Post-transcriptional gene silencing: Basic concepts and applications.

Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism wherein transcripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will be subjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance and cross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might have evolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA) molecules of 21-23 nucleotides length act as homology guides for triggering the systemic degradation of transcripts homologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouring chalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has been achieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of gene silencing has led to the development of several alternative strategies for inducing gene silencing in a precise and controlled way. This has paved the way for using PTGS as one ofthe chief functional genomicstools in plants and has helped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides, opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencing the deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in a new era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined the basics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, as case studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenic male sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress the production of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistance in sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify the concept and utility of gene silencing in crop plants.

Cloning, characterization, and expression of a new cry1Ab gene from DOR Bt-1, an indigenous isolate of Bacillus thuringiensis.

A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.