Figure 1.
Expression and purification of the LMX protein in E. coli.
A. Schematic representation of the expression vector pGEX-6P-1-LMX. B. Coomassie stained polyacrylamide gel (12%). M, molecular weight marker (SM0671, Fermentas); line 1–2, total protein extracts of pGEX-6P-1 without IPTG induction and with IPTG induction; line 3, total protein extracts of pGEX-6P-1-LMX without IPTG induction; line 4–5, supernatant and inclusion bodies of pGEX-6P-1-LMX with IPTG induction; line 6, purified sample of supernatant of pGEX-6P-1-LMX with IPTG induction. * represents the target band. C. Western blot probed with an antiserum raised against the purified GST-LMX fusion protein. The Molecular mass of polypeptides expressed by the GST-LMX recombinant construct was about 32.8 kDa.
Figure 2.
Structural analysis of LMX and LqhIT2 protein.
A. Comparison of the secondary structure of LMX and LqhIT2 protein by CD spectra. B. The homologous modeling of LMX was performed by SWISS-MODEL. The homologous modeling used the three-dimensional structure of LqhIT2 as a template.
Figure 3.
The LMX protein confers resistance to the rice leaf folder.
A. Damage to rice leaves daubed with 2.5 µg∼10 µg LMX fusion protein, caused by rice leaf folder. B. Ratio of the damaged leaf area to the whole leaf. C. Larvae lethality of the rice leaf folder fed with 2.5 µg∼10 µg bacterial-expressed LMX protein. For the control, the rice leaves were daubed with 1× PBS buffer and 10 µg GST tag protein. Pictures were taken and ratios of the damaged leaf area were measured after 72 h incubation with the rice leaf folder. Values are means ± SD. *** denotes 0.001 significant difference.
Figure 4.
Phenotype of the rice leaf folder larvae fed with LMX protein.
The larvae fed on 2.5 ∼7.5 µg LMX protein-applied rice leaves were treatment groups, while the larvae fed on PBS buffer-applied rice leaves and 7.5 µg GST tag protein-applied rice leaves were control groups. Pictures were taken at the start (0 days), 3 days, and 5 days.
Table 1.
Weight of surviving larvae of rice leaf folder on the 3rd and 5th day post-infestation (mg/larvae).
Figure 5.
Molting of rice leaf folder larvae fed with LMX protein.
A. Larvae fed with LMX-GST fusion protein delay molting. B. Ecdysone content in the larvae of rice leaf folder feeding on LMX fusion protein on the 3rd day post-infestation (ng/mg). The larvae fed on 2.5∼7.5 µg LMX protein-applied rice leaves were treatment groups, while the larvae fed on PBS buffer-applied rice leaves and 7.5 µg GST tag protein-applied rice leaves were control groups. Pictures were taken and ecdysone was measured after 72 h feeding. Values are presented as means ± SD of triplicates. * denotes p<0.05; *** denotes p<0.001. ns denotes no significant difference.
Table 2.
Enzyme activities in larvae feeding on LMX fusion protein on the 3rd day post-infestation.
Figure 6.
Molecular analysis of the LMX transgenic lines.
A. Schematic representation of the transgenic binary vector pCAMBIA 1301-LMX. B. Analysis of T0 independent transgenic plants expressing LMX by PCR. M, DL2000 marker; 1, Yuetai; 2, ddH2O; 3–12, T0 generation of transgenic plants expressing LMX gene. C. Southern-blot analysis of T0 transgenic lines with single copy insert. The DNA samples were hybridized with the prepared biotin-11-dUTP probe. D. qRT-PCR analysis of LMX expression in transgenic lines L-5, L-10, and L-22. The ubiquitin gene was used as the internal control. E. Western blot assay of T2 homozygous transgenic line L-10 using LMX antibody. P, purified LMX protein; 1, Yuetai; 2, empty vector transgenic line rbcS; 3–7, homozygous T2 LMX-10 transgenic lines.
Figure 7.
Leaves of the LMX transgenic rice plants demonstrate insect resistance.
A. Damage caused by rice leaf folder on the leaves of LMX transgenic rice plants. B. Ratio of the damaged leaf area to the whole leaf. C. Whole transgenic rice plants expressing LMX demonstrated improved resistance to rice leaf roller. Wild type plants and empty vector transgenic plants (rbcS plants) were used as the controls and homozygous LMX transgenic rice plants were used as the experimental group. Day 1 of 3rd instar rice leaf roller larvae were utilized in the bioassay. Leaves were photographed and measured after 72 h incubation with the rice leaf folder. Plants were photographed and measured after 7 days of incubation with the rice leaf folder larvae. Values are mean ± SD, *** denotes 0.001 significance value.
Table 3.
Damage analysis of LMX transgenic rice caused by rice leaf folders from 2011 to 2012.