Expression Of Stress Induced Jatropha Curcas Leaf Ribosome-inactivating Protein (Curcin 2) In Transgenic Tobacco

Ribosome-inactivating proteins (RIPs) are a family of plant enzymes. It wasdemonstrated that some type 1 RIPs have antiviral and/or antifungal activity in vitroand in vivo. In our study, the coding sequences of premature and mature curcin 2(cur2p and cur2m) were separately cloned into the pMD18-T vector (TaKaRa). Andthen were respectively subcloned into the plant transformation vector pBI121(Clontech) after digested with BamH I and Sac I. Thereupon we obtained twoconstructs, pBICP and pBICM, which were mobilized into Agrobacteriumtumefaciens strain EHA105 by the modified freeze-thaw method. According to theleaf-disk method, we obtained six and nine rooted transgenic plants which had beensuccessfully integrated the sequences of cur2p and cur2m. These transgenic plantswere named as TP1～6 and TM1～9.Expression of curcin 2 enhances resistance of tobacco plants against infectionby TMV. Transgenic plants TP1～6 and SR1 plants were mechanically infected withTMV by rubbing the virus suspension on the leaves with a sterile cotton swab. Afterabout 8 days, the SR1 tobacco plants exhibited symptoms of systemic infection, increased vein transparency followed with local discoloration on leaf surfaces. Thetransgenic lines (TP4, TP5, TP6) expressing high level curcin 2 appeared healthyafter 16 days, while the untransformed plants obtained apparent mosaic phenotypeon the top three fresh leaves. The time when the transgenic lines represented mosaicphenotype is. delayed as the increasing of the expression level of curcin 2. Althoughall tobacco plants infected by TMV exhibited symptoms of systemic infection after amonth, the transgenic plants exhibit much less phenotypic aberrations than controlSR1.Expression of curcin 2 enhances tolerance of transgenic plants againstRhizoctonia solani. Twenty plants from each independent transgenic line or controlSR1 were kept in a chamber for thirty days after infection. Each plant was assignedto a Dl-class ranging from 0 to 4. The mean values of the Dl-class number and theinfection rating by McKinney's formula for control SR1 and transgenic lines assayedfor tolerance to Rhizoctonia solani are calculated. The mean values of the Dl-classnumber for transgenic plants, which were on average 27.8% lower than for controlSR1 (1.53 vs 2.12), ranged from 1.87 (line TP3) to 1.33 (line TP6). The infectionrating by McKinney's formula for transgenic plants ranged from 46.8% (line TP3) to33.2% (line TP6), which correlated well with the relative levels ofimmunodetectable curcin 2 in the different lines. Fungal infection reduced plantfitness, which in turn reduced matter accumulation. These results indicate thataccumulation of curcin 2 increased the protection of transgenic tobacco plantsagainst infection by Rhizoctonia solani.