兰州大学机构库 >生命科学学院
高山离子芥MAP激酶基因CbMAPK3 的克隆及功能分析
Alternative TitleMolecular Cloning and Characterization of a Novel MAP Kinase Gene CbMAPK3 in Chorispora bungeana
张腾国
Thesis Advisor安黎哲
2006-03-06
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
KeywordRACE 促分裂原活化蛋白激酶 CbMAPK3 高山离子芥 基因克隆基因功能分析 环境胁迫
Abstract

为了适应各种生物胁迫和非生物胁迫,植物在长期进化过程中形成了完整的调节机制,感受外界刺激,调整基因的表达,调节代谢途径。通过完整的信号调节系统,激活或者抑制适当的基因以响应外界刺激。由促分裂原活化蛋白激酶(MAPK)、促分裂原活化蛋白激酶激酶(MAPKK)、促分裂原活化蛋白激酶激酶激酶(MAPKKK)组成的磷酸化级联途径在酵母和动物细胞中,起着重要的信号传递作用。植物中,鉴定出了多种 MAP 激酶基因,大量的研究表明植物 MAPK 激酶可以被激素,非生物胁迫,病原体侵染所激活,也可以在细胞分裂的特定时期被激活。
高山离子芥是稀有的高山冰缘植物,具有较高的适应寒冷环境的能力。由于高山离子芥是一种胁迫耐受植物,我们克隆得到了高山离子芥中与抗逆境胁迫有关的 MAP激酶基因 CbMAPK3 ,并通过 RT-PCR 和 Western blotting 分析等方法研究了 MAPK 在高山离子芥抵御非生物胁迫过程中所发挥的作用。研究结果如下:
(1)获得了高山离子芥 MAP 激酶基因 CbMAPK3 ,全长 1419bp,其中包括 1110bp 的开放阅读框(ORF),91bp 的 5`非翻译区(5`UTR),218bp 的 3`非翻译区(3`UTR),包括 15bp 的 polyA。该基因编码一个 369 个氨基酸的蛋白质,分子量42.5kDa,
等电点(P I )5.79。由于该基因与拟南芥 AtMPK3 同源性最高,达到了 90%以上,因此命名为 CbMAPK3 (genbank accession number: AY805424)。CbMAPK3 包含有MAP 激酶所具有的 11 个保守序列区,以及 MAP 激酶的磷酸化位点 TEY 基序。因此该基因很可能编码一个 MAP 激酶蛋白。
(2)序列分析结果显示,该蛋白与植物抗逆境胁迫有关的 MAP 激酶亚组 A 有很高的同源性, CbMAPK3 与 Arabidopsis AtMPK3 同源性最高,达到了 95%, 其次是Nicotiana tabacum NtWIPK (82%),  Capsicum annuum CaMPK1 (81%), PsMAPK3(80%),  Medicago sativa  MsMMK4 (80%),  Petrosecinum crispum PARSLEY MAPK (80%)。应用 SOPMA 在线分析将 CbMAPK3 预测蛋白序列进行二级结构预测,结果显示,CbMAPK3 预测蛋白序列包含 46% α-螺旋(alpha helix), 13% 伸展链(extended strand), 6% β-转角(beta turn), 35% 无规卷曲(random coil)。α-螺旋和无规卷曲为该蛋白的主要二级结构。(3)研究了 CbMAPK3 在高山离子芥不同的组织中的表达情况,结果表明, CbMAPK3的表达没有组织特异性。
(4)通过半定量 RT-PCR.分析,研究了高山离子芥 CbMAPK3 基因在低温,高盐胁迫以及 ABA 处理下转录水平的变化。结果表明,植物材料在 4℃处理下, CbMAPK3基因的转录水平迅速增长,并在处理 0.5 小时之后达到最高值,然后表达量逐渐降低,处理24h后,降低到了基础水平。当植物材料在-4℃下处理时, CbMAPK3激酶基因的转录水平同样迅速增长,并且在处理 24 小时时仍然保持在较高的
水平。 CbMAPK3 基因的转录也受到 ABA 以及高盐胁迫的诱导,在 ABA 的处理下,CbMAPK3 基因的转录在 30min 内明显升高,并在处理 12 小时之后逐渐降低。因此, CbMAPK3 可能属于 ABA 依赖型。在高盐胁迫的诱导下, CbMAPK3 基因的转录在 2 小时之后达到一个很高的水平,并且在 48 小时的处理时间内一直维持在一个较高的水平。综上所述, CbMAPK3 的转录受到低温、高盐以及 ABA 的诱导。胁迫条件下 MAP 激酶基因转录水平的增加可以相应的增加可供激活的蛋白激酶的量。因此,可以得出结论,虽然 MAP 激酶主要在翻译后被磷酸化激活以后起作用,但转录水平的调控在 MAP 激酶级联途径中同样发挥着重要作用。
(5)研究了 CbMAPK3 蛋白水平在低温以及其它环境胁迫下的变化。结果显示,高山粒子芥悬浮细胞在 4℃处理下,CbMAPK3 激酶蛋白质水平在处理前 3 个小时有所增加,并在 3 小时时达到最高值,然后逐渐降低,处理 24h 后,降低到了基础水平。当悬浮细胞在 0℃下处理时,CbMAPK3 激酶蛋白质水平只有很微弱的增加。当悬浮细胞在 150mM NaCL 下处理时,蛋白质水平在 3-12 小时内明显
高于基础水平,在处理 24 小时时又降低到了基础水平。胁迫条件下 MAP 激酶蛋白水平的增加同样可以相应的增加可供激活的蛋白激酶的量。因此,MAP 激酶蛋白水平的调控在 MAP 激酶级联途径中同样发挥着重要作用。
(6)为了进一步研究 CbMAPK3 本身是否具有抗渗透胁迫的功能。将 CbMAPK3 连接表达载体 pET-30a 中,转化 E. coli ( srl::Tn10 ),在 IPTG 的诱导下,检测转化菌株在在含有1M山梨醇的M9培养基上生长情况。结果显示,转化有CbMAPK3的 E. coli( srl::Tn10 )和没有转化 CbMAPK3 的 E. coli ( srl::Tn10 )的生长情况基本一致,没有明显的差异。因此,CbMAPK3 本身没有抗渗透胁迫的功能,可能作为信号传递物质在环境胁迫过程中发挥作用。       (7) CbMAPK3 在大肠杆菌 BL21 中的表达。 SDS-PAGE 电泳结果表明带有重组表达质粒的阳性菌株,经过诱导产生了约 46kD 的蛋白带,与由核苷酸序列推导的目的蛋白分子量大小相符。表达产物经SDS-PAGE电泳后转移至NC膜上进行免疫学反应,结果显示,CbMAPK3 能被抗拟南芥 MAP 激酶 AtMPK3 的抗体α-C-MPK3 特异地识别。从而验证了在高山离子芥全蛋白中可以被α-C-MPK3 抗体特异地识别的蛋白为CbMAPK3。同时,为进一步鉴定 CbMAPK3 激酶体外酶活性奠定了基础。我们从高山粒子芥中成功克隆了一个新的 MAP 激酶基因 CbMAPK3。该基因的转录受到低温、ABA 以及高盐的诱导,但没有组织特异性。CbMAPK3 蛋白水平在低温和高盐胁迫下有不同程度的增加。因此,CbMAPK3 在高山粒子芥响应环境胁迫过程中发挥着重要的作用。

Other Abstract

To survive biotic and abiotic stresses, plants have developed elaborate mechanisms to perceive external signals and adjust metabolic pathways by modulating the expression of genes. Activation and/ or inactivation of appropriate genes in response to particular stimuli are mediated through well-tuned signal transduction systems. The phosphorylation cascades including mitogen-activated protein kinases (MAPKs), MAPK kinases (MAPKKs), and MAPKK kinases (MAPKKKs) have been reported to function in various signal transduction pathways from yeasts to vertebrates. In plants, a variety of genes encoding MAPKs have been identified. Numerous studies shown that plant MAPK cascades are activated by hormones, abiotic stresses, pathogens and pathogen-derived elicitors, and also activated at specific stages during the cell cycle.Chorispora bungeana fisch. & C.A. Mey (Chorispora bungeana) is a rare alpine subnival plant species that is highly capable of resisting freezing environment. Since it is a stress-tolerant plant,  we have isolated from chorispora bungeana a new MAPK cDNA CbMAPK3 and investigated the participation of  CbMAPK3 as possible mediators of abiotic stresses by RT-PCR and Western blotting. We got results as below:
(1) Cloning and sequence analysis of the full-length cDNA of CbMAPK3. Based on cDNA sequences of the conserved regions of plant MAPK genes, we have isolated from chorispora bungeana a new MAPK cDNA CbMAPK3. The cloned full-length cDNA of CbMAPK3 was 1419bp with a polyA tail of 15bp. The cDNA contained a 1110bp ORF encoding a protein of 369 amino acids with a calculated molecular weight of about 42.5kDa and with a PI of 5.79. A 5` untranslated region (UTR) of 91bp was found upstream of the first ATG codon, and a 3` untranslated region (UTR) of 218bp was found downstream from the stop codon. The protein exhibit closest homology to a subgroup of plant MAPKs containing Arabidopsis AtMPK3 and we thus refer to them as CbMAPK3 (genbank accession number: AY805424). The CbMAPK3 protein contained all 11 conserved amino acid and peptide motifs characteristic of 11 subdomains of protein kinases with serin/threonine specificity. The TEY motif, whichincludes the threonin and tyrosine residues whose phosphorylation is necessary for MAP kinase activation and is a characteristic feature of MAP kinase, is also conserved in the CbMAPK3 protein sequence.
(2) Based on the phylogenetic tree of cloned plant MAPKs, CbMAPK3 can be grouped into subgroup A. Comparison of the predicted protein sequences of the CbMAPK3 with MAP kinases of other plants shows that CbMAPK3 is most homologous to the Arabidopsis AtMPK3 (95%), Nicotiana tabacum NtWIPK (82%), Capsicum annuum
CaMPK1 (81%), PsMAPK3 (80%), Medicago sativa MsMMK4 (80%), Petrosecinum crispum PARSLEY MAPK (80%). The secondary structure of the putative CbMAPK3 protein was analyzed by SOPMA. The result showed that the putative CbMAPK3 peptide contained 46% alpha helix, 13% extended strand, 6% beta turn, and 35% random coil. The alpha helix and random coil constituted interlaced domination of the main part of the secondary structure. From the above sequence analyses, CbMAPK3 was found to have many characteristics common to the MAPKs in plant and family A embers tend to share highly similar sequences.
(3) Investigate CbMAPK3 expression pattern in various tissues of chorispora bungeana, the result shown that its expression has almost no tissue specificity.
(4) CbMAPK3 expression patterns under cold and other abiotic stresses were analyzed by semi-quantitative RT-PCR. After exposure to 4℃, transcript levels of CbMAPK3 increased rapidly, maximum levels was observed 0.5 h after cold treatment, then, the CbMAPK3 transcript declined gradually and fell to the basal level at 24 h. When chorispora bungeana was exposure to -4℃, transcript levels of CbMAPK3 also increased rapidly, but the transcript maintained at a high levels up to 24h. To our knowledge, this is the first description of a plant MAPK gene whose expression is inducible at -4℃ treatment. The expression of CbMAPK3 could also be induced by salt stress and ABA treatment, under salt stress, the transcripts of CbMAPK3 increased to a high level after 2h and maintained high levels at 48h after treatment.
When treated with ABA, the transcripts of CbMAPK3 increased significantly after 30 min, and decreased after 12h. indicating that the CbMAPK3 gene was ABA-dependent.On the whole, the CbMAPK3 transcript levels were increased by cold (4℃ and -4℃), salt and ABA treatment. The increase in expression of MAPK gene may contribute to the stress response by increasing the amount of proteins available for activation.
Therefore, it can be concluded that MAPK not only become activated by posttranslational phosphorylation of the highly conserved threonine and tyrosine residue through upstream kinases, transcriptional control is also an important mechanism in MAPK signaling cascades in plant.
(5) Investigate CbMAPK3 protein level under cold and other abiotic stresses. Immunoblot analysis using α-C-MPK3 antibody show that the protein level of CbMAPK3 was
increased significantly and maximum levels was observed at 3h after 4℃ cold treatment, then, the CbMAPK3 protein level declined gradually and fell to the basal level at 24 h . but the protein level of CbMAPK3 was increased slightly by 0℃ cold treatment. When treat with 150mM NaCL, the protein level of CbMAPK3 increased after 3h and declined after 12h. The increase in protein level of MAPK may also contribute to the stress response by increasing the amount of proteins available for activation. Therefore, it can be concluded that protein level control is also an important mechanism in MAPK signaling cascades in plant.
(6) To comfirm whether CbMAPK3 has the osmo-protection function, the coding region was subcloned into prokaryotic expression vector. E. coli (srl::Tn10) cell transformed with this plasmid exhibitad no significantly better growth in the presence of 1M sorbitol, compared to E. coli (srl::Tn10) transformed with the vector only, after induction with IPTG.. we can conclude that CbMAPK3 is likely involved in abiotic stress responses as a signal transduction element , but itself did not has the function of osmo-protection.
(7) Expression of CbMAPK3 in E. coli BL21. The coding region of CbMAPK3 cDNA was subcloned into the bacterial expression vector pET30a, and transformed E. coli BL21.
After induced by IPTG, the expression protein had a molecular mass of about 46kD by SDS-PAGE, corresponding to the deduced amino acid sequence of CbMAPK3 fusion
protein. Western blotting analysis using α-C-MPK3 antibody showed that the BL21/ pET30a/CbMAPK3 samples had positive signal at 46kD position, where there is nosignal in BL21/ pET30a samples. This result confirmed that α-C-MPK3 antibody specifically reacted with CbMAPK3 protein. So, we can further conclude that the protein reacted withα-C-MPK3 antibody in the whole protein of  Chorispora bungeana is  CbMAPK3.
In conclusion, a novel MAPK gene CbMAPK3 was identified and characterized. The expression of CbMAPK3 was induced by cold, salt, and ABA but no tissue specificity.The protein level of CbMAPK3 increased when  treated with cold (4 and –4 °C) and salinity stress. These results indicate that the CbMAPK3 may play an important role in response to environmental stresses.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221374
Collection生命科学学院
Recommended Citation
GB/T 7714
张腾国. 高山离子芥MAP激酶基因CbMAPK3 的克隆及功能分析[D]. 兰州. 兰州大学,2006.
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