Physiology and Molecular Biology of Stress ... - KHAM PHA MOI

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A.K. Tyagi, S. Vij and N. Saini
The first large-insert library for raising transgenics was made for Arabidopsis.
The library was made in a BIBAC2, a plant-transformation-competent binary vector.
The library represents 11.5X coverage of the Arabidopsis genome and would greatly
aid gene identification through complementation screening (Chang et al., 2003). A largescale
gene inactivation through transgenic approach can be achieved by RNA mediated
gene silencing. As part of the European Consortia’s effort for Arabidospis functional
genomics, the AGRIKOLA (Arabidopsis Genomic RNAi Knock-Out Line Analysis)
has been set to make a collection of ~ 50,000 binary vectors for gene silencing using
RNAi for every Arabidopsis gene. The target will be to transform Arabidopsis with
around 5000 of these plasmids with the aim of targeted gene silencing; details of these
vectors can be obtained through www.agrikola.org (Hilson et al., 2003).
5. PROTEOMICS
The large-scale functional analysis of gene products, being expressed as proteins, is
called proteomics (Pandey and Mann, 2000). It involves identification, quantification,
activity and molecular interactions of all proteins from an organism. A proteome is a
total protein complement of a genome (Wasinger et al., 1995) and the analysis of a
functional proteome provides a powerful tool for linking gene expression to cell metabolism
and also to genetic maps (Burstin et al., 1993). Proteomics tells us what fraction
of the genome is functional and at what levels. Proteome analysis provides better
annotation of genome sequences by taking into account very small open reading frames
(ORFs) which are functional. It can also assign an unidentified ORF to a particular
protein product. Protein micro-characterization and their post-translational modifications
can be determined by proteome methodologies (Park, 2004).
Proteomics concept is based upon high quality separation of proteins. This
science of proteins dates back to late 1970’s with the advent of two-dimensional gel
electrophoresis (2DGE) (O’Farrell, 1975). The proteins are separated by 2DGE, estimated
using image analysis and then the mass spectrometer based identification of gel
separated proteins is carried out. Two-dimensional electrophoresis separates peptides
according to their molecular mass and isoelectric point. The 2DGE is the most powerful
technology in proteomics for separating a few thousands proteins simultaneously.
Proteomics approach is dependent on high purity and reproducibility. This approach
has been used to identify wound-response related proteins in rice (Shen et al., 2003).
Several computer softwares have been developed for the quantitative analysis
of complex 2D patterns but the major advancement in proteomics has been a combination
of 2D with mass spectrometry (MS) in 1990’s (Yates, 1998). MS technique has
replaced the sensitive methods of Edman’s protein microsequencing which has remained
time and cost intensive. MS is a high throughput technique making possible the
analysis of hundreds of proteins in short time and with picomole amounts of protein
(Burlingame et al., 1996; Blackstock and Mann, 2001; Mann et al., 2001; Mann and
Pandey, 2001). The basic principle is to generate ions from a source and analyze these