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Title of Thesis

Characterization of some Genes Related to the cotton(Gossypiam hirsutum L.) Fiber Quality

Author(s)

Hafiza Masooma Naseer Cheema

Institute/University/Department Details
Quaid-i-Azam University, Islamabad
Session
2010
Subject
Biotechnology
Number of Pages
212
Keywords (Extracted from title, table of contents and abstract of thesis)
Fiber, Strength, Utilization, Related, Cotton, Quality, Vector, Patterns, Characterization, Nucleotide, Genes, Gossypiam, Tissue, Analysis, Proteins

Abstract
EXPANSINs are the intrinsic proteins in a plant cell and are involved in disentangling the cellulosic microfibrils of the cell wall.The mechanism of EXPANSIN action is generally concerned with cellular expansion.This family of proteins has also been documented to have tissue specific members.Multiple variants of this protein have been identified in specific tissues, which are temporally regulated and functionally specific.The fiber specific EXPANSINs play a key role in the development of cotton fibers. Various isoforms of EXPANSINs were isolated by screening the cDNA libraries constructed at different developmental stages of cotton (Gossypium hirsutum) and Calotropis procera fibers.Nucleotide sequence analysis of the screened clones helped to identify two major variants in cotton (GhEXPA8 and GhEXPA15) and four EXPANSIN isoforms in fast elongating C. procera fibers.The C. procera CpEXPA3 was selected for further analysis on the basis of its close relatedness with cotton fiber EXPANSINs.The comparative analysis of these EXPANSINs with existing database of the gene family revealed that they belong to the third clade of EXPANSIN A family having two characteristic domains. Eight conserved cysteine residues were found in the N-terminal of the deduced amino acid sequence of GhEXPA8, GhEXPA15 and CpEXPA3, while one was in the signal peptide region.Five tryptophan residues were conserved in C-terminal region of these EXPANSINs.The Amino Acid sequences of GhEXPA8 and GhEXPA15 have 98 % identity, while they have 69.8 % and 69.6 % identity with CpEXPA3 respectively.The presence of the signal anchor sites, the hydrophobic regions and the transmembrane regions at the N-terminus suggested that these proteins are targeted to the cellulosic microfibrils through the secretory pathway.The expression of these variants in different tissues was quantified by real time PCR.The transcripts of GhEXPA8 and GhEXPA15 were observed only in fibers, while CpEXPA3 was found to be transcribed nonspecifically in all tissues of the respective plant.The different transcription patterns of GhEXPA8 and GhEXPA15 at various stages of fiber development indicated that they are functionally different genes.The real time PCR analysis indicated the presence of EXPANSIN variants in developing cotton fibers from 0-15 DPA.The RT-PCR demonstrated that transcripts of LTP3 gene could be detected in developing fibers from 0-20 DPA.A plant expression vector was constructed by fusing LTP3 promoter with a reporter gene (GUS with intron) for in vitro expression assay of the promoter strength and specificity in comparison with 2X35S.Transient expression studies on cultured cotton ovules, sepals, petals and stem revealed that the LTP3 promoter activity was confined only to the trichomes. The GUS gene in the expression cassettes was replaced with GhEXPA8, GhEXPA15 and CpEXPA3 generating six EXPANSIN genes constructs under the two promoters (2X35S and LTP3) with an aim to prolong the EXPANSIN gene expression in developing fibers.The construction of the expression cassettes was verified by DNA sequencing and the constructs were handed over to cotton transformation group at NIBGE for their utilization in fiber modification program.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 CONTENTS

 

 
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1

INTRODUCTION AND REVIEW OF LITERATURE

1.1 Introduction
1.2 History
1.3 Origin and Evolution
1.4 Cultivated Species of Cotton
1.5 Ecology
1.6 Cotton: A necessity of Humans
1.7 Cotton Plant
1.8 Cotton Fiber
1.9 Cotton Fiber Development
1.10 Cotton Fiber Physiology
1.11 Cotton Fiber: Model for Plant Cell Elongation and Cell Wall Biogenesis
1.12 In Vitro Fiber Growth (Ovule Culture)
1.13 Mechanism of Cotton Fiber Elongation
1.14 The Cotton Fiber Transcriptome
1.15 Cotton Fiber Specific Promoters
1.16 Objectives

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3 2 MATERIALS AND METHODS

2.1 Plant Growth
2.2 Fiber Developmental Studies
2.3 Construction of Fiber cDNA Libraries
2.4 Screening of cDNA Libraries for EXPANSINs
2.5 Molecular Analysis of the Sequencing Data using Bioinformatics Tools
2.6 Fiber Specific LTP3 Promoter Isolation
2.7 PCR Amplification of GUS with Intron
2.8 PCR Amplification of Full Length EXPANSIN Variants for Cloning in Plant Expression Vectors
2.9 Cloning of Cotton LTP3 Promoter
2.10 Cloning of GUS with Intron in pJIT166
2.11 Cloning of LTP3 Promoter in pGR1
2.12 Cloning of GhEXPA8, GhEXPA15 and CpEXPA3 in pGR5
2.13 Cloning of GhEXPA8, GhEXPA15 and CpEXPA3 in pGR1 under 2X35S Promoter
2.14 Cloning of Expression Cassette (LTP3 promoter EXPANSINs CaMV Terminator) in Binary Vector pGA482
2.15 Ovule Culture Establishment
2.16 LTP3 Promoter Specificity and Strength Evaluation
2.17 Real time PCR studies

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4 3 RESULTS

3.1 Cotton Boll and Fiber Developmental Stages
3.2 Calotropis procera Fruit and Fiber Developmental Studies
3.3 cDNA Libraries
3.4 Screening of cDNA Libraries for EXPANSIN
3.5 Molecular Analysis of the Sequencing Data Using Bioinformatics Tools
3.6 LTP3 Promoter Isolation from G. hirsutum
3.7 Construction of pGR1
3.8 Cloning of pGR5
3.9 Cloning of the Three Variants of EXPANSIN Gene in Derivatives of pJIT166
3.10 Cloning of Expression Cassettes Containing EXPANSIN Variants in pGA482
3.11 In Vitro Fiber Growth on Cotton Ovules
3.12 LTP3 Promoter Specificity and Strength Evaluation
3.13 Real Time PCR Studies

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5 4 DISCUSSION

 

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REFERENCES AND APPENDICES

 

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