Brinjal or eggplant (Solanum melongena L.) is an important solanaceous crop belonging to tropical and sub-tropical regions. The name brinjal popular in Indian subcontinents is derived from Arabic and Sanskrit whereas the name eggplant has been derived from the fruit shape of some varieties, which resemble in shape to chicken eggs. It is also known as aubergine in Europe.
Figure 2.1: Brinjal crop
It is a versatile crop adapted to different agro-climatic regions and can be grown all over the year. It is a perennial but grown commercially as an annual crop. It is an autogamous diploid with 12 chromosomes (2n = 24).
The varieties of brinjal exhibit a wide range of fruit shapes and colours. The fruit shape ranges from oval or egg-shaped to long club-shaped, whereas the fruit color varies from white, yellow, green and through degrees of purple pigmentation to almost black. Fruit length is between 4-45 cm and thickness 2-35 cm and weight ranging between 15-1500 g. The fruits are set as single or in clusters of up to 5 fruits. Physiologically ripened fruits become brown, red or yellow (Swarup, 1995).
Kingdom Plantae (plant)
Subkingdom Tracheobionta (vascular plants)
Superdivision Spermatophyta (seed plants)
Division Magnoliophyta (flowering plants)
Class Magnoliopsida (dicotyledons)
Family Solanaceae (potato family)
Genus Solanum (nightshade)
Table 2.1: Taxonomy of Brinjal
Brinjal having botanical name Solanum melongena L. belongs to the family Solanaceae, which is a very large plant family containing 2300 species, nearly one-half of which are placed under a single genus, Solanum (D'Arcy, 1991),which is one of the ten most species-rich genera of flowering plants (Frodin, 2004).
There are three main botanical varieties under the species melongena (Choudhury, 1976). The ordinary brinjal which has big, round or egg-shaped fruits are grouped under var. esculentum, the elongated slender types are grouped under var. serpentinum and the dwarf brinjal plants are placed under var. depressum.
The brinjal crop originated in India (Gleddie et al., 1986) and is cultivated as a vegetable crop in India, Japan, Indonesia, China, Bulgaria, Italy, France, USA and many African Countries (Kalloo, 1993).
At present brinjal, after potato and tomato, is the third most important crop of Solanaceae family, with an annual worldwide production of 48.42 million tones and 18.5x105 hectares area under its cultivation (FAO data, 2012).
India produces 12.2 million tones of brinjal from 7x105 hectares area, which is equivalent to one quarter of the worldwide production, making India the second largest producer of brinjal in the world, after China (FAO data, 2012). The brinjal producing states in India are Orissa, Bihar, Karnataka, West Bengal, Andhra Pradesh, Maharashtra and Uttar Pradesh. West Bengal is the largest producer of brinjal followed by Maharashtra and Bihar.
2.1.4. Nutritional value and uses
Brinjal is a good source of vitamins and minerals, providing a nutritional value comparable to that of tomato (Kalloo, 1993). It is particularly rich in iron. It has low calories and fats, and contains mostly water, proteins, fibre and carbohydrates.
Calories 24.0 Sodium (mg) 3.0
Moisture content (%) 92.7 Copper (mg) 0.12
Carbohydrates (%) 4.0 Potassium (mg) 2.0
Protein (g) 1.4 Sulphur (mg) 44.0
Fat (g) 0.3 Chlorine (mg) 52.0
Fiber (g) 1.3 Vitamin A (I.U.) 124.0
Oxalic acid (mg) 18.0 Folic Acid (??g) 34.0
Calcium (mg) 18.0 Thiamine (mg) 0.04
Magnesium (mg) 15.0 Riboflavin (mg) 0.11
Phosphorus (mg) 47.0 B-carotene (??g) 0.74
Iron (mg) 0.38 Vitamin C (mg) 12.0
Zinc (mg) 0.22 Amino Acids 0.22
Table 2.2: Composition per 100 g of edible portion of brinjal (National Institute of Nutrition, 2007)
Brinjal is known to have ayurvedic medicinal properties and is good for diabetic patients. A brinjal based diet with its high fiber and low carbohydrate content has been suggested for management of Type 2 diabetes (Kwon et al., 2008). It has also been suggested as an outstanding remedy for those suffering from liver complaints (Shukla and Naik, 1993).
Brinjal is also useful in the treatment of asthma, bronchitis, and cholera, and it has been suggested that the consumption of its leaves is beneficial for lowering blood cholesterol (Khan, 1979). A study evaluated methanol extracts from the peels of brinjal against five human cancer cell lines and the results showed moderate to potent activities against the tested cancer cell lines signifying a dose dependent anticancer activity (Shabana et al., 2013).
The fundamental concepts of heredity and thus genes can be traced back to 1865 in the studies of Gregor Mendel (Orel, 1995). Gregor Mendel formulated a set of rules to describe the inheritance of biological characteristics. The basic assumption of these rules is that each heritable property of an organism is controlled by a factor, now called gene that is a physical particle present somewhere in the cell.
In 1903, Sutton proposed that genes reside on chromosomes. He also received experimental support for his idea from T.H. Morgan in 1910. However, it was in 1944 that Avery, MacLeod and McCarty and in 1952, Hershley and Chase through their experiments showed DNA to be the genetic material (Avery et al., 1944; Hershey and Chase, 1952). Until then it was assumed that the genes were made of proteins.
Therefore a gene can be defined as the entire DNA that encodes for the primary sequence of some final gene product, which can be either a polypeptide or RNA with a structural or catalytic function. The term gene is also used to describe an open reading frame (ORF). Most ORFs have the same general format i.e., they start with a particular triplet of DNA bases ATG, called the start codon and end at another triplet of DNA bases TGA, TTA or TAG, called the stop codons.
2.2.1. Eukaryotic gene structure
Eukaryotic genes have a characteristic structural feature with their nucleotide sequences containing one or more intervening segments of DNA that do not code for the amino acid sequence of its polypeptide product. These non-translated regions interrupt the otherwise collinear relationship between the nucleotide sequence of the gene and the amino acid sequence of the polypeptide it encodes. Such non-coding DNA segments in genes are called intervening sequences or introns, and the coding segments are called exons.
The expression of eukaryotic genes requires an additional step that does not occur for uninterrupted prokaryotic genes, RNA splicing. The DNA gives rise to a precursor RNA that exactly represents the genome sequence. Then the introns are removed from the precursor RNA to form a messenger RNA that consists only of the series of exons. In higher eukaryotes, most genes are interrupted and the introns are generally much longer than exons, creating genes much larger than their coding regions.
All the genes present in an organism are not expressed constitutively in all the tissues. Different genes are expressed at different stages of development of an organism depending on the role they play. Some genes are tissue specific and are expressed in a particular tissue type only, thereby determining the specific function of that tissue. The expression of individual genes also varies widely. Highly abundant proteins are produced from highly expressed genes, while other proteins that are present at a lower level are often produced from genes that are expressed at a very low level.
A promoter is the central processor of gene regulation, comprising the 5' region of the transcribed sequence and located upstream from the transcription start site (TSS) of a gene. It contains binding sites for the protein complexes of RNA polymerases that are essential for gene transcription (Griffiths et al., 2000; Perier et al., 1998). Each promoter contains characteristic sets of short conserved sequences that are recognized by the appropriate class of transcription factors.
2.3.1. Eukaryotic promoter structure
Structurally, a promoter can be divided into proximal and distal regions. The proximal region includes the region adjacent to the TSS and covers approximately -250 to +250 nucleotides (Butler and Kadonaga, 2002). The region of the DNA that is necessary to correctly guide the initiation of transcription by cell machinery is called the core promoter, which includes the TSS with -35 to +35 nucleotides (Butler and Kadonaga, 2002).. This region generally contains a conserved sequence which is located at 25'30 base pairs from the TSS, called the TATA box.
The proximal promoter elements, also called cis-elements, are located 100 bp (CCAAT-box) and 200 bp (GC-box) upstream of the TSS (Griffiths et al., 2000). Other elements such as the initiator (Inr), B recognition element (BRE) and downstream promoter element (DPE) are usually conserved. The DPE is located at approximately 30 nucleotides downstream of the TSS of many TATA box lacking promoters (Burke and Kadonaga, 1996) and thus acts in conjunction with TSS to provide a binding site for the transcription factor TFIID.
Figure 2.2: Common structure of a eukaryotic gene and transcription control regions. (Klug and Cummings, 1997)
TATA box, the first element identified in eukaryotes (Goldberg, 1979; Breathnach and Chambon, 1981), is the only element having a relatively fixed location in relation to the TSS. The consensus sequence consists of approximately 8 bp and is entirely composed of A and T. The TATA box is usually surrounded by sequences rich in G and C (Lewin, 2001). The Inr element, which is also present in the core promoter of some genes, generally covers the TSS (Juven-Gershon and Kadonaga, 2010). It is found in promoters irrespective of the presence of TATA box. The BRE is usually located upstream of some TATA boxes.
In some cases, the eukaryotic promoters do not act alone, and the transcription rates of a gene are considerably increased or decreased by elements that are located at a range of distances from the core promoter elements. These elements comprise the distal part of the promoter and regulate the spatial and temporal expressions of a gene, such that the proteins acting on these elements are united for expression to occur only at requisite sites and within a specific time frame. These are known as the activation (enhancer) and repressor (silencer) regions (Fessele et al., 2002; Riethoven, 2010; Kolovos et al., 2012).
The enhancers and silencers need not to have fixed positions in relation to the core promoter, and thus they can function in two directions (Bulger and Groudine, 2011). They are approximately 100'200 bp and may be situated hundreds or thousands of bp from the TSS, both upstream and downstream or even in the introns (He et al., 2010; Lodish et al., 2000). An enhancer needs to be identified along with its core promoter to validate its specificity and function in transcription.
2.3.2. Types of Promoters
Promoters are categorized based on their activity that promotes gene expression in all tissues. Some promoters have elements that determine the strength of transcription taking in account the tissue, physiological conditions, age, and biotic and abiotic factors. These promoters are guided by specific transcription factors (Fickett and Hatzigeorgiou, 1997; Hochheimer and Tjian, 2003).
184.108.40.206. Constitutive Promoters
Genes under the control of constitutive promoters are expressed in all the cells throughout the development, although their expression levels depend on the cell type (Park et al., 2010). In vegetal transgenesis, the Cauliflower mosaic virus promoter (CaMV35S) is vastly used as it is active in most tissues and throughout the developmental stages of plants (Park et al., 2012; Ranjan et al., 2011).
220.127.116.11. Tissue-specific Promoters
Genes under the control of tissue-specific promoters are expressed only in the cells of tissue for which the promoter is specific. Understanding the role of promoters acting in specific organs is important for understanding the molecular mechanisms involved in gene expression and tissue differentiation. The availability of such promoters facilitates the acquisition of constructions that allow the expression of target genes in specific tissues where these promoters are active (Ortiz, 1998; Nain et al., 2008). Several research studies have identified potentially novel promoters that are expressed in specific tissues only.
2.4. Plant fruit specific genes and promoters
The identification and isolation of fruit specific genes and promoters of plants is essential for the manipulation of nutritional value and agronomic quality of fruits and vegetables by genetic engineering.
The study of fruit development and ripening has received great attention due to their uniqueness as plant developmental processes and because of the significance that fruits have in the human diet. Widespread genetic and molecular analyses have provided significant information about genes participating in numerous aspects of fruit ripening, such as the cell wall disassembly, variation in soluble sugars, pigment biosynthesis, and the production of antioxidants, vitamins, flavour, and aromatic volatiles (Giovannoni, 2001). In addition to elucidating the biochemical pathways that determine fruit ripening, the alteration of gene expression offers the potential to improve fruit quality by altering biochemical pathways that contribute to flavor, color, size and shape of fruits.
A number of fruit-specific genes that are activated and expressed during ripening have been isolated from tomato and other fruits (Chen et al., 2004; Karaaslan and Hrazdina, 2010). The promoters of these fruit-specific genes are also of great interest as they can be used to manipulate fruit metabolism and produce valuable proteins such as anti- body, biopharmaceuticals, and edible vaccines through genetic engineering.
Meyer et al have isolated and characterized a fruit-specific bell pepper cDNA that codes for a J1-1 protein the leve1 of which increases significantly in the fully ripe fruit (Meyer et al., 1996).
In 2002, the role of phosphoenolpyruvate carboxylase (PEPCase) in organic acid accumulation and tomato fruit development was investigated and it was reported that S1PPC2 gene was strongly and specifically expressed in fruit from the end of cell division to ripening (Guillet et al., 2002). This indicated that in developing tomato fruit, PEPCase is possibly important in permitting the synthesis of organic acids to provide the turgor pressure needed for cell expansion.
It was recently shown that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene confers fruit-specificity in transgenic tomato. The SlPPC2 promoter also responds to hormones and metabolites regulating fruit growth and metabolism (Guillet et al., 2012). Thus, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruits.
An expansin gene (CsExp) from Cucumis sativus has been identified to be specifically expressed in ripened fruit. The CsExp promoter also contains elements responsible for its fruit specific expression. Clear fruit specificity was observed for CsExp promoter in all the experiments when transient expression studies were conducted (Sindhu et al., 2012). Thus CsExp promoter from Cucumber is a good ca-didate to target expression of the foreign expression of the foreign genes to engineer fruit specific traits.
Agius et al analysed the expression of the GalUR promoter from strawberry. This gene encodes a D-galacturonic acid reductase, which is an enzyme involved in the biosynthesis of vitamin C in strawberry fruits (Agius et al., 2003). It was found that the expression of GalUR is fruit-specific and increases during the ripening process to reach very high level in mature red fruits. An expression level similar to that of the constitutive CaMV 35S promoter was indicated from the analyses of the GalUR promoter. Therefore, the promoter region of GalUR has a clear biotechnological interest (Agius et al., 2004).
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