Question

Cold acclimation can enhance the cold tolerance of a crop. Cold temperature induces phosphoproteome alteration and the cold-induced protein phosphorylation associates with cold tolerance. Describe a workflow (or steps) of 4C quantitative PTM proteomics used to identify phosphosites that associate with cold tolerance of a banana plant. These phosphosites might be used as biomarkers when people are trading banana seedlings on markets.

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Describe a workflow (or steps) of genetic engineering of a super- tomato plant, which can grow in a colder climate (being cold tolerant), have delayed senescence leaves (insensitive to ethylene), produce provitamin A-rich red ripe fruits and have a root system resistant to nematoes (or roundwarm).

Answer 1

Genetic engineering in plants:

The process of genetic engineering in plants involves the following steps:

DNA extraction- A sample is taken from the desired organism which contains the gene of interest. Further DNA from this sample is extracted.

Gene cloning- The DNA from the desired organism is extracted through amplification and the gene of interest is isolated from the DNA sample. Identical Copies of genes are produced.

Gene designing- Restriction endonuclease are used to genetically modify the gene of interest. A part of the gene is cut and a new part encoding for the desired feature is added to the gene.

Transformation- The newly modified gene is added to the cells of plants. vectors can be used to transfer the gene.This is done using methods like gene gun method, agrobacterium transfer, electroporation. This is because the cells are vast in numbers and it is practically impossible to transfer genes into all the cell nucleus. Once transferred these cells with the gene of interest grows into a transgenic plant.

Expression-The recombinant DNA expresses the protein from the gene of interest in the plant species.

Ethylene is a hormone responsible for ripening of tomatoes. DNA plant technology can be used to prevent formation of ethylene in tomatoes. As tomatoes reduces the level of ethylene by inhibiting the production of ACC synthase gene. Thereby, inserting an ACC deaminase gene blocks the activity of ACC synthase gene thus reducing the ethylene hormone.

An anti freeze gene called as afa3 gene can be derived from winter flounder and introduced in the plant cells. This will help increase the tolerance of the plant to cold climate.

A gene called as cysteine proteinase inhibitor gene derived from taro can be inserted into the plant genome. This gene is responsible for enhancing resistance from nematodes.

The nutritional levels in the tomato can be increased by using the process of genetic engineering. The level o pro vitamin A can be enhanced by inserting a bacterial gene encoding for phytoene desaturase.

4C quantitative PTM proteomics:

Post translational modification proteomics is used to examine changes in protein phosphorylation. Some species of banana are tolerant to cold while others are intolerant to cold temperatures. Membrane proteins gene are responsible for these fluctuation in temperature. Protein kinases regulates the process of protein phosphorylation. Liquid chromatography tandem mass spectroscopy is used to study the phosphorylated proteins and their counterparts. In this process the samples were labelled with TMT-6 plex complex and enriched with fe3+ and titanium di oxide based affinity chromatography. In order to study protein- protein based interactions expressed by different phosphoproteins a technique called as STRING V 10 was used. One dimensional isoelectric focusing can also be used to separate proteins and further proteins of interest can be studied by western blot.

Also the phosphorylated and non phosphorylated proteins can be separated and studied by using the method called acrylamide pendant phos-Tag separation. This method makes use of protein specific antibodies to study phosphorylated proteins. This is followed by SDS-PAGE and western blotting. Quantitative real time PCR can also be used to study the expression of gene tolerant to cold temperature.