【作者】 刘玉鲲；

【导师】 李德全；

【作者基本信息】 山东农业大学 ， 植物学， 2011， 博士

【摘要】 促分裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)级联途径是真核生物中广泛存在的信号转导途径。促分裂原活化蛋白激酶级联途径由MAPKKK-MAPKK-MAPK依次磷酸化传递信号。MAPK级联途径各激酶基因家族在不同植物物种中是保守的。目前,玉米基因组测序已经完成,在基因组水平上解析玉米MAPK家族的信息已经成为可能。MAPK是连接底物和上游信号的重要因子。以前的研究表明,MAPK级联途径参与植物的ABA信号转导。但是,关于ABA信号中特异的MAPK基因的研究仍然很有限。本研究根据植物MAPK基因的保守性鉴定了玉米MAPK家族的基因。对玉米MAPK基因家族的分类、蛋白生化特性、基因结构、染色体分布、蛋白的系统进化以及基因在幼苗根、茎、叶中的表达模式进行了系统分析。在此基础之上,选取了ZmMPK4基因进行功能研究。ZmMPK4为双拷贝基因,和ZmMPK3是基因对,ZmMPK4基因对参与ABA信号转导。主要研究结果如下:(1)利用网络数据库的BLAST和本地Stand-alone BLAST的方法,鉴定了19个玉米MAPK基因。19个MAPK基因的氨基酸长度在369到642之间,分子量在42.199 kDa到72.444 kDa之间,等电点在5.26到9.82之间。6号和8号染色体各有4个MAPK基因,5号和9号各3个,10号2个,1号、3号、4号各1个。与双子叶拟南芥和杨树比较,玉米MAPK基因和水稻MAPK基因的亲缘关系比较近。(2)ZmMPK1和ZmMPK2的序列高度相似,数据库搜索表明,没有EST序列特异的和ZmMPK1匹配。RT-PCR分析表明,在玉米幼苗根、茎、叶中检测不到ZmMPK1的表达,说明ZmMPK1可能是一个假基因。其它18个基因在玉米幼苗根、茎、叶中都能检测到。除了ZmMPK12、ZmMPK18、ZmMPK19没有明显的组织特异性外,其它15个基因在玉米幼苗根、茎、叶中的分布均不同。(3)PCR分析表明,ZmMPK4和文献报道的ZmMPK4(命名为ZmMPK4-2)的序列不完全一致。ZmMPK4-2是ZmMPK4的一个可变剪接产物。基因测序和玉米MAPK基因家族的分析表明,ZmMPK4是双拷贝基因,ZmMPK3和ZmMPK4是基因对。Southern实验验证了ZmMPK4的拷贝数。ZmMPK3和ZmMPK4的核苷酸相似度为92.1%,编码氨基酸的相似度为90.0%。ZmMPK3和ZmMPK4分别位于1号染色体短臂和9号染色体长臂。两个基因都能形成mRNA和蛋白质。(4)序列分析表明,ZmMPK4的3号内含子是GC-AG型内含子。ZmMPK4-2是保留了ZmMPK4的3号内含子形成的可变剪接产物。ZmMPK4-2的组成型表达量非常低,主要在叶中表达。ZmMPK4也可以剪切3号内含子形成正常的ZmMPK4-1,且为ZmMPK4基因表达的主要方式。ZmMPK3的3号内含子为GT-AG型内含子。(5)Northern杂交实验表明,ZmMPK3主要在生长5日的幼苗叶中表达,ZmMPK4主要在幼苗根中表达。ZmMPK3和ZmMPK4都受ABA(100?M)和NaCl(200mM)的诱导,但诱导时间和表达量不同。但是,Northern杂交不能区分ZmMPK4-1和ZmMPK4-2。利用RT-PCR的方法,进一步分析了ZmMPK4-1和ZmMPK4-2的特异表达,结果显示,ZmMPK4-2主要在叶中表达,即ZmMPK4在幼苗中的可变剪接主要在叶中进行。ZmMPK4的可变剪接受ABA或NaCl的调控。(6)制备了ZmMPK3的抗体(Anti-ZmMPK3)。因为ZmMPK3和ZmMPK4的氨基酸序列高度相似,无法设计特异抗体。ZmMPK3和ZmMPK4的混合蛋白(约43kDa)在生长5d的玉米叶片中组成型表达,ABA(100?M)可以轻微诱导ZmMPK3和ZmMPK4混合蛋白的表达。免疫沉淀分析表明,ABA可以激活ZmMPK3和ZmMPK4混合蛋白的激酶活性(0.5h和1h)。用烟草叶片瞬时转化法分别检测ZmMPK3、ZmMPK4-1和ZmMPK4-2的活性,检测不到ZmMPK4-2的活性,而ZmMPK3或ZmMPK4-1的激酶活性都可以被ABA诱导。(7)洋葱表皮表达分析表明,ZmMPK3或ZmMPK4-1的融合蛋白主要定位于细胞膜和细胞核,而ZmMPK4-2融合蛋白主要定位于细胞膜和细胞质。ZmMPK4-2中的30个氨基酸的插入可能影响ZmMPK4的细胞定位。(8)过表达ZmMPK4-1可以促进拟南芥提前抽薹。在发育后期,转基因拟南芥的主茎上可以形成莲座叶,并可继续抽薹。在正常MS培养基上,过表达ZmMPK4-1的拟南芥和野生型拟南芥的萌发率没有明显差别(约为100%)。0.5?M的ABA可以抑制转基因和野生型拟南芥种子的萌发,但对转基因拟南芥的抑制明显强于野生型。正常生长条件下,ZmMPK4-1在转基因拟南芥中具有激酶活性。ABA(0.5?M)可以增强转基因拟南芥中ZmMPK4-1的激酶活性。过表达ZmMPK4-1可以诱导拟南芥AtACT1的组成型表达,可以增强AtACT2、ABI3、ABI5基因对ABA(0.5?M)的响应。更多还原

【Abstract】 Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes. A MAPK cascade consists of MAPKKK-MAPKK-MAPK, that sequentially phosphorylate the corresponding downstream substrates. Each gene family of MAPKKK, MAPKK or MAPK is conserved in different plant species. The maize genome has been sequenced to date. It is possible to dissect the information of maize MAPK gene family based on the genomic sequence. MAPK links upstream components to downstream substrates. Previously, it has been reported that MAPK cascades participated in ABA signaling. However, the specific MAPK gene in ABA signaling is still sparse.In this study, we identified MAPK genes in the maize genome based on the conservation of plant MAPKs. The classification, the protein properties, the gene structure, the chromosomal distribution, the evolutionary relationship and the expression pattern in maize roots, stems and leaves of maize MAPK genes have been analyzed. Based on the analysis, we focused on the function of ZmMPK4. ZmMPK4 is a two-copy gene and exists as gene pair with ZmMPK3. ZmMPK4 plays a role in ABA signaling. The main results are as follows:(1) Using database BLAST and Stand-alone BLAST, we identified 19 MAPK genes in the maize genome. The amino acids of 19 MAPK proteins are between 369 and 642. The molecular weight of 19 MAPK proteins are between 42.199 kDa and 72.444 kDa. The pI of 19 MAPK proteins are between 5.26 and 9.82. Four of the 19 MAPK genes exist in chromosome 6 or 8. Three exist in chromosome 5 or 9. Two exist in chromosome 10. One exists in chromosome 1, 3 or 4. The maize MAPKs share more evolutionary relationship with rice MAPKs than Arabidopsis or poplar MAPKs.(2) ZmMPK1 shares a high sequence similarity with ZmMPK2. There is no EST sequence specifically matches ZmMPK1 sequence. RT-PCR analysis shows that there is not detectable mRNA in roots, stems or leaves of maize seedlings, suggesting ZmMPK1 might be a pseudogene. Other 18 genes can be detected in roots, stems or leaves of maize seedlings. In addition to ZmMPK12, ZmMPK18 and ZmMPK19, other 15 genes expressed in different level.(3) PCR analysis shows that the band of ZmMPK4 did not match the band of previously reported ZmMPK4 (ZmMPK4-2 in our study). ZmMPK4-2 is a alternative transcript of ZmMPK4 gene that can take alternative splicing. Gene sequence and MAPK gene family analysis revealed that ZmMPK4 is a two-copy gene and exists as gene pair with ZmMPK3. Southern blot showed ZmMPK4 is a two-copy gene. The nucleotide sequences of ZmMPK3 and ZmMPK4 share similarity of 92.1%. The amino acid sequences of ZmMPK3 and ZmMPK4 share similarity of 90.0%. ZmMPK3 and ZmMPK4 are located on the short arm of chromosome 1 and the long arm of chromosome 9, respectively. Both ZmMPK3 and ZmMPK4 could generate mRNA and proteins.(4) The third intron of ZmMPK4 gene is GC-AG type. ZmMPK4-2 is one alternative transcript result from retention of the third intron of ZmMPK4 gene. The transcript of ZmMPK4-2 is low and mainly found in maize leaves. Another alternative transcript, the main transcript, of ZmMPK4 gene is ZmMPK4-1 without the third intron. The third intron of ZmMPK3 gene is GT-AG type.(5) Northern blot analysis indicated that ZmMPK3 is mainly expressed in leaves of 5-d-old seedlings and ZmMPK4 in roots. ABA (100 ?M) or NaCl (200 mM) up-regulated the expression of both ZmMPK3 and ZmMPK4 with different patterns. Northern blot analysis did not differentiate the expression of ZmMPK4-1 and ZmMPK4-2. RT-PCR using specific primers revealed that the alternative splicing predominantly occurred in leaves of 5-d-old seedlings. The alternative splicing is regulated by ABA or NaCl.(6) ZmMPK3 antibody (Anti-ZmMPK3) was generated. Generation of specific antibodies for ZmMPK3 and ZmMPK4 is impossible because of the high similarity of amino acid. In leaves of 5-d-old seedlings, Anti-ZmMPK3 could detect protein band with an approximate molecular mass of 43 kDa. The protein amount could slightly be induced by ABA (100 ?M). Immunoprecipitation assay showed that ABA (100 ?M) stimulates a rapid activation of the 43 kDa protein in 0.5 h or 1 h. Transient expression in Nicotiana benthamiana was performed to specifically examine the kinase activities of ZmMPK3, ZmMPK4-1 or ZmMPK4-2. The results showed that ABA (100 ?M) activated ZmMPK3 or ZmMPK4-1, but not ZmMPK4-1 in 0.5 h, 1 h or 2 h.(7) Transiently expressing of 35S-GFP-ZmMPK fusion proteins suggest that ZmMPK3 or ZmMPK4-1 fusion protein localized in cell membrane and nucleus, and ZmMPK4-2 fusion protein localized in cell membrane and cytoplasm. These results indicate the insertion of 30 amino acids may disturb the localization of ZmMPK4 protein.(8) Overexpressing of ZmMPK4-1 in Arabidopsis accelerated stem development. In the adult plants, the rosette leaves arose from the stem and can form stem subsequently. The wild-type and ZmMPK4-1-overexpressing Arabidopsis showed no difference in germination (about 100%) on MS media without ABA treatment. ABA (0.5 ?M) inhibited the germination of both wild-type and ZmMPK4-1-overexpressing seeds. The inhibition effect of ABA on ZmMPK4-1-overexpressing seeds was stronger than that of ABA on wild-type seeds. Immunoprecipitation assay showed anti-p-ERK could detect kinase activity in ZmMPK4-1-overexpressing plants under normal growth conditions. ABA (0.5 ?M) stimulates activation of ZmMPK4-1 in 30 min. AtACT1 is upregulated in ZmMPK4-1-overexpressing plants under light growth conditions. Furthermore, in ZmMPK4-1-overexpressing plants, ABA-inducted AtACT2, ABI3 or ABI5 expression was enhanced.更多还原