RESUMEN
MAIN CONCLUSION: Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Syn vip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Syn vip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (syn bar) gene. Marker gene excision was achieved in hybrids with up to 41.18 % recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII + cre-syn bar), were established in subsequent generation through genetic segregation.
Asunto(s)
Proteínas Bacterianas/genética , Resistencia a los Insecticidas/genética , Oryza/genética , Enfermedades de las Plantas/genética , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Western Blotting , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos , Control de Insectos/métodos , Insectos/fisiología , Insecticidas/metabolismo , Oryza/parasitología , Enfermedades de las Plantas/parasitología , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribulosa-Bifosfato Carboxilasa/genéticaRESUMEN
Grass pea (Lathyrus sativus L.) is a worldwide popular pulse crop especially for its protein rich seeds with least production cost. However, the use of the crop became controversial due to the presence of non-protein amino acid, ß-N-oxalyl-L-α, ß-diaminopropionic acid (ß-ODAP) in its seed and leaf, which is known as the principle neurotoxin to cause neurolathyrism (a motor neurodegenerative disease of humans and animals) during prolonged consumption as regular diet. Till date, the knowledge on ß-ODAP biosynthesis in Lathyrus sp. is limited only to a small part of the complex bio-chemical steps involved including a few known sulfur-containing enzymes (viz. cysteine synthase, ODAP synthase etc.). In Lathyrus sativus, biosynthesis of ß-ODAP varies differentially in a tissue-specific manner as well as in response to several environmental stresses viz. zinc deficiency, iron over-exposure, moisture stress etc. In the present study, a novel cysteine synthase gene (LsCSase) from Lathyrus sativus L was identified and characterized through bioinformatics approaches. The bioinformatic analysis revealed that LsCSase showed maximum similarity with the O-acetyl serine (thiol) lyase of Medicago truncatula with respect to several significant sequence-specific conserved motifs (cysK, CBS like, ADH_zinc_N, PALP), sub-cellular localization (chloroplast or cytoplasm) etc., similar to other members of cysteine synthase protein family. Moreover, the tissue-specific regulation of the LsCSase as well as its transcriptional activation under certain previously reported stressed conditions (low Zn+2-high Fe+2, PEG induced osmotic stress) were also documented through quantitative real-time PCR analyses, suggesting a possible link between the LsCSase gene activation and ß-ODAP biosynthesis to manage external stresses in grass pea. This preliminary study offers a probable way towards the development of less toxic consumer-safe grass pea by down-regulation or deactivation of such gene/s (cysteine synthase) through genetic manipulations.