植物应激反应与分子生物学理论基础.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,Chaper2 植物应激反应与分子生物学理论基础,Adaptation of plants to nutrition starvation in Physiological and Molecular Levels,Development Adaptation,Nutrient signaling,Nutrient efficient activation,uptake&translocation,Molecular Adaptation,Efficient recycling utilization of nutrients inside the plants,Physiological Adaptation,Hormones,Regulation Factors,Energy、C/N metabolisms,Root forming,development&structures(nutritional capacity in rhizosphere),Root hairs induced by P-starvation,+P -P,Cluster roots induced by P-starvation,-P,Adaptation in root development,+P,-P,+P,50 mM N,3 mM N,Inhibited lateral root elongation by high N,Adaptation in molecular level,35S:OsPTF1,-P,WT 35S1 35S2,(Source:Wu P.et al.,Unpublished),Adaptation in Physiological level,80-55,陕213,85504,百泉3039,小偃6号,Different ability in acidifying rhizosphere of different genotypes of wheat,Low P,35S:CS,WT,(Source:Zhang Q.;CAS,Unpublished),Soybean,Wheat,Rice,-P,+P,-P,-P,+P,-P,Oil-seed rape,Different resistance between the genotypes of same crop to low P in soil,B10,B2,Wheat:小堰54,Germ plasma difference in resistance to low P、N,N starvation,-P,-P,Soybean,Seed Rape,Rice,In biological terms,the process by which a cell responds to signals(can be intracellular,extracellular).,Signal,Transduction,Input,Output,Signal Transduction,Signal Transduction in Plants,ALF3,High KNO,3,Emergence,Meristem,activation,Maintenance of,meristem function,ABA,auxin,？,Check-point of N-signaling/ABA interaction:using mutants of non-sensitive both to high N and to ABA,1.0 mM,50 mM,KNO,3,-,ABA,+ABA,Signal Transduction in Plants,In plants,as in animals,cells are,in constant,communication with one another.,Plant cells communicate to,coordinate,their activities in response to the changing conditions of nutrition,light,and temperature that guide the plants cycle of growth,flowering,and fruiting.,Plant cells also communicate to coordinate what goes on in their roots,stems,and leaves.,Like animals,plants make extensive use of,cell-surface receptors,.Plants seem to rely on a great diversity of,transmembrane receptor serine/,threonine,kinases,which have a typical serine/,threonine,kinase,cytoplasmic,domain and an,extracellular,ligand,-banding domain.,15.1&15.4,Two-Component Signal Transduction Pathways in,Planta,A membrane-localized His protein kinase,that senses the input signal,A,response regulator,a transcription factor,that mediates the output,(,The vertical bars represent transmembrane domains.H,His;D,Asp;P,phosphoryl group),Plant Physiol.2002 June;129(2):500515,prototypical two-component pathway uses a single phosphoryl transfer,multi-step His-to-Asp phosphorelay system,Transcription Factor,Model of the two-component signal transduction pathways in,Arabidopsis,BASIC MOLECULAR GENETIC MECHANISMS,1865,Genes are particulate factors,(by,Gregor Mendel,),1903,Chromosomes are hereditary units,(by,Walter Sutton,),1910,Genes lie on chromosomes,(by,Thomas Hunt,Morgan,),1913,Chromosomes contain linear arrays of genes,1927,Mutations are physical changes in genes,(,by Muller,),1931,Recombination is caused by crossing over,(,by Barbara McClintock,),1944,DNA is genetic material,(,by Oswald Theodore Avery,Colin McLeod and Maclyn McCarty,),1945,A gene codes for a protein,1953,DNA is a double helix,(,by James Watson and Francis Crick,),1958,DNA replicates semi-conservatively,(,by Meselson and Stahl,),1961,Genetic code is triplet,(,by Yanofsky and Brener,),1977,DNA can be sequenced,(,by Sanger and Gilbert,),1989,Polymerase Chain Reaction(PCR)technique first used,1995-1997,Genomes of 4 bacteria&yeast were sequenced,2000,Arabidopsis,genome was sequenced,2001,Human genome was sequenced,2002,Rice genome was sequenced,2004,Rat genome was sequenced,A brief history of genetics(some landmarks),1.1 Diversity of genome:,DNA,Gene,Chromosomes,Chromatin,The,genome,of an organism is the complete set of genes specifying how its phenotype will develop(under a certain set of environmental conditions).In this sense,then,diploid organisms(like ourselves)contain two genomes,one inherited from our mother,the other from our father.,DNA:,by two complementary strands of nucleotides guanine-cytosine(G-C)and adenine-thymine(A-T)base pairs.,Gene:,usually defined as A segment of DNA that contains the instructions for making a particular protein or a set of closely related proteins.Several%of genes produce a ribonucleic acid(RNA)molecule,instead of a protein,as their final product.Like proteins,these RNA molecules perform a diverse set of structural and catalytic functions in the cells.,Chromosome(染色体):,consists of a single,enormously long linear DNA molecular.Chromatids:染色单体,Chromatin(染色质):,Chromosome associated with proteins that fold and pack the fine DNA thread into a more compact structure.,Properties of the DNA Double Helix,Presentation in three ways,The two strands are held together by two forces:,the energy of,hydrogen bonding,between the complementary bases(A=T and G=C),the,hydrophobic effect,.,While the edges of the base pairs can make hydrogen bond contacts,the planar surfaces are relatively hydrophobic.Therefore,water is less ordered with the bases outside the helix(no base-paired)than when they are inside(base-paired),Which DNA molecule would have a lower melting temperature(T,m,)?,Pyrimidines:,Thymine(T)Cytosine(C),Purines:,Adenine(A)Guanine(G),(4.1),Organisms,Group,Chromosomes,Genome size(bp)*,Gene#,T4,Bacteriophage,1,2.0 x 10,5,200,E.Coli,Bacterium,1(2),4.5 x 10,6,4,000,Saccharomyces cerevisiae,Yeast,18,1.8 x 10,7,6.000,Neurospora crassa,Mo(u)ld(链孢霉),7,2.7 x 10,7,6,000,Dictyostelium discoideum,Slime mould,6,3.4 x 10,7,0.8-1x,10,4,Caenorhabdits elegans,Nematode,11/12(M/F),1.0 x 10,8,Arabidopsis thaliana,Plant,5,1.2 x 10,8,2.5 x 10,4,Drosophila melanogaster,Fruit fly,13,2.0 x 10,8,1.2 x 10,4,Oriza sativa,Plant(rice),12,4.5 x 10,8,4-5 x 10,4,Mus musculus,Mammal(小白鼠),20,3.0 x 10,9,Homo sapiens,Human,23,3.3 x 10,9,Zea mays,Plant(corn),10,5.4 x 10,9,Diversity of genome,*C-vale:,The total amount of DNA in the haploid genome is called its C value.The lack of a consistent relationship between the C value and the complexity of an organism(e.g.,amphibians vs.mammals)is called the,C value paradox,Model plants for studying plant molecular genetics,Arabidopsis,thaliana,(L.),Oriza sativa,(rice),Medicago sativa,(alfalfa),Lycopersicon esculentum,(tomato),Zea mays,L,(maize),,,zamir.sgn.cornell.edu/mutants/,,genome/,Genetic laws,G.Mendels“laws”,Law#1-The two members of a gene pair,segregate,from each other into the gametes,so that half the gametes carry one member of the pair and the other half of the gametes carry the other members of the pair.,Law#2-During gamete formation the segregation of the alleles of one gene is,independent,of the segregation of the alleles of another gene.,T.H.Morgans law,to Mendels original two:,Law#3-,linkage,(describes the tendency of genes to be inherited together as a results of their location on the same chromosome,measured by%recombination between loci:,cM,),Segregation and linkage of genes,Gene loci,allele,(等位基因),linkage,(连锁),Locus,(pl:,loci,):the position on a chromosome at which the gene for a particular trait resides;locus may be occupied by any one of the alleles for the gene.,Allele,:one of several alternative forms of a gene occupying a given locus on a chromosome.,Linkage group,:includes all loci that can be connected(directly or indirectly)by linkage relationships;equivalent to a chromosome.,Genetic linkage map,:A linear map of the relative positions of genes along a chromosome.Distances are established by linkage analysis,which determines the frequency at which two gene loci become separated during chromosomal recombination,Genetic linkage analysis,1.2 DNA replication,repairing and recombination,DNA semi-conservatively“discontinuous replication(53),During DNA replication inside a cell,each of the two old DNA strands serves as a temperate for the formation of an entire new strand.Because each of the two daughters of a dividing cell inherits a new DNA double helix containing one old and one new strand,the DNA double helix is said to be replicated“,semi-conservatively,”by a multi-enzyme complex that contains DNA polymerase.Although genetic variation is important for evolution,the survival of the individual demands genetic stability.,DNA discontinuous replication(5,3),(5.1-5.4),The DNA is always replicated from the 5 end to the 3 end.,Because the two strands are antiparallel,a leading strand just keeps on going,while a lagging strand consists of small(,Okazaki,)fragments that must later be linked together(by DNA ligase enzyme).,There are several enzymes that are involved in replication:,DNA,repairing,Accounts for 99%of all repairs,DNA repair systems,Because DNA damage occurs spontaneously and as a result to ubiquitous environmental agents,most organisms possess some capacity to repair their DNA.DNA is the only macromolecule which,IS,repaired by cells,.,(Fidelity:1 mistake/10,9,),Three categories of repair mechanisms:,1.,Damage reversal-,-simplest;enzymatic action restores normal structure without breaking backbone,2.*,Damage removal-,-involves cutting out and replacing a damaged or inappropriate base or section of nucleotides,3.,Damage tolerance-,-not truly repair but a way of coping with damage so that life can go on,DNA,general homologous,recombination-Holliday junction,DNA rearrangements occurs between DNA segments that are very similar in sequence.Although these rearrangements can result in the exchange of alleles between chromosomes,the order of the genes on the interacting chromosomes typically remains the same.,(5.5),DNA,site-specific,recombination*,alter gene order and add new information to the genome,In Prokaryotes:Bacteriophage,l,It occurs when a particular DNA that is not at all homologous to another DNA has a sequence that acts as a target for the recombination of that other DNA,Case 1:Bacteriophage,l,integrates its DNA into the,E.coli,chromosome.This recombinational event happens because there are sites in the bacteriophage DNA(att P)that have a sequence homology with sites in the bacterial DNA(att B),even though the two DNAs are completely different in all other sequences.,The enzyme that catalyzes this event is called integrase(Int),along with a host protein called IHF.When the bacteriophage DNA comes back out,it is excised by another viral protein called Xis.,DNA,site-specific,recombination,T,ransposons:mobile genetic elements(MGE),MGEs:,also called,Transposons,or,Transposable elements,(,TEs,),the sequences that move,between non-homologous sites within a genome,Three different mechanisms for transposition:,Conservative transposition,:The element itself moves from the donor site into the target site.,Replicative transposition,:The element moves a copy of itself to a new site via a DNA intermediate.,Retrotransposition,:The element makes an RNA copy of itself which is reversed-transcribed into a DNA copy which is then inserted,Short inverted repeats of transposon,The,Short inverted repeats,at the ends of the element are acted on by the,transposase,gene encoded for by the element.These inverted repeats act as the substrates for recombination reactions mediated by the transposase,Mitosis,(有丝分裂）,and Meiosis,（减数分裂）,1.3 From DNA to Protein:,Transcription and Translation,Basic steps of gene expression,ATG,STOP,Poly-A site,Promotor,EXONS,INTRONS,TRANSCRIPTION,5 capping&RNA splicing,&3 polyadenylation,AAAA,AAAA,EXPORT,mRNA,mRNA,Peptide,TRANSLATION,Primary RNA transcript,5 RNA cap,Genomic,DNA,CYTOPLASM,NUCLEUS,Protein,protein degradation,Initiation of protein synthesis,Protein folding&localization,The 5-methyl capping is the 1st modification of eucaryotic pre-mRNAs:,As soon as RNA pol II produces 25 nucleotides of RNA,the 5end of the new RNA molecule is capped by adding a methy group(CH3-)to guanosine.,The 5-methyl capping is the 1st modification of eucaryotic pre-mRNAs:,As soon as RNA pol II produces 25 nucleotides of RNA,the 5end of the new RNA molecule is capped by adding a methy group(CH3-)to guanosine.,Genetic codes for protein,Transcription(DNA,RNA)machines,(6.1-6.3),Translation(mRNA,protein),machines,T-shape of tRNA,RIBOSOME,mRNA,(6.4 6.5&3.10),Protein Structure,(3.1-3.4,),1.4 An Overview of Gene Control,The different cell types of a multi-cellular organism contain the same DNA,Different cell types synthesize different sets of proteins,A cell can change the expression of its genes in response to external signals,Gene expression can be regulated at many of the steps in the pathway from DNA to RNA to protein,Inactive protein,1.4 Control of Gene Expression,Structure of Genes In Eukaryotic Organisms,hnRNA,heterogeneous nuclear RNA,RNA splicing,Transcription,mRNA,Control Elements,genomic-DNA,Structure of Genes In Eukaryotic Organisms,Coding sequence can be discontinuous and the gene can be composed of many,introns,and,exons,The control regions(,=,operators)can be spread over a large region of DNA and exert,action-at-a-distance,Many different regulators,acting on a single gene,Alternate splicing can give rise to,more than one protein,product from a single gene,Predicting genes,(,introns,exons,and proper splicing)is very challenging,Because the control elements can be spread over a large segment of DNA,predicting the important sites and their effects on gene expression are not very feasible,Control of Eukaryotic Transcription Initiation,Control of Eukaryotic Transcription Initiation,Transcription of,RNAs,in eukaryotes:,RNA,pol,I synthesizes the,rRNAs,RNA,pol,II synthesizes the mRNAs and some,snRNAs,RNA,pol,III synthesizes the 5,S,rRNA,and the,tRNAs,2.,Majority of eukaryotic,RNAs,are subjected to post-transcriptional processing:,The most complex controls are those that regulate the expression of RNA,pol,II-transcribed genes,the mRNA genes.,Almost all eukaryotic mRNA genes contain a basic structure:coding,exons,non-coding,introns,basal promoters,+transcriptional regulatory domains.,The basal promoter elements are termed CCAAT-boxes(pronounced,cat,)and TATA-boxes because of their sequence motifs.The TATA-box resides 20 to 30 bases upstream of the transcriptional start site.,The switching devices for gene expression in regulatory region contain two types of fundamental components:,short stretches of DNA of defined sequence,gene regulatory proteins that recognize and bind to them,Chapter 11b Gene Regulation in Eukaryotes,(pages 479-505).,Use of reporter genes in promoter analysis,Transgenic plants-,assay GUS or GFP activity in low P conditions,P transporter promoter fragments,GUS/GFP reporter gene,2.0kb,1.5kb,1.1kb,0.6 kb,Transient expression following particle bombardment,(Schumann et al.,2004),Functional role of P1BS element of Pht1;1 promoter:Binding site with PHR1 transcription factor;Regulating P-starvation induced Pht1;1 expression,Gene regulatory proteins contain structural motifs that can read DNA sequences,The helix-turn-helix motif is one of the simplest and mo