上海交通大学：《植物生物技术》教学资源_Chloroplast Engineering_Leture 9-Chloroplast_Engineering

1日日日 SJTU Chloroplast Genetic Engineering: Recent Advances and Future Perspectives School of Life Science and Biotechnology Shanghai Jiao Tong University Dec 2009

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Chloroplast Genetic Engineering： Recent Advances and Future Perspectives School of Life Science and Biotechnology Shanghai Jiao Tong University Dec 2009

1日日日 Contents SJTU >What is a chloroplast? Structure,genomes,replication,translation,proteins >Chloroplast genetic engineering Advantages,genomes,applications,expression cassette, methods, Recent Advances Future Perspectives

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Contents Ø What is a chloroplast? structure, genomes, replication, translation, proteins Ø Chloroplast genetic engineering Advantages, genomes, applications, expression cassette, methods, Ø Recent Advances Ø Future Perspectives

Contents SJTU >What is a chloroplast? Structure,genomes,replication,translation,proteins >Chloroplast genetic engineering Advantages,genomes,applications,expression cassette, methods, Recent Advances Future Perspectives

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Contents Ø What is a chloroplast? structure, genomes, replication, translation, proteins Ø Chloroplast genetic engineering Advantages, genomes, applications, expression cassette, methods, Ø Recent Advances Ø Future Perspectives

Genomes in plant cells SJTU Nuclear-cytoplasmic system Mitochondria Plastids Plastids Etioplast Proplastid Chloroplasts Chromoplasts Amyloplasts Elaioplasts Proteinoplsts Chromoplast Chloroplast Leucoplast Proplastids Etioplasts Amyloplast Elaioplast Proteinoplast Statolith http://commons.wikimedia.org/

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Genomes in plant cells • Nuclear–cytoplasmic system • Mitochondria • Plastids Chloroplasts Chromoplasts Amyloplasts Elaioplasts Proteinoplsts Proplastids Etioplasts http://commons.wikimedia.org/

Structure of a chloroplast 1日日日 SJTU A typical plant cell might contain as many as 50 chloroplasts Outer membrane Inter membrane A system of thylakoid membranes Leaf cross section Vein Mesophyll 5 Chloroplast 7 Stomata CO2 02 Outer membrane Thylakoid Mesophyll cell Stroma Granum Thylakoid Intermembrane space space Inner membrane 5 um 1 um

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Structure of a chloroplast A typical plant cell might contain as many as 50 chloroplasts Outer membrane Inter membrane A system of thylakoid membranes

The chloroplast genome SJTU .A circular,self-replicating molecule that can vary in size from 120 to 220 kb depending upon species Conserved,with most land plant genomes composed of a single circular chromosome with a quadripartite structure Kiejeso!g pue K6ojo!g JeinoaloW queld that includes two copies of an inverted repeat (IR)that separate the large and small single copy regions (LSC and ssc) ~10000 copies per cell Photosynthesis TOBACCO (Nicotiana tabacum) Chloroplast DNA 军朝，观 Other biochemical pathways: 155,939bp 密 biosynthesis of amino acids,fatty acids,pigments,and vitamins 是鵝用1蘸0提美

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y The chloroplast genome • Conserved, with most land plant genomes composed of a single circular chromosome with a quadripartite structure that includes two copies of an inverted repeat (IR) that separate the large and small single copy regions (LSC and SSC) • A circular, self-replicating molecule that can vary in size from 120 to 220 kb depending upon species • ~10000 copies per cell • Photosynthesis • Other biochemical pathways: biosynthesis of amino acids, fatty acids, pigments, and vitamins

1日日日 Replication mechanism of chloroplast DNA SJTU Fig.2.The dual D-loop model for the initiation of chloroplast DNA replication.(A)Unidirectional elongation of nascent strand initiated from both origins.(B)Unidirectional fork movement toward each other.(C)Fusion of D-loops to Cairns-type intermediates.(D)Bidirectional,semi-discontinuous replication E.Resulting daughter molecules.The arrows denote the two D- loop initiation sites(origins). DNA replication in chloroplasts,Journal of Cell Science 104.1-9,1993

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Replication mechanism of chloroplast DNA DNA replication in chloroplasts, Journal of Cell Science 104. 1-9, 1993

Translation mechanism of chloroplast DNA 1日日日 SJTU In chloroplast,most start codon were ATG besides GUG,different mRNA has the different conservative demand of start codon Shine-Dalgarno'(SD)sequence may occur for some mRNAs The elongation was regulated by light RNA processing and editing of the initiation codon is required for translation of certain mRNAs stop PABP f23 processing 30S editing AUG SD Translation in chloroplasts,Biochimie 82 583-601,2000

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Translation mechanism of chloroplast DNA Translation in chloroplasts, Biochimie 82 583−601, 2000 • In chloroplast, most start codon were ATG besides GUG, different mRNA has the different conservative demand of start codon • Shine-Dalgarno’ (SD) sequence may occur for some mRNAs • The elongation was regulated by light • RNA processing and editing of the initiation codon is required for translation of certain mRNAs

Proteins in the chloroplast SJTU The majority of peptides in the chloroplast were encoded by the nuclear genes and synthesized on the ribosome in the cytoplasm The polypeptide precursor for chloroplast synthesized in the cytoplasm almost all contain a section of transit peptide Chloroplast genome encode hundreds of peptides and RNA by itself The peptides and a variety of RNA encoded by the Chloroplast DNA only play a role in the chloroplast Light was an important regulator for the accumulation of these proteins in the chloroplasts at different levels

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Proteins in the chloroplast • The majority of peptides in the chloroplast were encoded by the nuclear genes and synthesized on the ribosome in the cytoplasm • The polypeptide precursor for chloroplast synthesized in the cytoplasm almost all contain a section of transit peptide • Chloroplast genome encode hundreds of peptides and RNA by itself • The peptides and a variety of RNA encoded by the Chloroplast DNA only play a role in the chloroplast • Light was an important regulator for the accumulation of these proteins in the chloroplasts at different levels

Contents SJTU >What is a chloroplast? Structure,genomes,replication,translation,proteins >Chloroplast genetic engineering Advantages,genomes,applications,expression cassette, methods, Recent Advances Future Perspectives

SJTU Pla n t M ole c ula r Biolo g y a n d Bio s a f e t y Contents Ø What is a chloroplast? structure, genomes, replication, translation, proteins Ø Chloroplast genetic engineering Advantages, genomes, applications, expression cassette, methods, Ø Recent Advances Ø Future Perspectives