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Home > Cloning and Expression Studies on Abiotic Stress Tolerance Gene S in Solanum Tuberosum L .

Cloning and Expression Studies on Abiotic Stress Tolerance Gene S in Solanum Tuberosum L .

Thesis Info

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Author

Jahangir, Ghulam Zahara

Program

PhD

Institute

Lahore College for Women University

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/11828/1/ghulam%20Zahara%20jahangir%20biotechnology%202019%20lcwu%20lhr%20prr.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676725739598

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The three dehydrin genes of Eucalyptus species, the dehydrin-1 (EgDHN-1), dehydrin-2 (EgDHN-2) and dehydrin-10 (EgDHN-10) were isolated from local Eucalyptus globulus species for transformation in different varieties of potato for developing abiotic stress tolerance. These three lysine rich dehydrin genes of Eucalyptus have not been reported earlier to be manipulated for genetic engineering or for transformation into a crop plant. The genes were amplified from DNA as well as mRNA (through cDNA). The amplified genes were cloned in TA cloning vector. Cloned dehydrin genes were sequenced and detailed in silico analyses were conducted. Complete sequence of EgDHN-10 gene was obtained from DNA including introns. Coding sequence from its mRNA was also complete and found 92% similar with EgDHN-10 of Eucalyptus globulus after DNA BLAST analyses. The sequencing data of other genes obtained incomplete that showed 99% and 98% homology, after DNA BLAST analyses, with Predicted Eucalyptus grandis Rab18 gene and Eucalyptus globulus EgDHN-1 gene respectively. 297bp long sequence of EgDHN-10 that codes for mature peptide was further sub-cloned in E. coli expression vector pET30a. The sequence was submitted to the NCBI (MG948256.1) and it had extra integration of five electrically charged amino acids (two positively charged Histidine, one negatively charged Glutamate, and two negatively charged Aspartate residues) and one Lysine (K). The presence of one extra K residue is very important for enhanced stress tolerance activity of the transformed dehydrin protein due to its role in functions of K-segments. Presence of more charged amino acids in the binding surface of dehydrin protein enhances its capability to bind with other macromolecules, reactive oxygen species and free metal ions.IPTG induced successful expression of EgDHN-10, cloned in pET30a, was detected in E. coli BL21 ii DE3 strain by protein dot blot analysis. After getting excellent expression of Eucalyptus globulus EgDHN-10 gene outside Eucalyptus cell’s environment, the gene was sub-cloned in pCAMBIA 1301 plant binary vector and transformed into Agrobacterium tumefascience. The nodal regions of three potato cultivars, the Cardinal, Sante and Lady Rosetta, were infected with transformed Agrobacterium (through co-cultivation) carrying EgDHN-10. Putative transgenic plants were first screened by GUS assay and PCR. The successful integration of EgDHN-10 gene in potato genome was confirmed by DNA dot blot analysis. The EgDHN-10 gene’s expression was confirmed from potato mRNA by PCR and real time quantitative PCR. In some plants up to 12 folds DHN 10 expression was observed through qPCR. The acquired abiotic stress tolerance was quantified in stressed transformed potato plants by ion leakage assay (ILA) of cell rupture to select the stable transgenic plants. The stress tolerant transformed plants of Cardinal, Sante and Lady Rosetta were maintained in plant tissue culture setup and pots. The transgenic Sante variety was multiplied and maintained in CAMB trial field in an experiment of growth promoting effect of biofertilizer on trial field plants. The stress tolerant Sante plants showed significant difference over control plants in studied plant growth parameters like plant height, maximum root length, number of roots and number of multiple shoots formation, number and weight of tuber formation.
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در عدالتِ علے

در عدالت علے

                ’’در عدالت علے‘‘علی اکبر ناطق کی مناقب اہل بیت کا مجموعہ ہے۔مولا کی طرف سے خاص عطا کیا ہوا۔ ان کے کلام نے مولا علی کے قصائد آئمہ طاہرین کے حضور نذرانے کے طور پر کلام پیش کیا ہے۔قاری جب پڑھتا ہے تو بے اختیار پکار اٹھتا ہے کہ مصنف نے اس کا حق ادا کردیا ہے۔انہوں نے اس میں تاریخِ حق کو بیان کیا ہے کہ کس طرح  تاریخِ حق کو منقبت میں سمو دیا گیاہے۔در عدالت علے عکس پبلی کیشنز نے 2020ء میں شائع کی۔اس کا انتساب سید اظہر علی عابد کے نام ہے۔اس کتاب نے مذہبی اور ادبی حلقوں میں یکساں طور پر ارتعاش پیداکیا ہے۔ انہوں نے اس میں ایک نظم ’’یاعلی میں گداؤں کی بستی کا ساکن‘‘کے عنوان سے لکھی ہے۔جس سے ان کا علی سے عشق کی انتہا کا ثبوت ملتا ہے۔یہ نظم انسان کے دل ودماغ کی پاکیزگی اور اس کے کردار کو بلندی کی طرف مائل کرتی ہے۔وہ بتاتے ہیں کہ ایک دن وہ اسلام آباد میں تھے۔ظہور امام کے دن تھے۔وہ بتاتے ہیں کہ سید منظر نقوی کے ساتھ جامعہ صادق جی نائن اسلام آباد میں مجلس سننے کے واسطے پہنچا۔سید اظہر نے جیسے ہی مجھے قریب پایا تو مجھے سلام پڑھنے کیلئے کہہ دیا۔وہ کہتے ہیں کہ ان کے ہاتھ پاؤں پھول گئے کیونکہ کلام پاس نہیں تھااور لکھا ہوا کچھ یاد نہیں تھا۔خیر قدم اٹھتے چلے گئے اورایک نظم جو اسلامک کو ذہن میں رکھ کر لکھی تھی اسی کے شعر پڑھنے لگے ناطق بتاتے ہیں کہ انھیں خوب داد وصول ہوئی۔

                پھر انہوں نے ان کی مدح کے لیے بھی قلم اٹھایااور لکھنا شروع کیااور لکھتے ہی چلے  گئے۔ اس طرح انھوں نے در عدالت علے کو مکمل کیا۔اس کا انتساب اسی...

زواج المسيار: حقيقته و حكمه

The term “Nikah Misyar” (translated sometimes as “travellers’ marriage” or “marriage of convenience”) is not found in the Qur’an, Sunna or classical works of Islamic jurisprudence. It is a term that has been introduced recently. However, the concept can be found being discussed in the works of classical Muslim jurists (fuqaha). This is a marriage contract between a man and a woman, with the condition that the spouses give up one, two or several of their rights by their own free will. Some people consider that the misyar marriage can meet the needs of young people whose resources are too limited to settle down. However, there have been some (Sunni) scholars and organizations that have opposed the concept of Nikah Misyar altogether. As for the Islamic ruling concerning such marriages, there are two issues to consider: 1) Validity and permissibility; and 2) Appropriateness. Different scholars gave different opnions regarding these two imporatant issues. In this article, Nikah/zawaj Misyar was discussed in detail, describing different point of views and ruling in facvour and against Nikah Misyar.

Study of Inhibition of Carbonic Anhydrase Ii and Dpp-Iv As Possible Targets of Drug Discovery

Research on enzyme inhibitors has an enormous potential to introduce new drug candidates against enzyme related diseases. Keeping this in view, present study was designed to identify natural and synthetic compounds as leads against two clinically important enzymes, carbonic anhydrase-II (CA-II) and dipeptidyl peptidase-IV (DPP-IV). Carbonic anhydrases (CAs), and dipeptidyl peptidase-IV (DPP-IV) have pathological roles in the emergence of number of diseases, particularly glaucoma and diabetes, respectively. The results of the study are summarized below: PART A CA-II is an important enzyme for many physiological processes. Inhibitors of CA-II are used for the treatment of many diseases, such as epilepsy, mountain sickness, and glaucoma. During this study, over 350 fully characterized compounds were evaluated against BCA-II enzyme. Out of which 58 compounds showed a good inhibitory activity. Among these compounds, bisindoles and thiourea derivatives of bisindolyl showed the most significant activity with IC50 values in the range of 14.4 – 70.36 μM. To study the mechanism of action of inhibitors, most active inhibitors of these classes were further subjected to kinetic studies. Inhibition constants and type of inhibition were deduced by using Lineweaver-Burk plot, secondary re-plot of Lineweaver-Burk plot, and Dixon plot. Inhibition type and dissociation constants were deduced by Lineweaver-Burk plot, secondary re-plot, and Dixon plot. Bisindole derivatives, such as 23 (N-(4-(bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-2,4-dinitrobenzenesulfonamide), 28 (N-(4-(bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-3,5-dichloro-2-hydroxybenzenesulfonamide), and 38 (N-(4- (bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-2,4-dinitrobenzenesulfonamide) showed a significant inhibition of enzyme CA-II (IC50 = 15.6 – 28.86 μM). To assess their safety profile, cytotoxic studies were conducted on mouse fibroblast cell line (3T3). Fortunately some of the good active compounds were found non-cytotoxic, and thus can serve as leads for further studies on CA inhibitor drug design and development. PART B Epidemic prevalence of diabetes at national and global level emphasizes the need of urgent therapeutic intervention. In the second part of our work, we targeted an important enzyme of incretin pathway, dipeptidyl peptidase-IV (DPP-IV). Inhibitors of DPP-IV occupy center stage in the current anti-diabetic drug market. We screened over 1,800 fully characterized natural and synthetic compounds, through a mechanism-based colorimetric assay. This led to the identification of 87 new inhibitors. Significant inhibition was shown by the compounds of semicarbazones, thiosemicarbazone, benzophenone Schiff bases classes, and gold complexes. New inhibitors, identified through initial screening, were further subjected to mechanism-based kinetic studies. Lineweaver-Burk plot, secondary re-plot of Lineweaver-Burk plot, and Dixon plots were constructed to determine the type of inhibition, inhibition constant, and other kinetic parameters. Cytotoxic studies of active compounds were also conducted on mouse fibroblast cell line (3T3). Some potent inhibitors were also subjected to in situ DPP-IV inhibition assay by using Caco-2 cellular model. Synthetic compounds of different classes and Gold complexes were found to be active. Compounds (R)-2-phenyl-2,3-dihydrobenzo [d] imidazo[2,1-b] thiazole gold (I) triphenylphosphine tetrafluoro borate (204), (S)-2-phenyl-2,3-dihydrobenzo[d]imidazo [2,1-b]thiazole gold (I) triphenylphosphine tetrafluoro borate (207), and (S)-2-phenyl-2,3-dihydrobenzo[d] imidazo [2,1-b] thiazole gold (I) chloride (209) were identified as most potent inhibitors with IC50 values in the range of 22.0 – 35.6 μM. Earlier studies on DPP-IV inhibitors were restricted to selected number of compounds with limited structural variations. Present study presents comprehensive screening of different classes of synthetic compounds for the discovery of new inhibitors of DPP-IV. This is a cost effective, easy, reliable, and fast approach for the discovery of new drug candidates.