酵母双杂交筛选小麦TaMBD2互作蛋白
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摘要
为探讨小麦甲基结合域蛋白(TaMBD2)在植物生长发育过程中的调控功能,以TaMBD2基因的全长cDNA为模板,构建诱饵载体pGBKT7-TaMBD2,利用酵母双杂交系统从小麦cDNA文库中筛选TaMBD2互作蛋白。结果共筛选到91个菌斑显蓝色的克隆,对其进行菌液PCR检测并测序,然后在NCBI上进行BLAST比对分析,共获得8个可能与TaMBD2互作的蛋白,分别为二磷酸核苷激酶(NDPK)、ENTH结构域蛋白、SIAN蛋白、Agenet结构域蛋白、C2H2型锌指蛋白、磷酸激酶和2个假定蛋白,其中最有可能的TaMBD2互作蛋白为NDPK。这些候选蛋白主要参与细胞信号传导、抗逆、能量代谢和蛋白质运输等。其中,检测结果中参与植物抗逆胁迫的蛋白质为主要互作蛋白,所占比例为54.9%;参与蛋白质运输的互作蛋白所占比例为25.3%,其余互作蛋白所占比例较小。因此,推测TaMBD2可能主要参与植物对干旱、低温和高盐等非生物胁迫逆境的响应及调控。
In order to understand more regulatory functions of methyl-binding domain protein TaMBD2 during the growth and development in wheat,bait vector p GBKT7-TaMBD2 was constructed with the full length c DNA of TaMBD2 gene as template,and the interacted proteins of TaMBD2 from wheat c DNA library were screened by yeast two-hybrid system. The result showed that a total of 91 blue clones were screened,which were analyzed through PCR and homology analysis using the BLAST in NCBI,and eight possible interacted proteins of TaMBD2 were obtained,which were nucleoside diphosphate kinase(NDPK),epsin N-terminal homology domain-containing protein,seven in absentia family protein,Agenet domain-containing protein,zinc finger C2H2 type family protein,phosphate kinase and two hypothetical proteins. The most likely interacted protein of TaMBD2 was NDPK. These candidate proteins were related to mediate cell signal transduction,stress resistance,energy metabolism and protein transport,etc. Among them,the proteins involved in resistance to plant stress were the main interacted protein,the proportion was54. 9%; the proportion of proteins participated in protein transport was 25. 3%,and the proportion of the rest interacted proteins was smaller. Therefore,TaMBD2 may mainly participate in response and regulation to abiotic stress,such as drought,low temperature and high salt.
In order to understand more regulatory functions of methyl-binding domain protein TaMBD2 during the growth and development in wheat,bait vector p GBKT7-TaMBD2 was constructed with the full length c DNA of TaMBD2 gene as template,and the interacted proteins of TaMBD2 from wheat c DNA library were screened by yeast two-hybrid system. The result showed that a total of 91 blue clones were screened,which were analyzed through PCR and homology analysis using the BLAST in NCBI,and eight possible interacted proteins of TaMBD2 were obtained,which were nucleoside diphosphate kinase(NDPK),epsin N-terminal homology domain-containing protein,seven in absentia family protein,Agenet domain-containing protein,zinc finger C2H2 type family protein,phosphate kinase and two hypothetical proteins. The most likely interacted protein of TaMBD2 was NDPK. These candidate proteins were related to mediate cell signal transduction,stress resistance,energy metabolism and protein transport,etc. Among them,the proteins involved in resistance to plant stress were the main interacted protein,the proportion was54. 9%; the proportion of proteins participated in protein transport was 25. 3%,and the proportion of the rest interacted proteins was smaller. Therefore,TaMBD2 may mainly participate in response and regulation to abiotic stress,such as drought,low temperature and high salt.
引文
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[11]Kalthoff C,Alves J,Urbanke C,et al.Unusual structural organization of the endocytic proteins AP180 and epsin 1[J].Biol Chem,2002,277(10):8209-8216.
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[13]Xie Q,Guo H S,Dallman G,et al.SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals[J].Nature,2002,419:167-170.
[14]Welsch R,Maass D,Voegel T,et al.Transcription factor RAP2.2.and its interacting partner SINAT2:Stable elements in the carotenogenesis of Arabidopsis leaves[J].Plant Physiol,2007,145(3):1073-1085.
[15]Sakamoto H,Araki T,Meshi T,et al.Expression of a subset of the Arabidopsis Cys(2)/His(2)-type zinc-finger protein gene family under water stress[J].Gene,248(1/2):23-30.
[16]Kovarik A,Koukalova B,Bezdek M,et al.Hypermethylation of tobacco heterochromatic loci in response to osmotic stress[J].Theoretical and Applied Genetics,1997,95(1):301-306.
[17]Steward N,lto M,Yamaguchi Y,et al.Periodic DNA methylation in maize nucleosomes and demethylation by environmental stress[J].Journal of Biological Chemistry,2002,277:37741-37746.
[2]Jorgensen H F,Bird A.Me CP2 and other methyl-Cp G binding proteins[J].Ment Retard Dev Disabil Res Rev,2002,8:87-93.
[3]Berg A,Meza T J,Mahic M,et al.Ten members of the Arabidopsis gene family encoding methyl-Cp Gbinding domain proteins are transcriptionally active and at least one,At MBD11,is crucial for normal development[J].Nuc Acids Res,2003,31(5):5291-5304.
[4]Scebba F,De Bastiani M,Bernacchia G,et al.PRMT11:A new Arabidopsis MBD7 protein partner with arginine methyltransferase activity[J].The Plant Journal,2007,52(2):210-222.
[5]Yano A,Kodama Y,Koike A,et al.Interaction between methyl Cp G-binding protein and ran GTPase during cell division in tobacco cultured cells[J].Annals of Botany,2006,98(6):1179-1187.
[6]Li Y C,Meng F R,Yin J,et al.Isolation and comparative expression analysis of six MBD genes in wheat[J].Biochimica Biophysica Acta,2008,1779(2):90-98.
[7]孟凡荣,李占英,凌娜,等.Ta MBD2基因c DNA全长的克隆及其在小麦叶片和种子中的表达[J].麦类作物学报,2010,30(1):6-10.
[8]Im Y J,Kim J I,Shen Y,et al.Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling[J].Journal of Molecular Biology,2004,343(3):659-670.
[9]Fields S,Song O.A novel genetic system to detect protein interactions[J].Nature,1989,340:245-246.
[10]Zemach A,Grafi G.Characterization of Arabidopsis thaliana methyl-Cp G-binding domain(MBD)proteins[J].Plant J,2003,34:565-572.
[11]Kalthoff C,Alves J,Urbanke C,et al.Unusual structural organization of the endocytic proteins AP180 and epsin 1[J].Biol Chem,2002,277(10):8209-8216.
[12]Riezman H.Cell biology:The ubiquitin connection[J].Nature,2002,416(2):451-455.
[13]Xie Q,Guo H S,Dallman G,et al.SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals[J].Nature,2002,419:167-170.
[14]Welsch R,Maass D,Voegel T,et al.Transcription factor RAP2.2.and its interacting partner SINAT2:Stable elements in the carotenogenesis of Arabidopsis leaves[J].Plant Physiol,2007,145(3):1073-1085.
[15]Sakamoto H,Araki T,Meshi T,et al.Expression of a subset of the Arabidopsis Cys(2)/His(2)-type zinc-finger protein gene family under water stress[J].Gene,248(1/2):23-30.
[16]Kovarik A,Koukalova B,Bezdek M,et al.Hypermethylation of tobacco heterochromatic loci in response to osmotic stress[J].Theoretical and Applied Genetics,1997,95(1):301-306.
[17]Steward N,lto M,Yamaguchi Y,et al.Periodic DNA methylation in maize nucleosomes and demethylation by environmental stress[J].Journal of Biological Chemistry,2002,277:37741-37746.