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Characterization, Cloning And Expression Of Phenylalanine Ammonia-lyase From Jatropha Curcas L.

Posted on:2008-12-16Degree:MasterType:Thesis
Country:ChinaCandidate:S W ZhangFull Text:PDF
GTID:2120360242963687Subject:Botany
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Phenylalanine ammonia-lyase (PAL) play a crucial role in phenylpropanoidmetabolism pathway providing precursors of various phenylpropanoid compoundsincluding anthocyanin. Here, we report the cloning and functional characterization ofa phenylalanine ammonia-lyase(PAL) gene, designated JcPAL (GenBank accessionno. DQ883805), from Jatropha curcas. The full-length JcPAL cDNA contains anopen reading frame of 2142bp encoding 713 amino acid. The putative protein shareshigh identity to other homologues, especially Manihot esculenta, same categoryplants. Putative JcPAL protein is about 78KD in molecular weight and with pI ofabout 6.59, composing a tetrameric protein with four same subunits. Partial genomicDNA of JcPAL was cloned and showed that transcript is divided into two exons byone intron. The 1334bp 5'upstream region of the gene encoding phenylalanineammonia-lyase was also isolated from Jatrapha curcas L. by the DNA walkingtechnology. Sequence analysis results revealed that the JcPAL promoter sequencecontains not only CAAT and TATA basic modifs that are conserved in the eukaryoticgene promoter, but also various stress related cis-acting elements. Especially, manycis-elements observed in other phenylalanine ammonia-lyase promoters are alsofound in the JcPAL promoter. For further studying on function of JcPAL promoter, four JcPALP fragments might be used to investigate their corresponding expressionpattern.The corresponding fusion proteins were expressed in Escherichia coli andexhibited PAL activity with phenylalanine as substrate. The fragment encodingJcPAL protein was inserted into a prokaryotic expression vector pET-28a and wasoverexpressed in E. coli BL21. The positive clones were identified by colony PCRand restriction digestion. After induced by IPTG, SDS-PAGE analysis showed thatthe JcPAL protein of 80KD was expressed. In order to increase the cytosolicexpression, we lowered the incubation temperature to 20℃for 20h. The fusionproteins were found partly in an soluble formation and its content was about 50%among total cell protein by Gene Genius Bio Imaging System. The soluble fusionproteins exhibited the PAL activity as other plants PAL purified from native planttissue, but a slight low.The JcPAL expression pattern and corresponding activity changing wereinvestigated after inoculation with ethephon, a ethylene-releasing compound, andinteractions with light and exposure to temperature. The results showed thatethephon play roles in the increase of PAL acitivity, but the change is not dependantin light: high temperature reduces the rise in ethephon-induced PAL enzyme activityby reducing the translation of ethephon-induced PAL mRNA, or by increasing theturnover of the induced PAL protein; PAL mRNAs accumulate and increase of PALacitivity in leaves of Jatropha curcas (L.) upon exposure to high temperature in aethephon-and light-dependent manner.
Keywords/Search Tags:Jatropha curcas L., phenylalanine ammonia-lyase, gene clone, expression in E coli ethephon, light, high-temperature
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