Cloning and Characterization of the DHDPS Gene Encoding the Lysine Biosynthetic Enzyme Dihydrodipocolinate Synthase from Zizania latifolia (Griseb)

Abstract

Dihydrodipicolinate synthase (DHDPS) is the main enzyme of a specific branch of the aspartate pathway leading to lysine biosynthesis in higher plants. We have cloned and characterized the DHDPS gene from Zizania latifolia Griseb, which was named ZlDHDPS. Sequence analysis indicates that it contains an ORF of 954 bp interrupted by two exons and one intron encoding a polypeptide of 317 amino acids lacking a plastid transit peptide and a stop codon. The sequence of ZlDHDPS has high identity with known plant DHDPS in GenBank. Southern blotting analysis indicates that there are two copies of Z. latifolia DHDPS (ZlDHDPS) gene in the Z. latifolia nuclear genome. RT-PCR analysis shows the expression of ZlDHDPS is tissue specific and high level expression is present in fast-growing tissue and reproductive tissue. The 5′-regulatory sequence of ZlDHDPS contains a GT-1 box and a (CA)n element, which may play a role in regulating the expression of ZlDHDPS. The fusion construct of the ZlDHDPS sequence with the reporter gene GUS was transiently expressed in the onion epidermal cells by particle gun-mediated bombardment suggesting that ZlDHDPS was located in the cytoplasm, different from DHDPS gene of other species. Functional complementary analysis showed that ZlDHDPS can recover the DHDPS-deleted mutant of Escherichia coli.

Characterization of the LhcSR Gene Under Light and Temperature Stress in the Green Alga Ulva linza

“…The cDNA used for the 5′ and 3′ RACE was synthesized using a SMART RACE cDNA kit (Clontech Laboratories, Inc., Mountain View, CA, USA). The cDNA used for real-time PCR was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase (Promega Biotech Co., Madison, WI, USA; Kong et al 2009;Carzoli et al 2009). …”

Improving Nutritional Quality of Plant Proteins Through Genetic Engineering

“…Pyruvate and aspartate semi-aldehyde are catalyzed to 4-hydroxy-2,3,4,5-tetrahydrodipicolinate by DHDPS. Recombinant DHDPS from several plant species were characterized and the enzyme was found to be extremely sensitive to feedback inhibition by Lys even at very low levels [ 36 , 37 ]. Seed-specific overexpression of feedback-insensitive bacterial DHDPS in various plant species was shown to increase accumulation of Lys in seeds [ 36 , 38 ].…”

Short-term retention of kleptoplasty from a green alga (Bryopsis) in the sea slug Placida sp. YS001

“…The total RNA was subsequently extracted using Trizol Reagent according to the user's manual, after which it was dissolved in diethylpyrocarbonate-treated water. The cDNA used for PCR was synthesized using M-MLV reverse transcriptase and oligo d(T)18 (Promega Biotech Co., Madison, WI, USA; Kong et al 2009;Carzoli et al 2009). …”