Cloning And Functional Confirmation Of Lectin Genes From Pinellia Ternata

Commonly and frequently occurring aphids (Lipaphiserysimi) damage causes serious losses to agricultural production. With the development of plant transgenic technology, the gene engineering for resistance to aphids has become an effective way to prevent the insect damage in crop. In the paper, the five new lectin genes, named PPA-2, PPA-3, PPA-4, PPA-5 and PPA-6, was cloned from young leaf of Pinella pedatisecta using PCR method. According to bioinformatic analysis, the five lectin polypeptides encoded by the cloned genes contained one or two conserved domains, respectively, and each domain has a mannose-binding site (QXDXNXVXY). Then the five lectin genes were connected with constitutive promoter CaMV35S and Arabidopsis phloem protein-specific promoter (Atpp2) and ten plant expression vectors were constructed. In all,77 transgenic tobacco plants containing the five lectin genes via Agrobacterium-mediated leaf disc transformation of W38,45 plants with CaMV35S promoter and 32 with Atpp2 promoter, were confirmed by PCR analysis, YFP green fluorescent protein detection and Western-blot identification.The resistance of the above obtained transgenic tobacco plants to aphid were carried out with biological assay. Among the transgenic tobacco plants containing CaMV35S promoter, the highest efficiency of aphid resistance with PPA-5 gene reached 97.8%; followed by PPA-6 gene which efficiency was 97.4% and the lowest with PPA-2 gene was 81.4%. The resistance to aphid was also analyzed with high, medium and low exogenous protein expression transgenic tobacco plants according to Western-blot experiments. The preliminary results showed that the levels of aphid resistance efficiency of the five lectin genes were basically agreed with the abundance of exogenous protein expression in transgenic tobacco, namely, the plants with high expression of the exogenous protein had high efficiency of resistance to aphids, while low exogenous protein expression with low efficiency. For the transgenic tobacco plants containing Atpp2 promoter, the highest efficiency of aphid resistance with PPA-5 gene was 74.4% and the lowest with PPA-4 gene was only 57.5%. Generally, the aphid resistance level of transgenic plants with CaMV35S promoter was higher than those containing Atpp2 promoter based on our above results, and the resistant efficiency of the 5 lectin genes from high to low was PPA-5, PPA-6, PPA-3, PPA-4 and PPA-2.In the present study, five new lectin genes were cloned from Pinella pedatisecta, and their functions were, and these genes were showed a high efficiency of resistance to aphid and potential application identified by gene transformation with tobacco, which provides new gene resources for reisitance of crop breeding program to aphid with transgenic technology.