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Home > Expression and Binding Studies of Some Geminivirus Proteins

Expression and Binding Studies of Some Geminivirus Proteins

Thesis Info

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Author

Saiqa Andleeb

Program

PhD

Institute

Quaid-I-Azam University

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2014

Thesis Completion Status

Completed

Subject

Natural Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/2665/1/2887S.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726168730

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Geminiviruses are circular single-stranded DNA viruses and are classified into family Geminiviridae. The family consists of four genera Mastrevirus, Curtovirus, Topocuvirus and Begomovirus. Begomoviruses can be broadly divided into two groups, the Old World (eastern hemisphere, Europe, Africa, Australia, Asia) and the New World viruses (western hemisphere, the Americas). Their genomes have number of characteristics that distinguish Old World and New World viruses. All New World begomoviruses are bipartite, whereas both bipartite and monopartite begomoviruses are present in the Old World. Chilli leaf curl betasatellite (ChLCB) is a disease specific betasatellite and is found associated with monopartite as well as bipartite begomoviruses. On the other hand Tomato leaf curl New Delhi virus (ToLCNDV) is an Old World bipartite begomovirus whereas Cabbage leaf curl virus (CaLCuV) is New World bipartite begomovirus. The aim of research work presented here was to study the interaction of ChLCB with both the Old and New World bipartite begomoviruses and the identification of DNA A encoded proteins that contributed to symptoms induction, suppressor of gene silencing, and pathogenicity. The other objective was to find out the host genes that are up or down regulated due to the interaction of βC1 protein encoded by ChLCB. Differential display analysis was done to find out the host genes that are activated against βC1Chi, whereas gateway cloning technology and two expression systems i.e. prokaryotic (BL21 bacterial strain) and yeast (Pichia pastoris) were used for the expression analysis of geminivirus encoded proteins. Co-inoculation of the DNA ACa and DNA BCa yielded efficient infection, typical symptoms of leaf curling, leaf crinkling, mild yellow, leaf deformation and stunting after seven to eight days of inoculation and the levels of siRNA derived from CaLCuV correlated positively with the levels of viral DNA. It was observed that the inoculation of ACa + βChi showed mild symptoms and low levels of viral DNA ACa accumulation in systemic leaves as compared to both components of ACa + BCa. From this study it was concluded that betasatellite interacted with the New World begomovirus (CaLCuV), intensified symptoms and also helped in virus movement. These results confirmed that the interaction of betasatellites was not limited to monopartite begomoviruses and that betasatellites can complement movement of bipartite begomoviruses from both Old and New World. The role of DNA A encoded proteins of both Tomato leaf curl New Delhi virus and Cabbage leaf curl virus in viral pathogenicity was studied. For this study cloning and expression of AC2Ca, AC4Ca, and AV2To genes were carried out into pDONOR/Zeo entry vector and destination vectors p100, p201 and p202 having 35S promoter and different protein tags such as HA and FLAG through gateway technology. Floral dip transformation method was also used to express protein in Arabidopsis transgenic plants. It was found that AC2Ca was a suppressor of RNA silencing and its expression induced the hypersensitive response. Similarly, AC4Ca suppressed miRNA whereas AV2To was a weak suppressor of RNA silencing. Putative transgenic plants expressing AC2p201Ca, AC4p100Ca and AC4p201Ca showed distinct phenotype. AC4p100Ca and AC4p201Ca developed downward leaf curling, crumpling and stunted plants, whereas AC2p201Ca plants developed elongated leaves, rosette like pattern, slightly rolling and margins of the leaves were deformed. On the other hand, p202(AV2)To developed upward leaf curling and deformed leaves, AV2p100To and AV2p201To expression vectors showed scrunch and slightly deformed leaves. From all experimental data it can be concluded that these proteins are pathogenicity determinants and are involved in symptom induction and upon complementation of movement function by ChLCB were able to develop disease phenotype in the absence of DNA B Ca. In this regard efforts were made to express βC1Chi protein through both bacterial and yeast expression systems to understand their binding activity with DNA/RNA or protein-protein interaction through in vitro binding assays like pull down assay or co- immunoprecipitation. It was found that βC1Chi was lethal protein for bacterial and yeast expression system. The expression of βC1Chi from bacterial system was not detectable due to nucleotide mutations which resulted in failure of protein expression. Similarly, Pichia pastoris system was also failed to produce detectable expression and protein could not be purified. On the other hand AV2To was successfully expressed in BL21 system and indicated that bacterial system was working and the problem was occurred due to toxicity of βC1. Later βC1Chi was expressed in N. tabacum to isolate differentially expressed genes (DEGs) through differential display analysis during host protein interaction. It was observed that WRKY transcription factor IId-1 splice, putative Rieske iron-sulfur protein, NADH dehydrogenase subunit 1 and NADH dehydrogenase subunit 2, Quinonprotein alcohol dehydrogenase, trigger factor (chaperone in protein export), Arabidopsis thaliana calmodulin-binding receptor-like kinase, and Polyribonucleotide nucleotidyltransferase were isolated from N. tabacum in response of βC1Chi under 35 (CaMV) promoter after two and four days of infiltration. Delta, Livak and Pfaffi mathematical models of quantitative real time PCR gave an idea that these DEGs were up and down regulated. Interestingly these all DEGs are related to the chloroplast and mitochondrial genes, involved in plant development and growth processes, cell protection, replication mechanisms, act as carriers of ATP synthesis and electron transporters during photosynthesis and respiration processes. Kegg Orthology based annotation system indicated that Polynucleotide nucleotidyltransferase is a RNA binding protein, and involved in the purine and pyrimidine metabolic pathway that relates to RNA degradation. The data presented here show that the interaction of betasatellite is not limited to monopartite begomoviruses. The spread of begomovirus complexes that contain betasatellite thus pose a serious threat to global agriculture. It was concluded that AC2Ca, AC4Ca, AV2To, and βC1Chi proteins of both monopartite and bipartite begomoviruses involved in viral movement, symptom induction, and pathogenicity determinant.
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قندیلِ محبت کے دیے نعت کے الفاظ


قندیلِ محبت کے دیئے نعت کے الفاظ
چمکے ہیں دل و جاں میں مرے نعت کے الفاظ

آیاتِ مقدُس میں ملے نعت کے الفاظ
قرآن کے الفاظ بنے نعت کے الفاظ

دل اہلِ قلم کا ہوا سرشار خوشی سے
قرطاس پہ جس وقت لکھے نعت کے الفاظ

گل ہائے عقیدت کی ہے مہکار سرِ بزم
پھولوں کی طرح لب پہ کھلے نعت کے الفاظ

اس مشقِ سخن کی ہے جزا کوثر و تسننیم
میزان پہ خود بول پڑے نعت کے الفاظ

اعمال کے دفتر کی مجھے خوب خبر تھی
بخشش کا وسیلہ ہی بنے نعت کے الفاظ

الفاظ کی پرواز کوئی جامی سے پوچھے
خود جا نہ سکے اُڑ کے گئے نعت کے الفاظ

بوصیریٔ بیمار شفا یاب ہوئے تھے
جب جانِ مسیحاؐ نے سُنے نعت کے الفاظ

اللہ کی رحمت نے کیا صاحبِ دیوان
عرفانؔ کی خواہش تھی کہے نعت کے الفاظ

Shrinking Civic Space in Pakistan: Response and Strategies of the Civil Society

This paper focuses the issue of shrinking civic space in Pakistan. The study presents civil society activists’ responses on this issue, highlights their perception, present and future strategies to address this issue, and their effectiveness, as such, as well. Pakistan presents a unique case due to its involvement in the ‘war on terror’ and the consequences being faced by both the state and society at large. Present shrinking of civil society action is directly linked with it. In an attempt to grapple with the problems emerging from ‘war on terror’, the overgrown state is encroaching upon citizens’ space, thus denying the civil society to play its due role which can provide safety for the state as well. The preferred strategies of the NGOs include lobbying with parliamentarians and mobilizing the citizenry about the situation.

Genetic Architecture of Some Insect Resistant Traits in Upland Cotton

Insect pest infestation is a major constraint in cotton production and causes heavy yield losses every year. Some studies report that insect pests attack on genotypes/cultivars with the traits, frego bracts, okra leaf, and red leaf color is comparatively lower. The genotypes/cultivars with these traits were planted and separated by fine netting to assess insect pest infestation in comparison to commercial cultivars. The Gossypium arboreum cultivar, FH-170 had minimum number of population buildup of sucking and chewing insect pests. Among hirsutum genotypes/cultivars, minimum population buildup of thrips, mites, spotted bollworm and American bollworm was found on Gumbo Okra. Minimum aphid was recorded on Russian Red. PBG-Fb-5 showed the most resistant response against pink bollworm. Population buildup of thrips, mites and aphid was relatively higher under water deficit conditions compared to well watered conditions whereas, population buildup of jassid, and white fly was higher under well watered conditions but was non-significant. Populations of parents, F1 and segregation pattern of F2 and backcross populations of crosses involving okra leaf, frego bract and red leaf traits showed that the traits were monogenic, okra leaf type and red leaf color being incompletely dominant and frego bract as recessive trait. Gene action of agronomic traits (plant height, number of bolls, number of monopodial branches, number of sympodial branches, boll weight, lint percentage, seed index, fiber fineness, fiber strength, and fiber length) in the crosses involving okra leaf, frego bract and red leaf color were different. Heritability estimates for different agronomic traits in the crosses of okra leaf, frego bract and red leaf color varied from 0.26 to 0.95. These results showed the interaction of the insect resistant traits with agronomic traits. Correlation of the traits, okra leaf, frego bract and red leaf color with agronomic traits showed that okra leaf positively correlated with sympodial branches, lint percentage, fiber fineness (measurement scale has inverse relationship with fiber fineness) and fiber length whereas, negative correlation of okra leaf was observed with plant height and seed index. Frego bract had negative correlation with boll weight and lint percentage. Red leaf color had positive association with lint percentage and negative correlation with number of bolls and sympodial branches and fiber strength. Negative correlation of these insect resistant traits with some desirable agronomic traits reveal that very large F2 population would be required to find insect resistant recombinants with desirable agronomic attributes. The correlations among agronomic traits in the crosses involving different insect resistant traits were different which suggest that while incorporating each insect resistant trait into a strain, a different breeding strategy will have to be followed. Simple qualitative nature of the traits, okra leaf, frego bract and red leaf color reveals that the traits may easily be incorporated into commercial cultivars.