I= CLONING AND ANALYSIS OF A GERMIN-LIKE PROTEIN GENE(S) PROMOTER
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Title of Thesis
CLONING AND ANALYSIS OF A GERMIN-LIKE PROTEIN GENE(S) PROMOTER
Author(s)
Tariq Mahmood
Institute/University/Department Details
Department of Biochemistry/ University of Arid Agriculture Rawalpindi
Session
2007
Subject
Biochemistry
Number of Pages
130
Keywords (Extracted from title, table of contents and abstract of thesis)
germin-like protein, gene promoter, glp, plant microbe responses, osrglp2 promoter, agrobacterium, transgenic plants

Abstract
Germin and germin-like proteins (GLPs) are water soluble extra-cellular proteins reportedly expressed in response to some environmental and developmental signals. Some enzymatic activities have also been associated with germin/GLPs. However, their role in overall metabolism has not been fully understood. Significant insight into their function may also be gained by analysis of their promoter. A study was design to analyze the functional importance of a root expressed GLP gene promoter. During this study, about 1107 bp 5'region of OsRGLP2 gene was amplified, cloned, sequenced and was used for tobacco transformation. The sequence analysis by BLAST showed that this promoter sequence has five common regions (CRI-CR5) of different sizes (25-71 bp) which are repeated at 3-5 other locations in 30 Kb region in which this gene driven by its promoter is located. Interestingly all the genes driven by promoter harboring varying number of these' common regions are GLPs/putative germins. These common regions were analyzed and found to contain some important regulatory elements. Analysis of the 30 kb germin/GLP clustered region by GenScan detected each gene to have a putative 40 bp promoter containing TATA box and Dof factor which turned out to be a part of CR2. An attempt was also made to improve electroporation efficiency into EHA10l and LBA4404 strains of Agrobacterium by modifying the existing method for electroporation to improve electroporation efficiency. Multiple electric shocks ranging from 1-5 at a constant high voltage (20 KV/cm) for ~10ms with inter-pulse duration of -15 seconds was used and found to be more effective than increasing the voltage and/or time as later can cause decrease in cell viability resulting in subsequent low transformation efficiency. It was observed that both EHA10l and LBA4404 behaves equally well in response to multiple electric shocks. Cloned OsRGLP2 promoter region was ligated into p1391Z for expression vector construct preparation. The expression construct was transferred into ERA 101 by the modified method of electroporation. ERA101 harboring OsRGLP2 promoter construct was used for transformation of tobacco by leaf disks method. Transformed plants were analyzed under different stress conditions like temperature; dehydration, salt, and effect of plant growth regulator were also monitored. Transgenic plants were also used for the analysis of tissue specific activity of OsRGLP2 promoter. One of the striking and more important properties of OsRGLP2 promoter analyzed was the wound induced activity, which seems to increases with the passage of time. This is the first report about GLPs that they may have role in response to wounding. It was also observed that OsRGLP2 promoter is salt and dehydration inducible. Furthermore, temperature seems to have no effect on this promoter activity. Moreover, OsRGLP2 promoter is responsive to cytokinin, which is also a new property of a GLP promoter. In contrast to cytokinin, auxin has almost no effect on OsRGLP2 promoter induction. OsRGLP2 promoter activity was observed in inner and outer phloem of mid rib, at petiole stem junction, phloem, cortical cells adjacent to phloem, epidermal layer cells and epidermal hairs in stem, in young xylem cells, phloem cells and cortical cells adjacent to phloem in root, in petals veins while in sepals it was diffused and at the growing edges, no activity was observed in leaf mid rib, blade and apex of mature and young leaves. Microscopic analysis showed clear GUS expression in veins of leaf disks treated with salt stress while in all other treatments (temperature, dehydration, plant growth regulator) it was diffused. Conclusively, OsRGLP2 promoter seems to be a strong stress inducible promoter, capable of driving gene expression during wounding/mechanical stress, salt and dehydration.

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1610.2 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
184.24 KB
2 1 Introduction 1
47.02 KB
3 2 Review Of Literature 6
187.01 KB
  2.1 Origin Of Germin And GLPs 6
  2.2 Germin/Glps As Member Of Cupin Super Family 7
  2.3 Distribution 7
  2.4 In-Situ Localization 8
  2.5 Germin-Like Protein Are Expressed At Specific Development Stages In Plants 9
  2.6 Biochemical Properties And Structural Role 11
  2.7 Medicinal Importance 11
  2.8 Germin-Like Proteins Are Linked To Specific Plant Microbe Responses 13
  2.9 Sequence Based Phylogeny Of GLPs 18
  2.10 Mechanism Of Stress Tolerance By Glps 18
  2.11 Stress Inducted Expression 19
  2.12 GLP Promoter Analysis 21
4 3 Materials And Methods 23
149.94 KB
  3.1 Isolation Of Putative Promoter 23
  3.2 OSRGLP2 Promoter Construct Designing 28
  3.3 Standardization Of Electroporation Efficiency Into EHA101 And LBA4404 Strains Of Agrobacterium 30
  3.4 Plant Transformation 32
  3.5 Analysis Of Transgenic Plants 35
5 4 Results And Discussion 37
970.28 KB
  4.1 PCR Amplification 37
  4.2 Preparation Of OSRGP2 Construct 53
  4.3 Standardization Of Electroporation Efficiency In EHA101 And LB4404 By Multiple Electric Shocks 63
  4.4 Tobacco Transformation With Full Length Cloned Promoter Region 72
  4.5 Analysis Of Transgenic Plants 82
6 5 Summary 102
244.01 KB
  5.1 Literature Cited 105
  5.2 Appendices 126