Expression In Pichia Pastoris And Genetic Transformation Of TomloxD Gene From Tomato

Lipoxygenases (linoleate: oxygen oxidoreductase, EC 1.13.11.12; LOXs) are monomeric non-heme, non-sulfur iron dioxygenases, which catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids containing a cis, cis-1, 4-pentadiene moiety, such as linoleic acid(LA),α-linolenic acid(LeA), arachidonic acid,(AA). The function of various LOXs in plants has been implicated include wounding, pathogen attack, seed germination, fruit ripening, plant senescence, and synthesis of Abscisic acid (ABA) and Jasmonic acid (JA).There are five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD and TomloxE) present in tomato. TomloxA, TomloxB and TomloxE are 72% to 77% identical with each other at the amino acid level, while TomloxC and TomloxD show 42% and 47% identity, respectively, to the TomloxA protein, and 46% identity to each other. TomloxA, TomloxB and TomloxE are expressed at fruit. TomloxC is expressed during fruid ripening and also in leaf and flower, whereas TomloxD is mainly expressed at leaf. TomloxB and TomloxC expression is enhanced by the ripening hormone ethylene, whereas TomloxA expression declines. TomloxC and TomloxD contain chloroplast-targeting signals and are imported into chloroplasts in vitro. These results indicated that multiple isomers of LOX were regulated by different ways and have different functions in the growth and development of tomato. Nowadays, the fouction of TomloxD gene was not clear. To furtherly clarited the function of TomloxD gene in plant defense response, as follows:1. The full length of TomloxD gene, which was obtained from the plasmid pBluescript TomloxD by PCR, was inserted into the plasmid pDH5.1. and was then inserted into the plasmid pBINl9 that digested by EcoR I to construct the over-expression vector pBINl9-TomloxD. The conserved fragment of TomloxD, which was length of 290 bp, was inserted into the middle plasmid pDH5.2. The aim fragment was excised from pDH52-TomloxD by EcoR I, and was then inserted into the plasmid pBINl9 as above.2. The pBINl9-TomloxD and pBINl9-TomloxDi vector was transformed into Agrobactenum tumefacious strain LBA4404. Transformed tomato plants was obtained. Finally, we select the positive tomatos by PCR of NPTⅡgene.3. The tomato-specific gene expression of Actin as control, Semi-quantitative RT-PCR analysis showed that the the TomloxD gene was mainly expressed in young leaf, mature leaf and root, and nearly has no expression in fruits. The expression of the TomloxD gene was induced by wounding, and reached the sumit at 2 h.4. After AC++ tomato leaves were treated 2 h by wounding respose, crude enzymes were extracted and enzymes activity was determined. The result showed that activity of wounding treatment tomato leaf was higher than the control.5. The TomloxD-pPIC9K expression vector was constructed, which expressed the protein of TomloxD in Pichia pastoris GS115, lipoxygenase activity analysis also demonstrated that the TomloxD expressed protein has lipoxygenase activity.