2017 Solved Old Paper (BOT - 404D) New OK
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Nif genes:-
1. Introduction:-
> Nif means:-
Ni = Nitrogen
f = fixation
> These are genes encoding enzymes involved in the fixation of atmospheric nitrogen.
> Nif genes also encode a number of regulatory proteins involved in nitrogen fixation.
> The nif genes are found in both free-living and symbiotic nitrogen-fixing bacteria.
> The primary enzyme encoded by the nif genes is the nitrogenase.
2. Regulation of Nif genes:- In most bacteria, regulation is done by NifA protein.
i. When there is not enough fixed nitrogen, NtrC triggers NifA expression, and NifA activates the nif genes.
ii. When there is not enough fixed nitrogen, NifL inhibit NifA expression, and nif genes remain inactive.
Reversible ADP-ribosylation:- It is an additional regulation mechanism found in Rhodospirillum rubrum. Reversible ADP-ribosylation of a specific arginine residue in the nitrogenase complex. When reduced nitrogen is present, DraG and DraT catalyze the ribosylation of arginine residue in the nitrogenase. It causes a barrier in the electron transfer flow and thereby inactivates nitrogenase activity.
3. Expression of Nif genes:-
> There are total 20 nif genes.
> nifH, nifD, and nifK:- They encode the nitrogenase subunits.
> nifE, nifN, nifU, nifS, nifV, nifW, nifX, nifB, and nifQ:- They encode proteins involved the assembly and incorporation of Fe and Mo atoms into the nitrogenase subunits.
> nifF and nifJ:- They encode proteins related to electron transfer taking place in the reduction process.
> nifA and nifL:- They are regulatory proteins in charge of regulating the expression of the other nif genes.
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Translation:- It takes place at ribosomes. Includes 4 steps-
1. Charging of tRNA (aminoacylation of tRNA):-
· Formation of peptide bond requires energy obtained from ATP.
· For this, amino acids are activated (amino acid + ATP) and linked to their cognate tRNA in the presence of aminoacyl tRNA synthetase. So the tRNA becomes charged.
2. Initiation:-
· It begins at the 5’-end of mRNA in the presence of an initiation factor.
· The mRNA binds to the small subunit of ribosome. Now the large subunit binds to the small subunit to complete the initiation complex.
· Large subunit has 2 binding sites for tRNA- aminoacyl tRNA binding site (A site) and peptidyl site (P site).
· Initiation codon for methionine is AUG. So methionyl tRNA complex would have UAC at the Anticodon site.
3. Elongation:-
· At the P site the first codon of mRNA binds with anticodon of methionyl tRNA complex.
· Another aminoacyl tRNA complex with an appropriate amino acid enters the ribosome and attaches to A site. Its anticodon binds to the second codon on the mRNA and a peptide bond is formed between first and second amino acids in presence of an enzyme, peptidyl transferase.
· First amino acid and its tRNA are broken. This tRNA is removed from P site & send to E site and second tRNA at the A site is pulled to P site along with mRNA. This is called translocation.
· Then 3rd codon comes on A site and a suitable tRNA with 3rd amino acid binds at the A site. This process is repeated.
· A group of ribosomes associated with a single mRNA for translation is called a polyribosome (polysomes).
4. Termination:-
· When aminoacyl tRNA reaches the termination codon like UAA, UAG & UGA, the termination of translation occurs. The polypeptide and tRNA are released from the ribosomes.
· The ribosome dissociates into large and small subunits at the end of protein synthesis.
· An mRNA has additional sequences that are not translated (untranslated regions or UTR). UTRs are present at both 5’-end (before start codon) and 3’-end (after stop codon). They are required for efficient translation process.
Prokaryotic versus Eukaryotic Translation:-
1. Process:- In prokaryotes transcription and translation are continuous processes and occurs simultaneously in the cytoplasm. While in eukaryotes transcription and translation are separate process, transcription occurs in the nucleus whereas translation occurs in the cytoplasm.
2. Starting:- In prokaryotes 5' end of mRNA is immediately available for translation. While in eukaryotes the primary transcript is processed after transcription and then it is transported to the cytoplasm, then only the cytoplasmic ribosomes can initiate translation.
3. Ribosome:- 70S type ribosomes in prokaryotes. While 80S type ribosomes in eukaryotes.
4. Ribosome sub-units:- In prokaryotes 50S larger sub-unit and 305 smaller sub-unit. While in eukaryotes ribosome composed of 60S larger subunit and 40S smaller subunit.
5. rRNA of larger ribosome sub-unit:- In prokaryotes two rRNA molecules 5S and 23S rRNA. While in eukaryotes three rRNA molecules, 5S, 5.8S and 28S rRNA.
6. rRNA of smaller ribosome sub-unit:- In prokaryotes one rRNA molecule, 16S rRNA. While in prokaryotes one rRNA molecule18S rRNA.
7. Smaller ribosome sub-unit proteins:- In prokaryotes 21 proteins. While in eukaryotes 33 proteins.
8. Larger ribosome sub-unit proteins:- In prokaryotes 36 proteins. While in eukaryotes 49 proteins.
9. Ribosome mass:- In prokaryotes 2700 kd. While in eukaryotes 4200 kd.
10. Endoplasmic reticulum:- In prokaryotes it is absent and hence protein synthesizing ribosome freely distributed in the cytoplasm. While in eukaryotes Endoplasmic reticulum is present, protein synthesizing ribosome usually attached to the ER.
11. mRNA:- It is polycystronic in prokaryotes means mRNA can acts as the template for the synthesis of many polypeptides. While in eukaryotes it is always monocystronic means mRNA can act as the template for a single polypeptide.
12. Translation initiation:- In prokaryotes single type which is cap independent initiation. While in eukaryotes two types of translation initiation mechanisms- (i) Cap depended and (ii) Cap independent.
13. Start site:- Prokaryotes may have many start sites. While eukaryotes always have only one start site.
14. SD Sequence (Shine-Dalgarno sequence):- In prokaryotes SD sequence present 8 nucleotide upstream of start codon. SD sequence act as the ribosome binding site. Smaller subunit of ribosome (30S) recognize the SD sequence in the mRNA during translation initiation. While in eukaryotes SD sequence is absent in mRNA.
15. Kozak sequence:- In prokaryotes kozak sequence absent in mRNA. While in eukaryotes Kozak sequence is present in the mRNA which is located few nucleotide upstream of start site. Kozak sequence assists initiation process of translation. Smaller subunit of ribosome (40S) recognize the 5' cap of mRNA during initiation
16. Initiation codon:- In prokaryotes it is usually AUG, occasionally GUG or UUG. While in eukaryotes initiation codon is AUG, occasionally GUG or CUG.
17. First tRNA:- In prokaryotes first tRNA is special type namely Met-tRNAf. While in eukaryotes first tRNA is Met-tRNA.
18. First amino acid:- In prokaryotes first amino acid in the protein synthesis (methionine) will be formylated. While in eukaryotes no fomylation of methionine, the first amino acid, will occurs.
19. Initiation factors:- In prokaryotes only three initiation factors are required, they are IF1, IF2,IF3. While in eukaryotes seven types of initiation factors are required for translation, they are eIF1, eIF2, eIF3, eIF4, eIF5A, eIF5B, eIF6.
20. Elongation factors:- In prokaryotes two types of elongation factors, EF - Tu and EF -Ts. While in eukaryotes elongation factors are eEF1and eEF2.
21. Speed of translation:- In prokaryotes 20 amino acids / second. While in eukaryotes 1amino acid / second.
22. Termination:- In prokaryotes it is facilitated by three release factors, RF1, RF2, RF3. While in eukaryotes termination is facilitated by only one release factor eRF1.
23. Removal of first amino acid:- In prokaryotes only the formyl group from the first amino acid (methionine) is removed from the polypeptide after protein synthesis. While in ekaryotes usually the un-formylated first methionine as such is removed from the polypeptide after protein synthesis.
24. Life span of mRNA:- In prokaryotes it is short, few seconds to few minutes. While in eukaryotes
life span of mRNA is long, few hours to a day or sometimes more.25. Prevention of ribosomal association:- In prokaryotes IF3 prevents the association of ribosomal subunitsin the absence of initiation complex. While in eukaryotes eIF3 prevents the association of ribosomal subunits in the absence of initiation complex.
26. Post translational modifications:- In prokaryotes post translational modifications of proteins takes place in the cytoplasm. While in eukaryotes post translational modifications usually takes place in the endoplasmic reticulum or Golgi bodies or in the cytoplasm.
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Antisense - RNA:- It is single-stranded RNA molecule that can bind and inhibit specific mRNA translation to protein.
Principle:-
Sense RNA:-
> messenger RNA (mRNA) is single-stranded.
> Its sequence of nucleotides is called "sense" because it results in a gene product (protein).
> Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.
Antisense RNA:-
> RNA can form duplexes just as DNA does.
> All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand.
> The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense.
Example:-
5´ C A U G 3´ mRNA
3´ G U A C 5´ Antisense RNA
Antisense technology:-
> When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked.
> This may occur because the ribosome cannot gain access to the nucleotides in the mRNA or because the duplex RNA is quickly degraded by ribonucleases in the cell.
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> With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism.
Applications of Antisense technology:-
1. Treatment of viral infection:-
> Antisense oligonucleotides are selective and highly specific and binds with targeted viral mRNA and down regulate the expression of viral proteins, thus inhibit the virus replication.
> Thus this antisense technology can be used to design therapeutic compounds targeted to specific mRNA to treat various viral diseases.
> Vitravene (fomivirsen):- It is the first antiviral Antisense drugs used to treat cytomegalovirus retinitis. Other potential targets of antisense drugs include human papillomavirus, HIV, hepatitis-B virus, influenza A virus, and herpes simplex virus (HSV).
2. Treatment of cancer:-
> As antisense drugs are less toxic than conventional drugs, many antisense oligonucleotides are currently investigated to treat various cancers in humans or for the deactivation of oncogenes.
> For the first time an antisense oligonucleotide in combination with cisplatin was approved to treat bladder cancer.
3. Gene therapy:-
> Antisense oligonucleotides are being used in genetic research to treat various genetic disorders.
> In genetic disorders, some point mutations resulting in the formation of a defective mRNA and then defective protein.
> Antisense oligonucleotides blocks the formation of defective mRNA and then defective proteins.
> β-thalessemia:- A genetic blood disorder can be completely treated using antisense technology in which O-alkyl oligonucleotides or morpholino-oligonucleotides have been used.
4. Medical applications:-
> Antisense technology is used to investigate protein function in the living brain to study central nervous system (CNS) proteins such as transmembrane receptors, ion channels, transporters, G proteins, and growth factors.
> Antisense oligonucleotides can be used to inhibit expression of a particular enzyme. For eg. Inhibition of acetyl cholinesterase enzyme is the molecular target for the treatment of diseases like Alzheimer’s disease.
> In inflammatory diseases, antisense oligonucleotides is used to demonstrate the relative importance of various signaling components at the molecular level.
> A novel respirable antisense drug called RASONS is used to treat various respiratory diseases including asthma, influenza, bronchitis, pulmonary fibrosis, pneumonia, and lung cancer.
> Antisense oligonucleotide can be used target specific membrane component that influences the pathophysiological mechanisms in renal and cardiovascular disorders.
5. Flavr Savr tomatoes:-
> Most tomatoes that have to be shipped to market are harvested before they are ripe. Otherwise, ethylene synthesized by the tomato causes them to ripen and spoil before they reach the customer.
> Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.
> The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe.
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Male Sterility:- When the pollens of a plant are dead and inactive, while the female gametes are alive and active, it is called male sterility. This occurs very rarely. This occurs due to mutation. There are three types of male sterility: -
1. Genetic Male Sterility (GMS)
2. Cytoplasmic Male Sterility (CMS)
3. Cytoplasmic Genetic Male Sterility (CGMS)
1. Genetic Male Sterility (GMS):- When male sterility is controlled by a recessive gene present in the nucleus, it is called genetic male sterility (GMS).
MS gene = For male fertility
ms gene = For male sterility
MS MS = Male fertile plant
MS ms = Male fertile plant
ms ms = Male sterile plant
Maintenance:- For maintaining GMS, male sterile plants are cross heterozygous male fertile plants. As a result, 50 percent male sterile plants are obtained in each generation.
Application in hybrid seed production:- For hybrid seed production, the male sterile line is used as the female parent and crosses with the appropriate male parent line. Hybrid seeds are produced using GMS in Castor and Pigeon pea crops.
EGMS (Environment sensitive GMS):- The environment has a great impact on genetic male sterility. Genetic male sterility is determined by photo period and temperature. This property is used in hybrid seed production in paddy crop. Thus, there are two types of EGMS:
i. TGMS (Temperature sensitive GMS)
ii. PGMS (Photoperiod sensitive GMS)
i. TGMS (Temperature sensitive GMS):- Temperature has an effect on genetic male sterility in paddy crop. At temperatures below 28 ° C, paddy plants are completely male fertile while at temperatures above 30 °C, paddy plants are completely male sterile.
Maintenance of TGMS line:- For this, paddy plants are grown in an area where the day temperature remain below 28 ° C. At this temperature, paddy plants are fully male fertile. Now allow self-pollination in plants, resulting in the production of TGMS line seeds and then plants.
Hybrid Seed Production:- For this, paddy plants are grown in an area where the temperature is above 30 ° C. At this temperature paddy plants are fully male sterile which can be used as female parent. Now these male sterile female parent plants are crossed with the appropriate male parent plant, resulting in the production of hybrid seeds.
ii. PGMS (Photoperiod sensitive GMS):- Photo period also has an effect on genetic male sterility in paddy crop. Paddy plants are fully male fertile at 10 hours of light period, while at 14 hours of light, paddy plants are completely male sterile.
Maintenance of PGMS line:- For this, paddy plants are grown in an area where light period remain available for 10 hours. Paddy plants are fully male fertile in this condition. Now allow self-pollination in plants, resulting in the production of PGMS line seeds and then plants.
Hybrid Seed Production:- For this, paddy plants are grown in an area where the light period lasts for 14 hours. Paddy plants are fully male sterile in this condition which can be used as female parent. Now these male sterile female parent plants are crossed with the appropriate male parent plant, resulting in the production of hybrid seeds.
2. Cytoplasmic Male Sterility (CMS):- When male sterility is controlled by cytoplasm, it is called cytoplasmic male sterility (CMS). When fertilization occurs, only the nucleus comes from the male parent, while both the nucleus and cytoplasm come from the female parent. So when the male sterile female parent is crossed with the male parent, all the plants produced from the hybrid seeds are male sterile as shown in the diagram below.
Hybrid Seed Production:- CMS cannot be used for hybrid seed production in crops in which seed is of economic importance. But in crops where a somatic part is of economic importance rather than seed, CMS is used for hybrid seed production. Such as onion, garlic, carrot, radish etc.
3. Cytoplasmic Genetic Male Sterility (CGMS):- When male sterility is controlled by both nucleus and cytoplasm, it is called cytoplasmic genetic male sterility (CGMS). Cytoplasm is the main controller. The dominant gene present in the nucleus is called restorative gene, which acts to restore male fertility by preventing the male sterile effect of cytoplasm. We denote this as R letter. While r is the inactive gene.
R gene = For male fertility
r gene = For male sterility
RR = Male fertile plant
Rr = Male fertile plant
rr = Determined by cytoplasm.
Different combinations of nucleus and cytoplasm are given in the following diagram: -
Hybrid Seed Production:- CGMS is most commonly used in hybrid seed production. A three line system has been developed for hybrid seed production using CGMS which consists of the following three lines:
i. A – line:- It is a male sterile line that is used as a female parent.
ii. B – line:- It is a male fertile line which is used as a male parent. It is the maintainer line that works to maintain the male sterile A - line.
iii. R – line:- It is a male fertile line which is used as a male parent. It is a restore line that works to restore the male fertility in hybrid plants.
To maintain the A-line, its cross is made with the B-line. For hybrid seed production, the A-line crosses with the R-line as shown in the diagram below.
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Molecular farming:- It describes the production of recombinant proteins and other secondary metabolites in plants. This technology depends on a genetic transformation of plants.
The strategies of plant transformation:- Molecular farming depending on the production of transgenic plants. It has been operated by two general methods:
1. Stable or permanent expression systems:-
a. Stable nuclear transformation:-
> It refers to the integration of foreign genes into the nuclear genome of plants, which results in the change of genetic structures and consequent expression of transgenes after integration with the host genomes.
> The largest amount of recombinant proteins has been produced by one of the most common methods of stable nuclear transformation.
> A method exploited for aggregating proteins in dried beans of maize culminates in a long-term storage in the beans at the room temperature without decomposition of proteins.
> In addition, it has a considerable potential for producing crops like cereals that actually grow everywhere.
> Limitations:-
i. A long production cycle for some crops.
ii. Potential collisions of some crops with natural species or food products.
b. Stable plastid transformation:-
> Numerous advantages:-
i. Preventing transgene escape through amphimixis, because plastids are inherited through the maternal tissue in the majority of species.
ii. Absence of chloroplasts in pollen and consequent improbability of their transfer, which reduces environmental concerns.
> The transformed transgenic plants with homoplasmic chloroplasts (all chloroplasts carry transgenes) were selected after several generations of plant regeneration from bombarded leaf explants.
> Selection was conducted on a medium containing spectinomycin or combined with streptomycin.
> The researchers have already extracted a human pharmaceutical protein, more than 3% to 6%, from the total soluble proteins in the chloroplasts of tobacco.
> Recently, Oey reported a very high level (70% of an entire soluble protein) for a protein antibiotic with the chloroplast system, which, till today, has been the highest concentration of recombinant proteins.
> Functional limitation:-
i. Chloroplast transformation only in tobacco is practically possible, but unfortunately this plant is inedible and full of poisonous alkaloids.
ii. Long lasting storage in refrigerators will bring about changes in protein stability.
c. Plant cell suspension culture:-
> This method involves the removal of cell walls and gene transfer to the obtained protoplasts and suspension culture.
> The purification system and its downstream processing are cheaper and easier.
> In addition, the use of suspension culture can decrease heterogeneity in proteins and sugar (N-glycans) regarding the uniformity of the type and size of cells.
> Furthermore, as a fast system there is no need for the production of transgenic plants.
> Some samples of plant suspension cultures can be used for producing biomedicines, including:-
i. Vaccines of Newcastle disease virus of chicks
ii. Recombinant human glucocerebrosidase for treating diabetes.
> Though this method is cheaper, safer, and easier in comparison to the other methods of genetic manipulation, cell suspension has not yet been suggested as an optimal production choice of production in plant systems.
> Limitation:- It is believed that the ultimate products and their usability are constrained by reducing the level of recombinant proteins during the stationary phase because of the enhanced proteolytic activity.
2. Temporary or transient expression systems:-
a. Agrobacterium transformation method:-
> Infiltration of recombinant agrobacterium suspension into tobacco leaf tissue is achieved without stable gene transfer, which facilitates the transfer of T-DNA to a very high percentage of cells, where the transgenes are expressed at a high level without a stable transfer of genes.
> Presently, this method has been very efficient for the production of clinical biomedicines with a fast expansion.
b. Viral infection methods:- The viral infection method depends on the capability of plant viruses, such as tobacco mosaic virus and X potato virus, which functions as a vector to convey foreign genes into plant genomes without fusing with the genome of that plant.
c. Magnifection system:-
> Expression systems based on viral vectors and agrobacterium methods suffer from some constraints for the co-expression of two or more polypeptides required for the production of hetero-oligomeric proteins.
> Thus, a new transient expression system known as MagnICON technology has been developed by Icon Genetics Company.
> This method includes removing coat proteins (responsible for systemic movement) of non-competitive virus stains and systemic delivery of the derived viral vectors to all of the plants using agrobacterium as the medium of primary infection.
> This method not only optimizes the infection but also significantly increases proliferation, and finally results in the co-expression of several polypeptides and the rise of functional protein production more than 100 times.
The advantages of utilizing transgenic plants as bioreactors:- Comparison of different expression systems reveals the advantages of plants in comparison with other expression systems as follows:
i. The healthiness of derived products (plants cannot be the host of human pathogens and bacterial toxins).
ii. Capability of post-translational processing (respecting the features of eukaryotic cells).
iii. The possibility of using breeding methods and sexual crosses to obtain active recombinant multi-chain proteins (therefore, there is the possibility of producing antibodies without application of a double transformation).
iv. Reducing the costs of production (plants can produce biological materials by the use of carbon dioxide, solar energy, and inorganic materials. Moreover, the scale of production can be manipulated regarding scalability).
v. Reducing the costs of storage and transportation of recombinant proteins (when they are produced in dry textures like grains).
vi. Removing the purification step (when the plant tissues containing recombinant protein are edible).
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Antisense - RNA:- It is single-stranded RNA molecule that can bind and inhibit specific mRNA translation to protein.
Principle:-
Sense RNA:-
> messenger RNA (mRNA) is single-stranded.
> Its sequence of nucleotides is called "sense" because it results in a gene product (protein).
> Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.
Antisense RNA:-
> RNA can form duplexes just as DNA does.
> All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand.
> The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense.
Example:-
5´ C A U G 3´ mRNA
3´ G U A C 5´ Antisense RNA
Antisense technology:-
> When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked.
> This may occur because the ribosome cannot gain access to the nucleotides in the mRNA or because the duplex RNA is quickly degraded by ribonucleases in the cell.
> With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism.
Applications of Antisense technology:-
1. Treatment of viral infection:-
> Antisense oligonucleotides are selective and highly specific and binds with targeted viral mRNA and down regulate the expression of viral proteins, thus inhibit the virus replication.
> Thus this antisense technology can be used to design therapeutic compounds targeted to specific mRNA to treat various viral diseases.
> Vitravene (fomivirsen):- It is the first antiviral Antisense drugs used to treat cytomegalovirus retinitis. Other potential targets of antisense drugs include human papillomavirus, HIV, hepatitis-B virus, influenza A virus, and herpes simplex virus (HSV).
2. Treatment of cancer:-
> As antisense drugs are less toxic than conventional drugs, many antisense oligonucleotides are currently investigated to treat various cancers in humans or for the deactivation of oncogenes.
> For the first time an antisense oligonucleotide in combination with cisplatin was approved to treat bladder cancer.
3. Gene therapy:-
> Antisense oligonucleotides are being used in genetic research to treat various genetic disorders.
> In genetic disorders, some point mutations resulting in the formation of a defective mRNA and then defective protein.
> Antisense oligonucleotides blocks the formation of defective mRNA and then defective proteins.
> β-thalessemia:- A genetic blood disorder can be completely treated using antisense technology in which O-alkyl oligonucleotides or morpholino-oligonucleotides have been used.
4. Medical applications:-
> Antisense technology is used to investigate protein function in the living brain to study central nervous system (CNS) proteins such as transmembrane receptors, ion channels, transporters, G proteins, and growth factors.
> Antisense oligonucleotides can be used to inhibit expression of a particular enzyme. For eg. Inhibition of acetyl cholinesterase enzyme is the molecular target for the treatment of diseases like Alzheimer’s disease.
> In inflammatory diseases, antisense oligonucleotides is used to demonstrate the relative importance of various signaling components at the molecular level.
> A novel respirable antisense drug called RASONS is used to treat various respiratory diseases including asthma, influenza, bronchitis, pulmonary fibrosis, pneumonia, and lung cancer.
> Antisense oligonucleotide can be used target specific membrane component that influences the pathophysiological mechanisms in renal and cardiovascular disorders.
5. Flavr Savr tomatoes:-
> Most tomatoes that have to be shipped to market are harvested before they are ripe. Otherwise, ethylene synthesized by the tomato causes them to ripen and spoil before they reach the customer.
> Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.
> The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe.
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1. Origin of plant tissue:- The genetics of the plants have a high influence on the production of secondary metabolites. For example, high fielding cultivars produce high nicotine content compared to low fielding cultivars.
2. Culture Media:- The composition of the culture medium influences both, biomass production and the synthesis of secondary metabolites. So, ideal conditions are required to be maintained to keep a balance between these two.
3. Growth Regulators:- They have a significant effect on the production of secondary metabolites, as they can either be synergistic or antagonists in their actions. For example, the addition of gibberellic acid to the Taxus cuspidate’s medium increases the taxol production.
4. Carbon source in the medium:- Sucrose is one the most widely used carbon source in the culture media. However, there are also some others, which include glucose, fructose, and galactose.
5. Temperature:- A higher temperature is reported to enhance the production of secondary metabolites.
6. pH:- A perfect pH between 5-6 is required to be maintained for the production of secondary metabolites.
7. Light intensity:- Apart from stimulating the production of secondary metabolites, light intensity also has a role in determining the amount of the production.
8. High-cell Density culture:- A high-density culture increases the production of secondary metabolites. The case is reported in Japonic cells where they have grown up to 759 \ L of cell mass to increase productivity.
9. Elicitation:- Elicitors are compounds of biological origin involved in plant microbe interaction. Elicitors are considered as mediator compounds which induce secondary metabolites formation in cells cultures. Varieties of elicitors have been used for production of secondary metabolites. Elicitation improves the efficiency of Sec. Product accumulation in plant cell culture by:
i. Minimizing up on time
ii. Avoiding change of media
iii. Induction of enzymes involved in biosynthetic pathway
iv. Inducing excretion of metabolites into the medium
Some of the biotic elicitor- induced products
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10. Precursor feeding:- Under in vitro circumstances, often the precursors of metabolite die and can’t carry out the reaction. This can be overcome by supplying enough precursors to cells, which can be done by two methods:
a. Add precussors:- By adding precursors in the medium so that their availability is not limited.
b. Prevent resistant precursor analogs:- By preventing resistant precursor analogs and increasing the intracellular levels of precursors for the metabolite production. Eg.-
i. Production of shikonin increased three-fold after the addition of L-Phenylalanine to the medium in Lithospermum species.
ii. Datura sp. cell suspension cultures are supplemented with hydroquinone, in traces, the arbutin synthesis increase considerably.
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Transgenic Plant:-
• Definition:- Stable transformation is achieved when a desired gene is integrated into the genome of a plant, this plant is called transgenic plant.
• These transgenic plants are developed for the following purposes:-
1. Insect Resistance
2. Virus Resistance
3. Seed Protein Quality
4. Gene Silencing
5. Male Sterility
6. Biochemical Production
1. Insect Resistance:-
cry gene transfer:- The cry gene is found in the plasmid of Bacillus thuringiensis (Bt) bacteria. This cry gene makes Crystal protein which is insecticidal.
Ø The cry gene was isolated from the plasmid of Bt and integrated into the cotton genome to form a transgenic plant called Bt-cotton.
Ø This Bt - cotton is resistant to boll worm.
Ø Crystal protein perforates the larva's alimentary canal leading to its death.
2. Virus Resistance:-
cp – gene transfer:- The coat protein is transferred from the TMV virus to the tobacco plant.
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Ø The RNA of TMV virus is not come out in the presence of cp-protein. Due to which the tobacco plant becomes resistant to mosaic disease.
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Ø Other examples: - Tomato, Alfalfa, Beetroot, Potato
3. Seed Protein Quality:-
SFA8 gene transfer:- In sunflower seeds, the SFA8 gene produces a protein that contains an excess of sulfur-rich amino acids methionine and cysteine. While the proteins present in pea seeds lack methionine and cysteine amino acids.
Ø Therefore the SFA8 gene is transferred to pea seeds.
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4. Gene Silencing:-
Slow ripening tomatoes:-
Ø PG (Poly Galacturonase) enzyme digest the pectin. Due to which fruits become soft and fluffy quickly and ripen quickly.
Ø Anti-sense genes are integrated into the genome of tomato plants against genes that code PG enzymes. This inhibits the expression of PG gene and the tomato fruit ripens late.
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Ø Antisense gene made against PG gene inhibits its expression, which causes tomato to ripen late and can be easily exported.
5. Male Sterility:-
Producing male sterility:-
Ø Flavonoid are essential for maturation of pollens. If we stop its formation then male sterility can be created in the plant.
Ø The CHS (Chalcone Synthase) enzyme is required for flavonoid synthesis.
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Ø Anti-sense genes are integrated into the plant's genome against genes that code CHS.
Ø Absence of a flavonoid causes pollen to become inactive. The plant becomes male sterile.
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6. Biochemical Production:-
• PHB (Poly Hydroxy Butyrate):- It is prepared from Acetyl CoA. From this bio-degradable plastic is made.
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• The transgenic plant is developed by isolating the phb - B and phb - C genes from the Alcaligenes eutrophus bacterium and transfer to Arabidopsis thaliana. Now PHB is obtained from this plant.
Golden Rice:-
• The genes that form β - carotene are integrated into the embryo of rice within the genome to prepare golden rice.
• 2 genes are isolated from Daffodils plant and 1 gene from Erwinia uredovora bacterium.
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• β - carotene is yellow in color, which makes rice yellow.
• β - carotene is the precurssor of vitamin A.