过量表达CDPK9对拟南芥耐逆性的影响

【作者】 张茂杰；

【导师】 王增兰；

【作者基本信息】 山东师范大学 ， 发育生物学， 2007， 硕士

【摘要】 CDPK(calcium dependent protein kinase)是在植物中首先发现的一种钙依赖型蛋白激酶,属于Ser/Thr型蛋白激酶,分子量40～90kDa,Mg2+是该酶的重要辅因子。该蛋白质在其氨基端有一个激酶活性域,在其羧基端有一个类似CaM的结构区域,在两者之间还有一个自我抑制域。当类似CaM的结构域上的钙离子结合位点与Ca2+结合后,抑制被解除,酶即被活化。钙离子是细胞信号转导中重要的第二信使,CDPK作为钙离子的受体蛋白和效应蛋白在信号转导过程中起着关键的作用。CDPK家族在调节信号转导中具有多样性和可选择性。近年来通过对拟南芥、水稻等植物研究表明CDPK家族有多个成员,其中拟南芥含有12个亚家族34个成员,水稻有27个成员(Harper et al., 2004),而且它们大多与逆境信号转导有关。已经证实,拟南芥AtCDPK1基因和AtCDPK2基因在干旱和盐诱导条件下,表达量增强(Urao et al., 1994);另外, Sheen(1996)证实拟南芥AtCPK10和AtCPK30参与了对ABA和非生物胁迫的信号转导。盐芥是一种与拟南芥近缘的十字花科植物,具有很多与拟南芥相同的优点:如类似的形态、小的基因组、短的生活史、丰富的种子和易于被转化等。但是,盐芥对多种胁迫的耐受能力远远高于拟南芥,它在短时间内能耐受高达500mMNaCl的冲击,在盐适应前后既不产生盐腺也没有复杂的形态上的变化,因此,对盐芥钙依赖的蛋白激酶的功能研究对于阐明植物的抗逆生理机制,揭示植物的耐逆机理都具有重要意义。本实验在盐芥EST库中得到CDPK9基因。通过构建过量表达载体,并用农杆菌介导的花序侵染法侵染拟南芥,得到5个转基因的纯合株系。CDPK基因家族蛋白成员主要存在于植物细胞质膜、叶绿体中,不同的定位与其相应的功能相关。为了解CDPK9的生物学功能,我们首先构建了CDPK9-GFP融合基因表达载体,并用农杆菌介导的花序侵染法侵染拟南芥,通过共聚焦显微镜检测对CDPK9进行了初步的亚细胞定位。同时构建了CDPK9-GST原核表达载体,在DE3大肠杆菌中表达融合蛋白,通过SDS-PAGE确知该蛋白以包涵体的形式存在.另外,分别利用拟南芥CDPK9的全长及N端特异性较强部分为探针,对得到的纯合的转基因株系Northern bloting,可能是由于杂交条件或者提取的RNA纯度不高的原因,没有得到理想的杂交条带。更多还原

【Abstract】 CDPK is a kind of Calcium-dependent protein kinases which is first found in plants.It belongs to Ser/Thr protein kinases family,CDPK has a molecular weight of 40～90kDa,Mg2 + works as its assist member。CDPKs are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor.The CDPK family represents diversity and choosable during its signal transduction gateway.Rencently lots of investigations into rice and arabidopsis represent that the CDPK family is comprised of multiplegene family. Arabidopsis includes 12 subfamilies consisted of 34 members. As to rice,27 members. Furthermore most of them involve in adversity signal transduction.we have validated the expression of arabidopsis genes AtCDPK1 and AtCDPK2 are toned up in drought and salt conditions(Urao et al., 1994). In addition, Sheen(1996) make sure that the arabidopsis genes AtCPK10 and AtCPK30 involve in ABA and abiological signal transduction.Thellungiella halophila is close relative to Arabidopsis which is also belong to Cruciferae and has good genetic features such as similar morphology, small genome size, short life cycle, high seed number and an efficient transformation method. However, Thellungiella halophila is able to withstand dramatic salinity shock up to 500mM NaCl. This plant does not produce salt glands or other complex morphological alterations either before or after salt adaptation. Therefore, the study of function of thioredoxin from Thellungiella halophila is quite significant for expatiating plant response mechanism of oxidative stress, understanding the metabolism alteration and adjustment of plant under stresses and expounding the stress tolerance metabolism.The CDPK9 gene was obtained from ESTs (expressed sequence tags) acquired fromλZap-cDNA library of Thellungiella halophila in our lab.Through the expression in vitro and activity experiment we try to prove that CDPK9 did have kinases activity. The family members of CDPK9 exist not only in cytoplasm membrane but also in chloroplast, and different localizations affect their potential functions.By constructing the GFP expression vector of CDPK9 and transforming it into the Arabidopsis anthotaxy, CDPK9 is mostly localized in cytoplasm membrane by laser scanning confocal microscope. Only a few fraction is localized in chloroplast . The subcellular localization results were in accordance with the result of iPSORT software. At the same time ,the CDPK9-GST vector was constructed, amalgamation protein was expressed in DE3 Ecol, Our results showed that the amalgamation protein forms inclusion during its expression. Besides, by constructing the over-expression vector of CDPK9 ,we attained 5 lines of transgene Arabidopsis. and the phenotype of the 5 lines was analyzed. Besides,we also did the Northern bloting experiment,fallaciously,we did not get the hybridize signals on the hybridize membrane.更多还原