Increasing lysine levels in pigeonpea (Cajanus cajan (L.) Millsp) seeds through genetic engineering

Abstract

In higher plants the essential amino acids lysine, threonine, methionine and isoleucine are synthesised through a branched pathway starting from aspartate. The key enzyme of lysine biosynthesis in this pathway-dihydrodipicolinate synthase (DHDPS)-is feedback-inhibited by lysine. The dhdps-r1 gene from a mutant Nicotiana sylvestris, which encodes a DHDPS enzyme insensitive to feedback inhibition, was used to improve the lysine content in pigeonpea seeds. The dhdpsr1 coding region driven by a phaseolin or an Arabidopsis 2S2 promoter was successfully overexpressed in the seeds of pigeonpea by using Agrobacterium transformation and particle bombardment. In 11 lines analysed, a 2-to 6-fold enhanced DHDPS activity in immature seeds at a late stage of maturation was found in comparison to wild type. The overexpression of dhdps-r1 led to an enhanced content of free lysine in the seeds of pigeonpea from 1.6 to 8.5 times compared with wild type. However, this was not reflected in an increase in total seed lysine content. This might be explained by a temporal discrepancy between maximal expression of dhdps-r1 and the rate of amino acid incorporation into storage proteins. Assays of the lysine degradative enzyme lysineketoglutarate reductase in these seeds showed no co-ordinated regulation of lysine biosynthesis and catabolism during seed maturation. All transgenic plants were fertile and produced morphologically normal seeds.

Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea

“…Between 100 and 500 mg of fresh leaf material was collected and amino acid composition determined as described in Thu et al. (2007).…”

Stacking Multiple Transgenes at a Selected Genomic Site via Repeated Recombinase-Mediated DNA Cassette Exchanges

“…Complex feedback controls on key enzymes in the pathway, such as dihydrodipicolinate synthase (DHPS) and cystathionine g-synthase (CGS), maintain a dynamic balance of the amino acids levels (Chiba et al, 1999;Falco et al, 1999;Falco, 2006). The overexpression of a feedbackinsensitive DHPS gene alone, or combined with the knockout of Lys catabolism key enzymes Lys ketoglutarate reductase/saccharopine dehydrogenase and the overexpression of a feedback-insensitive CGS gene, can dramatically increase the levels of free Lys or both Lys and Met Galili, 2003, 2004;Hacham et al, 2007;Thu et al, 2007). But the increased levels of free amino acids may not necessarily be stored unless enough sink, such as in the barley (Hordeum vulgare) high Lys (BHL8), a small storage protein engineered to contain high levels of Lys and Met, is provided to capture the increased free amino acids in seeds (Roesler and Rao, 2000).…”

Advances in Genetic Engineering of Primary Grain Legume Crops: a Review Study

“…The key enzyme of the lysine biosynthetic pathway, dihydrodipicolinate synthase (DHDPS), is inhibited by lysine through feedback regulation in pigeon pea. The transgenic pigeon pea with dhdpr-r 1 was developed for enhancement of lysine content in the seed protein [36,37]. Besides these, edible vaccine genes such as haemagglutinin gene of rinder pest virus and haemagglutinin neuraminidase gene of peste des petits ruminant's virus (PPRV-HN) were also integrated into pigeon pea to immunize goat and sheep to render pest virus and peste des petits ruminants virus, respectively [38,39].…”