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Expression and Transformation Studies of Antifreeze Protein Gene of Carrot in Potato

Thesis Info

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Author

Saleem, Muhammad Zafar

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2018

Thesis Completion Status

Completed

Subject

Molecular Biology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10700/1/Muhammad%20Zafar%20Saleem_Mol.%20Bio_2018_UoPunjab_PRR.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726169853

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The Antifreeze protein gene (1.022Kb) from local of D.carrota cultivar T29 was amplified from four weeks old seedling leave tissue genomic DNA. The PCR amplified gene was cloned in TA-Cloning vector pCR2.1. The cloned DcAFP gene was then sequenced, the DNA BLAST homology with wild carrot AFP was 96.1%. The 999bp gene of DcAFP from T29 cultivar was translated into amino acid sequence, the translated amino acid sequence when aligned with wild carrot AFP gene of amino acid sequence. The gene has 13 amino acid variability. The Asparagine amino acid residues which play an important role as an ice interacting/ice binding sites were intact as in wild species of carrot amino acid sequence, but extra three Asparagine amino acid were noted in 332 amino acid gene sequence as in the case of DcAFP (T29). The 944bp DNA sequence that code the mature peptide of DcAFP was cloned in pET15b E.coli expression vector. The IPTG induced 36kDa protein detected from the BL21 expression host transformed with expression plasmid. The 36kDa recombinant protein inclusion refolded and purified for polyclonal antibody production. Purified IgG of DcAFP was used in western blot, protein dot blot and ELISA analysis of transgenic potato lines. The 999bp full length gene was ligated in pCAMBIA-1301 without selection marker. Plant expression construct pCAMBIA1301hph - DcAFP (T29) was transformed in Agrobacterium host strain LBA4404. Agrobacterium with pCAMBIA construct was transformed in potato nodal explant. The transgenic plant first selected by PCR, DNA dot blot and GUS reporter assay. Stable integration confirmed by southern blot analysis. The DcAFP mRNA detected by Northern blotting after cold induction of transgenic potato plants. DcAFP protein detected by protein dot blot and western blotting methodologies. The sandwich ELISA was successfully done for expressed protein estimation in transgenic potato lines. The stable potato transgenic lines were tested with ILA (Ion Leakage Assay), which results are significant when compared with the control plants. It was noted that due to DcAFP protein expression in transgenic potato lines demonstrating that transgenic plants are tolerating the frost temperature.
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کتابیات

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بنیادی ماخذات
٭ شاہد رضوان ،ادھوری کہانی کی تصویر،دانیال پبلشرز، چیچہ وطنی،2020ء
٭ شاہد رضوان ،آوازیں،سانجھ پبلشرز ،لاہور، ،2015ء
٭ شاہد رضوان،پتھر کی عورت ،دانالل پبلشرز،چچہا وطنی،جنوری2010ء
٭ شاہد رضوان،پہلا آدمی،دانا،ل پبلشرز،چچہ، وطنی،2013ء
٭ مہر غلام فرید کاٹھاد، لمحوں کی قدل،محکمہ ادبی مرکز،شادمان ٹاؤن،ساہویال2008ء
٭ مہر غلام فرید کاٹھا،،سرسوں کے پھول،ناصر بک سنز ،لاہور،2019ء
٭ مہر غلام فرید کاٹھا،،سفدو تتلونں کا ہار،ساہومال2021ء
ثانوی ماخذات
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٭عائشہ سلطانہ ، ڈاکٹر، مختصر اردو افسانے کا سماجیاتی مطالعہ (1947ء سے تا حال) ، دہلی ، ایجوکیشنل پبلشرز ہاؤس
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حدود التفسير الموضوعي

This study deals with the limitations of thematic exegesis of the Qur’ān. The main place where exegesis of the Qur’ān can be found remains the commentaries. In the majority of cases such commentaries start from the beginning of the first sūrah. In order to acknowledge the inputs and outputs in the Qur’ānic studies; explanation of the literary aspects of the verse, an account of its background and occasionally an interpretation of it with the help of another verse will be reviewed in this paper. In order to achieve the objectives of this study, scientists’ quotes will be critically examined.

Analytical Characterization of Lipids and Antioxidant Components in Flaxseeds Linum Usitatissimum

Flaxseed or linseed (Linum usitatissimum) belonging to family lineaceae is an ancient crop and has been grown for its fiber (linen) and oil seeds since 9000 to 8000 B.C. Flaxseed is cultivated in almost fifty countries of the world including Pakistan. Flaxseed is a rich source of alpha linolenic acid, very efficient antioxidant system and dietary fiber. These characteristics and the health benefits associated with these characteristics of flaxseed have made it a great attraction for the researchers in the field of functional foods and neutraceuticals. Keeping in view the immense importance of flaxseed as neutraceutical and functional food, the present study was planned to assess the nutritional composition, oil characteristics and antioxidant potential of different flaxseeds cultivars grown in Pakistan. Moreover qualitative and quantitative analysis of the flaxseed phenolics was done by HPLC and LC-MS. The proximate analysis of flaxseed that the oil, moisture, crude protein, fiber and ash contents in the seeds of different cultivars of flaxseed were observed to range from 33.25 to 38.38%, 5.98 to 6.22%, 16.02 to 18.50%, 23.30 to 26.80% and 3.21 to 3.60% respectively. Gamma tocopherol was observed to be the major tocopherol present in the oil with minor contents of alpha tocopherol. The unsaturated fatty acids in the oils extracted from different cultivars were observed to be in the range of 88.79 to 89.78%, with alpha linolenic acid with the highest contents and ranged from 44.51 to 54.87%. From the data for the physicochemical characteristics of the oils it was observed the oils were up to the mark in quality as compared to the international standards. 80% aqueous ethanol and methanol were the most effective solvents for recovering antioxidant components from flaxseed while the pure ethanol and acetone had shown the least efficacy towards isolation of flaxseed antioxidants as established by the antioxidant activity analysis by different methods. Furthermore, the sonication assisted shaking proved to be superior in extraction of antioxidant components. HPLC and LC-MS analysis established that SECO (secoisolariciricinol) was the major phenolic (lignan) and dominated all the other phenolic compounds. SDG, ferulic acid, coumaric acid, chlorogenic acid and gallic acids were the other phenolic compounds detected in the flaxseed. The data presented would certainly help to ascertain the potency of the flaxseeds grown in Pakistan, as viable source of natural antioxidants and alpha linolenic acid, thus suggest their uses for nutraceutical and functional food applications.