【作者】 王明帮；

【导师】 罗利军； 张端品；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 硕士

【摘要】 促分裂原活化蛋白激酶（Mitogen-activated protein kinase,MAPK）途径在植物生长发育和生物与非生物胁迫响应方面起到了重要的作用。在水稻中,促分裂原活化蛋白激酶激酶基因（MAPKKs）的抗逆功能依然不清楚。本研究通过表达序列标签（EST）和cDNA的数据比对的方法搜寻水稻数据库,得到了9个水稻促分裂原活化蛋白激酶激酶基因（OsMKKs）。本研究的主要内容如下:1、OsMKKs在不同器官下的特异表达研究:通过荧光定量PCR检测了OsMKKs在日本晴不同器官（叶,根和花）下的表达情况,发现其表达具有组织特异性。OsMKK1,OsMKK3,OsMKK4,OsMKK5,OsMKK6和OsMKK10-2在叶,根和花中都有表达,在根中表达有差异。而OsMKK8,OsMKK9和OsMKK10-1在不同器官中的表达丰度很低,主要在根和花中表达。2、OsMKKs在生物或非生物胁迫下的表达研究。分别用双氧水（H₂O₂）,水杨酸（Sa]icylic acid,SA）,脱落酸（abscisic acid,ABA）,甲基茉莉酸（methyl jasmonic acid,Me-JA）,甘露醇,氯化钠和低温（4℃）模拟各种不同的逆境,采用荧光定量PCR检测OsMKKs在不同逆境处理下的表达情况。发现不同OsMKKs的表达具有时空特异性。全基因组表达分析表明:OsMKK1在七种逆境信号处理下均有上升表达,且30分钟之前到达最高表达,具有早期表达特性。在H₂O₂,SA,Mannitiol,NaCl诱导下在根中启动表达要早,且表达量较高。而在ABA,Me-JA,低温诱导下则相反。OsMKK3在七种逆境信号处理下表达情况比较复杂,在Mannitiol,NaCl和低温诱导下表达量大幅度上升,在SA,ABA和Me-JA诱导下叶和根中表达模式不同,在SA诱导的早期,根中表达上升而叶中表达下降,ABA和Me-JA诱导下则相反。在H₂O₂处理下早期下降表达。OsMKK4在H₂O₂,MeJA和Mannitiol诱导下早期有上升表达,在ABA,NaCl和低温（4℃）诱导下早期在叶中有上升表达,在根中则相反。在SA诱导下早期下降表达。OsMKK5在H₂O₂,SA,ABA,Me-JA,Mannitiol和NaCl诱导下有上升表达。其中在H₂O₂诱导下晚期表达,NaCl和ABA诱导下早期表达;在SA,Mannitiol诱导下在根中早期表达,叶中晚期表达,而Me-JA诱导下则相反。低温（4℃）诱导下在叶中有上升表达,在根中下降表达。OsMKK6在H₂O₂、SA、ABA、Me-JA、Mannitiol、NaCl和低温诱导下均有上升表达。其中SA,NaCl和低温诱导下早期表达,在H₂O₂诱导下晚期表达;在ABA,Mannitiol诱导下在根中早期表达,叶中晚期表达,而Me-JA诱导下则相反。OsMKK8,OsMKK9在不同处理下均没有检测到表达。OsMKK10-1在H₂O₂,SA,NaCl和低温诱导下均有上升表达。其中在H₂O₂诱导下晚期表达,SA,NaCl和低温诱导下早期表达。OsMKK10-2在H₂O₂诱导下有上升表达,其中在H₂O₂诱导下根中早期表达,叶中晚期表达。在低温诱导的早期下降表达,在SA,ABA,Me-JA,Mannitiol和NaCl诱导的早期在叶和根中的表达情况不同,其中在ABA,Me-JA诱导下叶中上升表达,在根中下降表达;其他则相反。3、克隆了OsMKK3,构建了超表达和抑制表达载体,并分别被用于转化水稻、烟草和拟南芥。更多还原

【Abstract】 Mitogen-activated protein kinase（MAPK） cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses.In rice（Oryza sativa）,a monocot model and economically important cereal crop,only two MAPKKs were characterized.In this study.We have identified 9 members of the rice MAPKKs family through an EST and cDNA hitting search of rice genome databases.Results of the experiment is as following.1.Upon real time PCR,we analyze the difference of expression of OsMKKs under different organ（leaf,root and flower）.The result indicate that OsMKKI,OsMKK3, OsMKK4,OsMKK5,OsMKK6 or OsMKK10-2 confer expression in shoot root and flower, and the expression is varied in root.OsMKK8,OsMKK9 or OsMKK10-1 confer low expression in diferent organ,they expressed mainly in root and flower.2.Upon elicitor inducing with the different signal molecules such as H₂O₂,salicylic acid（SA）,abscisic acid（ABA）,methyl jasmonic acid（MeJA）,Mannitiol,NaCl and cold（4℃） treatment.8 of 90sMKKs genes were found to be induced at the mRNA level during either early,late,or both stages of treatment.The genome-wide expression analysis suggests that:OsMKK1 confers strong expression under the induction of H₂O₂,SA,ABA,MeJA, Mannitiol,NaCl or Cold（4℃）and early expression before 30 minutes.When treated with H₂O₂,SA,Mannitiol,NaCl,the expression of OsMKK1 in the root was earlier and higher than in the shoot.But the expression of OsMKK1 under the induction of ABA,Me-JA is on the contray.OsMKK3 confers complex expression under the induction of H₂O₂,SA,ABA, MeJA,Mannitiol,NaCl or Cold（4℃）.OsMKK3 confers strong expression under the induction of Mannitiol,NaCl or Cold（4℃）.OsMKK3 confers different expression under the induction of SA,ABA and Me-JA between leaves and roots of,in the early time when treated with SA,the expression rised in the root but declined in the shoot,the expression of ABA and Me-JA in on the contrary.OsMKK3 confers fall expression when treated with H₂O₂.OsMKK4 confers early strong expression under the induction of H₂O₂,MeJA and Mannitiol.The early expression of OsMKK4 treated with ABA,NaC1 and low temperature（4℃） is different between shoot and root,it rised in the root and declined in the shoot OsMKK4 confers early fall expression when treated with SA.OsMKK5 confers early strong expression under the induction of H₂O₂,SA,ABA, Me-JA,Mannitiol and NaCl.It expressed late when treated with H202,and early when treated with ABA and NaCl.When treated with SA and Mannitiol,it expressed early in the root and late in the shoot,it expressed on the contray when treated with Me-JA.When treated with low temperature（4℃）,it confers rise expression in the shoot and fall expression in the root.OsMKK6 confers strong expression under the induction of H₂O₂,SA,ABA,MeJA, Mannitiol,NaCl or Cold（4℃）.It expressed late when treated with H₂O₂ and early when treated with SA,NaCl or Cold（4℃）.It expressed different between shoot and root when treated with the ABA or Mannitiol.OsMKK8 and OsMKK9 confers no expression under the different inductionOsMKK10-1 confers strong expression under the induction of H₂O₂,SA,NaCl and low temperature,It expressed late when treated with H₂O₂ and early when treated with SA, NaCl or low temperature（4℃）.OsMKK10-2 confers strong expression under the induction of H₂O₂,it expressed early in the root and late in the shoot.It confers decline expression under the induction of low temperature（4℃）.It expressed different between shoot and root when early treated with SA,ABA,Me-JA,Mannitiol and NaCl,its expression rised in the shoot and falled in the root when treated with ABA or Me-JA,its expression rised in the root and failed in the shoot when treated with SA,Mannitiol and NaCl.3.One of rice MAPKKs which known as OsMKK3 was cloned and analyzed by gene overexpression and silencing ways.更多还原