Chymosin (also known as rennin) plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA). The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen.

Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins.

Plant polyploidization represents an effective means for plants to perpetuate their adaptive advantage in the face of environmental variation. Numerous studies have identified differential responsiveness to environmental cues between polyploids and their related diploids, and polyploids might better adapt to changing environments. However, the mechanism that underlies polyploidization contribution during abiotic stress remains hitherto obscure and needs more comprehensive assessment. In this study, we profile morphological and physiological characteristics, and genome-wide gene expression between an autotetraploid rice and its diploid donor plant following saline stress. The results show that the autotetraploid rice is more tolerant to saline stress than its diploid precursor. The physiological characteristics were rapidly responsive to saline stress in the first 24 h, during which the elevations in sodium ion, superoxide dismutase, peroxidase, and 1-aminocyclopropane-1-carboxylic acid were all significantly higher in the autotetraploid than in the diploid rice. Meanwhile, the genome-wide gene expression analysis revealed that the genes related to ionic transport, peroxidase activity, and phytohormone metabolism were differentially expressed in a significant manner between the autotetraploid and the diploid rice in response to saline stress. These findings support the hypothesis that diverse mechanisms exist between the autotetraploid rice and its diploid donor plant in response to saline stress, providing vital information for improving our understanding on the enhanced performance of polyploid plants in response to salt stress.

A novel rice lesion mimic mutant (LMM) was isolated from the mutant population of Japonica rice cultivar Hitomebore generated by ethyl methane sulfonate (EMS) treatment. Compared with the wild-type (WT), the mutant, tentatively designated E40, developed necrotic lesions over the whole growth period along with detectable changes in several important agronomic traits including lower height, fewer tillers, lower yield, and premature death. To understand the molecular mechanism of mutation-induced phenotypic differences in E40, a proteomics-based approach was used to identify differentially accumulated proteins between E40 and WT. Proteomic data from isobaric tags for relative and absolute quantitation (iTRAQ) showed that 233 proteins were significantly up- or down-regulated in E40 compared with WT. These proteins are involved in diverse biological processes, but phenylpropanoid biosynthesis was the only up-regulated pathway. Differential expression of the genes encoding some candidate proteins with significant up- or down-regulation in E40 were further verified by qPCR. Consistent with the proteomic results, substance and energy flow in E40 shifted from basic metabolism to secondary metabolism, mainly phenylpropanoid biosynthesis, which is likely involved in the formation of leaf spots.