拟南芥TUA2基因参与ABA胁迫下种子萌发过程的研究
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摘要
微管蛋白是一种在真核细胞中普遍表达的蛋白,由α微管蛋白(TUA)和β微管蛋白(TUB)组成的微管蛋白异二聚体构成。在拟南芥中共有6种不同的TUA基因亚型和9种不同的TUB基因亚型。许多研究证实,不同的微管蛋白基因亚型在特定的发育阶段或组织细胞中高效表达,随着外界环境胁迫表达量也会发生变化。因此,微管蛋白基因不仅仅是持家基因,也参与众多的信号应答反应。
本研究以TUA2基因亚型表达变化的突变体为材料。利用反向遗传学的方法,运用遗传转化,Tail PCR,半定量PCR等技术进行TUA2参与ABA胁迫下种子萌发过程的研究。
主要结果如下:
1.通过生理检测我们在若干个T-DNA插入突变体中发现了一个对ABA敏感的突变体。运用Tail PCR的方法找到了T-DNA的插入位点位于TUA2基因上游启动子区,并发现该位点的插入导致突变体中TUA2基因表达量提高。另外我们在SALK库也找到了相应位点的突变体。并通过遗传转化的方法得到了三种不同株系的TUA基因超表达体。
2.构建了TUA2基因的亚细胞定位转基因表达载体,运用基因枪转化的方法将TUA2基因和GFP融合基因在洋葱表皮瞬时表达,结果显示TUA2主要在细胞膜上分布。
3.通过对ABA胁迫下种子萌发率的检测发现TUA2基因突变体,超表达体均出现了种子萌发的延迟。推测TUA2可能参与了ABA对种子萌发的调控过程。
4.通过RT-PCR的方法对ABA途径相关基因表达量进行分析的结果显示,与野生型相比突变体的HAB和ABI基因在ABA处理下的表达量明显降低,暗示了TUA2可能参与了ABA信号系统的负调控过程。
Tubulin is a kind of protein which generally expresses in eukaryocyte , and it is made up of tubulin heterodimers composed byαtubulin andβtubulin.There are 6 different TUA subgroups and 9 different TUB subgroups in Arabidopsis . Many experimental results showed that different tubulin subgroups not only expressed effectively in specified developmental stage and tissue , but also change their expression level under environmental stresses. Thus , tubulin is a kind of housekeeping gene , and it participated in masses of singal respond reaction .
Our research used the tua2 mutant of TUA2 subgroups as material , and we undertaked the research of germination under the TUA2-participated ABA stress by reverse genetics , genetic transformation , Tail PCR , semiquantitative-PCR and other technology.
The major results showed as follow :
1. We found an ABA-sensitive mutant from several T-DNA insertion mutants by phaenotype observation . Then we found the T-DNA insertion site ( located in the upstream promoter region of TUA2 ) with Tail PCR ,and the level of TUA2 genetic expression raised in this T-DNA insertion mutant. In addition we obtained the mutant inserted in the corresponding site form the SALK library ,and got three overexpression mutants in different strains by genetic transformation.
2. We constructed TUA2 transgenic expression vector for subcellular localization , transiented expressed the fusion gene of TUA2 and GFP by particle gun transformation . The results showed that the TUA2 mainly distributed at cellular membrane.
3. By observation of germinationrate under ABA stress , the TUA2 mutant is found , and the overexpressionmutant all emerged a phaenotype of germination delayed. We supposed that TUA2 may participate in the germination regulating process of ABA.
4. We analyzed the expression level of ABA pathway related gene by RT-PCR, found that the expression level of mutant had been depressed compared to HAB and ABI in wild type under ABA stress. It implied that TUA2 may participated in ABA negative regulation process.
本研究以TUA2基因亚型表达变化的突变体为材料。利用反向遗传学的方法,运用遗传转化,Tail PCR,半定量PCR等技术进行TUA2参与ABA胁迫下种子萌发过程的研究。
主要结果如下:
1.通过生理检测我们在若干个T-DNA插入突变体中发现了一个对ABA敏感的突变体。运用Tail PCR的方法找到了T-DNA的插入位点位于TUA2基因上游启动子区,并发现该位点的插入导致突变体中TUA2基因表达量提高。另外我们在SALK库也找到了相应位点的突变体。并通过遗传转化的方法得到了三种不同株系的TUA基因超表达体。
2.构建了TUA2基因的亚细胞定位转基因表达载体,运用基因枪转化的方法将TUA2基因和GFP融合基因在洋葱表皮瞬时表达,结果显示TUA2主要在细胞膜上分布。
3.通过对ABA胁迫下种子萌发率的检测发现TUA2基因突变体,超表达体均出现了种子萌发的延迟。推测TUA2可能参与了ABA对种子萌发的调控过程。
4.通过RT-PCR的方法对ABA途径相关基因表达量进行分析的结果显示,与野生型相比突变体的HAB和ABI基因在ABA处理下的表达量明显降低,暗示了TUA2可能参与了ABA信号系统的负调控过程。
Tubulin is a kind of protein which generally expresses in eukaryocyte , and it is made up of tubulin heterodimers composed byαtubulin andβtubulin.There are 6 different TUA subgroups and 9 different TUB subgroups in Arabidopsis . Many experimental results showed that different tubulin subgroups not only expressed effectively in specified developmental stage and tissue , but also change their expression level under environmental stresses. Thus , tubulin is a kind of housekeeping gene , and it participated in masses of singal respond reaction .
Our research used the tua2 mutant of TUA2 subgroups as material , and we undertaked the research of germination under the TUA2-participated ABA stress by reverse genetics , genetic transformation , Tail PCR , semiquantitative-PCR and other technology.
The major results showed as follow :
1. We found an ABA-sensitive mutant from several T-DNA insertion mutants by phaenotype observation . Then we found the T-DNA insertion site ( located in the upstream promoter region of TUA2 ) with Tail PCR ,and the level of TUA2 genetic expression raised in this T-DNA insertion mutant. In addition we obtained the mutant inserted in the corresponding site form the SALK library ,and got three overexpression mutants in different strains by genetic transformation.
2. We constructed TUA2 transgenic expression vector for subcellular localization , transiented expressed the fusion gene of TUA2 and GFP by particle gun transformation . The results showed that the TUA2 mainly distributed at cellular membrane.
3. By observation of germinationrate under ABA stress , the TUA2 mutant is found , and the overexpressionmutant all emerged a phaenotype of germination delayed. We supposed that TUA2 may participate in the germination regulating process of ABA.
4. We analyzed the expression level of ABA pathway related gene by RT-PCR, found that the expression level of mutant had been depressed compared to HAB and ABI in wild type under ABA stress. It implied that TUA2 may participated in ABA negative regulation process.
引文
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[3] Wasteneysg O,Galwaym E.Remodeling the cytoskeleton for growth and form: An overview with some new views[J].Plant Biol,2003,54:691~722.
[4] Steven D.Kopczak,Nancy A,et al.The small genome of Arabidopsis contains at least six expressed a-Tubulin genes[J].The Plant Cell,1992,5(4):539~547,
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[6] Steven D.Kopczak,Nancy A,et al.The small genome of Arabidopsis contains at least six expressed a-Tubulin genes[J].The Plant Cell,1992,5(4):539~547
[7] Sandra N,Anne B,Thomas C,et al.A reference map of the Arabidopsis thaliana mature pollen proteome[J] . Biochemical and Biophysical Research Communications 2005,337:1257~1266
[8] Ce′line S,Luc N,Thierry B,et al.Proteomic analysis of differentmutant genotypes of Arabidopsisled to the identification of 11 proteins correlating with adventitious root development[J]. Plant Physiology,2006,2(140):349~364
[9] Jordan B,Sottosanto,Angie G,et al. DNA array analyses of Arabidopsis thaliana lacking a vacuolar Na+/H+ antiporter : impact of AtNHX1 on gene expression[J].The Plant Journal 2004,40:752~771
[10] Renata F D,Kathleen F K,Paul F,et al.The Arabidopsis thalianaran scriptome in response to agrobacterium tumefaciens[J].MPMI,2006,6(19):665~681
[11] Cao D,Cheng H,Wu W,et al.Gibberellin mobilizes distinct DELLA-Dependent transcriptomes to regulate seed germinationand floraldevelopment in Arabidopsis[J].Plant Physiology,2006,10(140):509~525.
[12] Tang W,Deng Z,Juan A,et al.Proteomics studies of Brassinosteroid Signal transduction using prefractionation and Two-dimensional DIGE[J].Molecular and Cellular Proteomics,2008,7(4):728~738.
[13] Mazars C,Thion L,Thuleau P.Organization of cytoskeleton cont rolst he changes in cytosolic calcium of coldshocked Nicotiana plumbagini folia protoplasts[J].Cell Calcium,1997,22:413~420.
[14] Wang C , Li J J , Yuan M . Salt tolerance requires cortical microtubulen reorganization in a Arabidopsis[J]. Plant cell physiology , 2007 , 48(11) :1534~1547.
[15] Huang R F,Wang X C,Lou C H.Stomatal opening induced by acetyl choline is associated with cytoskeletal components[J].Acta Botanica Sinica,2000,6: 12~16.
[16]魏宁,郑慧琼.细胞骨架和高等植物的向重力性[J].自然杂志,2007,29(6): 338~347.
[17]王超,侍福梅.植物微管动态的研究进展[J].安徽农业科学,2009,37(26): 12501~12502.
[18]张永梅,吴忠义,王学臣,等.拟南芥保卫细胞微管骨架的重排参与NO诱导的气孔关闭[J].科学通报,2008,53 (3): 293~298.
[19] Carpenter J L,Kopczak S D,Snustad D P,et al.Semi-constitutive expression of an Arabidopsis thaliana alpha-tubulin gene[J]. Plant Mol Biol.1993,4,21(5):937~942.
[20] Hu W,Wang Y,Bowers C,et al.Isolation,sequence analysis,and expression studies of florally expressed cDNAs in Arabidopsis [J].Plant Molecular Biology,2003,53:545~56
[21] Clayton L,Black C M,Lloyd C W.Microtubule nucleating sites in higher plant cells identified by an auto–antibody against peri–centriolar material[J].Cell Biol,1985,101:319~324
[22]简令成,孙龙华,林忠平.微管的冷稳定性与植物抗寒性关系的研究[J].植物学报,1989,31:737~741.
[23]刘海龙.拟南芥AtPLC3和AtPLC4参与盐胁迫应答的功能研究[D].河北:河北业大学,2008.
[24] Stevenson J M,Perera I Y,Heilmann I,et al.Inositol signaling and plant grow- -th.Trends Plant Sci[J].2000,5(6):252~258.
[25] Murashige T,Skoog F.A revised medium for rapid growth and bioassay with tobacco tissue cul tures[J].Physiol Plant,1962,15:473~497.
[26]张震祯,徐煜泉,黄海.拟南芥一把通往生命科学奥秘大门的钥匙[J].生命科学,2006,18(5):442~446.
[27]赵镝,徐春晖.拟南芥基因组测序工作圆满完成[J].山东大学学报,2001,36 (1):89~93
[28] Exton J H.Phosphatidylcholine breakdown and signal transduction[J].Biochim Biophys,1994,1212:26~42.
[29] Maria S , Iwonna R S . Biological role of phosphatidylcholine -specifi c phospholipase C in mammalian cells[J].Postepy Hig Med Dosw (online),2008,62:593~598.
[30] Yuki N,Koichiro A, Tatsuru M, et al.A novel phosphatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis[J].The Journal ofBiological Chemisitry,2005,280(9):7469~7476.
[31] Maxim G,Anireddy S.A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination[J].PNAS,2003,100(18):10558~10563.
[32] Morten S,Shawn M R,Lone B,et al.A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization[J].PNAS,2004,101(25):9502~9507.
[33] Megumi I,Hiroshi S,Teruhiko M,et al.Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis[J].Plant Physiology,2004,136:3562~3571.
[34] Pierson E S,Miller D D,Callaham D A,et al.Tip-localized calcium entry fluctuates during pollen tube growth[J].Dev Biol,1996,174:160~173.
[35] Barend H,Alice Y,Tatyana A,et al.Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in Tobacco[J].The Plant Cell,2005,17:2564~2579.
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