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Effect of Heat Stress on Protein Expression in Potential BARI Released Tomato Varieties (2017--2018)

S. Islam
Biotechnology Division, BARI, Joydebpur, Gazipur

M.R. Kabir
Biotechnology Division, BARI, Joydebpur, Gazipur

D. Khanam
Biotechnology Division, BARI, Joydebpur, Gazipur

M.A.Y. Akhond
Biotechnology Division, BARI, Joydebpur, Gazipur

Abstract/ Executive Summary:

The experiment was conducted based on heat shock protein in BARI released tomato varieties under heat stress conditions. Four BARI released tomato varieties and four parental lines were selected for this experiment. Attempts were made to identify heat shock protein and to differentiate the heat-tolerant tomato varieties in the context of overly expressed heat shock protein under heat stress conditions. Total plant protein including large and small heat shock proteins was extracted and SDS-PAGE was done to get an idea about the extracted proteins. Results obtained from this experiment can be used for further experiments in this field.

Keywords: Heat shock protein, Tomato, Heat stress

Location:

Start Date: 00-00-2017

End Date: 00-00-2018

Program Area: Variety and Species

Commodity: Tomato

Objectives:

To identify heat shock protein and to differentiate the heat-tolerant tomato varieties in the context of overexpression of heat shock protein under heat stress conditions.

Methodology:

In this experiment, BARI Hybrid Tomato-4 and its parental lines, BARI Hybrid Tomato-8 and its parental lines, BARI Tomato- 14, BARI Tomato-15 were used as germplasm. Heat shock proteins (HSP) are a family of proteins that are produced by cells in response to exposure to stressful conditions. They were first described in relation to heat shock. With this point of view, tomato germplasms were exposed to heat shock at 40 ºC. Tomato seeds were surface-sterilized with Ridomil gold MZ 68 WG, Tween 20and 60% clorox and then germinated on sterile water-soaked paper in Petridis for 3 days. Soon after germination, each seedling was transferred to a plastic recipient, containing soil and sand (2:1) treated with Formaldehyde and maintained in a greenhouse at 28 ºC with the growth period. 50 days old plants, grown at 28 ºC, were submitted to 2-4 h of heat shock treatment at 40 ºC in a growth chamber. Leaves were collected after 10 and 30 min and 1, 2 and 4h of stress. Following the end of the heat shock treatment (4 h), the plants were maintained at 28 ºC for a recovery period of up to 4h and leaves were again collected after every hour. The leaves were immediately frozen at –70 ºC. Protein was extracted from leaf tissue of those 8 genotypes, using different protein precipitation protocol. Extracted protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 13% polyacrylamide gel and stained with Coomassie Brilliant Blue staining solution and analyzed under white light.

Source of Information: Annual Research Report 2017-2018, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Firstly, the protein was extracted from leaf tissue of those 8 genotypes, using TCA/Acetone Protein Precipitation Protocol. Precipitating proteins from homogenized tissue or cells with TCA (10% w/v) in acetone is a commonly used protein extraction protocol that was developed initially by Damervalet al. to extract seedling proteins. The protocol is based on protein denaturalizing under acidic and/or hydrophobic conditions that help to concentrate proteins and remove contaminants. Different from direct TCA precipitation of protein extracts, TCA/ acetone extraction involves the cleanup of tissue powder with 10% TCA/acetone, which is usually more effective than either TCA or acetone alone. Since it is less time-consuming and easier to perform than the phenol-based protocol, TCA/acetone extraction was used as a starting protocol for plant protein extraction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protocol has been done to separate extracted protein.SDS-PAGE was done with the purpose of identifying the difference among the BARI released tomato varieties at protein expression level for differentiating heat tolerant and heat susceptible varieties. By using this protocol poor quality of protein and no significant difference was found among the tomato varieties at the protein expression level. Another protein extraction protocol is needed to standardize to extract better quality of protein and for finding out the difference among the tomato varieties based on the level of heat shock protein (Hsps) expression. For this reason, another protein extraction protocol such as TCA/acetone precipitation/phenol extraction protocol was adopted to get better quality protein. The protocol holds the merits of both TCA/acetone precipitation (for removal of phenolics, lipids, and pigments) and phenol extraction (for removal of phenolics, lipids and pigments, polysaccharides, nucleic acids, and salts). In the TCA/acetone/phenol extraction protocol, tissue powder purged by TCA/acetone is used as the starting material in phenol extraction of proteins. The protocol has been proved to be very effective to handle recalcitrant tissues and provides enhanced 2-DE-based proteomic analysis of plant tissues. The protocol has been proved to provide high-quality protein samples. In these cases, TCA/acetone cleanup steps remove most of the interfering compounds, facilitating the subsequent phenol extraction of proteins. Although the TCA/acetone/phenol extraction is time-consuming and laborious compared to both the TCA/acetone precipitation and the phenol-based extraction, it has the potential to generate high-quality protein samples. The conventional protein extraction method, TCE/Acetone followed by Phenol extraction did not give good results of electrophoretic separation due to less amount of protein. To solve this problem I moved to Plant Protein Extraction Kit bought from the company. The Plant Protein Extraction Kit provides rapid recovery (as few as 20 minutes) of soluble proteins from plant tissue samples. The final protein extract is compatible with many downstream applications, including SDS-PAGE, 2-D gel electrophoresis, Western blotting, activity assays and affinity purification. The Kit has been tested on leaves, roots, flowers and seeds from various species, including Arabidopsis, tobacco, spinach, peas and soybeans. The amount of protein extracted by using a plant protein extraction kit was not sufficient enough to make the desired bands visible in Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and for finding out the difference among the tomato varieties based on the level of heat shock protein (Hsps) expression. Further research is necessary to extract better quality of protein and for finding out the difference among the tomato varieties, especially at the tomato heat shock protein (Hsps) expression level.

Knowledge Management: Report/Proceedings