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Use of a Plant Virus Vector for Screening of Gene Constructs With Potential Insecticidal Activity

Thesis Info

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Author

Shaheen, Musarrat

Program

PhD

Institute

Pakistan Institute of Engineering and Applied Sciences

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2017

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/13013/1/Musarrat%20Thesis%2014-7-2017.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727642998

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Extending the novel implications of plant viruses as invaluable genetic tools in the field of transgenic plant technology, Potato Virus X (PVX) based transient gene expression is a simple rapid, inexpensive methodology employed for preliminary screening and evaluation of insecticidal gene constructs in plant sucking insect species. In this study, PVX vector was employed to achieve high throughput expression of two-candidate insecticidal proteins viz. Hvt, (Hydronyche versuta spider) and ACA Lectin (Allium cepa agglutinin) in tobacco plants. The Reverse transcriptase (RT-PCR) revealed a high throughput expression of 117 bp Hvt, 325 bp ACA Lectin in tobacco were bioassayed in Phenacoccus solenopsis Tinsley (P. solenopsis Tinsley), Aphis gossypii Glover, (A. gossypii Glover), M. persicae Sulzer (M. persicae Sulzer) and herbivorous insect species of Helicoverpa virescens (H. virescens), Spodoptera littoralis (S. littoralis). The results revealed severe insecticidal and antagonistic effect of Hvt in terms of significant insect mortality p<0.01%, reduced population survival and nymph production in P. solenopsis Tinsley, A. gossypii Glover and M. persicae Sulzer. ACA Lectin also exerted significant anti-feedent and insecticidal characteristics at p<0.01% on colonization and population survival in tested insects and larval mortality and lower dry weight gain in herbivorous insect species of H. virescens and S. littoralis. The practical application of PVX vector was extended through PVX- based VIGS mediated production of dsRNAs of two partial insect gene transcripts i.e. Voltage Gated Calcium Channel (PSCaVα1) and Elongation Factor (PSEF-1α) in tobacco plants and RNAi response in P. solenopsis Tinsley. The Reverse Transcriptase (RT-PCR) revealed a high throughput expression of 117 bp Hvt, 325 bp ACA Lectin and plants dsRNAs of 152 bp PSCaVα1 and 325 bp PSEF-1α in tobacco used in plant feeding bioassays. The results revealed significant insect mortality 96% PSCaV-α1 and 46% PSEF-1α and several allied phenotypic deformities like stiffing, melanization and shedding of cuticle, arrested growth and reduced fecundity in P. solenopsis Tinsley were consonant with declined expression of targeted genes in insects feeding corresponding dsRNAs duly elaborated by a semi-quantitative RT-PCR. The specificity of dsRNAs 152 bp PSCaV-α1 and 325 bp PSEF-1α dsRNAs derived from P. solenopsis Tinsley and 402 bp Arginine kinase (AK) from A. gossypii Glover was checked through feeding bioassays in non-target insect species like M. persicae Sulzer, H. virescens and S. littoralis. The results revealed non-significant effect of 152 bp PSCaV-α1 and 325 bp PSEF-1α and significant effect of AK in adult aphid in terms of mortality, nymphs produced and survived, larval mortality and dry weight gain in tested insect species. The cumulative results of this study highlighted the significance of PVX vector as valuable genetic tool for preliminary screening and evaluation of candidate insecticidal proteins and RNAi gene constructs. This study will enhance the efforts of biologists in a way to screen the candidate insect genes and design the complementary transgenic crop protection strategies.
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فصل سوم: آیاتِ استفہام میں فہم عبادات

ایمان لانے کے بعد انسان پر سب سے پہلے عبادت کا ادا کرنا لازم ہے ہر مذہب میں عبادت کا ایک خاص طریقہ ہوتا ہے جو مخصوص طریقے کے ساتھ ادائیگی کا حکم دیا جاتا ہے اسی طرح اسلام میں بھی نماز، روزہ، حج اور زكوة عبادات کی مختلف طرق ہیں اصل عبادت کی غایت یہ ہے کہ معبود صرف اللہ تبارک وتعالیٰ ہی کو ماننا ، صرف اسی کی عبادت کرنا ہر چیز میں اسی سے مدد طلب کرنا اسی کو حاجت روا اور مشکل کشا سمجھنا اسی کو مالک، خالق اور رب تسلیم کرنا اسی سے التجاء کرنا، ہر چیز کے لئے اسی کو پکارنا اور یہ یقین رکھنا کہ اللہ کے سوا کسی کے دائرہ اختیار میں کوئی چیز نہیں ہے اگر وہ نفع پہنچانا چاہے تو اسے کوئی روکنے والا نہیں ہے اور اگر ضرر پہنچائے تو اس کو کوئی ہٹانے والا نہیں ہے ہر طرح کی عبادت مثلاً قیام، رکوع، سجدہ صرف اسی کے لئے خاص ہے اور کسی اور کے سامنے جھکنا جائز نہیں۔

 انسانوں سے اللہ تعالیٰ نے انکی تخلیق سے پہلے ایک وعدہ لیا تھا جس کا ذکر قرآن مجید میں یوں مذکور ہے:

"اَلَسْتُ بِرَبِّكُمْ، قَالُوْا بَلٰي، شَہِدْنَا"۔[[1]]

" کیا میں تمہارا رب نہیں ہوں؟ اس وقت سب نے یہ کہا کیوں نہیں اے ہمارے رب!"۔

 سب نے اس وقت اللہ کی ربوبیت کا اقرار کیا تھا گویا کہ اللہ تعالیٰ کی ربوبیت کا اقرار و اعتراف انسانوں کی فطرت میں داخل اور انکے وجدان میں شامل ہے۔

اللہ تعالیٰ کی ربوبیت کا مطلب اور اس کا تقاضا کیا ہے ؟اسکے جواب کے بارے میں بشیر احمد لودھی یوں رقمطراز ہیں:

 " انسان ازخود پیدا نہیں...

الفكر السياسي الإسلامي وتطوره من الشورى إلى الديمقراطية

There has been a tussle going on between Islam and democracy for many decades in the Muslim world which has benefited most certainly the anarchic factions like kings, lords and army dictators. Though democracy is close to Islam and thought to be the best of all types of governments as majority is authority in it that may make or mar the government. In a democratic setup, the individual and personal rights and freedom are considered which is why it is preferred more these days. Many as Muslim thinkers are also in favor of it, but still there is a large faction of clerical scholars who deem democracy as the root of all evils since it empowers general public as an authoritative and decisive force. However with the passage of time, the Islamic counseling system has evolved and come very close to the spirit of democracy. Therefore, may contemporary scholars hold Islamic counseling and democracy almost similar. This article aims to present a preview of democracy and counseling in the light of what the Ulema (Scholars) say about it.

Synthesis and Characterization of Novel Heterocycles: Pyrazoles, Dihydropyrimidinones, Iminothiazolidinones, Quinolinyloxy Oxa/Thiadiazolamines and Triazolones /Thiones and Study of Palladium 0 -Catalyzed Cross- Coupling Reactions

This thesis describes the synthesis of various classes of heterocyclic compounds like, pyrazoles, dihydropyrimidinones, iminothiazolidinones, quinolinyloxy oxa/thiadiazol amines and triazolones/thiones and in addition study of palladium(0)-catalyzed cross- coupling reactions. Michael addition of some substituted anilines to methyl acrylate in acidic medium afforded the methyl 3-(substituted anilino)propionates (96-104), which on treatment with hydrazine hydrate in methanol were converted into corresponding 3-(substituted anilino) propionohydrazides (105-113) in good yields. Microwave irradiation of the latter with pentane-2,4-dione afforded 1-(3,5-dimethyl-1H-pyrazol-1-yl)-3-(substituted anilino) propan-1-ones (114-122) under solventless conditions. A series of new 5-substituted thiobarbituric acid derivatives 5-acetyl-3-aryl-2-thioxo- dihydropyrimidine-4,6(1H,5H)-diones (133-140) were synthesized by cyclocondensation of 1-aroyl-3-arylthioureas with malonic acid in the presence of acetic anhydride. These compounds exist in equilibrium with their enolic tautomeric form 5-(1-hydroxy ethylidene)-3-aryl-2-thioxo-dihydropyrimidine-4,6-(1H,5H)-diones. Plausible mechanism for the formation of products was proposed. Compounds (133-140) were evaluated for their preliminary antibacterial activity against a representative panel of Gram positive and Gram negative bacteria and were found to exhibit promising activity towards the tested microorganisms using levofloxacin as the reference drug. Of these compounds, the derivative (134) having methoxy group at ortho position of phenyl ring showed the highest effect on pathogenic bacteria and was identified as the lead molecule for further structural modifications. An efficient, synthesis of some 1-aroyl-3-aryl thioureas (123-132) was carried out. Substituted aroyl chlorides were treated with an equimolar quantity of potassium thiocyanate in acetone to afford the corresponding isothiocyantes which were not separated followed by reaction with an equimolar amount of substituted anilines to furnish the 1-aroyl-3-arylthiourea derivatives (123-132) which were assayed in vitro for their antimicrobial activity against Gram positive and Gram negative bacteria and were found to exhibit moderate to potent activity towards the tested microorganisms, compared to the standard drugs Tetracycline, Penicillin and Metronidazole.An efficient, regioselective synthesis of some 2-aroylimino-3-aryl-thiazolidin-4-ones (141-150) involving base-catalyzed cyclization of 1-aroyl-3-aryl thioureas with chloroacetyl chloride in dioxane is reported. Compounds (141-150) were assayed in vitro for their antimicrobial activity against Gram positive and Gram negative bacteria and were found to exhibit promising activity towards the tested microorganisms, comparable to and in some cases better than those of the standard drugs. The cyclic condensation of 1-aroyl-3-aryl thioureas with dimethyl acetylene dicarboxylate (DMAD) in methanolic solution at room temperature resulted in the formation of methyl [4-oxo-2-(substituted benzoylimino)-3-(substituted phenyl) thiazolidin-5-ylidene] acetates (151-164) in good to excellent yield. The synthesis of bis(thioureas) was carried out by treatment of aroyl/alkyl chloride with potassium thiocyanate and as a result corresponding isothiocyanates were obtained using acetone as solvent. These isothiocyanates were treated with 1,2-phenylene diamine and 1,2-bis(thioureas) (165-172) were obtained in good yield. Similarly, these isothiocyanates were treated with 1,4-phenylene diamine and resulted in 1,4-bis(thioureas) (173-180) synthesis. These bis-(thioureas) were also treated with dimethyl acetylene dicarboxylate (DMAD) and corresponding 1,2-bis(iminothiazolidinone) acetates (181-188) and 1,4- bis(iminothiazolidinone) acetates (189-196) were synthesized in good yield. A series of quinolinyloxy oxa/thiasemicarbazide derivatives (200-207) were synthesized. Intramolecular cyclization of these quinolinyloxy oxa/thiasemicarbazide derivatives in the acidic medium of poly phosphoric acid (PPA) resulted in synthesis of quinolinyloxy 1,3,4-oxa/thiadiazolamine derivatives (208-215) while in basic medium of sodium hydroxide intramolecular cyclization of these quinolinyloxy oxa/thiasemicarbazide derivatives resulted in synthesis of quinolinyloxy 1,2,4- triazolones/thiones derivatives (216-223). A series of 1,2-difluoro-3,4,5,6-tetra substituted phenylbenzene derivatives (226-228) was reported through Suzuki-Miyaura coupling-reaction of 1,2-difluoro-3,4,5,6- tetraiodobenzene with various boronic acid derivatives using tetrakis(triphenyl phosphine) palladium(0) as catalyst. The substrate 1,2-difluoro-3,4,5,6-tetraiodobenzene (225) was also synthesized by treatment of 1,2-difluoro-benzene with molecular iodine in the presence of K2S2O8, trifluoro acetic acid and sulphuric acid.