Mathematically modeling photosynthesis helps to interpret gas exchange in a plant and estimate the photosynthetic rate as affected by environmental factors. Notably, the photosynthetic rate varies among leaf vertical positions within a single plant. The objective of this study was to measure the distinct photosynthetic rate of lily (Lilium Oriental Hybrid ‘Casa Blanca’) at the upper, medium, and basal leaf positions. Subsequently, the FvCB (Farquhar-von Caemmerer-Berry) photosynthesis model was employed to determine the parameters of the model and compared it with a rectangular hyperbola photosynthesis model. The photosynthetic rates were measured at different intracellular CO2 concentrations () and photosynthetic photon flux density (PPFD) levels. SPAD values significantly decreased with lowered leaf position. The photosynthetic rates at the medium and basal leaves were lower compared with the upper leaves. FvCB model parameters,  and   , showed no significant difference between the medium and basal leaves. Estimated photosynthetic rates from derived parameters by the FvCB model demonstrated over 0.86 of R2 compared with measured data. The rectangular hyperbola model tended to overestimate or underestimate photosynthetic rates at high  with high PPFD levels or low  with high PPFD levels, respectively, at each leaf position. These results indicated that the parameters of the FvCB model with different leaf positions can be used to estimate the photosynthetic rate of lily.

Salinity stress is a major threat to plant growth and development, affecting crop yield and quality. This study investigated the effects of different salinity levels on photosynthetic responses and bulb growth of Lilium LA hybrid “‘Serrada’.” Plants were irrigated with 1 L of 0, 200, and 400 mM NaCl solutions every two weeks for 14 weeks in a greenhouse. At the end of the cultivation period, the substrate pH decreased, and electrical conductivity increased with increasing salinity. Regardless of salinity levels, the days to flowering and number of flowers were similar among treatments. In contrast, the flower width, plant height, number of leaves, and leaf area decreased with increasing NaCl concentrations. Although there were no differences in the photosystem II (PSII) operating efficiency and maximum quantum yield of PSII, net CO2 assimilation rates (An) and stomatal conductance (gs) were significantly reduced at 200 and 400 mM NaCl solutions compared to the control. At 400 mM NaCl solution, bulb diameter and weight significantly decreased at the end of the experiment. These results suggest that bulb growth inhibition could be attributed to limiting photosynthetic rate and stem growth. This finding suggests that salinity mitigation is necessary to maintain plant growth and photosynthetic capacity in lily cultivation on salt-affected soils.

In order to establish symbiotic host-bacterial relationships, symbionts in insects evolved a mechanism to overcome host immune responses. Here we provide the resistance of symbiotic bacteria on the insect immune system. As a result, through the transposon mutagenesis, we found a salivary gland (SG) susceptible mutant. The disrupted gene was identified as nlpB involved in lipoprotein synthesis. The nlpB, bla double deletion mutant was sensitive to SG like nlpB-Tn5 inserted mutant. This mutant increases outer membrane permeability. It provides an explanation for SG susceptibility, because the antimicrobial peptide in SG would be able to translocate across the outer membrane more easily than in the wild type. These results indicate that nlpB and bla are likely to be important factors in terms of determining resistance against SG of Riptortus that is connected with the successful colonization of the Riptortus midgut.

Biological properties of antimicrobial peptides (AMPs) of hemimetabolous insect are poorly characterized in innate immunity field. To investigate the biochemical properties of hemimetabolous insect’s AMPs, we purified the pyrrhocoricin-like AMP from the hemolymph of Riptortus pedestris and then named as riptocin. We successfully determined the primary protein structure and its cDNA sequence. Interestingly, the determined cDNA revealed that riptocin precursor is composed of 12 repeating units of active riptocins, which implied that riptocin precursor might require to be processed to generate active riptocins by several unidentified processing enzymes. In order to characterize the bio-processing mechanisms of riptocin precursor, we generated the antibody against active riptocin. Using quantitative PCR and Western blot analyses, we showed that gene of riptocin was started to express from the fatbody after three hours post bacterial infection. To address our hypothesis that active riptocin is generated from riptocin precursor by several processing enzymes, we need to obtain the riptocin precursor. Currently, we are expressing the recombinant riptocin precursor using in vitro translation system. Meanwhile, we investigated whether naive hemolymph (naive HL), which may contain precursor riptocin, can generate active riptocin when riptocin precursor was co-incubation with bacteria-challenged hemolymph (active HL), which may contain all processing enzymes. Actually, when naive HL was incubated with active HL, antimicrobial activity was dramatically increased, suggesting that processing enzymes in active HL may induce processing of riptocin precursor to generate active riptocins.

The Riptortus-Burkholderia symbiosis is a newly emerging insect-bacterium symbiotic system. This symbiosis system has a good merit as an experimental model system to produce the non-symbiotic (apo) and symbiotic (sym) host insect. In recent reported papers, the symbionts play important biological roles for the host insects. Meanwhile, juvenile hormone (JH) is one of major hormone synthesized corpora allata(CA) to control many physiology of insect. However, the study for cross-talk mechanism between symbionts and host hormones to control important physiological phenomenon of insects is almost none. In this study, we found that Riptortus speed up adult emerging and increase egg laying on presence of symbiont Burkholderia. Also we found that hexamerin proteins, which were controlled the expression by JH, were accumulated in sym-Riptortus hemolymph compare with apo-Riptortus. According as combined results, we hypothesized that the gut symbiont Burkholderia can control JH titer to conclude out beneficial effects such as development and reproduction of R. pedestris. To verify this hypothesis, we examined measurement of JH titer, expression of hexamerins as JH response genes and RNAi for hexamerin protein during whole Riptortus life on presence or absence of symbiont Burkholderia. All results demonstrated that gut symbiont controlled JH titer of Riptortus. Controlled JH amount by symbiont Burkholderia in host midgut regulated hexamerin protein expression for speeding up adult emerging and increasing egg production.

The Riptortus (stinkbug) has a specialized symbiotic organ, M4 midgut, to harboring symbiont Burkholderia. M4 midgut is located in abdomen and surrounded with insect hemolymph. Recently our group demonstrated that symbiotic Burkholderia showed different physiology after adapting in M4 gut compare with in vitro cultured Burkholderia. And population of symbiotic Burkholderia in the M4 midgut is regulated by special organ. However, the molecular mechanism to prevent spreading and migrating symbiont bacteria to other host tissues from symbiotic organ is not clear. Therefore, we assumed that symbiont Burkholderia are susceptible to host humoral immunity after established infection in M4 midgut to prevent spreading and migrating into the other host tissues through Riptortus hemolymph. To prove this assuming, we tested the susceptibility and survival rate of symbiont Burkholderia in hemolymph of Riptortus in vitro and in vivo. We also examined the susceptibility of symbiont Burkholderia using purified antimicrobial peptides (AMP), pyrrhocoricin-like, thanatin-like and defensin-like AMPs. Finally, we tested inducing ability for AMPs by systemic infection of symbiotic Burkholderia. Gene expression of purified AMPs was not different after systemic infection of both symbiont and in vitro cultured Burkholderia. Surprisingly, in vitro cultured Burkholderia resisted on bacteria injected hemolymph and purified AMPs but symbiont Burkholderia were highly susceptible in bacteria injected hemolymph and purified AMP. These results suggest that symbiont Burkholderia can't survive in the hemolymph after escaping symbiotic organ. Moreover, humoral immunity of host Riptortus is important to prevent spreading and migrating symbiont Burkholderia into the other host tissue or organ from symbiotic organ.

Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Aujeszky's disease (AD), also called pseudorabies, is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.