Chapter 15 Gene Cloning & DNA Analysis in Agriculture

Transcription

1 Chapter 15 Gene Cloning & DNA Analysis in Agriculture 1. The gene addition approach to plant genetic engineering 2. Gene subtraction 3. Problems with genetically modified plants Agriculture or cultivation plants = world s oldest biotechnology Human have constantly searched for improved varieties of their crop plants Better nutritional qualities, higher yields Features aid cultivation & harvesting Most sophisticated breeding program retains an element of chance dependent on random merging of parental characteristics in hybrid offspring Display a precise combination of desired characteristics A lengthy & difficult process 1

2 Gene cloning provides a new dimension to crop breeding Enable directed changes to be made to genotype of a plant Circumvent random processes inherent in conventional breeding Two general strategies Gene addition: cloning is used to alter characteristics of a plant Gene subtraction: gene engineering techniques are used to inactivate one or more of plant s existing genes 15.1 The gene addition approach to plant genetic engineering Gene addition Use of cloning techniques to introduce into a plant one or more new genes coding for a useful characteristic that plant lacks A good example Development of plants that resist insect attack by synthesizing insecticides coded by cloned genes 2

3 A number of projects are being carried out around the world A representative selection of these projects Plants that make their own insecticides Most conventional insecticides e.g. Pyrethroids & organophosphates Relative non-specific poisons that kill a broad spectrum of insects High toxicity-some also have potentially harmful side effects for other members of local biosphere Exacerbate ( )-need to apply them to plants surfaces by spraying Subsequent movement of them in ecosystem cannot be controlled Insects that live within plant, or on undersurfaces of leaves, can sometimes avoid toxic effect altogether 3

4 What features would be displayed by ideal insecticide? Must be toxic to insects which it is targeted Toxicity should be highly selective Harmless to other insects & not poisonous to animal & human Should be biodegradable Do not persist long enough to damage environment Possible to apply in such a way that all parts of crop Ideal are insecticide protect against has not insect yet been attack discovered Closest: Not just upper δ-endotoxins surface of produced plants by soil bacterium Bacillus thuringiensis The δ-endotoxins of Bacillus thuringiensis B. thuringiensis: during sporulation Intracellular crystalline bodies contain an insecticidal protein (δ-endotoxins) Highly poisonous to insects More toxic than organophosphates (80,000X) Relatively selective (Table 15.1) Different strains of bacterium synthesizing proteins effective against larvae of different groups of insects 4

5 Table 15.1 The range of insects poisoned by various types of B. thuringiensis δ-endotoxin type Effective against CryI CryII CryIII CryIV CryV CryVI Lepidoptera (moth & butterfly) larvae Lepidoptera & Diptera (twowinged fly) larvae Lepidoptera larvae Diptera larvae Nematode worms Nematode worms First patent: 1904 Biodegradability: a disadvantage Must be reapplied at regular intervals Increasing costs Do not require regular application Protein Engineering: modifying its structure => more stable To engineer crop to synthesize its own toxin Figure 15.1 Mode of action of a δ-endotoxin 5

6 Cloning a δ-endotoxin gene in maize A major pest ( ): European corn borer ( ) (Ostrinia nubilialis) Tunnel into plant from eggs laid on undersurfaces of leaves Evading effects of insecticides applied by spraying First attempt at countering this pest Engineer maize to synthesize δ-endotoxin 1993, CryIA(b) protein: 1155 AA (toxin activity: AA) Fig 15.2 Procedure used to obtain GM maize plants expressing an artificial δ- endotoxin Improve its gene expression in maize plants GC content: 38% => 65% from cauliflower mosaic virus 6

7 Cloning a δ-endotoxin gene in maize ligated artificial gene into a cassette vector introduced into maize embryos by bombardment with DNA-coated microprojectiles grow into mature plant Transformants identified by PCR Cloning a δ-endotoxin gene in maize Immunological test: if δ-endotoxin was being synthesized Artificial gene was indeed active But: vary from plant to plant (250 ~ 1750 ng toxin/mg total protein Fig 15.3 Positional effects Larval tunnels: 40.7 cm => 6.3 cm A very significant level of resistance 7

8 Nuclear cloned gene might escape from engineered plant to a weed species Cloning δ-endotoxin genes in chloroplasts Tobacco: CryIIA(a2) gene A broader toxicity spectrum: two-winged fly as well as Lepidopterans Fig 15.4 CryIIA(a2) operon One advantage: chloroplasts like bacteria is able to express all genes in an operon Cloning δ-endotoxin genes in chloroplasts Introduce CryIIA(a2) operon into tobacco leaf cells by biolistics Attach chloroplast DNA seq. to operon Ensure insertion into chloroplast genome Selection: kanamycin resistance marker leaf segments: agar containing kanamycin>13 wks transgenic shoots ( ) medium induced root formation plants grown Amounts of CryIIA(a2) protein 45% of total soluble protein 8

9 Cloning δ-endotoxin genes in chloroplasts Plant: extremely toxic to susceptible insect larvae 5 days: all cotton bollworm ( ) & beet ( ) armyworm ( ) larvae were dead Toxin not to affect plants themselves Attempts: maize, cotton, & other useful crops Countering insect resistance to δ-endotoxin crops Crops synthesizing δ-endotoxin might become ineffective after a few seasons Resistance among insect populations Express both CryI & CryII genes (Fig 15.5a) Difficult to develop resistance to both types However, some strain may--resist both Risky: base a counter-resistance strategy on assumed limitations to genetic potential of insect pests 9

10 Fig 15.5 Three strategies for countering the development of insect resistance to δ-endotoxin crops Countering insect resistance to δ-endotoxin crops Synthesis occur only in those parts that need protection (Fig 15.5b) Maize: some damage to non-fruiting parts of plant could be tolerated Expression of toxin only occurred late in plant life cycle Might delay onset of resistance, but it is unlikely to avoid it altogether 10

11 Countering insect resistance to δ-endotoxin crops Mix GM plants w/ Non-GM plants Non-GM plants act as a refuge for insects Insect population continually includes a high proportion of non-resistant individuals Trials have been carried out to identify most effective mixed planting strategies δ-endotoxin production crops This success of GM projects with plants clearly depends Maize, on much cotton, more rice, than potato, cleverness & tomato of genetic Not engineers most wide-spread GM crops today Herbicide resistant crops : Most important transgenic plants: Those have been engineered to withstand herbicide glyphosate :, Widely used by farmers & horticulturists ( ) Environmentally friendly: non-toxic to insects & animals; a short residence time in soils; breaking down over a period of a few days into harmless products Glyphosate kills all plants (both weeds & crops) a less rigorous ( ) & hence less expensive herbicide application 11

12 Roundup Ready crops by Monsanto Co. First crops for glyphosate resistance Modified genes - enzyme enolpyruvyl-shikimate-3- phosphate synthase (EPSPS) Fig 15.6 Glyphosate competes with phosphoenol pyruvate (PEP) in EPSPS catalysed synthesis of enolpyruvylshikimate-3- phosphate, & hence inhibits synthesis of tryptophan, tyrosine & phenylalanine Without these amino acids, plants quickly dies Roundup Ready crops GM plants: greater than normal amounts EPSPS Withstand higher doses of glyphosate Unsuccessful: 80X EPSPS; increase in glyphosate tolerance was not sufficient to protect these plants from herbicide application Petunia Agrobacterium strain CP4 Whose EPSPS is resistant to glyphosate inhibition EPSPS is located into plant chloroplasts Not use chloroplast transformation GM plants were found to have a threefold increase in EPSPS genes was cloned in Ti vector as a fusion herbicide protein with resistance a leader sequence Biolistics: introduce vector into soybean callus culture 12

13 Roundup Ready crops Recent years: a variety of crops soybean & maize USA & other part of the world They do not actually destroy glyphosate Herbicide can accumulate in plant tissues Not poisonous to human or other animals Interfere with reproduction of plant Some crops: notably wheat Degree of resistance: too low to provide a major economic benefit A new generation of glyphosate resistant crops Recently a few reports Organisms can actively degrade glyphosate Relatively common among genus Bacillus Possess an enzyme: glyphosate N- acetyltransferase (GAT) (Fig 15.7a) Detoxify glyphosate by adding an acetyl group Most active detoxifier: a strain of Bacillus licheniformis Rates are too low to be of value if transferred to a GM crop 13

14 Fig 15.7 Use of glyphosate N-acetyltransferase to generate plants that detoxify glyphosate (a) GAT detoxifies glyphosate by adding an acetyl group (blue). (b) Creation of a highly active GAT enzyme by multigene shuffling. Is it possible to increase activity of GAT synthesized by B. licheniformis? Multigene shuffling: a type of directed evolution Bacterium possesses 3 related genes (Fig 15.7a) Take parts of each member of a multigene family & reassembling these parts to create new gene variants Most active genes are identified Clone all variants in E. Coli & assay recombinant colonies for GAT activity As substrates for next round of shuffling 11 rounds: a gene specifies a GAT w/ 10,000X activity GM maize: 6X in glyphosate tolerance w/o any reduction in productivity of plant 14

15 Other gene addition projects (Table 15.2) An alternative: confer insect resistance Genes coding for proteinase inhibitors Small polypeptides disrupt activities of enzymes in insect gut; prevent or slow growth Produced naturally by legumes ( ) Cowpeas & common beans Their genes: successfully transferred to other crops inhibitors Particularly effective against beetle ( ) larvae that feed on seeds A better alternative than δ-endotoxin for plants whose seeds are stored for long periods 15

16 Other gene addition projects Improve nutritional quality of crop plants content of essential AAs Change plant biochemistry More available nutrients can be utilized during digestion by human or animals Ornamental plants w/ unusual flower colors Transfer genes for enzymes involved in pigment production from one species to another Most successful: antisense technology 15.2 Gene subtraction Misnomer: modification does not involve actual removal of a gene, merely its inactivation 16

17 Antisense RNA & engineering of fruit ripening ( ) in tomato Commercial tomatoes & other soft fruits Usually picked before they are completely ripe Allow time to transported to marketplace before they begin to spoil Most immature fruits do not develop their full flavor GM tomato by antisense technology (2 ways) Fruit ripening process is slowed down Leave fruits on plant until they ripen to stage where flavor has fully developed Still being time to transport & market crop before spoilage ( ) sets in Timescale for development of a fruit Tomato: 8 wks (from start to finish) After 6 wks: Color & flavor changes associated w/ ripening beginning Genes are switched on polygalacturonase slowly break down polygalacturonic acid component of cell walls in fruit pericarp ( ; ) Softening makes fruit palatable ( ), but if taken too far results in a Fig 15.8 Increase in polygalacturonase squashy ( ), spoilt ( ) gene expression seen during later tomato stages of tomato fruit ripening 17

18 Using antisense RNA to inactivate polygalacturonase gene Partial inactivation of polygalacturonase time between flavor development & spoilage of fruit 730 bp restriction fragment Orientation was reversed Cauliflower mosaic virus promoter Plant poly(a) signal Ti plasmid pbin19 Agrobacterium tumefaciens Kanamycin resistant transformants develop into mature plants Fig 15.9 Construction of an antisense polygalacturonase gene Using antisense RNA to inactivate polygalacturonase gene Effect of antisense RNA synthesis Northern hybridization with SS DNA probe specific Transformed sensefruits: store for a prolonged mrna period before beginning to spoil Result: less mrna Amounts The GM of tomatoes enzyme - marketed under PAGE trade name FlavrSavr Activity One of the first GM plants to be Fig Differences in approved for sale polygalacturonase to public (1994) activity in normal tomato fruits & in fruits expressing its antisense RNA 18

19 Using antisense RNA to inactivate ethylene synthesis Ethylene: a gas, acts as a hormone Switch on gene involved in later stage of tomato ripening 2nd way: delaying plant ripening Engineer plant: not synthesize ethylene Unable to complete ripening process Artificial ripening Spraying tomatoes with ethylene Using antisense RNA to inactivate ethylene synthesis SAM: S-adenosylmethionine ACC synthease inactivation A truncated ACC synthase gene in reverse orientation was cloned into tomato An antisense version of ACC synthase mrna GM Summer plants: only 2% ethylene produced These tomatoes - marketed as Endless 19

20 Other examples of using antisense RNA in plant genetic engineering A growing number of plant biotechnology projects Likely to increase in importance 15.3 Problems with genetically modified plants Ripening-delayed tomatoes Among 1st GM whole foods: approved for marketing Safety & ethical issues: Our ability to alter genetic makeup of living organisms Good or otherwise?? GM crops might have on local farming practices in developing world Not in this book: We can & should look at biological issues 20

21 Safety concerns with selectable markers Possible harmful effects of marker genes Kanamycin resistance (kan R gene): nptii c Enzyme: neomycin phosphotransferase II, might be toxic to human It allay ( ) by tests with animal models 1. Could kan R gene contained in a GM foodstuff be passed to bacteria in human gut, making these resistant to kanamycin & related antibiotics? 2. Could kan R gene be passed to other organisms in environment & would this result in damage to ecosystem? Safety concerns with selectable markers Neither question can be fully answered with our current knowledge 1. The chance would be very small. Nevertheless, the risk factor is not zero. 2. kan R genes are already common in natural ecosystems. Future occurrence of some unforeseen ( ) & damaging event cannot be considered an absolute impossibility => Devise ways of removing these genes from plant DNA after transformation event has been verified 21

22 Fig DNA excision by Cre recombinase enzyme Two cloning vectors 1.Gene with marker 2.Cre gene Cre: an enzyme from bacteriophage P1 catalyses a recombination event excises DNA fragments flanked by specific 34 bp recognition sequences What if Cre gene is itself hazardous in some way? Two vectors would probably integrate their DNA fragments into different chromosomes Random segregation during sexual reproduction F1: contained one integrated fragment but not the other A plant that contains neither Cre gene nor kan R selectable marker, but does contain the important gene that we wished to add to the plant s genome, can therefore be obtained. 22

23 The terminator technology Market GM crops companies Protect their financial investment Ensure farmers must buy new seed every year Ensure 2nd generation seed cannot be grown by farmers RIP gene - promoter is active only during Genes embryo for ribosome development inactivating Cutting protein one (RIP) Plants grow normally but rrna unable into to two segments produce seeds Embryos die before they develop into seeds Fig (a) RIP gene codes for a protein that blocks protein synthesis How are the 1st generation seeds, those sold to farmers, obtained? RIP gene is nonfunctional Disrupted by a segment of non-rip DNA Flank by 34 bp recognition seq. for Cre recombinase Once seeds have been obtained Supplier activates the Cre recombinase by placing the seeds in a tatracycline solution Fig The terminator technology 23

24 Possibility of harmful effects on environment 1999, UK Government commissioned an independent investigation How herbicide resistant crops, whose growth in UK was not at that time permitted, might affect the abundance & diversity of farmland wildlife UK research team reported in field trials throughout England, Wales, & Scotland Glyphosate resistant sugar beet (maize) & spring rape engineered for resistance to a 2nd herbicide (glufodinate-ammonium) Differences in abundance of wildlife: GM v.s. conventional crop fields C beet & spring rape: better for many groups of wildlife More insects (butterflies & bees) More weeds to provide food & cover More weed seeds (important in diet of some animals - birds) GM maize: better for many groups of wildlife More weeds & more weed seeds More butterflies & bees at certain times of the year 24

25 The possibility of harmful effects on the environment These GM crops give farmers new options for weed control Use different herbicides & apply them differently Growing GM crops could have implications for wider farmland biodiversity Other issues will affect medium- & long-term impacts Areas & distribution of land involved How land is cultivated & how crop rotation are managed? Hard to predict it with any certainty Other management decisions will continue to impact on wildlife The End 25