Answer To: Task: Genetic reprogramming of the production of the valuable chemical in plants
Dr Shweta answered on May 23 2022
RMIT Classification: Trusted
1. The first aim is to design a strategy for the building of a transgene used for the production of X chemical in leaves and to show its promoter region, coding region and terminator region in a transgene map. So, for this firstly we understand what a transgene is? And how the transgenic plants generally produced? Comment by Aidyn Mouradov: To design a strategy Comment by Aidyn Mouradov: Comment by shweta kamthan: Added
A transgene is defined as an artificial gene which is constructed in the molecular biology lab by incorporating a desired foreign gene and all the necessary components (promoter region, coding region and terminator region) essential for gene expression in the suitable vector. The foreign gene may belong to either the same or different species. To select the recombinant vectors containing properly inserted transgene from the non-recombinant one’s suitable selectable markers like antibiotic resistance gene (kanamycin resistance gene, tetracycline resistance gene etc.) metabolic selectable markers (beta galactosidase gene, etc. are also added. The general structure of transgene is shown as below:
Comment by Aidyn Mouradov: This is a vector, not a transgene. Transgene is a promoter-coding region and terminator. Comment by shweta kamthan: Corrected
Figure 1 Image courtesy: https://www.slideserve.com/ginger/plant-genetic-engineering-plant-transformation
Using this strategy, different transgenic plants having one or more desired characters can be artificially synthesised. Using recombinant DNA technology, one or more desired gene specific to a particular character of interest is inserted into the existing genome of plant so that along with the existing features new characters also gets incorporated in it. For example, a normal rice is well known for its rich starch content but with the intention of enriching rice with vitamin A content, using recombinant DNA technology, the beta globin gene of carrot is extracted from the carrot and inserted into rice to generate a vitamin A rich ‘Golden rice’. Here, the foreign beta-globin gene inserted is known as the ‘Transgene’ and the existing rice genome is known as the ‘Host genome’ and the rice plant is now known as the ‘Transgenic plant’. This entire process is known as Transgenesis. The transgenic plants are the genetically modified organisms. In this technology, the desired gene is cut and excised from the parent genome via restriction endonuclease enzymes known as the molecular scissors. Restriction endonucleases enzymes have the unique ability to cut DNA at a specific site so that the desired gene can be extracted precisely. Later, it is joined with the homologous segments of host genome using DNA-ligase enzymes and then transferred into the host genome via desired cloning vector like Ti/Ri plasmid or virus-based vectors. Once entered into host cell the desired gene recombines with the host genome via the homologous segment and the desired gene now becomes a part of this host genome. Since, along with the insertion of desired gene, our primary aim is to obtain the protein product of the included gene, instead of simple cloning vectors, expression vectors are used. Expression vectors have specific promoter, operator and terminator sequences that helps in the transcription and translation of inserted gene and provides its suitable protein product in functional form. Since, the inserted gene, the vector, the plant genome all are of micron size and invisible to naked eye suitable selection strategies like antibiotic resistance method, fluorescent markers etc. will be used to check proper insertion and gene-expression.
To understand the entire process more clearly, let us take an example of complete synthesis of a transgenic plant in which we will insert gene TFX in the genome of leaves and let say that this gene is capable of producing chemical X then the steps we will follow are as follows:
1. The DNA of the host leaf is isolated using standard isolation procedure. In this firstly leaf cells are ruptured with cellulases, pectinases and detergents to removed its cell wall and cell membrane. Later, it is treated with proteases and RNase to lyse the RNA and proteins. Now, pure DNA is extracted via chilled ethanol extraction method.
2. Similarly, the genome having the desired TFX gene will be extracted by cutting it with suitable restrictions enzyme and separated using gel electrophoresis. Then using the cloning method or polymerase chain reaction method multiple copies of the desired gene and vector genome will designed so that if some handling issue occur still, we don’t lose our vector or gene sample.
3. Now, a suitable vector, for instance here we are taking Ti vector,...