Araceae Lectins And Amaryllidaceae Lectin Gene Cloning And Insect

Developing crops which can resist insects or fungal diseases by genetic engineering is one of the current research focuses in plant biotechnology area. Some plant lectins have been successfully used in the genetic engineering to develop pest-resistant or fungal-resistant crops. Some plant lectins have been found to confer enhanced resistance to many insect pests belonging to different orders including homopteran insects. In order to identify more potential insect resistance genes, the full-length cDNAs of Arisaema heterophyllum agglutinin (AHA) and Lycoris radiata agglutinin (LRA) were cloned by RACE-PCR technique. Comparative analysis revealed that AHA and LRA had significant similarities at both the nucleotide level and amino acid level with lectins from other plant species of Araceae, Orchidaceae and Amaryllidaceae. The coding sequence of the mature Arisaema heterophyllum agglutinin was constructed into the bacterial expression vector pQE-30. The resulting recombinant vector was then transformed into the expression host strains M15. The recombinant protein was produced by the induction of IPTG and purified. Artificial diet assay by feeding aphids with artificial diet containing the purified AHA protein showed that AHA could inhibit aphids'survival and reproducibility.In order to test the potential application of aha in improving insect resistance of plants by genetic engineering, a plant expressing vector pBI121-AHA containing aha was constructed by substituting the report gene (gusA) in pBI121 with the coding sequence of aha. The recombinant vector was then introduced into Agrobacterium tumefaciens strain EHA105 with freeze-thaw method. The leaf discs of tobacco plants were transformed with A. tumefaciens EHA105 (pBI121-AHA). The regenerated kanamycin-resistant plants were tested by PCR, Southern blot and Northern blot for the presence and expression of the introduced aha gene. PCR and Southern blot analysis confirmed that the aha gene has been integrated into tobacco genome. Northern blot analysis revealed the aha expression in transgenic plants at various levels. The feeding assay showed that trangsgenic plants expressing aha had significant inhibition to the survival and reproducibility of aphids. The current study demonstrated that AHA could confer enhanced resistanceto homopteran pests such as aphids, The aha gene thus represents a promising candidate to be used in genetic engineering for enhanced insect resistance and may make a valuable contribution to crop protection.