Cloning And Expression Of5-Aarinoievuiinate Dehydratase Gene And Its Relationship To Garlic Greening

Garlic (Allium sativum L.) is a perennial herb of the Liliaceae Allium, which has highnutritional value, unique flavor, and a wide range of use. Greening often occurs during thefurther production processing, which seriously affects the appearance and quality of theproduct. It also results serious economic losses in garlic processing enterprises. At present,some researches used vitro chemical synthesis and physiological or biochemical methods toreveal the mechanism of garlic greening. However, these methods can not effectively explainthe biosynthetic pathways of garlic greening. The studies have shown that the garlic greeningpigment has pyrrole ring, but the source of pyrrolyl compounds is unclear. Therefore, it is ofgreat significance to elucidate the mechanism of greening by molecular methods studying thepyrrole compounds synthetase gene.5-aminolevulinic acid dehydratase generates pyrrolylcompounds PBG by catalyzing two molecules ALA. Using garlic bulb as the raw material, wesuccessfully cloned5-aminolevulinic acid dehydratase partical-length cDNA sequence fromPizhou White garlic. To better understand the relationship between the enzyme ALAD andgarlic greening at the molecular level, we compared the relationship among ALAD geneexpression, the enzyme activity, PBG content and the strength of garlic greening duringdifferent temperatures and with different varieties. The main results were as follows:1. Primers were designed based on the conserved amino acid sequences of differentplants in GenBank. We cloned partical5-aminolevulinic acid dehydratase gene from PizhouWhite garlic by RT-PCR and RACE, the partical sequence of ALAD was1251bp, encoding400amino acids.2. We used Pizhou White garlic and Cangshan Puke garlic as materials. At roomtemperature(23~25℃), we determined the ALAD activity and gene relative expression. Itshowed that: it had no obivous distinction in the ALAD gene expression, but it had adistinctive difference in ALAD activity between the two varities of garlic. The ALAD activityof Pizhou White garlic was higher than that of Cangshan Puke garlic. The lack of obviouschange in mRNA levels encoding the ALAD gene suggested that the regulation of ALADactivity likely occurred at the post-transcriptional level, either at the levels of translation orenzyme activation and existed varities’ difference. 3. We used Pizhou White garlic and Cangshan Puke garlic as raw materials. During thedifferent storage temperatures, we determined the ALAD activity, gene relative expression,PBG content and greening strength, and the results showed that: under the low temperature(4℃), there was a certain effect on theALAD gene expression. TheALAD gene expressionof Pizhou White garlic increased14.2-fold, while Cangshan Puke garlic increased1.9-fold.The ALAD activity had the same variation tendency with the content of porphobilinogen andstrength of garlic greening, so we suggested that ALAD which generated pyrrolyl compoundsPBG had a great relationship to garlic greening.