Cloning and Characterization of a Differentially Expressed Phenylalanine Ammonialyase Gene （liPAL） After Genome Duplication from Tetraploid Isatis indigotica Fort.

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase （PAL） catalyzes the first step in the biosynthesis of phenylpropanoids. In the present study, we isolated a novel phenylalanine ammonialyase gene （designated as liPAL） from tetraploid Isatis indigotica Fort. by rapid amplification of cDNA ends （RACE）, which was a cultivar from the diploid plant by genome duplication. The full-length cDNA of liPAL was 2 530-bp long with an open reading frame （ORF） of 2 178 bp encoding a polypeptide of 725 amino acid residues. Analysis of liPAL genomic DNA revealed that it was structurally similar to other plant PAL genes, with a single intron at a conserved position, and a long highly conserved second exon. Semi-quantitative RT-PCR revealed that the liPAL expression in roots and leaves from a tetraploid sample was higher than that in diploid progenitor, whereas expression of liPAL in stems was almost the same as each other. Furthermore, the highest expression of liPAL in tetraploid plant was found in roots, which was found in stems in diploid plants. Further expression analysis revealed that gibberelUn （GA3）, abscisic acid （ABA）, methyl jasmonate （MeJA） and cold treatments could up-regulate the liPAL transcription in tetraploid plants. All our findings suggest that liPAL participates not only in the defense/stress responsive pathways, but also probably in the polyploidy evolution of L indigotica.

Synthesis of Self-assemble pH-responsive Cyclodextrin Block Copolymer for Sustained Anticancer Drug Delivery

Well-defined pH-responsive poly（e-caprolactone）-graft-β-cyclodextrin-graft-poly（2-（dimethylamino）ethyl- methacrylate）-co-poly（ethylene glycol） methacrylate amphiphilic copolymers （PCL-g-β-CD-g-P（DMAEMA-co-PEGMA）） were synthesized using a combination of atom transfer radical polymerization （ATRP）, ring opening polymerization （ROP） and ＂click＂ chemistry. Successful synthesis of polymers was confirmed by Fourier transform infrared spectroscopy （FTIR）, proton nuclear magnetic resonance （^1H-NMR）, and gel permeation chromatography （GPC）. Then, the polymers could self- assemble into micelles in aqueous solution, which was demonstrated by dynamic light scattering （DLS） and transmission electron microscopy （TEM）. The pH-responsive self-assembly behavior of these copolymers in water was investigated at different pH values of 7.4 and 5.0 for controlled doxorubicin （DOX） release, and these results revealed that the release rate of DOX could be effectively controlled by altering the pH, and the release of drug loading efficiency （DLE） was up to 88% （W/W）. CCK-8 assays showed that the copolymers had low toxicity and possessed good biodegradability and biocompatibility, whereas the DOX-loaded micelles remained with high cytotoxicity for HeLa cells. Moreover, confocal laser scanning microscopy （CLSM） images revealed that polymeric micelles could actively target the tumor site and the efficient intracellular DOX release from polymeric micelles toward the tumor cells further confirmed the anti-tumor effect. The DOX-loaded micelles could easily enter the cells and produce the desired pharmacological action and minimize the side effect of free DOX. These results successfully indicated that pH-responsive polymeric micelles could be potential hydrophobic drug delivery carriers for cancer targeting therapy with sustained release.