Cloning and Genes Expression Analyses of 2-Acetyl-1-Pyrroline Biosynthesis-Related Enzymes in Pandanus amaryllifolius

• Main Authors: Yi-Jyun Ke, 柯怡君
• Other Authors: Tzou-Chi Huang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/63061502583327533845

碩士 === 國立屏東科技大學 === 食品科學系 === 94 === Pandanus amaryllifolius Roxb. (Pandan) grows in Southeast Asia and is the only reported pandanus species with scented leaves. The aroma source is mainly 2-acetyl-1-pyrroline. The nitrogen source of pyrroline of 2-AP structure was proline. Proline metabolism may play a crucial role in 2-AP formation. On the other hand, methylglyoxal (MG) may derive from dihydroxyacetone phosphate (DHAP) was proposed to be responsible for 2-AP formation. This study probed into the formation mechanism of 2-AP in pandan under light or dark. In the preliminary experiment, the level of 2-AP was higher in pandan leave under light than under dark. The mRNA expression of the 2-AP synthesis related genes were analyzed with gene-specific primers by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) in pandan under light and dark. MG, P5C and 2-AP contents in pandan leaves under either light or dark were examined. The levels of expression of GapC mRNA under light were the lowest. GapC exists in the cytosol and is responsible for the metabolism of GAP. The lower activity of GapC in lighted pandan leave may be attributed to the accumulation of MG. The levels of both P5C and MG were higher in pandan leave under light than that in dark were found to be parallel to the 2-AP level. A formation mechanism of 2-AP from interation between P5C and MG was proposed. In addition, the key enzymes from pandan for MG biosynthesis, including the subunit A (GapA), B (GapB) and C (GapC) of GAPDH were cloned. Overexpression of GAPDH of the recombinant proteins exhibit a molecular mass of 43.3KDa, 34.3KDa and 36.7KDa, respectively. Specific activities of GapC of the recombinant proteins purified by Ni2+ affinity column were determined as 5.11 U/mg.