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Home > Molecular Cloning and Overexpression of Heat Shock Transcription Factor Hsfa1d in Tobacco for Enhancement of Thermotolerance

Molecular Cloning and Overexpression of Heat Shock Transcription Factor Hsfa1d in Tobacco for Enhancement of Thermotolerance

Thesis Info

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Author

Shah, Zamarud

Program

PhD

Institute

University of Agriculture

City

Faisalabad

Province

Punjab

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Applied Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/8214/1/Zamarud%20thesis%20for%20final%2024-06-2016-.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726696109

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Among abiotic stresses, heat stress has the most devastating impact on plant growth. In the present study, heat shock transcription factor HsfA1d was isolated from Arabidopsis thaliana and cloned into an in-house constructed gateway compatible cloning vector pUC57GW-CmRccdB through infusion cloning. Entry clone was confirmed through restriction digestion and sequencing analysis. For subcellular localization, HsfA1d was cloned in-frame with yellow florescent protein (YFP) in pGWB442 through LR clonase reaction resulted in YFP-HsfA1d chimeric gene construct. Homodimerization of HsfA1d was studied using Bimolecular Florescence complementation (BIFC) assay. For BIFC, HsfA1d was cloned in-frame with sequences that codes for N and C termini of yellow florescent protein, into 2 in-house constructed vectors pGSA002-nYFPn and pGSA002-nYFPc respectively. After confirmation through restriction enzyme digestion, the newly constructed vectors were transformed into Agrobacterium tumefaciens GV3101 for transient expression in tobacco (Nicotiana benthamiana) through syringe-infiltration. Subcellular localization of YFP-HsfA1d in DAPI-stained cells was evaluated using confocal microscopy. For plant transformation, HsfA1d was cloned into 2 plant expression vectors pGWB402Ω and pGWB442 through LR clonase reaction. After confirmation through RFLP analysis, the vectors were transformed into Agrobacterium GV3101 for tobacco leaf disc infection. Putative transgenic plants were selected and regenerated on appropriate selection media using different growth regulators. After PCR and confocal-based confirmation, transgenic plants were evaluated for their response to heat stress. HsfA1d, HSP70 and HSP90 exhibited 4.8, 2.8 and 2 folds increase respectively in their expression under heat stress compare to room temperature. HsfA1d was found to significantly enhance the expression of downstream gene HSP70 while no such effect on the expression of HSP90 was recorded. The plants transformed with HsfA1d were found to retain more water and accumulate more proline under heat stress. The transgenic plants exhibited efficient protective system, causing less 8 electrolyte leakage and less chlorophyll damage under heat stress. It is concluded that HsfA1d plays a vital role in thermotolerance enhancement and hence recommended for increasing plant thermotolerance.
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صلاح الدین اویسی

جناب صلاح الدین اویسی مرحوم
جناب غلام محمود بنات والا کی فرقت کا صدمہ ابھی کم نہ ہوا تھا کہ مجلس اتحاد المسلمین کے قائد، فرزند دکن اور سالار ملت، سلطان صلاح الدین اویسی بھی اس دنیا سے رخصت ہوگئے، امت ایک اور ہوش مند، جرأت مند اور حوصلہ مند راہبر سے محروم ہوگئی، وہ عرصے سے صاحب فراش تھے، عمر مستعار کم نہ تھی، وقت موعود آیا اور رمضان المبارک کے نہایت مبارک اور آخری عشرے میں یعنی ۲۸؍ رمضان کو وہ اپنے مالک حقیقی کی رحمتوں سے جاملے ، اناﷲ وانا الیہ راجعون۔
قریب ساٹھ ستر سال پہلے جب مجلس اتحاد المسلمین کے بانی نواب بہادر یار جنگ کا ۱۹۴۴؁ء میں انتقال ہوا تھا تو اس وقت مولانا سید سلیمان ندوی نے معارف میں ان کا ماتم کرتے ہوئے لکھا تھا کہ ’’ان کا سا آدمی صدیوں میں پیدا ہوتا ہے اور جب پیدا ہوتا ہے تو انقلاب انگیز ہوتا ہے، ان کی ذات سے امت اسلامیہ کو بڑی بڑی امیدیں قائم تھیں اور خصوصیت کے ساتھ دکن کے مسلمانوں کے حق میں ان کا وجود آب حیات کا حکم رکھتا تھا‘‘۔
۱۹۴۴؁ء اور ۲۰۰۸؁ء کا زمانی فرق، تغیرات احوال کے لحاظ سے زمین آسمان کا فرق ہے لیکن صلاح الدین اویسی مرحوم کی ہستی کے لیے ان الفاظ کی حقیقت میں ذرا فرق نہیں۔
سلطنت آصفیہ اسلامیہ میں مجلس اتحاد المسلمین کا قیام، قومی تخیل اور سیاسی جذبوں کو نئی زندگی دینے کے ساتھ مسلمانوں کی تعلیمی اور معاشرتی ترقی کے لیے ہوا تھا، نصب العین تو ’’وسیع تر، عظیم تر، بیرونی و اندرونی مداخلتوں سے پاک اور کامل آزاد حیدرآباد تھا‘‘ اس وقت اس مجلس کے حوصلوں کو دیکھ کر کہا گیا تھا کہ دکن کے مسلمانوں نے صدیوں کے آرام کے بعد کروٹ لی ہے لیکن چند برسوں کے بعد تد اول...

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