摘要
叶绿体是植物非常重要的细胞器,除了进行光合作用外,还参与了其他的生物代谢.在模式植物拟南芥中,叶绿体基因组编码87 个叶绿体蛋白,核基因组编码约4255 个叶绿体蛋白.绝大部分叶绿体蛋白的功能通过序列同源性分析确定,但是仍有约25%的蛋白质功能尚不清楚.本论文利用生物信息学方法并结合分子生物学方法对拟南芥叶绿体蛋白质功能进行研究.首先利用三种生物信息学的方法构建了叶绿体蛋白质相互作用数据库.其中利用平移法,将5 个物种的蛋白质相互作用数据平移到拟南芥中,共获得拟南芥叶绿体蛋白质相互作用8157对;利用系统进化谱法,共获得可能的叶绿体相互作用的数据19,652 对;利用基因芯片表达谱法,对两个基因表达系数为0.90 以上数据分析,共获相互作用数据65,299 对.然后我们对以上构建的拟南芥叶绿体蛋白相互作用数据进行分析。这些蛋白质相互作用对参与多种代谢途径,如蛋白质的转运,淀粉代谢,脂肪酸代谢,光合作用等.预测结果表明,多个未知蛋白质与光合作用四个复合体中的亚单位相互作用;叶绿体中FtSH 家族金属蛋白酶7 个成员之间有相互作用;与光合作用蛋白质磷酸化作用的激酶STN7 和STN8 相互作用的蛋白质多为蛋白质激酶,多数蛋白质激酶的生物功能未知.最后我们通过酵母双杂交实验验证部分蛋白质的相互作用。从所构建的拟南芥叶绿体蛋白质相互作用数据库中,选取了46 对与光合作用有关的蛋白质相互作用对进行实验验证,初步实验结果表明,一个未知蛋白质C 与光合系统I 的蛋白psaD2 存在相互作用,因此,该未知蛋白质C 可能与光合作用有关.该数据库将有助于我们深入开展拟南芥叶绿体系统生物学研究以及光合作用相关蛋白的功能挖掘.
Chloroplast is a very important organelle of plant cells. Besides photosynthesis,it also carries out other essential metabolism. In model plant Arabidopsis thaliana, atotal of 87 chloroplast proteins are encoded by plastomes, and about 4255nulcear-encoded proteins are predicted to be located in chloroplast. The functions ofmost chloroplast proteins are identified based on sequence homology, and about 25%of chloroplast proteins are still unknown. In this dissertation, chloroplast proteinfunctions in Arabidopsis thaliana were investigated, using both bioinformatics andmolecular biology techniques. Firstly, a chloroplast protein interaction database wasconstructed using three bioinformatics methods. A total of 8157 pairs of chloroplastprotein interactions were predicted based on protein interlogs from data of other 5species; a total of 19652 pairs were predicted based on the phylogenetic profile; and atotal of 65299 pairs were predicted based on the expression level of genes from 79gene chips data downloaded from TAIR with the coefficient of the gene co-expressionmore than 0.9000. After the chloroplast protein interactions database was constructed,protein interactions in the database were analyzed. The protein pairs in the databasewere involved in metabolic pathways such as protein transport, starches, lipidmetabolites and photosynthesis. Our database also showed that some unknownproteins interact with subunits of four major complexes in photosynthesis, and mostproteins interacting with STN7 or STN8, which are responsible for thephosphorylation in photosynthesis were protein kinases. Finally, a total of 46 proteininteraction pairs were validated by two-hybrid experiments. Our results indicated thatone unknown protein C interacts with psaD2, a protein of photosystem I. Thus, it isquite possible that the unknown protein C is involved in photosynthesis. The databasewould facilitate us the further investigation of the chloroplast systems biology andfunctional mining of proteins related to photosynthesis in Arabidopsis thaliana.
引文
姜泊, 张亚历, 周殿元(1995) 分子生物学常用实验方法. 北京: 科学出版社.
C W 迪芬巴赫, G S 德维克斯勒(1998) PCR 技术实验指南. 北京: 科学出版社.
卢圣栋(1993) 现代分子生物学实验技术. 北京: 高等教育出版社.
史雨红,郑滔,陈剑平(2003) 酵母双杂交系统在植物病毒学中的应用. 浙江农业学报15(1):42-46.
石晓冰,魏家绵,沈允钢(2001) 叶绿体ATP 合酶CF_1 与CF_0 亚基间的相互作用. 科学通报46:1550-1554.
万群,吴洁,宋明(2004) 表达谱基因芯片及其在植物研究中的作用. 四川农业科技4:5-6.
王建,张令强(2003)蛋白质间连锁图的建立. 钱小红,贺福初主编,蛋白质组学:理论与方法. 北京:科学出版社,p242-265.
吴乃虎(1998) 基因工程原理. 北京:科学出版社.
吴志聪, 王一飞(2003) 酵母双杂交系统. 生物技术13(5):43-44.
张迪,霍克克,赵翔等(2000) 酵母双杂交技术研究进展. 高技术通讯, 10 (11 1): 98-101.
朱金鑫,李小方(2004) 酵母双杂交技术及其在植物研究中的应用植物生理学通讯40(2):235-240.
Abdallah F, Salamini F, Leister D (2000) A prediction of the size and evolutionary origin of the proteome of chloroplasts of Arabidopsis. Trends Plant Sci 5(4):141-2.
Adam Z, Clarke AK (2002) Cutting edge of chloroplast proteolysis. Trends Plant Sci. 7(10):451-6.
Adam Z (2000) Chloroplast proteases: possible regulators of gene expression? Biochimie 82(6-7):647-54.
Adam Z, Adamska I, Nakabayashi K et al (2001) Chloroplast and mitochondrial proteases in Arabidopsis. A proposednomenclature. Plant Physiol 125: 1912–1918
Andrade MA, Sander C (1997) Bioinformatics: from genome data to biological knowledge. Curr Opin Biotechnol 8(6):675-83.
Baginsky S, Gruissem W (2004) Chloroplast proteomics: potentials and challenges. J Exp Bot 55(400):1213-20.
Bellafiore S, Barneche F, Peltier G et al (2005) State transitions and light adaptation require chloroplast thylakoid protein kinase STN7. Nature 24;433(7028):892-5.
Bornke F (2005) The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system. J Plant Physiol 162(2):161-8.
Birnbaum K, Shasha DE, Wang JY et al (2003) A gene expression map of the Arabidopsis root. Science 302(5652):1956-60.
Bork P, Ouzounis C and Sander C (1994) From genome sequences to protein function. Curr Opin Struct Biol 4:393-403.
Bork P, Dandekar T, Diaz-Lazcoz Y et al (1998) Predicting function: From genes to genomes and back. J Mol Bio 283:707-725.
Brenner SE (1999) Errors in genome annotation. Trends Genet 15:132-133.
Budziszewski GJ, Lewis SP, Glover LW et al (2001) Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning. Genetics 159(4):1765-78.
Choi G, Yi H,Lee J et al (1999) Phytochrome signaling is mediated through nucleoside diphosphate kinase2. Nature 401:610-613.
Davis SJ, Kurepa J, Vierstra RD (1999) The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases. Proc Natl Acad Sci U S A 96(11):6541-6.
de Folter S, Immink RG, Kieffer M, et al (2005) Comprehensive Interaction Map of the Arabidopsis MADS Box Transcription Factors. Plant Cell 17:1424–1433.
Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8(5):978-84.
Emanuelsson O, Nielsen H, Brunak S et al (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300(4):1005-16.
Felder S, Meierhoff K, Sane AP et al (2001)The nucleus-encoded HCF107 gene of Arabidopsis provides a link between intercistronic RNA processing and the accumulation of translation-competent psbH transcripts in chloroplasts. Plant Cell 13(9):2127-41.
Ferro M, Salvi D, Brugiere S, Miras S et al (2003) Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana. Mol Cell Proteomics 2:325-345.
Fraster CM, Gocayne JD,White O et al (1995) The minimal gene complement of Mycoplasma genitalium. Science 270:397-388.
Fraser CM, Norris SJ, Weinstock GM et al (1998) Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 281(5375):375-88.
Friso G, Giacomelli L, Ytterberg AJ et al (2004) In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database. Plant Cell 16(2):478-99.
Fankhauser C,Yeh KC, Lagarias et al (1999) PKS1, a Substrate Phosphorylated by Phytochrome That Modulates Light Signaling in Arabidopsis. Science 284: 1539-1541
Giot L, Bader JS, Brouwer C, Chaudhuri A et al (2003) A protein interaction map of Drosophila melanogaster. Science 302(5651):1727-36.
Gupta R, He Z, Luan S (2002) Functional relationship of cytochrome c(6) and plastocyanin in Arabidopsis. Nature 417(6888):567-71.
Ge H, Liu Zh,George M et al (2001) Correlation between transcriptome and interactome mapping data from saccharomyces cerevisiae. Nature Genetics 29:482-486.
Guevara-Garcia A, San Roman C, Arroyo A et al (2005) Characterization of the Arabidopsis clb6 Mutant Illustrates the Importance of Posttranscriptional Regulation of the Methyl-D-Erythritol 4-Phosphate Pathway. Plant Cell 17(2):628-43.
Gutierrez-Nava Mde L, Gillmor CS, Jimenez LF, et al (2004) CHLOROPLAST BIOGENESIS genes act cell and noncell autonomously in early chloroplast development. Plant Physiol 135(1):471-82.
Gutierrez RA, Green PJ, Keegstra K et al (2004) Phylogenetic profiling of the Arabidopsis thaliana proteome: what proteins distinguish plants from other organisms? Genome Biol 5(8):R53.
Haldrup A, Lunde C, Scheller HV (2003) Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition, have unstable photosystem I complexes, and altered redox homeostasis in the chloroplast stroma. J Biol Chem. 29;278(35):33276-83.
Hegyi H and Gerstein M (2001) Annotation transfer for Genomics:Measuring functional divergence in muti-domain proteins. Genome Res 11:1632-1640.
Heinemeyer J,Eubel H,Wehmhoner D et al (2004)Proteomic approach to characterize the supramolecular organization of photosystems in higher plants. Phytochemistry 65(12):1683-92.
Honma T, Goto K (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409(6819):525-9.
Huala E, Dickerman AW, Garcia-Hernandez M et al (2001) The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant. Nucleic Acids Res 29(1):102-5.
Ichimura K, Mizoguchi T, Irie K et al (1998) Isolation of ATMEKK1(a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis. Biochem Biophys Res Commun 253:532-543.
Ihnatowicz A, Pesaresi P, Varotto C et al (2004) Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions. Plant J 37(6):839-52.
Ito T, Chiba T, Ozawa R, Yoshida M et al (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A 98(8):4569-74.
Jarillo JA, Capel J, Tang RH et al (2001) An Arabidopsis circadian clock component interacts with both CRY1 and phyB. Nature 22;410(6827):487-90.
Kubis S, Baldwin A, Patel R, et al (2003) The Arabidopsis ppi1 mutant is specifically defective in the expression, chloroplast import, and accumulation of photosynthetic proteins. Plant Cell 15(8):1859-71.
Kieselbach T, Bystedt M, Hynds P, et al (2000) A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen. FEBS Lett 480(2-3):271-6.
Koncz C, Chua N H, Schell J (1992) Methods in Arabidopsis Research. World Scientific publishing Co Pte Ltd.
Leister D (2003) Chloroplast research in the genomic age. Trends Genet.19(1):47-56.
Lezhneva L, Meurer J (2004) The nuclear factor HCF145 affects chloroplast psaA-psaB-rps14 transcript abundance in Arabidopsis thaliana. Plant J 38(5):740-53.
Lennartz K, Plucken H, Seidler A et al (2001) HCF164 encodes a thioredoxin-like protein involved in the biogenesis of the cytochrome b(6)f complex in Arabidopsis. Plant Cell 13(11):2539-51.
Li S, Armstrong CM, Bertin N et al (2004) A map of the interactome network of the metazoan C. elegans. Science 303:540-543.
Lockhait DJ, Winzeler EA (2000) Genomics,gene expression and DNA arrays. Nature 405:827-836
Ma L, Li J, Qu L et al (2001) Light control of Arabidopsis development entails coordinated regulation of genome expression and cellular pathways. Plant Cell 13(12):2589-607.
Mandel MA, Feldmann KA, Herrera-Estrella L et al (1996) CLA1, a novel gene required for chloroplast development, is highly conserved in evolution. Plant J 9(5):649-58.
Marcotte EM, Pellegrini M, Thompson MJ et al (1999) A combined algorithm for genome-wide prediction of protein function. Nature 402:83-96.
Martin W, Rujan T, Richly E et al (2002) Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc Natl Acad Sci U S A 99(19):12246-51.
Meyerowitz E M (1987) Arabidopsis thaliana. Annu Rev Genet 21: 93-111.
Meurer J, Plucken H, Kowallik KV et al (1998) A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana. EMBO J 17(18):5286-97.
Moller SG, Kunkel T, Chua NH (2001) A plastidic ABC protein involved in intercompartmental communication of light signaling. Genes Dev 15(1):90-103.
Motohashi R, Nagata N, Ito T et al (2001) An essential role of a TatC homologue of a Delta pH- dependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana. Proc Natl Acad Sci U S A 98(18):10499-504.
Munekage Y, Hashimoto M, Miyake C et al (2004) Cyclic electron flow around photosystem I is essential for photosynthesis. Nature 429(6991):579-82.
Ni M, Tepperman JM, Quail PH (1999) Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. Nature 400(6746):781-4.
Ni ZL, Shi XB, Wei JM (2004) Functional consequences of N-or C-terminal deletions of the delta subunit of chloroplast ATP synthase. Biochemistry 43(8):2272-8.
Ort, D.R., and Yocum, C.F (1996) Oxygenic Photosynthesis: The Light Reactions. (Dordrecht, The Netherlands: Kluwer Academic Publishers).
Park SW, Yu SH, Kim MII et al (2000) Interaction of PRK1 receptor-like kinase with a putative eIF2b β-subunit in tobacco. Mol Cells 10:626-632.
Pellegrini M, Marcotte EM, Thompson MJ et al (1999) Assigning protein functions bycomparative genome analysis: protein phylogenetic profiles. Proc Natl Acad Sci U S A13;96(8):4285-8.
Peltier JB, Friso G, Kalume DE et al (2000) Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins. Plant Cell 12(3):319-41.
Peltier JB, Emanuelsson O, Kalume DE et al (2002) Central functions of the luminal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction. Plant Cell 14(1):211-36.
Peltier JB, Ytterberg AJ, Sun Q et al (2004) New Functions of the Thylakoid Membrane Proteome of Arabidopsis thaliana Revealed by a Simple, Fast, and Versatile Fractionation Strategy. J. Biol. Chem 279(47): 49367 -49383.
Pesaresi P, Varotto C, Richly E et al (2001) Functional genomics of Arabidopsis photosynthesis. Plant Physiol.Biochem 39:285-294.
Pyke, KA (1999) Plastid division and development. Plant Cell 11:549-556.
Qiao H, Wang F, Zhao L et al (2004)The F-box protein AhSLF-S2 controls the pollen function of S-RNase-based self-incompatibility. Plant Cell 16(9):2307-22
Quail PH (2000) Phytochrome-interacting factors. Seminas Cell Dev Biol.11:457-466.
Raynaud C, Cassier-Chauvat C, Perennes C et al (2004)An Arabidopsis homolog of the bacterial cell division inhibitor SulA is involved in plastid division. Plant Cell 16(7):1801-11.
Reiter RS, Coomber SA, Bourett TM et al (1994) Control of leaf and chloroplast development by the Arabidopsis gene pale cress. Plant Cell 6(9):1253-64.
Rochaix, J.-D., Goldschmidt-Clermont, M., and Merchant, S (1998).The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, Advances in Photosynthesis and Respiration, Vol. 7. (Dordrecht, The Netherlands: Kluwer Academic Publishers).
Rujan T, Martin W (2001) How many genes in Arabidopsis come from cyanobacteria? An estimate from 386 protein phylogenies. Trends Genet 17(3):113-20.
Richly E, Leister D (2004) An improved prediction of chloroplast proteins reveals diversities and commonalities in the chloroplast proteomes of Arabidopsis and rice. Gene 329:11-6.
Sambrook J, Russell D V ( 2001) Molecular Cloning: A Laboratory Mannual, 3rd ed. Cold Spring Harbor Laboratory Press.
Sang Y, Li QH, Rubio V et al (2005) Arabidopsis CRYPTOCHROME 1 N-Terminal Domain-Mediated Homodimerization Is Required for Its Photoreceptor Activity. Plant Cell 17: 1569-1584.
Sakamoto W, Zaltsman A, Adam Z et al (2003) Coordinated regulation and complex formation of yellow variegated1 and yellow variegated2, chloroplastic FtsH metalloproteases involved in the repair cycle of photosystem II in Arabidopsis thylakoid membranes. Plant Cell 15(12):2843-55.
Scheller HV, Jensen PE, Haldrup A et al (2001) Role of subunits in eukaryotic Photosystem I. Biochim Biophys Acta 1507(1-3):41-60.
Schubert M, Petersson UA, Haas BJ et al (2002) Proteome map of the chloroplast lumen of Arabidopsis thaliana. J Biol Chem 277(10):8354-65.
Seki M, Narusaka M, Ishida J et al (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J 31(3):279-92.
Shi XB, Wei JM, Shen YK (2001) Effects of sequential deletions of residues from the N-or C-terminus on the functions of epsilon subunit of the chloroplast ATP synthase. Biochemistry 40(36):10825-31.
Sternberg PW (2001)Working in the post-genomic C.elegans world. Cell 105,173-176.
Stockel J, Oelmuller R (2004) A novel protein for photosystem I biogenesis. J Biol Chem 279(11):10243-51.
Streatfield SJ, Weber A, Kinsman EA, et al (1999) The phosphoenol pyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression. Plant Cell 11(9):1609-22.
Sundberg E, Slagter JG, Fridborg I, et al (1997) ALBINO3, an Arabidopsis nuclear gene essential for chloroplast differentiation, encodes a chloroplast protein that shows homology to proteins present in bacterial membranes and yeast mitochondria. Plant Cell 9(5):717-30.
The Arabidopsis Genome Initiative (2000)Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796-815.
Uetz P, Giot L, Cagney G et al (2000) A comprehensive analysis of protein-protein interactions