Expression of two insecticidal genes in cotton
Başlık çevirisi mevcut değil.
- Tez No: 402996
- Danışmanlar: DR. TAYYAB HUSNAIN
- Tez Türü: Doktora
- Konular: Mikrobiyoloji, Microbiology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2010
- Dil: İngilizce
- Üniversite: University of the Punjab
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 172
Özet
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Özet (Çeviri)
Agriculture serves as a jugular vein in the economy of Pakistan and a good chunk of the population in the country depends directly and indirectly on agriculture. It is the source of livelihood of almost 44.7 percent of the total employed labour force in the country. With the present contribution to GDP at 21.8 percent, agriculture sector is the mainstay of the rural economy around which socio-economic privileges and deprivation revolve. The importance of cotton can hardly be over emphasized in the economy of Pakistan. Pakistan is one of the ancient home of cultivated cotton, 5th largest producer of cotton, the 3rd largest exporter of raw cotton, 4th largest consumer of cotton, and a leading exporter of yarn in the world. Transgenic cotton expressing Bt (Bacillus thuringiensis) toxins is currently cultivated on a large commercial scale in many countries, but data has shown that it behaves variably in toxin efficacy against target insects under field and green house conditions. The present study was conducted to determine stable integration and spatio -temporal expression of two insecticidal genes (Cry1Ac and Cry2A) in advance lines of transgenic cotton variety CIM-482. The quantitative levels of these insecticidal genes were determined. Seasonal decline in expression differed significantly among different cotton lines tested in green house and field conditions., the aim of study was to quantify the expression of these insecticidal genes with the age of plants as well in different plant parts because the sustainable expression of Bt genes is crucial for its effectiveness to control the lepidopteron insects pest especially American bollworms. The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, anthers, petals and bolls. Expression of Bt genes decreased consistently with the age of plants. Compared with the temporal or spatial-specific expression of the toxin, constitutive expression of foreign proteins in transgenic plants may cause adverse effects, such as the metabolic burden imposed on plants for constant synthesis of foreign gene products, and these may increase the potential risk of resistance of the target insects to Bt. There is also concern about the food safety of genetically modified plants (Kuiper et al. 2001; Shelton et al. 2002; Conner et al. 2003). Therefore, in certain circumstances, it is desirable to use expression-specific promoters which only express the foreign gene in specific plant tissues or organs (Cai et al. 2007). Generally Genetically Modified (GM) crops contain the viral 35S CaMV promoter to drive insecticidal and herbicidal resistance genes. In cotton the reductions in the efficacy of Bacillus thuringiensis toxin (Bt) expressing plants has been attributed to reductions in promoter activity. Sustained expression of these endotoxins through out the life of cotton plant is possible by using a promoter with robust activity. For this purpose, a promoter from RuBisCo (Ribulose-1,5-bisphosphate carboxylase oxygenase) was isolated from Gossypium arboreum. RuBisCo is the bifunctional enzyme found in the chloroplasts of plants that catalyzes the initial carbon dioxide fixation step in the Calvin cycle and functions as an oxygenase in photorespiration. RuBisCo is the most abundant protein found in plant leaves, representing up to 50% of the soluble protein. These promoters and their transit peptides are attractive candidates for expression of genes at high levels. A construct was developed by isolating RbcS (RubisCo small subunit) promoter from Gossypium arboretum. This RbcS promoter was fused with Cry1Ac gene; followed by NOS terminator (construct was named as Rb- Ac in which Cry1Ac had been cloned under RbcS promoter). After the formation and confirmation of construct, a local cotton variety NIAB-846 was transformed with this construct using Agrobacterium tumefaciens strain LB4404. Same cotton variety was transformed with another construct pk2Ac harboring Cry1Ac under 35S promoter. Transformation efficiency remained x 0.20% in case of Rb-Ac construct while it was 0.15% in case of pk2Ac plants. The putative transgenic plants were screened using GUS assay. Furthermore, these transgenic plants were subjected to PCR and southern blot to confirm gene integration. The comparative study for insecticidal gene expression in Rb-Ac and pk2Ac plants showed that RbcS is efficient promoter to drive the expression of Cry1Ac gene consistent throught out the life of cotton plant as compared to 35S promoter. As promoter activity would be restricted to green tissues, there would no expression of Bt gene in soil and seed; hence no threat to soil organisms, environment and to users of cotton products and by products.
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