Cloning And Expression Of A Lectin Gene From Narcissus Tazzetta Var. Chinensis

Insect pests do great harmfulness to agriculture. It had provided a significant effect by using pesticide to cope with pests, however, this means had also brought many problems such as pesticide residue, enviroment pollution and ecological imbalance. In recent years, a new established molecular biology method that clone and transfer toxic gene into crop plants provides a new option to control pests. Lectins from Amaryllidaceae family have excellent anti-insect ability, and snowdrop lectin, one of them, have been used in related breeding work.Lectin from Narcissus tazetta var.chinensis had been reported great inhabitation to many piercing-sucking insects. Therefore, it could provides valuble imformation to the breeding work on transgenetic anti-insect crops by clonig this gene and study its function.A lectin gene had been successfully cloned from Narcissus tazetta var.chinensis and then construced into an eukaryotic expression vector which had been transferred into Arabidopsis thaliana and tobacco; a prokaryotic expression vector had been constructed and got high level expression in E. Coli.. The major findings wer described as the following:1. A new lectin gene (NTL1) has been cloned from Narcissus tazetta var. chinensis'Jinzhanyintai'through RT-PCR technology. The results of sequence analysis indicated that the cDNA is 609 bp in length with an open reading frame(ORF) which is capable to encoding 172 amino acids; the protein precursor , with a molecular weight 18.7 kD, composed of a signal peptide which is 25 amino-acid residues in length and a mature protein which is 147 amino-acid residues in length. The homologous analysis showed that the identity between NTL1 and Narcissus hybrid cultivar lectin, Galanthus nivalis lectin and Lycoris radiata lectin was 83%, 81% and 77% respectively. Characteristic prediction results indicated that the secondary structure of NTL1 mainly includedα-helix, random coil and extended strand. Its three-dimensional structure prediction indicated that NTL1 is a mannose specific binding lectin which is strongly resembled the snowdrop's.2. The tissue-specific expression pattern was studied by RT-PCR method by using 18 S rRNA as the reference gene. The results revealed that the transcript of NTL1 was detectable in root, shoot, leaf and flower.3. The expression vector of pBI121-NTL1 with 35S sense orientation was construced through the process that add restriction sites directly. Transgentic A. thaliana and tobacco were generated via Agrobacterium tumefaciens mediated flower-dipping and leaf disk transformation methods. The transgenic offsprings have got an preliminary prove through anti-body selection and PCR check.4. A prokaryotic expression vector has been constructed by vector pET-23a and NTL1 sequence without the guide peptide. Under induction of IPTG, NTL1 got highly expression (more than 40% total protein) in E. coli. BL21 system in a similarity molecular weight to the predicted protein and formed inclusion body. Through these work, A solid foundation for further study of its charactors and functions has been established.