Molecular Cloning Of Zephyranthes Candida(Lindl.)Herb Agglutinin Gene(zca), Structure Analysis, Vectors Construction And Transformation

Homoptera, the very important groups of insects that suck the phloem of plants (including aphids and planthoppers) have proved difficult to control by conventional plant breeding, a matter made worse by their importance as vectors of plant viruses, up to now, most of the lectin genes that show more or less inhibition to sap-sucking insects including aphids and planthopper by artificial diet assays were cloned from species of Amaryllidaceae, among which the lectin (GNA) from snowdrop (Galanthus nivalis) is the most toxic and has been most widely used in plant insect-resistant breeding. Transgenic plants expressing GNA showed significant insecticidal activity towards homoptera insects such as aphids and brown planthopper in bioassay and feeding tests. As Zephyranthes Candida belongs to Amaryllidaceae, it is speculated that Zephyranthes Candida lectin also has similar inhibitory effect on sap-sucking insects like other lectins from Amaryllidaceae and may play a role in controlling sap-sucking insects by genetic engineering.In this paper a lectin gene was firstly isolated from leaves of Zephyranthes Candida by useing rapid amplification of cDNA ends (RACE) technique. Characterization, functional and advanced structure prediction, construction of plant expression vector and transformation of this novel gene were studied. The analyzing results of the geneare very helpful to functional expression and potential application in insect resistance breeding. The major experiment results from this work were as follows:1. Primers were designed based on conserved sequences of other lectin genes from Amaryllidaceae. A novel lectin gene was isolated from Zephyrathes Candida. The full-length cDNA of Zephyrathes Candida lectin was 647bp, containing an open reading frame (477bp) encoding a peptide of 159 amino acids. (China patent public No. 1323887).2. The signal cleavage site prediction, advanced structure prediction, blocks' analysis and sugar-binding sites were analyzed. Its deduced protein (ZCA) showed 69% similarity to GNA, and its cDNA sequence showed 76% similarity to gna ,which inferred that it possessed similar function to GNA. at the same time, cDNA sequence and protein of zca were campared with another species belonging to the same genus , it showed that zca was very similar to zga.3. Based on the cloning of Zephyrathes Candida agglutinin gene, two plant expression vector p2301-zca and p!305-zca was successfully constructed and introduced into Agrobacterium tumefaciens ERA 105.4. The plasmid p2301-zca carrying zca was transferred into wild type tobacco via Agrobacrium-mediaed by using leave disk, PCR and Western blot confirmed that transgenic tobacco were obtained, further bioassay is undergoing .5. Mature embryo derived callus elite from japonica rice cv. EY1 105 were used for transformation, via Agrobacriue-mediating p1305-zca carrying zca were transferred into rice, preliminary results by PCR showed 6 transgenic rice plants out of 15 that had been tested were obtained, further study is undergoing.