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Home > Cloning and Transformation of Sucrose Synthase Sus Gene in Cotton Gossypium Hirsutum L.

Cloning and Transformation of Sucrose Synthase Sus Gene in Cotton Gossypium Hirsutum L.

Thesis Info

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Author

Mukhtar Ahmed

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Molecular Biology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10795/1/Mukhtar_Ahmed_Molecular_Biology_2019_UoP_Punjab_PRR.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676725741118

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Long staple length, optimum micronaire with good strength is the highest demand of the textile industry in Pakistan. Cotton fiber quality is a multifactorial trait controlled by different genes that play a decisive role in different stages of fiber development. However, only a few genes have been explored yet that are crucial in fiber development and some of them have shown a positive effect on fiber yield and quality in transgenic cotton. Sucrose synthase (SuS) is among the important factors involved in inter-conversion of sucrose to fructose and UDP-glucose that serve as a precursor for the synthesis of cell wall cellulose. In the current study, an attempt was made by the introduction of synthetic SuS gene under the control of a CaMV35S promoter. The cassette was cloned in pCAMBIA 1301 vector with BstX1 and Xho1 restriction sites. The recombinant plant expression vector pCAMBIA-SuS was introduced into local non-transgenic cotton (Gossypium hirsutum) variety CEMB-00 through Agrobacterium-mediated transformation method. The transformation efficiency in this study remained 1.02%. Molecular analyses of putative transgenic cotton plants were performed to verify their transgenic nature. Young leaves of T0 plants were used for histochemical GUS assay to confirm the transformation event. Amplification of 813bp fragment by using gene-specific internal detection primers confirmed the successful integration of SuS gene in the cotton genome. Maximum mRNA expression of SuS gene was obtained in MA0023 and MA0034 as compared to other transgenic cotton plants. Four transgenic plants were selected after PCR analysis for molecular and biochemical studies in advanced generations. SuS gene mRNA expression in fiber was significantly higher at 15 DPA and 20 DPA as compared to 8 DPA in all transgenic lines. The Leaf SuS activity in transgenic lines increased up to 54% as in the case of MA0023. SuS activity at the reproductive stage (15DPA) was also increased up to 40% in case of MA0034. In fiber of transgenic lines decrease in sucrose contents while the increase in total soluble sugars contents was evident from elongation (15DPA) towards secondary wall synthesis and maturation (40DPA). Cleavage of sucrose by SuS catalytic activity was found to be higher in transgenic cotton plants as compared to non-transgenic control plants. The mRNA expression was found positively correlated with SuS activity and cellulose contents. Fiber analyses of transgenic plants showed an increase in fiber length up to 11.7%, fiber strength 18.65% and 28% increase in cellulose contents. Improvement in micronaire value up to 3.00 was observed in transgenic line MA0023. SEM analyses revealed that fibers from transgenic plants were smooth, highly spiral and fiber twist number increased per unit length when compared with control. Improvement in fiber length, micronaire value and surface smoothness led to producing better quality fiber for textile purposes. Genetic modification of cotton with SuS gene also resulted in improvement in some morphological and agronomic traits like plant height increased up to 88.76%, number of bolls 87.80% and GOT increased up to 22.08 % as compared to control plants. FISH analysis showed single copy number at chromosome number 9 and no signal was detected in control plants. The results depict that increased SuS activity, fiber properties, morphological and agronomic traits in transgenic lines are caused by genome integration and constitutive expression of SuS gene in cotton. From the results, it is clear that the SuS gene holds the potential to improve fiber quality and also provide more positive effect when combined with other potential fiber trait. Stacking of fiber-related genes into a single plant could be a better way to improve fiber quality in local cotton varieties to meet the standards of the modern textile industry.
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روش صدیقی

روش صدیقی
افسوس ہے گزشتہ ماہ جناب روش صدیقی بھی رہ گزائے عالم جادوانی ہوگئے۔ مرحوم بلند پایہ اورصاحب فن شاعرتھے۔ان کی شہرت کا آغاز رومانی نظموں سے ہوا جو اس زمانہ کے مشہور ادبی رسالوں میں بڑے اہتمام سے چھپتی تھیں اورجنہیں وہ اپنی خاص پرجوش آواز میں لہرالہراکر پڑھتے تھے۔بعد میں ان کی شاعری حکمت وفلسفہ اورانسانی ووطنی مسائل وآلام کی ترجمان بن گئی لیکن ان کا کلام غامض اور دقیق ہوتاتھااورالفاظ اورتراکیب پرشکوہ وباوقار،طبیعت میں روانی اورجدت پسندی بلا کی تھی۔اخلاقی اعتبار سے بڑے باوضع،ملنسار اورمذہبی حیثیت سے صوم وصلوٰۃ کے اورارادو وظائف تک کے پابند تھے۔انتقال سے چھ سات روزپہلے(۱۴/جنوری)کوشام کے وقت نئی دہلی کے ریلوے اسٹیشن پر اچانک ملاقات ہوگئی توحسب معمول بڑے تپاک سے ملے اورمعانقہ کیا۔کافی ہشاش بشاش اورمگن تھے۔اس وقت اس کاوہم وگمان بھی نہیں ہو سکتا تھا کہ اس عالم آب وگل میں وہ بس اب چندروز کے اورمہمان ہیں اوران سے یہ آخری دید و شنید ہے۔شاہ جہاں پور کے ایک مشاعرے میں گئے تھے وہیں دل کادورہ ہوا اور جاں بحق ہوگئے۔اﷲ تعالیٰ ان کی قبر کو ٹھنڈی رکھے اوران کے پسماندگان کا حامی وناصرہو۔آمین [فروری۱۹۷۱ء]

 

Is History a Threat? (Comment)

Erick Hobsbawm (d.2012) was a Marxist historian, a professor of history at Birkbeck College, University of London, where he taught till the end of his life. Birkbeck is an evening college where most of the students belong to the working class and continue with studies to improve their educational qualifications. Conscious of the condition of students who attended his lectures after a hard day of physical labour, he devised lectures on the history of Europe to create emancipatory thought. His four books use history as a tool to comprehend present-day Europe. They are titled The Age of Capital, The Age of Industrial Revolution, The Age of Revolution and The Age of Extreme. Besides these four books, he has published a number of papers and essays that attempt to build a new historical consciousness. There are other progressive historians besides Hobsbawm who believe that history can be liberating if it is not written in the service of the rulers of the time. It is also not a static and depressing account of the past but is dynamic and constantly changing. While history tells the story of unjust rulers and the institutions they created, it also reveals how the rulers and their unjust institutions meet their end. The reign of absolute kingship where the king was considered the Viceregal God, brutal military dictatorships, and exploitative economic systems that lasted for centuries with the aim of torturing and subduing the common working people were all destroyed over time, especially when people joined forces and resisted them to gradually establish a just order.

The Ecology of Algal Flora from Kalar Kahar Lake and its Associated Tributaries

Algae are thaloid organisms comprising of simple photosynthetic organism having unicellular to multicellular organization. Being lower organism, little attention has been paid to document algal resources across the lakes in particular and country in general. The present study was aimed to collect, identify and ecology of the algal flora of Kalar Kahar Lake and its Tributaries. A total of 341 species distributed across 89 genera, 53 families, 29 order, 11 classes and 7 phyla were identified from the study area. Highest number of families were contributed by phylum Bacillariophyta (24), followed by phylum Chlorophyta (13 families), Cyanophyta (11 families). Overall, 32 new taxa were recorded from the study area, out of which, 29 belonged to Bacillariophyta, 02 Cyanophyta and one Chlorophyta. Species richness (SR) was highest in the Lake in July during both the years, followed by August. While in the Tributaries, maximum SR was recorded in August and July during 2010 and 2011 respectively. Overall, 2011 was rich in algal diversity in both the Lake and the Tributaries. Similarly, cell density (D) was higher in 2011 than 2010. Bacillariophyta and Cyanophyta were constantly present in all months in the Lake and the Tributaries. Chlorophyta was present in all months except November in the Lake while in the Tributaries it was absent in January, February and November. Charophyta was present in October, November 2010 and May to July and October during 2011. 12 The highest number of species (α-diversity) was recorded during 2011 from both the Lake and the Tributaries (193 and 175 respectively), followed by the Tributaries (161 spp.) and the Lake (159 spp.) in 2010. There was opposite trend in the case of beta diversity (β) in which highest value was recorded during 2010. Maximum β-diversity was recorded from the Lake (2.14), followed by the Tributaries (2.12). Highest species were shared by Lake 2010-Lake 2011 pair (122 species), followed by Tributaries 2010-Tributaries 2011 (106 species), Tributaries 2010- Lake 2010 (83) and Tributaries 2011- Lake 2011(67 species). Amongst habitat pairs, Lake 2010-Lake 2011 possessed highest similarity Index (0.69) with least β-diversity followed by Tributaries 2010-Tributaries 2011 (0.63), Tributaries 2010-Lake 2010 (0.52) and Tributaries 2011- Lake 2011 (0.36). The Shannon–Weaver index in the Lake and the Tributaries varied from 2.14 to 3.42 and from 2.47 to 3.49 respectively suggesting that the water quality is moderately polluted to clean. Based on Importance Value Index (IVI), Microcystis–Nitzschia-Cyclotella formed algal community in the Lake during 2010, while Microcystis-Cladophora- Chara in 2011. In the Tributaries, Chara-Nitzschia-Microcystis and Microcystis- Phormidium-Nitzschia constructed dominant communities during 2010 and 2011 respectively. Water quality parameters viz., temperature, pH, dissolved oxygen, total dissolved solids , conductivity, total hardness, nitrates, phosphate, sulphate, light transparency, turbidity, iron, copper, zinc, lead and nickel varied significantly (P3 <0.05) amongst all the months during 2010 and 2011. Environmental variables such as dissolved oxygen, turbidity, phosphate, nitrate and total hardness significantly affected cell density (P <0.05) in the Lake, while water temperature, turbidity, phosphate and lead significantly (P <0.05) affected density in the Tributaries. The relationship of algal species with environmental data set was determined by Canonical Correspondence Analysis (CCA) using CANOCO software (version 4.2). Overall, environmental data sets such as nitrate, turbidity, copper, transparency, dissolved oxygen (DO) and zinc were found positively correlated in the distribution of specific taxa, while total hardness, water temperature, conductivity, total dissolved solids (TDS), and pH were negatively correlated for distribution of various taxa in the Lake during 2010-2011. In the Tributaries, environmental variables such as phosphate, total hardness, zinc, nickel, transparency, chlorophyll a, sulphate, lead, conductivity, TDS, copper and pH had positive relationship with algal composition during both years. DO in 2010 while nitrate and iron during 2011 exhibited negative correlation for algal distribution.