Analysis Of The Expressed Sequence Tag Of The CDNA Library From Macleaya Cordata Leaves And Recombinant Expression Of Defensins

Macleaya cordata belongs to the Papaveraceae family, is a traditional Chinese herbal medicine for antibacteria and antiinsect. Because M. cordata and other Papaveraceae family plants contain abundant alkaloids, the current research about Papaveraceae family plants mainly focuses on alkaloids. In order to promote the development and utilization of M. cordata, the elucidation of the molecular diversity of M. cordata is of great importance.The transcriptome of M. cordatahas been studied by our team. The data from leaf cDNA library expressed sequence tags(ESTs) and Illumine sequencing provide a soundly base for the molecular research of M. cordata. In present study, we analyzed the ESTs from the leaf cDNA libraryof M. cordata.Sequences from 511 high-quality ESTs were grouped as 78 clusters and 286 singlets. By searching against non-redundant protein sequences (nr) database of NCBI, and using Gene Ontology(GO), Cluster of Orthologous Groups (COGs), Kyoto Encyclopedia of Genes and Genomes (KEGG), the non-redundancy sequences were annotated and classified. Some deduced proteins from the ESTs were predicted to plant pathogenesis-related (PR) proteins (PR proteins). These proteins include chitinase, (S)-norcoclaurine synthase, major allergen protein, defensin, and non-specific lipid transfer protein, and belong to the PR-3 family, PR-10 family, PR-12 family and PR-14 family respectively. The results illustrate that M. cordata own not only alkaloids, but also large quantity of moleculars about plant disease-resistance. All the PR proteins were studied in the level of bioinformatics in present study. We focused on the defensins from M. cordata, and discovered 5 defensins, named McDefl, McDef2, McDef3, McDef4 and McDef5. Our analyses revealed the main classes of plant defensive peptides are represented by a/β-defensins and lipid-transfer proteins according to amino acid sequence homology. This study elucidated the transcriptome ESTs of M. cordata leaf, provided new insight into the antimicrobial components from M. cordata. The results present a promising application of M. cordata in agriculture, medicine and industry.Based on the deduced amino acid sequences of defensins we identified from M. cordata, a species of Papaveraceae family,31 novel defensins in Papaveraceae family are identified by searching on the all registered Expressed Sequence Tags (ESTs) from the whole family Papaveraceae in the NCBI database. The molecular weight, pI value, net charge, the average hydrophobicity, protein-binding Potential (Boman index), signal peptide, subcellular localization and tertiary structure of the novel identified defensins are predicted and analyzed. The properties of the difensins from Papaveraceae are summarized, and the genetic evolution is analyzed. This study provides a new perspective to study the antibacterial substances in Papaveraceae.To further study the characteristic of the defensins from M. cordata, we did the work of the recombinant expression of M. cordata defensins. We used the vector pGAPZa A and pPICZa A, and established constitutive and inducible eukaryotic expression system for the defensins; we also used the vectors pET-22b, pET-28a and pET-32a to establish prokaryotic expression system for the defensins. We obtained abundant fusion proteins of the defensins, by using vector pET-32a. We set up the basic methods for the recombinant expression of M. cirdatadefensins.