I= IDENTIFICATION AND CLONING OF ANTIFUNGAL GENES
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Title of Thesis
IDENTIFICATION AND CLONING OF ANTIFUNGAL GENES

Author(s)
Shahina Bano
Institute/University/Department Details
National Centre of Excellence in Molecular Biology University of the Punjab, Lahore
Session
1998
Subject
Molecular Biology
Number of Pages
180
Keywords (Extracted from title, table of contents and abstract of thesis)
CLONING, ANTIFUNGAL GENES, pathogens, insect pests, Chickpea, Ascochyta isolates, chickpea blight, Bacillus subtilis, B. cereus, garlic, Alium sativum, Ascochyta Rabiei

Abstract
The application of molecular genetic approaches has revolutionised the study of plant pathogen interaction. Diseases caused by fungi pathogens and insect pests constitute a major problem in agriculture. Chickpea, an important food crop, is susceptible to attack by different species of fungal pests. According to the environmental situation in Pakistan, the most destructive chickpea disease is the blight caused by Ascochyta rabiei. This situation is also due to the scarcity or complete absence of genetic resistance in the available commercial cultivars. Therefore, efforts were made to utilise available sources for the development of genetic resistance in agronomically important crops against pathogenic fungi. Microsatellite fingerprinting and amplification techniques were used to produce and characterise a mapping population among different isolates of A. rabiei. Considerable genetic differences between Ascochyta isolates collected from different regions have been detected using oligonucleotide fingerprinting. It was also observed that all the tested motifs are obviously present in the genome of all A. rabiei however, their organization and relative abundance appeared to be somewhat different. Binary character matrix and two phenograms clearly differentiated A. rabiei isolates from other species of Ascochyta. The extent of polymorphism among all the A. rabiei isolates has revealed the most suitable probes. These probes can be used as molecular markers for the identification bf A. rabiei.

In order to find out antifungal protein/s or antifungal gene/s to control chickpea blight, different plant species and bacteria were tested for antifungal activity against A. rabiei. This activity was found to be present in garlic (Alium sativum) and in two bacterial species Bacillus subtilis and B. cereus. The antifungal compound with high fungicidal activity was isolated and identified ir as 'allicin' using different biochemical and spectrophotomytric techniques Another antifungal molecule was isolated and characterized from Bacillus cereus culture filtrate. This compound was characterized was an antibiotion (consisting of two polypeptide chains and one organic compound.

Besides the above basic research, efforts were also utilised to clone two known antifungal genes: chi26 (encoding chitinase) and bg/32 (encoding) β-1, 3-glucanase) in fusion protein expression vectors pGEX-2T and pGEX-5X2. Both genes were successfully cloned into pGEX vectors using E.coR1 cloning site. New clones were confirmed and studies were carried out to check the expression levels for both plant genes in E. coli for the use of biochemical and for other detailed analysis about the protein function and specificity. High levels of protein expression were observed in each case. Expression studies also revealed that the plant proteins have such structural configurations that facilitate cotranslational cleavage of the fusion peptide. These studies and the antifungal effect of these proteins against different fungal pathogens has revealed that these cloned genes can be used to develop genetically engineered crops like chickpea, rice and wheat to protect them from their fungal pathogens.

It was also concluded that new molecular markers could help in the identification of fungal pathogens in order to take necessary precautions before the spread of disease in crop fields. By using this technique, it would also be possible to check seeds and field soil to detect the, presence of pathogenic fungi before sowing. Moreover, biopesticidal formulations can be made by using garlic antifungal component and bacterial co pound to stop the fungal infection in chickpea growing fields. In addition, new antifungal genes can be transformed genetically into chickpea plants that would provide stable and maximum resistance against A. rabiei. Therefore, It is suggested that the present approach will delay or, perhaps in combination with other integrated pest management practices, prevent the damage caused by fungal pathogens to the food crops.

Download Full Thesis
1957.87 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
92.3 KB
2 1 General Introduction 1
41.41 KB
  1.1 Plants And Pathogens 1
  1.2 Disease Symptoms 2
  1.3 Molecular Basis Of Host Resistance 3
  1.4 Aims Of This Study 5
3 2 Historical Review 7
168.99 KB
  2.1 Ascochyta Rabiei 7
  2.2 Mechanism Of Fungal Virulence 11
  2.3 Mechanism Of Plant Resistance 14
  2.4 Bacterial Proteins/ Antibiotics 19
4 3 Characterization Of Ascochyta Rabiei 22
492.42 KB
  3.1 Summary 22
  3.2 Introduction 23
  3.3 Materials And Methods 26
  3.4 Results And Discussion 30
5 4 Detection, Purification And Characterization Of Antifungal Activity From Plants And Bacteria 39
638.43 KB
  4.1 Summary 39
  4.2 Introduction 41
  4.3 Materials And Methods 45
  4.4 Results And Discussion 52
6 5 Cloning And Expression Of Chitinase And ’-1 ,3 -Glucanse Antifungal Fusion Genes 60
281.29 KB
  5.1 Summary 60
  5.2 Introduction 62
  5.3 Materials And Methods 65
  5.4 Results And Discussion 72
7 6 General Discussion, Conclusion And Prospects 82
41.69 KB
8 7 References 87
181.54 KB