proc Natl Acad Sci USA, 2000,97(26):14784-14788.
Mason M G, Botella J R. Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with Gbeta. Biochim Biophys Acta,2001,1520(2):147-153.
Mizoguchi T, Ichimura K, Shinozaki K. Environmental stress response in plants:the role of mitogen-activated protein kinases. Trends Biotechnol,1997,15(1):15-19.
Mizoguchi T, Irie K, Hirayama T, et al. A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc Natl Acad Sci USA, 1996,93(2):765-769.
Monroe-Augustus M, Zolman B K, Bartel B. IBR5, a dual-specificity phosphatase-like protein modulating auxin and abscisic acid responsiveness in Arabidopsis. Plant Cell,2003,15(12):2979-2991.
Nagata T, Takebe I. Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta,1970,92(4):301-308.
Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol, 2005,56:165-185.
Neel B G, Tonks N K. Protein tyrosine phosphatases in signal transduction. Curr Opin Cell Biol, 1997,9(2):193-204.
Nehra N S, Chibbar R N, Kartha K K, et al. Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants.Plant Cell Rep,1990a,9(1):10-13.
Nehra N S, Chibbar R N, Kartha K K, et al. Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system. Plant Cell Rep,1990b,9(6):293-298.
Nuhse T S, Peck S C, Hirt H, et al. Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK6. J Biol Chem,2000,275(11):7521-7526.
Ochatt S J, Caso O J. Shoot regeneration from leaf mesophyll protoplasts of vild pear.Plant Physiology.1986,122:243-249.
Ortiz-Masia D, Perez-Amador M A, Carbonell J, et al. Diverse stress signals activate the Cl subgroup MAP kinases of Arabidopsis. FEBS Lett,2007,581(9):1834-1840.
Ou-Lee T M, Turgeon R, Wu R. Expression of a foreign gene linked to either a plant-virus or a Drosophila promoter, after electroporation of protoplasts of rice, wheat, and sorghum. Proc Natl Acad Sci USA,1986,83(18):6815-6819.
Owens C L, Iezzoni A F, Hancock J F, et al. CBF1 orthologs in sour cherry and strawberry and the heterologous expression of CBF1 in strawberry. J Amer Soc Hort Sci,2002,127(4):489-494.
Owens C L, Iezzoni A F, Hancock J F. Enhancement of freezing tolerance of strawberry by heterologous expression of CBF1. Acta Hort.2003,626:93-100.
Pandey S, Nelson D C, Assmann S M. Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell,2009,136(1):136-148.
Parihar D S,maheshwari S C.Influence of heat shock and UV irradiation on PEG-mediated DNA uptake and transient expression of NPT Ⅱgene in protoplasts of Brassicanapas.Indian J of Expbio,1998, 36(10):1002-1006.
Park S Y, Fung P, Nishimura N, et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science,2009,324(5930):1068-1071.
Parry A D, Babiano M J, Horgan R. The role of cis-carotenoids in abscisic acid biosynthesis. Planta, 1990,182(1):118-128.
Petersen M, Brodersen P, Naested H, et al. Arabidopsis map kinase 4 negatively regulates systemic acquired resistance. Cell,2000,103(7):1111-1120.
Qin X, Zeevaart J A. The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean. Proc Natl Acad Sci USA,1999,96(26):15354-15361.
Quettier A L, Bertrand C, Habricot Y, et al. The phs1-3 mutation in a putative dual-specificity protein tyrosine phosphatase gene provokes hypersensitive responses to abscisic acid in Arabidopsis thaliana. Plant J,2006,47(5):711-719.
Rakotondrafara A M, Jackson J R, Kneller E P, et al. Preparation and electroporation of oat protoplasts from cell suspension culture. Curr Protoc Microbiol,2007,Chapter 16:13D-16D.
Rasmussen J O,Rasmussen O S.PEG mediated DNA uptake and transient GUS expression in carrot,rapeseed and soybean protoplasts.Plant Sci,1993(89):199-207.
Rodrigo M J, Alquezar B, Zacarias L. Cloning and characterization of two 9-cis-epoxycarotenoid dioxygenase genes, differentially regulated during fruit maturation and under stress conditions, from orange (Citrus sinensis L. Osbeck). J Exp Bot,2006,57(3):633-643.
Rodrigo M J, Marcos J F, Alferez F, et al. Characterization of Pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content. J Exp Bot,2003,54(383):727-738.
Rudnicki R. Biosynthesis and metabolism of abscisic acid in plants. Postepy Biochem,1971, 17(1):43-55.
Sakamoto S, Fujikawa Y, Esaka M. Analysis of ascorbic acid biosynthesis using a simple transient gene expression system in tomato fruit protoplasts. Biosci Biotechnol Biochem,2013,77(3):673-675.
Santiago J, Rodrigues A, Saez A, et al. Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. Plant J,2009,60(4):575-588.
Schaffer R, Landgraf J, Accerbi M, et al. Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. Plant Cell,2001,13(1):113-123.
Schaffner A R, Sheen J. Maize rbcS promoter activity depends on sequence elements not found in dicot rbcS promoters. Plant Cell,1991,3(9):997-1012.
Schoenbeck M A, Samac D A, Fedorova M, et al. The alfalfa (Medicago sativa) TDY1 gene encodes a mitogen-activated protein kinase homolog. Mol Plant Microbe Interact, 1999,12(10):882-893.
Sheen J. Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell,1991,3(3):225-245.
Sheen J. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol, 2001,127(4):1466-1475.
Sheen, J.2002. A transient expression assay using maize mesophyll protoplasts.
Shimomura O, Johnson F H, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol, 1962,59:223-239.
Shinozaki K, Yamaguchi-Shinozaki K, Seki M. Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol,2003,6(5):410-417.
Spolaore S, Trainotti L, Casadoro G. A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium. Journal of Experimental Botany,2001,52:845-850.
Stone R L, Dixon J E. Protein-tyrosine phosphatases. J Biol Chem,1994,269(50):31323-31326.
Tan B C, Joseph L M, Deng W T, et al. Molecular characterization of the Arabidopsis 9-cis-epoxycarotenoid dioxygenase gene family. Plant J,2003,35(1):44-56.
Tan B C, Schwartz S H, Zeevaart J A, et al. Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA,1997,94(22):12235-12240.
Tanoue T, Adachi M, Moriguchi T, et al. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat Cell Biol,2000,2(2):110-116.
Tena G, Asai T, Chiu W L, et al. Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol,2001,4(5):392-400.
Thompson A J, Jackson A C, Symonds R C, et al. Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J, 2000,23(3):363-374.
Tonks N K, Neel B G. Combinatorial control of the specificity of protein tyrosine phosphatases. Curr Opin Cell Biol,2001,13(2):182-195.
Tonks N K. Protein tyrosine phosphatases:from genes, to function, to disease. Nat Rev Mol Cell Biol,2006,7(11):833-846.
Ulm R, Ichimura K, Mizoguchi T, et al. Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1. EMBO J,2002,21(23):6483-6493.
Van Vactor D,0,Reilly A M,Neel BG.Genetic analysis of Protein tyrosine phosphatase.Curr Opin Genet Develop,1998,8:112-126.
Wang J L, Ge H B, Peng S Q, et al. Transformation of strawberry (Fragariax ananassa Duch.) with late embryogenesis abundant protein gene. J Hort Sci Biotech,2004,79(5):735-738.
Wang X Q, Ullah H, Jones A M, et al. G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science,2001,292(5524):2070-2072.
Weiss C A, Garnaat C W, Mukai K, et al. Isolation of cDNAs encoding guanine nucleotide-binding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc Natl Acad Sci USA, 1994,91(20):9554-9558.
Weiss C A, Huang H, Ma H. Immunolocalization of the G protein alpha subunit encoded by the GPA1 gene in Arabidopsis. Plant Cell,1993,5(11):1513-1528.
Wenzler H C, Mignery G A, Fisher L M, et al. Analysis of a chimeric class-I patatin-GUS gene in transgenic potato plants:High-level expression in tubers and sucrose-inducible expression in cultured leaf and stem explants. Plant Mol Biol,1989,12(1):41-50.
Winge P, Brembu T, Bones A M. Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana. Plant Mol Biol,1997,35(4):483-495.
Winge P, Brembu T, Kristensen R, et al. Genetic structure and evolution of RAC-GTPases in Arabidopsis thaliana. Genetics,2000,156(4):1959-1971.
Xiong L, Schumaker K S, Zhu J K. Cell signaling during cold, drought, and salt stress. Plant Cell, 2002,14 Suppl:S165-S183.
Xiong L, Yang Y. Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell,2003,15(3):745-759.
Xu Q, Fu H H, Gupta R, et al. Molecular characterization of a tyrosine-specific protein phosphatase encoded by a stress-responsive gene in Arabidopsis. Plant Cell,1998,10(5):849-857.
Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol,2006,57:781-803.
Yang Z. Small GTPases:versatile signaling switches in plants. Plant Cell,2002,14 Suppl:S375-S388.
Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis. Nat Protoc,2007,2(7):1565-1572.
Yoshida R, Umezawa T, Mizoguchi T, et al. The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABIl and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J Biol Chem,2006,281(8):5310-5318.
Yuasa T, Ichimura K, Mizoguchi T, et al. Oxidative stress activates ATMPK6, an Arabidopsis homologue of MAP kinase. Plant Cell Physiol,2001,42(9):1012-1016.
Zhai Z, Sooksa-nguan T, Vatamaniuk O K. Establishing RNA interference as a reverse-genetic approach for gene functional analysis in protoplasts. Plant Physiol,2009,149(2):642-652.
Zhang H M, Yang H, Rech E L, et al. Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Rep,1988,7(6):379-384.
Zhang M, Yuan B, Leng P. The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit. J Exp Bot,2009,60(6):1579-1588.
Zhang S, Klessig D F. MAPK cascades in plant defense signaling. Trends Plant Sci, 2001,6(11):520-527.
Zheng Z L, Nafisi M, Tam A, et al. Plasma membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis. Plant Cell, 2002,14(11):2787-2797.
Zheng Z L, Yang Z. The Rop GTPase:an emerging signaling switch in plants. Plant Mol Biol, 2000,44(1):1-9.
Zhu J K. Salt and drought stress signal transduction in plants. Annu Rev Plant Biol, 2002,53:247-273.