Lecture 11 Promoters
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Lecture 11 Promoters and marker genes Neal Stewart 2016 Discussion questions: promoters • What heterologous promoters are used to make transgenic plants, and why? • Why are constitutive promoters so popular? • What other kinds of promoters (besides constitutive) are needed and why? Discussion questions: marker genes 1. Why use marker genes for producing transgenic plants? 2. What are some differences between selectable markers and scorable markers? 3. What are the relative merits of and enzymatic marker such as GUS and an in vivo marker such as GFP? 4. What are the advantages, if any, for the use of the manA gene over the nptII gene as a selectable marker for food and feed crops, and would the use of the manA gene overcome public concern over the use of the nptII gene? Conversely, what are the disadvantages? Figure 7.9 Figure 7.7 What would be the properties of an ideal promoter? • Broad range of expression in a number of diverse species • Of plant origin • Maybe constitutive • High expression • Tissue specificity • Developmental specificity • Inducibility • Compact—short sequence Figure 10.1 Figure 10.1 Schemes of a gene with a 5’ and 3’untranslated region (UTR) and a TATA-box promoter (a) and with a synthetic promoter (b). The TATA-box promoter is located upstream of the transcription start site (TSS), and contains a TATA-box in the core region, a few key motifs in the proximal region and many more distant motifs in the distal region. These motifs are the binding sites for various transcription factors, activators or repressors, and can be used together with a core promoter (TATA-box) to engineer a synthetic promoter. The motif selection, motif number, arrangement and space as well as the selection of 5’ and 3’ UTR in synthetic promoters are subject to the expected promoter functions and optimization. TABLE 10.1 The most widely used tissue-specific promoters in plants. Promoter Promoter Type Name Green tissue Vascular Gene Function Species Reference Cab3 Chlorophyll a/b-binding protein Arabidopsis Mitra et al., 1989 rbcS Ribulose bisphosphate carboxylase small subunit Arabidopsis De Almeida et al., 1989 PEPC Phosphoenolpyruvate carboxylase Maize Ku et al., 1999 PP2 Phloem protein 2 Pumpkin Guo et al., 2004 Pfn2 Profilin 2 Arabidopsis Christensen et al., 1996 EIR1 Ethylene insensitive root1 Arabidopsis Luschnig et al., 1998 NAC10 NAM, ATAF1-2, CUC2 Rice Jeong et al., 2000 Lat52;59 Late anthogenesis Tomato Twell et al., 1991 TA29 Tobacco anther-specific protein TA29 Tobacco Koltunow et al., 1990 Zm13 Pollen specific Maize Hamilton et al., 1998 napA Napin storage protein Brassica napus Rask et al., 1998 GluB-1 Glutelin storage protein Rice tissue Root Pollen Seed Wu et al., 2000 TABLE 10.2 The most widely used inducible promoters in plants. Promoter Inducibility Promoter Name Gene Function Species Reference Pathogen PR1 Pathogenesis-related 1 Lebel et al., 1998 NPR1 Nonexpressor of PR1 Arabidopsis Arabidopsis VSP1 Vegetative storage protein 1 Arabidopsis Guerineau et al., 2003 PcPR1-1 Pathogenesis-related 1 Parsley Rushton et al., 2002 PcPAL1 Phenylalanine ammonia-lyase 1 Parsley Lois et al., 1989 PR2-d Pathogenesis-related 2-d Tobacco Shah et al., 1996 NtGlnP Glucanase 2 Tobacco CHS Chalcone synthase Parsley Weisshaar et al., 1991 LHCP Light-harvesting chlorophyll a/b protein Pea Simpson et al., 1985 Rca Rubisco activase Spinach Orozco & Ogren 1993 MPI Maize proteinase inhibitor Maize Cordero et al., 1994 Pin2 Proteinase inhibitor II Potato Thornburg et al., 1987 Drought ERD1 Early responsive to dehydration stress 1 Arabidopsis Tran et al., 2004 Salt RD29A,B Responsive to desiccation 29A, B Arabidopsis Yamaguchi-Shinozaki & Shinozaki 1994 Cold Cor15A Cold-regulated 15A Arabidopsis Stockinger et al., 1997 CBF2/DREB1C C-repeat/DRE binding factor 1 Arabidopsis Zarka et al., 2003 HVA22 ABA-inducible Wheat Shen et al., 1993 Osem Rice homolog of Em Rice Hattori et al., 1995 ABA, RD22 Responsive to desiccation Arabidopsis Abe et al., 1997 Drought Em Late embryogenesis Wheat Guiltinan et al., 1990 RD29A,B Responsive to desiccation 29A, B Arabidopsis Yamaguchi-Shinozaki & Shinozaki 1994 AlcA Alcohol-regulated Asperqillu nidulans Caddick et al., 1998 Light Wound ABA Ethanol Yu et al., 2001 Selectable markers • Typically used to recover transgenic plant cells from a sea of non-transgenic cells • Antibiotic resistance markers and herbicide resistance markers are most common Scorable markers (reporter genes) • Can help visualize transient expression • Can help visualize if tissue is stably transgenic • Useful for cellular and ecological studies Figure 10.8 TABLE 10.4 Categories of Marker Genes and Selective Agents Used in Plants Category Marker Genes Selectable marker genes: Antibiotic resistant nptII, neo, aphII hpt, hph, aphIV Herbicide resistant bar pat CP4 EPSPS Nutritional inhibitor manA related xylA Hormone related Ablation Reporter genes: aka scorable marker genes Enzymatic ipt codA uidA, gusA Luc Fluorescent proteins gfp pporRFP mOrange Source of Genes Selective Agent Escherichia coli Tn5 (bacteria) E. coli (bacteria) Streptomyces hygroscopicus (bacteria) Kanamycin Hygromycin Phosphinothricin S. viridochromogenes (bacteria) Phosphinothricin Agrobacterium sp. strain CP4 (bacteria) E. coli (bacteria) S. rubiginosus; Thermoanaerobacterium Thermosulfurogenes (bacteria) Agrobacterium tumefaciens (bacteria) E. coli (bacteria) Glyphosate Mannose D-xylose E. coli (bacteria) MUG, X-gluc Aequorea victoria (jellyfish) Porites porites (hard coral) Discosoma sp. (soft coral) N/A N/A N/A N/Aa 5-Fluorocytosine Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed Figure 10.2 Figure 10.5 Barnase kills tapetum cells (and pollen)— non-conditional selection useful to engineer male-sterility Common reporter genes • Beta glururonidase (GUS) uidA protein from Escherichia coli– needs the substrate X-gluc for blue color • Luciferase proteins from bacteria and firefly yields light when substrate luciferin is present. • Green fluorescent protein (GFP) from jellyfish is an example of an autofluorescent protein that changes color when excited by certain wavelengths of light. • Red and orange fluorescence proteins—RFP and OFP. Figure 10.9 Figure 10.9 Luminescence detected in transgenic tobacco transformed with the firefly luciferase gene driven by the 35S promoter and watered with a solution of luciferin, the luciferase substrate. [Reprinted with permission from Ow et al. (1986), copyright 1986, AAAS.] Figure 10.6 GUS positive plants and cells Figure 10.8 http://www.youtube.com/watch?v=90wpvSp4l_0&feature=related 35S:GFP canola White light UV light in a darkened room agged GFP—segregating 1:1 GFP-tagged pollen on a bee leg. Hudson et al 2001 Mol Ecol Notes 1:321 Green (and other color) fluorescent proteins • • • • • FP properties Detection and measurement Anthozoan FPs Why red is better than green Why orange is best of all! What is fluorescence? Excitation 475 nm Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852 Stokes shift* x Emission 507 nm Quantum yield = Brightness % light fluoresced Horseweed transformation with GFP Blue Light with GFP Filter White Light Blue Light with GFP Filter White Light Transgenic flower cross section Transgenic versus wild-type flowers Relative fluorescence In planta fluorescence ex = 395 nm Wavelength (nm) LIFI-laser induced fluorescence imaging—for stand-off detection of GFP and other flourescence Journal of Fluorescence 15: 697-705 Canola LIFS 200000 180000 A1 160000 A2 Water Raman Peak 140000 A3 A4 Intensity 120000 A5 100000 A6 80000 A7 60000 A8 A9 40000 20000 0 400 450 500 550 600 Nanom eters (nm ) 650 700 750 800 A brief FP history Patterson Nature Biotechnol. (2004) 22: 1524 Anthozoan FPs in transgenics Wenck et al Plant Cell Rep 2003 22: 244 Soybean ZsGreen Wheat leaf DsRed Rice callus ZsGreen Corn callus AmCyan Cotton AmCyan Cotton ZsGreen Cotton callus AsRed DsRed tobacco Fluorescence Excitation 475 nm Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852 Stokes shift* x Emission 507 nm Quantum yield % fluoresced = Brightness Species and FP name Ex max nm (Ext Coef) Em max nm (103 M-1 cm-1) Reference (Quantum yield %) Aequorea victoria GFP 395 (27) 504 (79) Tsien 1998 A. victoria GFP S65T 489 (55) 510 (64) Tsien 1998 A. victoria EGFP 488 (56) 508 (60) Tsien 1998 A. victoria GFP “Emerald” 487 (58) 509 (68) Tsien 1998 A. victoria GFPYFP “Topaz” 514 (94) 527 (60) Tsien 1998 A. victoria GFPYFP “Venus” 515 (92) 528 (57) Nagai et al. 2002 Zoanthus sp. ZsGreen 497 (36) 506 (63) Matz et al. 1999 Zoanthus sp. ZsYellow 528 (20) 538 (20) Matz et al. 1999 Anemonia majano AmCyan 458 (40) 486 (24) Matz et al. 1999 Heteractis crispa t-HcRed1 590 (160) 637 (4) Fradkov et al. 2002 Discosoma sp. DsRed 558 (75) 583 (79) Matz et al. 1999 Discosoma sp. mRFP1 584 (50) 607 (25) Discosoma sp. dimer2 552 (69) 579 (29) Campbell et al. 2002, Shaner et al. 2004 Campbell et al. 2002, Shaner et al. 2004 Discosoma sp. mOrange 548 (71) 562 (69) Shaner et al. 2004 Discosoma sp. dTomato 554 (69) 581 (69) Shaner et al. 2004 Discosoma sp. tdTomato 554 (138) 581 (69) Shaner et al. 2004 620 633 646 620 633 646 607 594 581 568 555 542 529 516 503 490 477 464 451 438 425 400000 350000 300000 250000 200000 150000 100000 50000 0 Wavelength 375 nm excitation 425 nm excitation 525 nm excitation 550 nm excitation 475 nm excitation Nicotiana tabacum leaf fluorescence 400000 350000 300000 250000 200000 150000 100000 50000 Wavelength 607 594 581 568 555 542 529 516 503 490 477 464 451 438 0 425 CPS Excitation scan: Nontransgenic leaf fluorescence—why red fluorescence is better than green CPS Brassica napus leaf fluorescence With GFP Brassica napus leaf fluorescence 400000 350000 300000 CPS 250000 200000 150000 100000 50000 646 633 620 607 594 581 568 555 542 529 516 503 490 477 464 451 438 425 0 Wavelength 375 nm excitation 425 nm excitation 475 nm excitation 525 nm excitation 550 nm excitation GFP 375 nm excitation Nicotiana tabacum leaf fluorescence 400000 350000 300000 200000 150000 100000 50000 646 633 620 607 594 581 568 555 542 529 516 503 490 477 464 451 438 0 425 CPS 250000 Why RFP is better– less fluoresc ence “noise” in the red More colors in fluorescent proteins discovered (mostly from corals…then improved) http://www.photobiology.info/Zimmer_files/Fig6.png Relative Brightness (% of EGFP) 250 200 GFP 150 100 50 EBFP EBFP2 Azurite mTagBFP mTurquoise mECFP Cerulean ECFP CyPet TagCFP AmCyan1 mTFP1 (Teal) Midor-Ishi Cyan TurboGFP Azami Green TagGFP AcGFP ZsGreen EGFP Emerald Superfolder GFP mWasabi T-Sapphire TagYFP EYFP Topaz Venus mCitrine Ypet PhiYFP ZsYellow1 mBanana Kusabira Orange mOrange Kusabira Orange2 mOrange2 dTomato dTomato dTomato-Tandem DsRed2 DsRed Ta gRFP Ta gRFP-T DsRed-Express(T1) mTangerine DsRed-Monomer mApple AsRed2 mStrawberry mRuby mRFP1 jRed mCherry HcRed1 dKeima-Tandem mRaspberry HcRed-Tandem mPlum AQ143 300 Brightness of Fluorescent Proteins 445 Jennifer Hinds 489 510 539 Emission Maximum (nm) 584 Orange Fluorescent Protein 0 610 Orange Fluorescent Protein (OFP) An old trick: ER targeting Signal transit 5’ GFP HDEL 3’ peptide Signal peptide directs GFP to endoplasmic reticulum for secretion But HDEL tag sequesters assembled GFP in ER—protected environment allows more accumulation. Haseloff et al 1997 PNAS 94: 2122. ER retention dramatically improves OFP brightness (monomers) 3x brighter! Big Orange Fluorescent Proteins Mann et al. 2012 Red foliage as output Arabidopsis MYB transcription factor PAP1 regulates the expression of anthocyanin biosynthesis genes: overexpression of PAP1 results in a red-plant phenotype
