Callus Induction And Molecular Cloning Of Phenylalanine Ammonia-lyase Gene Fragments In Asarum Heterotropoides

Asarum heterotropoieds was an important herbal medicine in northeast region while methyl eugenol was the main active ingredient. Currently Asarum heterotropoieds wild resources decreased dramatically, and it would take long time for seed reproduction, so tissue culture with high breeding speed and reproduction coefficient became the best way for seedling large-scale production. Then improving the active ingredient and increasing medicinal value was the another effective method to resolve the problem of asarum resource decreased.Effects of differrent explants, media types and plant growth regulators on callus induction of Asarum heterotropoides were studied by using the petioles and leaves as explants. Also we aimed to discover morphology characteristics of callus in different periods through paraffin method. Subsequently these callus could be differentiated to develop the regeneration plantlet. At the same time,CIoning and bioinformatics analysis of the key enzyme phenylalanine ammonia lyase which connect the primary metabolism and secondary metabolic pathways aimed to regulate and control the key enzymes to increase the yield of methyl eugenol, improve the utilization rate of the Asarum heterotropoieds.The results were as followed.1. The efficient callus induction medium was1/2MS+0.6mg·L-16-BA+0.15mg·L-1NAA while using petioles and leaves during young period as explants.Using70%ethanol solution sterilize30s then0.1%mercuric chloride solution sterilize8min for explant sterilization.2. Callus browning could be effectively prevented by adding2g·L-1activated carbon or1.5g·L-1PVP.3. Morphological characteristics of callus in different periods found that callus would be originated from the vascular tissue near the parenchyma cells of petiole and leaf. Meristematic cells became organized callus with nest-like structure4. Two phenylalanine ammonia-lyase gene fragments AhPALl and AhPAL2were cloned with882bp and879bp coding about294and293amino acids,respectively.5. AhPAL1、AhPAL2were the members of asarum PAL gene family by bioinformatics analysis. Blast results showed that AhPAL1and AhPAL2were similar in gene sequence with other species. Phylogenetic tree analysis revealed that PAL gene sequence between different genera differed but it was similar in the same genera. These results indicated that PAL had strong conservatism in the process of evolution of. AhPALl and AhPAL2were typical matrix proteins without transmembrane domain.