Isolation of novel genes is primarily the foremost part of the molecular biology to understand molecular control of fundamental plant processes that will facilitate their modification and use in crop improvement through genetic engineering technologies. In plants, the genes which govern the genetic basis of development and stress-response will provide opportunities to optimize agricultural productivity in terms of increased yield, decreased negative environmental consequences and reduced ecological repercussions. Gene isolation can be followed by two broad strategies, the reverse genetics (from gene to phenotype) and the forward genetics (from phenotype to gene). Some of the commonly used approaches under these two broad strategies include screening of genomic/cDNA libraries, differential expression profiling, functional complementation, insertional mutagenesis and positional cloning of the genes.
(a) Genomic/cDNA library screening
Genomic library is made by cloning genomic DNA fragments obtained by either restriction digestion or mechanical shearing of the genomic DNA. The library represents the genome regardless of the cell type or developmental stage from which the DNA is isolated. The cDNA (complementary DNA) libraries are generated by cloning DNA obtained by reverse transcribing the cellular mRNA. It thus represents the genes that are transcribed in a tissue from which mRNAs are derived. Plant genes are isolated from genomic and cDNA libraries by screening the clones with an appropriate hybridization probe of known sequence from a heterologous system or through differential hybridization technique. Besides, variations on normal hybridization-based library screening protocol include subtracted cDNA libraries preparations, which are enriched with differentially expressed cDNA clones. A recent development in differential screening has come in the form of cDNA array, suppression subtractive hybridization (SSH) and representational difference analysis (RDA). In cDNA microarrays, cDNA clones are transferred to a miniature solid support in a dense grid pattern (cDNA chip), and screened simultaneously with complex probes from two sources, which are labeled with two different fluorochromes.
(b) Differential display
Identification of differentially expressed genes in various cells or under diverse stress situations are one of the core areas of molecular biology research. Liang and Pardee (1992) developed a new PCR-based technique called Differential Display (DDRTPCR). The method is based on detection of the differentially expressed cDNAs from two or more samples that are separated on adjacent lanes in sequencing gels. The differentially expressed bands can readily be cloned and used as probes in northern or southern (DNA) blots to isolate the genes from cDNA or genomic libraries. A number of technical modifications have been made to reduce the problem of false positives and to increase the reproducibility of the technique (Zhao et al. 1995, Huang et al. 1996, Jones et al. 1997, Doss 1996). Modifications that allowed the display of longer cDNAs have also been reported (Averboukh et al. 1996). Therefore, with properly designed primers and controls, DDRT-PCR results will truly reflect the pattern of gene expression in different tissues.
(c) Functional cloning or functional complementation
The alternative approach in isolating plant genes involves the exploitation of the biochemical or physiological activity of the gene product through production and analysis of mutants generated by ultraviolet light, ethyl methane sulphonate and X-rays. However, in plants, this powerful technique has had only limited success and complementation has been reported only for genes that had previously been characterized. For example, complementation of a flower pigment mutation of Petunia hybrida with a maize gene previously cloned. In this case, the maize gene was able to catalyze the conversion of an intermediate metabolite in anthocyanin biosynthesis which accumulated in the mutant plant thus changing the flower color of the plant from white to brick red (Meyer et al. 1987).
(d) Insertional mutagenesis
The use of insertional mutagenesis in principle provides a more rapid way to clone a mutated gene. DNA elements such as transposons or the T-DNA of Agrobacterium tumefacians (Azpiroz-Leehan and Feldmann 1997) that are able to insert in random manner within chromosomes, can be used as mutagens to create loss of function mutations in plants. This approach has the additional advantage that an altered phenotype might provide clues to the function of the product of the gene in question as well as facilitating its isolation. In this tagging approach, first the mutation caused by the insertion sequence has to be identified through phenotypic screening. Second, co-segregation of the insertion sequence with the mutant phenotype has to be verified. Subsequently, the gene can be isolated molecularly by cloning the DNA sequences flanking the insertion element.
Map-based/positional gene cloning
This is another forward genetics approach of the gene isolation where markers on the genetic map are used for gene cloning. The identification of a gene affected by natural variation or through chemically or radiation-induced mutation requires map-based cloning (MBC) approach in which markers linked to the mutated gene are used to define the region containing the gene of interest. The rapid advances in sequencing projects, the abundance of various marker systems and the progress made in methods to detect DNA polymorphisms facilitate fast map-based cloning (MBC) of genes, although this is presently true only for a few of the plant species where sequence information and markers are available such as Arabidopsis and rice. For example, in case of rice, six bacterial blight resistance genes against Xantomonas oryzae pv. oryzae, Xa1, xa5, xa13, Xa21, Xa26 and Xa27 (Chu et al. 2006, Iyer and McCouch 2004, Sun et al. 2004, Gu et al. 2005) have been successfully cloned by using the map base cloning approach. With the recent advancement in genome sequencing projects, we will have more sequence and marker availability in future which will facilitate gene cloning by this method in other plant species.
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