Avutu S. Reddy

Isolation of a Δ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp.strain PCC 7120

The enzyme Δ6-desaturase is responsible for the conversion of linoleic acid (18:2) to γ-linolenic acid (18:3γ). A cyanobacterial gene encoding Δ6-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking Δ6-desaturase. Expression of the Synechocystis Δ6-desaturase gene in Anabaena resulted in the accumulation of γ-linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis Δ6-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a Δ12-desaturase gene linked to the Δ6-desaturase gene. Expression of Synechocystis Δ6-and Δ12-desaturase in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and γ-linolenic acid.

A High Through-put Procedure for Capturing Microsatellites from Complex Plant Genomes

A method is outlined for large-scale isolation and characterization of microsatellite sequences from complex plant genomes. The method presented here differs from the previously published procedures in the use of randomly sheared (nebulized) genomic DNA for adapter-ligation, rigorous removal of biotinylated oligos, and high-density colony blots for constructing enriched libraries. Using this method we have constructed cotton microsatellite enriched libraries with over 20% (high stringency screening) or 75% (by random sequencing). Thus far we have identified and sequenced over 500 cotton microsatellites using this procedure. The procedure can be used to generate enriched SSR libraries from genomic DNA in about one week. High throughput screening and automated DNA sequencing can be accomplished in less than one month.

Sugarcane DIRIGENT and O-METHYLTRANSFERASE promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Ο-METHYLTRANSFERASE (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the β-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both ProDIR16:GUS and ProOMT:GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. ProDIR16 and ProOMT will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Production of γ-Linolenic Acid by Transgenic Plants Expressing Cyanobacterial or Plant △6-Desaturase Genes

△6-desaturase genes were isolated from cyanobacteria and Borago officinalis. Introduction of these genes into tobacco under the control of the cauliflower mosaic virus 35S promoter resulted in γ-linolenic acid (GLA) production in leaf lipids. The use of seed specific promoters also resulted in significant levels of GLA in seed lipids.