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Home > Isolation, Identification and Characterization of Osrglp2 Promoter Binding Protein S

Isolation, Identification and Characterization of Osrglp2 Promoter Binding Protein S

Thesis Info

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Author

Deeba, Farah

Program

PhD

Institute

Pir Mehr Ali Shah Arid Agriculture University

City

Rawalpindi

Province

Punjab

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Natural Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/9223/1/Farah_Deeba_Biochemistry_2016_HSR_PMAS_13.02.2017.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726552052

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Germin and germin-like proteins (GLPs) belong to cell wall glycoproteins that have shown asignificant resistance to detergent action, denaturation by heating, and degradation by proteases. GLPs expression is reportedly modulated during exposure to pathogens and abiotic stresses. Yet, little is identified about the regulatory mechanism of the GLP genes. The promoter of OsRGLP2 gene was isolated and cloned by Mahmood et al. (2007). This promoter showed strong expression ofthe GUS gene in transgenic tobacco during salinity, dehydration and wounding stresses. In this study, the regulatory cis-elements and their binding proteins for OsRGLP2 promoter are characterized. Various putative stress responsive cis-regulatory motifs and their specific binding proteins were identified by in silico analysis. DNA binding domains of OsWRKY71, OsDOF18 and OsMYB1 were cloned, overexpressed in E. coli and then purified by affinity chromatography using Glutathione Sepharose resin followed by cationic exchange chromatography. Electrophoretic mobility shift assays (EMSAs) have shown that recombinant OsWRKY71, OsDOF18 and OsMYB1 proteins were capable to interact with DIG labeled fragments of OsRGLP2 promoter containing W-box, AAAG and WAACCA motifs respectively. Binding was further confirmed by competitor EMSA and EMSA with mutant oligonucleotides. These regulatory elements were also active in binding with nuclear factors from rice nuclear proteins extract in vitro as confirmed by competitive EMSA. Expression analysis was performed to check the level of OsWRKY71, OsDOF18 and OsMYB1 against salt, cold, heat, wounding and drought stresses. Expression of OsWRKY71 was found to be induced in case of salt and cold stresses while OsDOF1 was expressed at relatively high level in response to salinity and drought. OsMYB1 expression was 23 fold higher in response to wounding which demonstrates the valueofOsMYB1 up-regulation in response to wounding stress inrice. In order to investigatethe functionof OsWRKY71, OsMYB1 and OsDOF18againstdiverse abiotic stresses, recombinant plasmids were subjected to transformationin E. coli and their effect on E. coli growth was analyzed. The E. colicells containing pGEX-OsWRKY71, pGEX-OsDOF18 and pGEX-OsMYB1 has shown different levels of tolerance against salt, drought, cold and heat stresses as compared to empty pGEX-4T1 vector. In silico characterization suggested the involvement of these proteins in protein-protein interaction. In conclusion, the positive response of OsWRKY71, OsDOF18 and OsMYB1 in abiotic stresses suggests their association with OsRGLP2 promoter and the importance of these proteins in providing protection to plants under abiotic stresses. Overexpression of OsWRKY71, OsDOF18 and OsMYB1 genes in crop plants may help in obtaining stress tolerant lines.
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۳۔ سمندر کا قان

سمندر کا قانون

سڑک کے ساحلی کنارے پر

چھوٹی مچھلیاں قسمت کی ریڑھی پر ضرورت کے کھلونے سجائے

کنڈی سے لالچ کا چارہ لگائے

قدرِ زر میں اضافے کے گُر آزما رہی ہیں

بڑی مچھلیاں سڑک کے سمندر میں

تیز رو گاڑیوں میں بہتی

ان کو اُچکتی، نگلتی اور روندتی

ہارن کے ڈکار بجاتی ہوئی

بے سمت سفر پر رواں ہیں

سمندر کا یہ قانون اب خشکی پر بھی چل رہا ہے

الٰہی کوئی سونامی۔۔۔۔!

کوئی صر صر۔۔۔۔!

کوئی انقلاب۔۔۔!

دراسة مقارنة في تعيين الزمان والمكان والأعلام عند شراح البخاري (الكرماني وابن حجر نموذجاً)

Sahih al-Bukhari by Imam Abu Abdullah Mohammad al-Bukhari comes second to the Holy Quran as a fundamental source of Islam. It is a most authentic collection of sayings and deeds of Prophet Mohammad (PBUH), which covers almost all aspects of life in providing proper guidance from the Messenger of Allah. The Muslim scholars of past and present time gave great importance to this book by making their efforts to extract the hidden treasures from this book and to present the different approaches and benefits of this book. “Al Kawakib Ad Durari” by Imam Alkirmani is an old explanation of Sahih Bukhari. During studying “Fath al Bari” I found that Alhafiz Ibne Hajar has consulted “Al Kawakib Ad Durari” and quoted Imam Alkirmani’s commentary and added it. Alhafiz Ibn e Hajar differs at many times with the opinions of Imam Alkirmani about specific time, place, different tribes and personalities mentioned in the text of traditions. I wanted to study such analysis to check the right opinion after comparing statements of both Imams and by consulting with the statements of other scholars of this field. This article approves that judgments of Ibn e Hajar about specific time, place, different tribes and personalities are more authentic than opinions of imam Alkirmani.

Studies on Antihyperlipidemic and Vasomodulating Effects of Eruca Sativa, Erucin, Hedera Helix and Hederacoside C

Aerial parts of Eruca sativa (E. sativa) and leaves of Hedera helix (H. helix) are popular remedies for the treatment of cardiovascular diseases in humans. Erucin and hederacoside C (HDC) are the important constituents of E. sativa and H. helix, respectively. Literature lacks pharmacological investigation on these plants and the constituents in hyperlipidemia and hypertension. This study aimed to investigate the E. sativa and H. helix, erucin and HDC effect in hyperlipidemia, hyperlipidemiainduced vascular dysfunction and hypertension. Crude extracts of both plants (30, 100 and 300 mg/kg), erucin (1 and 3 mg/kg) and HDC (2.5 and 5 mg/kg) were tested in tyloxapol and high fat diet (HFD)-induced hyperlipidemic Sprague-Dawley (SD) rats. Biochemical evaluation of lipid profile was carried out on blood collected from all groups. Histopathological and vascular dysfunction studies were performed on aortae isolated from normal, hyperlipidemic and treated rats. The antihypertensive effect was investigated in both normotensive and hypertensive rats. The mean arterial pressure (MAP) was measured in both groups. The mechanisms were investigated using isolated rat aorta and atria. Both extracts and compounds significantly reduced total cholesterol and triglycerides (p < 0.001), compared to lovastatin in tyloxapol-induced hyperlipidemia. In high fat diet-induced hyperlipidemia, both extracts significantly (p < 0.001) reduced TC, LDL and increased (p < 0.05) HDL levels at higher dose. Erucin and HDC also significantly (p < 0.001) decreased TC and LDL levels. Extract of H. helix was more potent (p < 0.001) in decreasing the atherogenic index in both hyperlipidemic models, compared to E. sativa. The data thus shows that extracts of both plants and compounds are antihyperlipidemic agents. Further in-vitro studies were carried out to explore the role of these agents on vascular endothelium disruption (dysfunction). The thoracic aortae from HFD rats were used for histopathological and vascular reactivity studies. Extract of E. sativa reversed endothelial dysfunction in HFD-induced hyperlipidemic rats in-vitro by inhibiting macrophages infiltration and reducing endothelial disruption. Extract of H. helix markedly preserved endothelial dysfunction by improving the architecture of vascular wall. Both compounds also improved endothelial disruption. The vascular dysfunction xi study in the aortic rings from hyperlipidemic rats treated with both extracts and compounds showed that acetylcholine caused complete relaxation against phenylephrine (PE) precontractions. This indicates that extract and compounds are effective remedies in improving disrupted vascular architecture due to hyperlipidemia. To see effect on blood pressure, extracts, fractions and compounds were tested in normotensive, normotensive atropinized and hypertensive rats. Extract of E. sativa and fractions, dose-dependently decreased mean arterial pressure (MAP) that was significantly (p < 0.001) reduced with atropine (1 mg/kg) pretreatment in normotensive rats. Extract of E. sativa and fractions also decreased MAP in hypertensive rats. The effects of H. helix extract on MAP in both normotensive and hypertensive rats were greater than E. sativa. The antihypertensive effect of extract and fractions of H. helix remained unchanged in the presence of atropine in normotensive rats excluding the involvement of muscarinic receptors. Erucin and HDC also induced antihypertensive effect in normotensive rats (unaffected by atropine) and hypertensive rats in-vivo. The underlying mechanisms of antihypertensive effect of extracts and compounds were further investigated in invitro experiments in rat aorta and atria. In rat aorta, extract and fractions of E. sativa produced vasorelaxant effect that was partially inhibited with L-NAME and atropine pretreatment indicating role of muscarinic receptor-linked nitric oxide (NO) pathway. This effect of extract and fractions was also partially eliminated with denudation of endothelium and aortic rings from hypertensive rats, also suggesting role of vascular endothelium. The vasorelaxant effect of n-hexane fraction was least, indicating that it might be due to presence of vasocontractile constituents, which may have role in vasomodulation. Erucin also produced incomplete relaxation in normotensive rat aorta, suggesting that it may be one of the constituents involved in vasomodulation. The vasorelaxant effect of H. helix and HDC was inhibited with L-NAME pretreatment and denudation but did not change with atropine pretreatment excluding role of muscarinic receptors. The extracts of both plants, erucin and HDC produced vasorelaxant effect against high K+ precontractions like verapamil. Extract of E. sativa and H. helix, fractions and compounds suppressed PE peak formation; erucin was less potent than HDC. In isolated at atrial strips, E. sativa and erucin induced negative inotropic and chronotropic effects with a positive inotropic effect by the nhexane fraction, which was not affected by atropine pretreatment, suggesting that cardiac muscarinic receptors are not involved. The extract, fractions of H. helix and xii HDC caused depression of force and rate of atrial contraction which remained unchanged in the presence of atropine. To have possible chemical profile of the extract, spectrophotometric and HPLC analysis were carried out that showed the presence of quercetin and erucin in crude extract of E. sativa and HDC in H. helix. According to acute toxicity test, crude extract of E. sativa and H. helix were safe at 3 and 5 g/kg, respectively. In conclusion, the findings of present study indicated that E. sativa and H. helix are effective antihyperlipidemic and antihypertensive remedies. Both extracts and important constituent’s erucin and hederacoside C significantly reduced TC and LDL and preserved the endothelial disruption evident by histopathological and vascular dysfunction studies in-vitro. The preservation of endothelial dysfunction is due to decrease in LDL. The antihypertensive effect of E. sativa and H. helix extracts is possibly due to vasodilatory and cardiac effects. The endothelium-independent mechanisms involved inhibitory effect on calcium influx and release. Endothelium-dependent mechanisms involved muscarinic receptor linked NO mediated pathway. Erucin acted through endothelium-independent mechanism mediated by calcium antagonism. E. sativa and erucin showed negative inotropic and chronotropic effects, possible due to calcium channel blockade. Antihypertensive effect of H. helix extract and HDC are mediated through NO release inhibiting calcium release from stores and entry via VDCs also decrease cardiac rate and force of contractions. This data provide pharmacological base to medicinal use of E. sativa and H. helix in hyperlipidemia and hypertension. The presence of erucin in E. sativa and HDC in H. helix further support the findings and this study identified erucin and HDC as important constituents for the management of hyperlipidemia and hypertension.