Aphid And Disease Resistant Functional Analysis Of Lectin Genes From Pinellia Pedatisecta

Pinellia Pedatisecta Agglutinin (PPA) belongs to mannose-binding lectin muti-family. The transgenic tobacco plants containing five different kinds of PPA genes under the control of CaMV 35S promoter and Arabidopsis thaliana phloem promoter AtPP2 were used to perform the functional analysis of PPA. The results are as follows:In order to obtain the single locus inserted transgenic tobacco plants, Basta resistance and molecular identification were used to screen the positive transgenic PPA tobacco plants. In total, we obtained three 35S-PPA-2 transgenic lines, three 35S-PPA-3 transgenic lines, seven 35S-PPA-4 transgenic lines, nine 35S-PPA-5 transgenic lines, five 35S-PPA-6 transgenic lines, and four AtPP2-PPA-3 transgenic lines, three AtPP2-PPA-4 transgenic lines, three AtPP2-PPA-5 transgenic lines and three AtPP2-PPA-6 transgenic lines.Further screening in T1 generation of above transgenic tobacco lines, we identified ten 35S-PPA-5 and six AtPP2-PPA-5 homozygous transgenic lines with single locus inserted and high expression level of PPA in T3 generation.In order to know whether PPA-5 could confer the same resistance to aphid as other mannose-binding lectins, the homozygous transgenic lines were classified as high, moderate and low protein expression levels detected with western-blotting and aphid bioassay. The result of aphid bioassay showed that the both 35S-PPA-5 and AtPP2-PPA-5 transgenic plants had significantly insecticidal activity against the tobacco aphids (Myzus nicotianae). The average inhibition rate against aphid of 35S-PPA-5 transgenic plants is 84.8%, and the inhibition rate reach up to 99.37% in transgenic line with high protein expression; while the average inhibition rate against aphid of AtPP2-PPA-5 transgenic plants is 53.78%, the highest inhibition rate is 59.95%. The inhibition in AtPP2-PPA-5 transgenic lines is much lower than in 35S-PPA-5 ones. These finding suggested that PPA-5, which has high resistant effect to aphid, is a suitable candidate gene for insect resistance in the control of sap-sucking insects through a transgenic approach.Some of Mannose-binding lectins have not only anti-insect activity, but also anti-pathogen and anti-nematode activity. In order to know whether PPA has the same anti-pathogen activity as other mannose-binding lectins, the homozygous transgenic 35S-PPA-5 tobacco plants were infected with Verticillium wilt, and the result showed that the area of disease spot was obviously smaller in the transgenic plants than in wild plants, indicating that PPA protein may have the inhibitive activity against Verticillium wilt.At present, the research about PPA prokaryotic expression is rare. In order to characterize the function of PPA-5, the signal peptide sequence in PPA-5 was knocked out according to bioinformatic analysis, and the modified PPA-5 was named APPA-5. The APPA-5 sequence was inserted into prokaryotic expression vector pGEX-6p-1 digested with restriction enzymes EcoR I and Xho I, and was then overexpressed in E. coli Rosetta (DE3) cells by IPTG induction. The purified PPA protein was used to antifungal test with Verticillium wilt, and the result further confirmed the inhibitive activity of PPA protein against Verticillium wilt.