转OsMAPK4、OsCDPK7基因耐盐碱水稻的筛选与抗性分析
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摘要
水稻是世界上重要的粮食作物,世界上3/4以上的人口以水稻为主食,但是低温、干旱、盐碱等因素严重影响水稻产量和品质,每年都会造成不同程度的损失。据统计,世界干旱、半干旱地区占地球陆地面积的33%,其危害相当于其它自然灾害之和。盐碱地占地球陆地面积的7.6%。另外,还有许多地区经常受低温冷害的影响。而我国约有670万公顷盐碱土耕地,还有2000万公顷盐碱荒地尚待开发。黑龙江省的盐碱地集中在松嫩平原地区,分布面积广,盐渍化程度高,其中尤以三肇、安达、大庆等地盐碱地分布最为集中,大面积的盐碱地严重影响农作物的产量,对黑龙江省的经济造成极大的损失。随着工业现代化的发展,灌溉地和塑料大棚面积不断扩大,土壤次生盐渍化日趋严重。随着人口的不断增加、耕地面积的日益减少和生态环境的急剧恶化,粮食供求矛盾将日益凸现,因此,如何提高作物抗干旱、盐渍和低温的能力是一个全世界都亟待解决的问题。
传统育种学家已经在提高植物抗逆性方面进行了长期的探索与研究,但常规育种方法存在着工作量大、周期长、耗资多、效率低、可利用基因资源有限等弊端,而通过基因工程手段进行分子育种则避免了以上缺点。近年来,随着对植物逆境胁迫的分子反应和信号传递机理研究的逐渐深入,研究者已发现从改良或增强一个关键调控基因的调控能力着手,也许是提高作物抗逆性更为有效的方法和途径,因此,进行这些调控基因的克隆和功能鉴定是植物抗逆基因工程研究的重要前提条件,筛选并获获得具有抗逆形状的转基因植株植物,为改良不断恶化的生态环境提供了重要依据。
本研究以本实验室获得的转钙依赖的蛋白激酶基因OsCDPK7和促分裂素原活化蛋白激酶基因OsMAPK4基因的T2代和T3代水稻作为试材,对T2代植株进行了耐盐碱的初步筛选,并研究了株高和死亡率；将收获的T3水稻,进行了纯合体的筛选及耐盐碱的抗逆性检测,同时研究了外源基因的表达情况和主要的耐盐碱性状。通过本研究可以获得具有耐盐碱的转基因水稻,用于黑龙江省盐碱地的改良,研究结果将为植物基因工程改良盐碱地提供了新思路,具有一定的理论和应用价值,为最终培育可在盐碱地正常生长的转基因水稻提供了重要资源。主要研究结果如下所示：
1转OsMAPK4、OsCDPK7基因水稻植株的遗传稳定性分析
1.1转OsMAPK4、OsCDPK7基因水稻植株PCR检测
对转OsMAPK4、OsCDPK7基因水T2代植株和T3代植株进行了PCR检测,PCR检测结果表明,转OsMAPK4、OsCDPK7基因水稻T2代植株和T3代植株均能扩增出目的基因；初步证明转基因水稻T2代植株和T3代植株中均含有目的基因,目的基因已经稳定遗传到后代。
1.2转基因抗性植株Southern杂交检测
为了进一步验证转OsMAPK4、OsCDPK7基因水稻的遗传稳定性,Southern杂交此部分试验正在进行中。
2转OsMAPK4、OsCDPK7基因水稻T：植株遗传特性的分析
对转OsMAPK4、OsCDPK7基因水稻T2代植株进行了耐盐碱的初步抗性检测,调查和分析了株高和死亡率,结果表明,转基因水稻的株高变化量大于对照水稻株高的变化量,转基因水稻的死亡率也低于对照水稻；其中E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株株高变化量大于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株株高变化量,E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株死亡率低于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代死亡率,转OsMAPK4、OsCDPK7基因水稻T2代植株具有较高的抗盐碱性。
3转OsMAPK4、OsCDPK7基因水稻T3植株遗传特性的分析
对转OsMAPK4、OsCDPK7基因水稻T3代植株进行了耐盐碱的抗性检测,调查和分析了株高和盐碱伤害指数,结果表明,转基因水稻T3代植株的株高变化量大于对照水稻株高的变化量,转基因水稻的盐碱伤害指数也明显低于对照水稻；E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株株高变化量大于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株株高变化量,E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株盐碱危害指数低于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代盐碱危害指数,具有较高的耐盐碱性,转OsMAPK4、OsCDPK7基因水稻T3代植株具有较高的抗盐碱性。
Rice is the world's important food crops,the 3/4 people of the world eat rice.But salinity, drought and low temperature and other abiotic stresses is the most harmful rice production and quality every year. According to statistics, the arid area and Semi-Arid Area accounts for 33 percent of the world.The damage is equivalent to the total of other natural disasters. Saline accounted for 7.6 percent of the land area. In addition, there are many areas often affected by low temperature stress.In China a total area of saline-alkali land is about 6.7 million hectares, includingmillion hectares potentials only saline-alkali land in Heilongjiang Province reached more than 9,000 hectares, has seriously hampered agricultural production in Heilongjiang Province. Relative to the changing external environment, enhance their salt and alkali resistance of plants to be more effective mitigation of the impact of salinity on the protection of the natural environment and the maintenance of world food production has an important role. Therefore, how to improve the ecological environment of plants to survive, how to improve the crop salinity, to achieve rapid agriculture, sustainable development has become an urgent need to resolve key issues.
Traditional breeders have carried out long-term exploration and study to improve plant resistance,but traditional breeding methods has the problem of huge workload, long growth periods and cost more,low efficiency, the genetic resources available, through such means of genetic engineering molecular breeding avoid above shortcomings.In recent years, with studying the stress on plant molecular reactions and adversity signal transmission mechanism,researchers have found that gradually improved or increase ability to control gene regulation, is perhaps the methods and ways to more effectively improve crops resistance, the regulation of gene cloning and function is the prerequisites of plant gene engineering research, screening and obtain the transgenic plants with resistibility,and provides an important basis for the worsening ecological environment.
In our study, utilized rd29A and E12 respectively promoter regulation from rice transformed with OsCDPK7 gene and OsMAPK4 gene of T2 and T3 transgenic plant as the tested varieties.Studying the preliminary screening of T2 generation of plants on salt or alkali stress for plant growth and plant death..homozygote.。Harvesting T3 transgenic plant,to the screening of homozygote and salt and alkali resistance testing, at the same time, research gene expression and the main characters of the salt and the alkali.Through this research we can be obtained transgenic rice with the ability to resist the alkali and saline, used in in Heilongjiang province. The results showed that provided a new idea to improve for plant genetic engineering modified saline and alkali, has certain theoretical and practical value. Providing an important resource for the final cultivation of normal growth of transgenic rice.The major findings are as follows:
1.Genetic Stability Analysis of Transgenic Rice
1.1 Genetic stability analyses of target genes in T2 generation and T3 generation
The genome DNA of T2 generation and T3 generation were determined by PCR analysis.The target genes were amplified in transgenic rice with PCR; which validated that the transgenic plants T2 generation and T3 generation co-integrated with OsMAPK4 and OCCDPK7 genes.
1.2.Southern blot
Southern blot, this part of its research is in progress.
2. Genetic Character Analysis to Transgenic Rice of T2 generation
Studying the preliminary screening of T2 generation of plants on salt or alkali stress for plant height and plant mortality homozygote.The results showed that the variation of height of transgenic rice more than the control and the mortality rate is below the control rice Which the variation of plant height of transgenic rice which is the regulation of E12 promoters OsMAPK4,OsCDPK7 gene more than the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.the plant mortality of transgenic Rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene was lower the transgenic Rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.
3. Genetic Character Analysis to Transgenic Rice of T3 generation
Studyed the screening of homozygote of transgenic rice of T3 generation and the screening of transgenic rice on salt or alkali resistance testing for the plant height and the index of salt or alkaline tolerance. The results showed that the variation of height of transgenic rice more than the control and the index of salt or alkaline tolerance.was below the control rice Which the variation of plant height of transgenic rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene more than the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene, the index of salt or alkaline tolerance.of transgenic rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene was lower the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.
传统育种学家已经在提高植物抗逆性方面进行了长期的探索与研究,但常规育种方法存在着工作量大、周期长、耗资多、效率低、可利用基因资源有限等弊端,而通过基因工程手段进行分子育种则避免了以上缺点。近年来,随着对植物逆境胁迫的分子反应和信号传递机理研究的逐渐深入,研究者已发现从改良或增强一个关键调控基因的调控能力着手,也许是提高作物抗逆性更为有效的方法和途径,因此,进行这些调控基因的克隆和功能鉴定是植物抗逆基因工程研究的重要前提条件,筛选并获获得具有抗逆形状的转基因植株植物,为改良不断恶化的生态环境提供了重要依据。
本研究以本实验室获得的转钙依赖的蛋白激酶基因OsCDPK7和促分裂素原活化蛋白激酶基因OsMAPK4基因的T2代和T3代水稻作为试材,对T2代植株进行了耐盐碱的初步筛选,并研究了株高和死亡率；将收获的T3水稻,进行了纯合体的筛选及耐盐碱的抗逆性检测,同时研究了外源基因的表达情况和主要的耐盐碱性状。通过本研究可以获得具有耐盐碱的转基因水稻,用于黑龙江省盐碱地的改良,研究结果将为植物基因工程改良盐碱地提供了新思路,具有一定的理论和应用价值,为最终培育可在盐碱地正常生长的转基因水稻提供了重要资源。主要研究结果如下所示：
1转OsMAPK4、OsCDPK7基因水稻植株的遗传稳定性分析
1.1转OsMAPK4、OsCDPK7基因水稻植株PCR检测
对转OsMAPK4、OsCDPK7基因水T2代植株和T3代植株进行了PCR检测,PCR检测结果表明,转OsMAPK4、OsCDPK7基因水稻T2代植株和T3代植株均能扩增出目的基因；初步证明转基因水稻T2代植株和T3代植株中均含有目的基因,目的基因已经稳定遗传到后代。
1.2转基因抗性植株Southern杂交检测
为了进一步验证转OsMAPK4、OsCDPK7基因水稻的遗传稳定性,Southern杂交此部分试验正在进行中。
2转OsMAPK4、OsCDPK7基因水稻T：植株遗传特性的分析
对转OsMAPK4、OsCDPK7基因水稻T2代植株进行了耐盐碱的初步抗性检测,调查和分析了株高和死亡率,结果表明,转基因水稻的株高变化量大于对照水稻株高的变化量,转基因水稻的死亡率也低于对照水稻；其中E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株株高变化量大于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株株高变化量,E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代植株死亡率低于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T2代死亡率,转OsMAPK4、OsCDPK7基因水稻T2代植株具有较高的抗盐碱性。
3转OsMAPK4、OsCDPK7基因水稻T3植株遗传特性的分析
对转OsMAPK4、OsCDPK7基因水稻T3代植株进行了耐盐碱的抗性检测,调查和分析了株高和盐碱伤害指数,结果表明,转基因水稻T3代植株的株高变化量大于对照水稻株高的变化量,转基因水稻的盐碱伤害指数也明显低于对照水稻；E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株株高变化量大于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株株高变化量,E12启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代植株盐碱危害指数低于rd29A启动子调控的转OsMAPK4、OsCDPK7基因水稻T3代盐碱危害指数,具有较高的耐盐碱性,转OsMAPK4、OsCDPK7基因水稻T3代植株具有较高的抗盐碱性。
Rice is the world's important food crops,the 3/4 people of the world eat rice.But salinity, drought and low temperature and other abiotic stresses is the most harmful rice production and quality every year. According to statistics, the arid area and Semi-Arid Area accounts for 33 percent of the world.The damage is equivalent to the total of other natural disasters. Saline accounted for 7.6 percent of the land area. In addition, there are many areas often affected by low temperature stress.In China a total area of saline-alkali land is about 6.7 million hectares, includingmillion hectares potentials only saline-alkali land in Heilongjiang Province reached more than 9,000 hectares, has seriously hampered agricultural production in Heilongjiang Province. Relative to the changing external environment, enhance their salt and alkali resistance of plants to be more effective mitigation of the impact of salinity on the protection of the natural environment and the maintenance of world food production has an important role. Therefore, how to improve the ecological environment of plants to survive, how to improve the crop salinity, to achieve rapid agriculture, sustainable development has become an urgent need to resolve key issues.
Traditional breeders have carried out long-term exploration and study to improve plant resistance,but traditional breeding methods has the problem of huge workload, long growth periods and cost more,low efficiency, the genetic resources available, through such means of genetic engineering molecular breeding avoid above shortcomings.In recent years, with studying the stress on plant molecular reactions and adversity signal transmission mechanism,researchers have found that gradually improved or increase ability to control gene regulation, is perhaps the methods and ways to more effectively improve crops resistance, the regulation of gene cloning and function is the prerequisites of plant gene engineering research, screening and obtain the transgenic plants with resistibility,and provides an important basis for the worsening ecological environment.
In our study, utilized rd29A and E12 respectively promoter regulation from rice transformed with OsCDPK7 gene and OsMAPK4 gene of T2 and T3 transgenic plant as the tested varieties.Studying the preliminary screening of T2 generation of plants on salt or alkali stress for plant growth and plant death..homozygote.。Harvesting T3 transgenic plant,to the screening of homozygote and salt and alkali resistance testing, at the same time, research gene expression and the main characters of the salt and the alkali.Through this research we can be obtained transgenic rice with the ability to resist the alkali and saline, used in in Heilongjiang province. The results showed that provided a new idea to improve for plant genetic engineering modified saline and alkali, has certain theoretical and practical value. Providing an important resource for the final cultivation of normal growth of transgenic rice.The major findings are as follows:
1.Genetic Stability Analysis of Transgenic Rice
1.1 Genetic stability analyses of target genes in T2 generation and T3 generation
The genome DNA of T2 generation and T3 generation were determined by PCR analysis.The target genes were amplified in transgenic rice with PCR; which validated that the transgenic plants T2 generation and T3 generation co-integrated with OsMAPK4 and OCCDPK7 genes.
1.2.Southern blot
Southern blot, this part of its research is in progress.
2. Genetic Character Analysis to Transgenic Rice of T2 generation
Studying the preliminary screening of T2 generation of plants on salt or alkali stress for plant height and plant mortality homozygote.The results showed that the variation of height of transgenic rice more than the control and the mortality rate is below the control rice Which the variation of plant height of transgenic rice which is the regulation of E12 promoters OsMAPK4,OsCDPK7 gene more than the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.the plant mortality of transgenic Rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene was lower the transgenic Rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.
3. Genetic Character Analysis to Transgenic Rice of T3 generation
Studyed the screening of homozygote of transgenic rice of T3 generation and the screening of transgenic rice on salt or alkali resistance testing for the plant height and the index of salt or alkaline tolerance. The results showed that the variation of height of transgenic rice more than the control and the index of salt or alkaline tolerance.was below the control rice Which the variation of plant height of transgenic rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene more than the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene, the index of salt or alkaline tolerance.of transgenic rice which is the regulation of E12 promoters OsMAPK4, OsCDPK7 gene was lower the transgenic rice which is the regulation of rd29A promoters OsMAPK4, OsCDPK7 gene.
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张树珍,王自章.2001.植物耐旱的分子基础及植物耐旱基因工程的研究进展.生命科学研究.5(1)：134～139.
张秀海,黄丛林,沈元月,曹鸣庆.2001.植物抗旱基因工程研究进展.生物技术通报.4：21～25.
赵恢武,陈杨坚.2000.干旱诱导性启动子驱动的海藻糖-6-磷酸合酶基因载体的构建及转基因烟草的耐旱性.植物学报.42(6)：616～619.
赵可夫.2002.植物对盐渍逆境的适应.生物学通报.37(6)：7-10.
周志琦,刘强.1998.真核生物的MAPK级联信号传递途径.生物化学与生物物理进展.25(6)：496～503.
Abbasi F, Onodera H, Toki S, Tanaka H, Komatsu S.2004. OsCDPK13,a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath. Plant Mol Biol.55(4):541-52.
Agrawal GK,Rakwal R,Iwahashi H.2002.Isolation of novel rice multiple stress responsive MAP kinase gene,OsMSRMK2,whose mRNA accumulates rapidly in response to environmental cues.Biochem Biophys Res Commun.294(5):1009-1016.
Anil VS,Harmon AC,Rao KS.2000.Spatic-temporal accumulation and activity of calcium-dependent protein kinase during embrycgenesis,seed development and germination in sandalwood.Plant Physiol.122:1035-1043.
Anil VS,Rao KS.2001.Calcium-mediated signal transduction in plant:a perspective on the role of Ca2+ and CDPKs during early plant development.J Plant Physiol.158: 1237～1256.
Anil VS,Rao KS.2001.Purification and characterization of a Ca2+-dependent protein kinase from sandalwood(Santalum album L):evidence for Ca2+-induced conformational changes.Phytochemistr.58:203-212.
Atsushi Sakamoto and Norio Murata.2000.Genetic engineering of glycinebetaine synthesis in plants current status and implications for enhancement of stress tolerance.Journal of Experimental Botany.51:81～88.
Cao Zhifang,Chen Feng,Li Jie.2001.TINY,a DREB-Like Transcription Factor Binding to DRE Element.Tsinghua science and technology.6(5):432-437.
Cheng SH,Willmann MR,Chen HC,Sheen J.2002.Calcium signaling through protein kinase.The Arabidopsis calcium-dependent protein kinase gene family.Plant Physiol.129: 469-485.
Chico JM,Raices M,Tellez-Inon MT,Ulloa RM.2002.A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants.Plant Physiol.128:256-270.
Choi TY,Oh Y,Yoo MA,Matsukage A,Ryu Y Han K,Yoon J,Baek K,Chc NY.2000.The DNA replication-related element(DRE)—DRE-binding factor(DREF) systemmay be involved in the expression of the Drosophila melanogaster TBP gene.FEBS Letters.483(1):71-77.
Harmon AC,Gribskov M,Harper JF.2000.CDPK——a kinase for every Ca2+ signal? Trends Plant Sci.5:154-159
Harmon AC,Gribskov M,Gubrium E,Harper JF.2001.CDPK superfamily of protein kinases.New Phytol.151:175-183
Harper JF,Braton G,Harmon A.2004.Decoding Ca2+ Signal through plant protein kinases.Annu Rev Plant Biol.55:263-288
Kasuga M,Liu Q,Miura S,et al.1999.Improving plant drought,salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor.Nature Biotechnology.17:287-292.
Kodama H.1994.Genetic enchancement of cold tolerance by expression of a gene for chloroplastco-3 fatty acid desaturase in transgenic tobacco.Plant Physiol.105:601-610.
Lee JH,Montagu MV,Verbruggen N.1999.A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells.Proc Natl Acad Sci USA.96: 5873-5877.
Lilius G,Holmberg N,Bulow L.1996.Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase.Bio Technology.14:177-180.
Liming Xiong,Manabu Ishitani,and Jian-Kang Zhu.1999.Interaction of Osmotic Stress,Temperature,and Abscisic Acid in the Regulation of Gene Expression in Arabidopsis.Plant Physiol.119:205～212
Liu L,White MJ,MacRae TH.1999.Transcription factors and their genes in higher plants.Eur J Biochem.262:247-257
Liu Q,Kasuga M,Sakuma Y,et al.1998.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis.Plant Cell.10:1391-1406
M Ishitani,L Xiong,B Stevenson and JK Zhu.1997.Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis;interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways.Plant Cell.9:1935～1949
Martin ML,Busconi L.2000.Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation.Plant J.24:429～435
McGrath RB,Echer JR.1998.Ethylene signaling in Arabidopsis:events from the membrane to the nucleus.Plant Physiol Biochem.36(1-2):103～113
Mckersie BD,Chen Y,Beus M.1993.Superoxide dismutase enhance tolerance of freezing stress in transgenic alfalfa.Plant Physiology.103:1155～1163
Meskiene I,et al.2000.MAP kinase pathways:molecular plug-and-play chip for the cell.Plant Mol Biol.42:791-806
Ning J, Yuan B,Xie KB,Hu HH, Wu CQ, Xiong LZ.2006.Isolation and identification of SA and JA inducible protein kinase gene OsSJMKl in rice. Yi Chuan Xue Bao.33(7):625-33.
Nakashima K,Sakuma Y,Seki M,Miura S,Shinozaki K, Yamaguchi-Shinozaki.2000.Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration-and high-salinity-responsive gene expression.Plant Molecular Biology.42(4):657～665
Patharkar OR,Cushman JC.2000.A stress-induced calcium-dependent protein kinase from Mesombryanthemum crystallinum phosphoryltes a two-component pseudo-response regulator.Plant J.24:679～691
Qiang Liu,Jin Li,Guiyou Zhang.1999.Isolation of cDNAs Encoding Two Distinct Transcription Factors Binding to DRE cis-acting Element Involved in Cold-and drought-induced Expression of Arabidopsis rd29A Gene.Tsinghua science and technology.4 (3):1551-1559
Romeis T,Ludwig AA,Martin R,Jones JDG.2001.Calcium-dependent protein kinase play an essential role in a plant defense response.EMBOJ.20:5556～5567
Romeis T,Piedras P,Jones JDG.2000.Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response.Plant Cell.12:803～815
Saijo Y,Hata S,Kyczuka J,Shimancto K,Izui K.2000.Over-expression of a singal Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plant.Plant J.23:319-327
Saijo Y,Kinoshita N,Ishiyama K,Hata S,Kyozuka J,Hayakawa T,Nakamura T, Shimamoto K,Yamaya T,Izui K.2001.A Ca2+-dependent protein kinase that endows rice plant with cold-and salt-stress tolerance functions in vascular bundles.Plant Cell Physiol.42: 1228-1233
Schenk PW,Snaar-Jagalska BE.1999.Signal perception and transduction:the role of protein kinase.Biochim Biophys Acta.1449:1-24
Schwechheimer C,Bevan M.1998.The regulation of transcription factor activity in plant.Trend in Plant Science.3(10):278-283
Thomashow MF.1999.Plant clod acclimation:Freezing Tolerance Genes and Regulatory Mechanisms.Annu. Rev. Plant Physiol Plant Mol.Biol.50:571～599
Urao T,Yakubov B,Satoh R,Yamaguchi-Shinozaki K,Seki M,Hirayama T,Shinozaki K.1999.A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor.Plant Cell.11(9):1743～54
Urao T,Yamaguchi-Shinozaki K,and Shinozaki K.2000.Two-component systems in plant signal transduction.Trends Plant Sci.5:67～74
Xu D,Duan X,Wang B,Hong B,Ho T,Wu R.1996.Expression of a Late Embryogenesis Abundant Protein Gene,HVA1,from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice.Plant Physiol.110(1):249～257
Yamaguchi-shinozaki K,et al.1998.Stress response of photo syntheticorganisms.New York:Academic Press.13:158～163
杜秀敏,殷文璇,赵彦修,张慧.2001.植物中活性氧的产生及清除机制.生物工程学报.17(3)：121～125.
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侯丙凯,于惠敏,夏光敏.2002.用于叶绿体遗传转化的表达载体.遗传.24(1)：100-103.
胡风庆.2002.MAPK对高等植物细胞分裂和生长的调节.中国生物工程杂志.22(5)：58～64.
江香梅,黄敏仁,王明庥.2001.植物抗盐碱、耐干旱基因工程研究进展.南京林业大学学报(自然科学版).25(5)：57～62.
焦芳婵,毛雪,李润植.2001.转基因改良植物的胁迫耐性.生物技术通讯.12(2)：135～139.
李秋莉,杨华,等.2002.植物甜菜碱合成酶的分子生物学和基因工程.生物工程进展.22(1)：84-86.
李一琨,王金发.1998.高等植物启动子研究进展.植物学通报.15(增刊)：1-6.
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刘贯山,陈珈.2003.钙依赖蛋白激酶(CDPKs)在植物钙信号转导中的作用.植物学通报.20(2)：160～167.
刘强,张贵友,陈受宜.2000.植物转录因子的结构与调控作用.科学通报.45(14)：1465～1474.
刘强,张勇,陈受宜.2000.干旱、高盐及低温诱导的植物蛋白激酶基因.科学通报.45(6)：561～566.
刘强,赵南明等.2000.DREB转录因子在提高植物抗逆性中的作用.科学通报.45(1)：11～16.
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彭志红,彭克勤等.2002.渗透胁迫下植物脯氨酸积累的研究进展.中国农学通报.18(4)：80～83.
邱全胜.2000.双组分系统—细胞识别渗透胁迫信号的感应器.生物化学与生物物理进展.27(6)：593～596.
萨姆布鲁克,拉塞尔.2002.分子克隆实验指南.黄培堂等译.科学出版社.
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苏金,朱汝财.2001.渗透胁迫调节的转基因表达对植物抗旱耐盐性的影响.植物学通报.18(2)：129～136.
王关林等.2002.植物基因工程(第二版).科学出版社.
熊清,王伯初,段传人.2000.植物抗脱水胁迫的分子机制.生物化学与生物物理进展.27(3)：247～250.
颜华,贾良辉,王根轩.2002.植物水分胁迫诱导蛋白的研究进展.生命的化学.20(2)：165～168.
杨洪强,梁小娥.2001.蛋白激酶与植物逆境信号传递途径.植物生理学通讯.37(3)：185～191.
杨洪强,梁小娥.2003.高等植物中的CDPK及其生理生化功能.山东农业大学学报(自然科学版).34(2)：284～288.
余光辉,李玲,曾福华.2002.水分胁迫的基因表达和信号转导(综述).亚热带植物科学.31(1)：57～62.
张树珍,王自章.2001.植物耐旱的分子基础及植物耐旱基因工程的研究进展.生命科学研究.5(1)：134～139.
张秀海,黄丛林,沈元月,曹鸣庆.2001.植物抗旱基因工程研究进展.生物技术通报.4：21～25.
赵恢武,陈杨坚.2000.干旱诱导性启动子驱动的海藻糖-6-磷酸合酶基因载体的构建及转基因烟草的耐旱性.植物学报.42(6)：616～619.
赵可夫.2002.植物对盐渍逆境的适应.生物学通报.37(6)：7-10.
周志琦,刘强.1998.真核生物的MAPK级联信号传递途径.生物化学与生物物理进展.25(6)：496～503.
Abbasi F, Onodera H, Toki S, Tanaka H, Komatsu S.2004. OsCDPK13,a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath. Plant Mol Biol.55(4):541-52.
Agrawal GK,Rakwal R,Iwahashi H.2002.Isolation of novel rice multiple stress responsive MAP kinase gene,OsMSRMK2,whose mRNA accumulates rapidly in response to environmental cues.Biochem Biophys Res Commun.294(5):1009-1016.
Anil VS,Harmon AC,Rao KS.2000.Spatic-temporal accumulation and activity of calcium-dependent protein kinase during embrycgenesis,seed development and germination in sandalwood.Plant Physiol.122:1035-1043.
Anil VS,Rao KS.2001.Calcium-mediated signal transduction in plant:a perspective on the role of Ca2+ and CDPKs during early plant development.J Plant Physiol.158: 1237～1256.
Anil VS,Rao KS.2001.Purification and characterization of a Ca2+-dependent protein kinase from sandalwood(Santalum album L):evidence for Ca2+-induced conformational changes.Phytochemistr.58:203-212.
Atsushi Sakamoto and Norio Murata.2000.Genetic engineering of glycinebetaine synthesis in plants current status and implications for enhancement of stress tolerance.Journal of Experimental Botany.51:81～88.
Cao Zhifang,Chen Feng,Li Jie.2001.TINY,a DREB-Like Transcription Factor Binding to DRE Element.Tsinghua science and technology.6(5):432-437.
Cheng SH,Willmann MR,Chen HC,Sheen J.2002.Calcium signaling through protein kinase.The Arabidopsis calcium-dependent protein kinase gene family.Plant Physiol.129: 469-485.
Chico JM,Raices M,Tellez-Inon MT,Ulloa RM.2002.A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants.Plant Physiol.128:256-270.
Choi TY,Oh Y,Yoo MA,Matsukage A,Ryu Y Han K,Yoon J,Baek K,Chc NY.2000.The DNA replication-related element(DRE)—DRE-binding factor(DREF) systemmay be involved in the expression of the Drosophila melanogaster TBP gene.FEBS Letters.483(1):71-77.
Harmon AC,Gribskov M,Harper JF.2000.CDPK——a kinase for every Ca2+ signal? Trends Plant Sci.5:154-159
Harmon AC,Gribskov M,Gubrium E,Harper JF.2001.CDPK superfamily of protein kinases.New Phytol.151:175-183
Harper JF,Braton G,Harmon A.2004.Decoding Ca2+ Signal through plant protein kinases.Annu Rev Plant Biol.55:263-288
Kasuga M,Liu Q,Miura S,et al.1999.Improving plant drought,salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor.Nature Biotechnology.17:287-292.
Kodama H.1994.Genetic enchancement of cold tolerance by expression of a gene for chloroplastco-3 fatty acid desaturase in transgenic tobacco.Plant Physiol.105:601-610.
Lee JH,Montagu MV,Verbruggen N.1999.A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells.Proc Natl Acad Sci USA.96: 5873-5877.
Lilius G,Holmberg N,Bulow L.1996.Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase.Bio Technology.14:177-180.
Liming Xiong,Manabu Ishitani,and Jian-Kang Zhu.1999.Interaction of Osmotic Stress,Temperature,and Abscisic Acid in the Regulation of Gene Expression in Arabidopsis.Plant Physiol.119:205～212
Liu L,White MJ,MacRae TH.1999.Transcription factors and their genes in higher plants.Eur J Biochem.262:247-257
Liu Q,Kasuga M,Sakuma Y,et al.1998.Two transcription factors,DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis.Plant Cell.10:1391-1406
M Ishitani,L Xiong,B Stevenson and JK Zhu.1997.Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis;interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways.Plant Cell.9:1935～1949
Martin ML,Busconi L.2000.Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation.Plant J.24:429～435
McGrath RB,Echer JR.1998.Ethylene signaling in Arabidopsis:events from the membrane to the nucleus.Plant Physiol Biochem.36(1-2):103～113
Mckersie BD,Chen Y,Beus M.1993.Superoxide dismutase enhance tolerance of freezing stress in transgenic alfalfa.Plant Physiology.103:1155～1163
Meskiene I,et al.2000.MAP kinase pathways:molecular plug-and-play chip for the cell.Plant Mol Biol.42:791-806
Ning J, Yuan B,Xie KB,Hu HH, Wu CQ, Xiong LZ.2006.Isolation and identification of SA and JA inducible protein kinase gene OsSJMKl in rice. Yi Chuan Xue Bao.33(7):625-33.
Nakashima K,Sakuma Y,Seki M,Miura S,Shinozaki K, Yamaguchi-Shinozaki.2000.Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration-and high-salinity-responsive gene expression.Plant Molecular Biology.42(4):657～665
Patharkar OR,Cushman JC.2000.A stress-induced calcium-dependent protein kinase from Mesombryanthemum crystallinum phosphoryltes a two-component pseudo-response regulator.Plant J.24:679～691
Qiang Liu,Jin Li,Guiyou Zhang.1999.Isolation of cDNAs Encoding Two Distinct Transcription Factors Binding to DRE cis-acting Element Involved in Cold-and drought-induced Expression of Arabidopsis rd29A Gene.Tsinghua science and technology.4 (3):1551-1559
Romeis T,Ludwig AA,Martin R,Jones JDG.2001.Calcium-dependent protein kinase play an essential role in a plant defense response.EMBOJ.20:5556～5567
Romeis T,Piedras P,Jones JDG.2000.Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response.Plant Cell.12:803～815
Saijo Y,Hata S,Kyczuka J,Shimancto K,Izui K.2000.Over-expression of a singal Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plant.Plant J.23:319-327
Saijo Y,Kinoshita N,Ishiyama K,Hata S,Kyozuka J,Hayakawa T,Nakamura T, Shimamoto K,Yamaya T,Izui K.2001.A Ca2+-dependent protein kinase that endows rice plant with cold-and salt-stress tolerance functions in vascular bundles.Plant Cell Physiol.42: 1228-1233
Schenk PW,Snaar-Jagalska BE.1999.Signal perception and transduction:the role of protein kinase.Biochim Biophys Acta.1449:1-24
Schwechheimer C,Bevan M.1998.The regulation of transcription factor activity in plant.Trend in Plant Science.3(10):278-283
Thomashow MF.1999.Plant clod acclimation:Freezing Tolerance Genes and Regulatory Mechanisms.Annu. Rev. Plant Physiol Plant Mol.Biol.50:571～599
Urao T,Yakubov B,Satoh R,Yamaguchi-Shinozaki K,Seki M,Hirayama T,Shinozaki K.1999.A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor.Plant Cell.11(9):1743～54
Urao T,Yamaguchi-Shinozaki K,and Shinozaki K.2000.Two-component systems in plant signal transduction.Trends Plant Sci.5:67～74
Xu D,Duan X,Wang B,Hong B,Ho T,Wu R.1996.Expression of a Late Embryogenesis Abundant Protein Gene,HVA1,from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice.Plant Physiol.110(1):249～257
Yamaguchi-shinozaki K,et al.1998.Stress response of photo syntheticorganisms.New York:Academic Press.13:158～163