Cloning And Expression Of The Gene Encoding A Lysine Decarboxylase And Activity Of The Enzyme In Detoxifying Organic Acid Toxin Produced By Xanthomonas Oryzae Pv.oryzae And A Preliminary Study On Detoxification Of Oxalate Toxin By A Bacterial Strain

As an important pathogenic factor, phytopathogens-produced toxins play an important role in the occurrence and development of plant diseases. Detoxification of these toxins is one of biological control strategies for controlling plant diseases. Organic acids, small molecular metabolites produced by phytopathogens, are one common class of these pathogenic toxins. In this paper, I studied the activities of some bacteria and enzymes to detoxify the organic acid toxins from Xanthomonas oryzae pv.é­Šyzae(the pathogenic bacteria of the rice leaf blight) and the oxalic acid from many pathogenic fungi, and evaluated their application potentialities in biological control of plant diseases in which organic acids are involved in pathogenesis. The previous studies in our laboratory indicated that Xanthomonas oryzae pv.oryzae not only produces the organic acid toxin which can cause the death of tobacco leaves, but also produces an organic acid- detoxifying protein that has decarboxylase activity. Based on these findings, I set out to test the organic acid-degrading activities of five commercial decarboxylases. The enzymes are lysine decarboxylase, argine decarboxylase, oxaloacetic decarboxylase, glutamate decarboxylase, fumarase, and their gene sequences are available in GenBank. All the five decarboxylases were separately incubated with the organic acid toxin from JXOIII for a certain time under optimum conditions. Then the reaction mixtures were injected into tobacco 104 leaves, and the death of the treated tobacco leaves were observed two weeks later. The results showed that lysine decarboxylase has significant organic acid-detoxifying activities. Oxaloacetic decarboxylae has some detoxification activities (but unstable). Other three decarboxylases have no obvious activities. In order to study application value of detoxifying enzyme, an engineered bacterium that produces lysine decarboxylase was constructed. By PCR technique, 2.2kb DNA fragment that encodes lysine decarboxylase was amplified using wild-type Hafnia aivei DNA as templates. The DNA fragment was cloned into an expression vector, pTrc99A, to construct recombinant plasmids. The recombinant plasmids were used to transform E. coil HB1O1, and 8 recombinant clones were obtained from 156 transformants. Further screening characterized 1 positive clone from the 8 recombinant clones and named as HB1O1/ pLD151. SDS-PAGE analysis indicated that the expressed lysine decarboxylase in HBIO1/ pLD151 accounted for 16.5 % of the total proteins. Biochemical analysis showed that lysine decarboxylase activity of HB1O1/ pLD151 is 10 times of that of the parental strain. Bioassay indicated that HB1O1/ pLD151 is able to cancel the death of tobacco leaf and the inhibition of rice seedling growth induced by the toxin of Xanthomonas oryzae pv.oryza. These results proved that the lysine decarboxylase is able to detoxify the toxin of Xanthomonas oryzae pv.oryza at the molecular genetics level. The molecular weight of the expressed recombinant lysine decarboxylase is about 53 kd, much smaller than the expected 83 kd. Sequence analysis showed that this is because the expression of lysine decarboxylase gene in the positive clone terminates at 1422 bp. A N- terminal 1.4kb fragment of lysine decarboxylase gene was cloned, expressed and functionally analyzed. The results confirmed that the N- terminal 1.4kb of lysine decarboxylase gene have complete gene...