| As an animal cells cytokines, interferon (IFN) possesses broad-spectrum antiviral activity and the role of immune protection. With the intensification and commercialization of poultry industry, large amounts of interferon were needed for effective prevention against viral diseases. Conventionally, expression of recombinant IFN-γrelies mainly on microbial and mammalian cell expression systems in which the post-treatment process brings about cumbersome problem, resulting in susceptibility to proteolysis, shorter survival times and high production costs. However, plant bioreactor expression systems have demonstrated an advantage over other in vitro expression systems because it may provide an attractive and large production capacity, and inexpensive alternative to conventional production system in terms of low costs. In addition, plant-based vaccines can be directly used as direct oral advantages. Therefore, elucidation of the IFN expression characteristics and biological activity in plants may be highly important for both theoretical and practical benefits.Based on the sequence of the ChIFN-γgene from GenBank, a coding sequence for this gene was optimized by modification of codon usage of Brassica napus. The synthetic ChIFN-γgene is about 614bp along with a Napin signal peptide, Kozak sequence and an endoplasmic reticulum retention signal (SEKDEL).With the utility of specific primers a specific promoter about 1,158 bp of Napin rape seeds and NOS terminator (around 220 bp) were obtained from Brassica napus genome via PCR amplification. Sequence analysis of napin gene showed that it contain promoter basic element, specific element of ABREs and RY motif. Using isocaudamer enzyme ligation method, plant expression vector pSH-NGN, pSH-NGNL and pSH-IFNG are constructed for the genetic transformation of tobacco, lettuce and rape, respectively.In order to verify the correctness of design and construction about plant expression vector, transient expression system was employed to detect lettuce (Lactuca sativa L.) vacuum-infiltrated with Agrobacterium tumefaciens containing plasmid pSH-NGN, pSH-NGNL or pSH-IFNG The ELISA detection showed that three expression levels of ChIFN-7 in lettuce were 0.6757, 0.6761 and 0.5195μg.g-1 fresh leaf weight (FLW), respectively. To simplify the method for extracting protein, and decrease the degradation rate of target protein, different protein extract buffers were used to extract ChIFN-γprotein. Extract solution (pH7.5) containing 50 mM PBS, 1% SDS, 0.25 M NaCl, 1%β-mercaptoethanol, and 0.05% Tween-20 demonstrated the highest extraction efficiency of ChIFN-γprotein via ELISA test, whose yield was as high as 1.2μg.g-1 FLW. The result of western blot tests showed that the molecular weight of the protein was approximately 30 kDa.Agrobacterium tumefaciens strain EHA105 harboring pSH-IFNG and pSH-NGNL were used to carry out the genetic transformation in tobacco herein. A total of 24 transgenic ChIFN-γtobacco clones were obtained. Evidences from PCR, RT-PCR, GUS histochemical staining and ELISA quantitative tests had substantially proved that ChIFN-γgene had been integrated in the genome of tobacco, and it could transcribe and express correctly. The expression level of ChIFN-γin tobacco leaves was up to 20μg.g-1 FLW, which is much higher than the common level (0.000017% to 0.001%). Bands of approximately 30 kDa and 40 kDa were detected by Western analysis in transgenic tobacco leaves using an anti-chicken IFN-γantibody. The molecular weight of ChIFN-γby SDS-PAGE was higher than the predicted molecular weight, possibly duo to the different glycosylation among the lines. According to the observation of agronomic traits in the transgenic tobacco, it has been found that the character differences between the transgenes and wild type are not significant, except for the more leaves stickiness, smaller flower bud, less kernal number as well as shallower colors of the seed in the the former.Also Agrobacterium tumefaciens strain EHA105 harboring pSH-IFNG and pSH-NGNL were genetically transformed into Brassica napus. A total of 25 transgenic rape lines integrated with ChIFN-γwere obtained by screening. Plants transformed with the napin promoter targeting ChIFN-γto the nucleus, the expression content of ChIFN-γprotein could reach to 58μg.g-1 FLW, which was around 2.46 times higher than that of transgenic plants using the constitutive cauliflower mosaic virus 35S promoter. It showed that the startup ability of in leaves of Brassica napus with the expression box containing napin promoters, and its signal peptide was obviously higher than the ones with constitutive promoters 35S. Napin promoter of 'leakage' phenomenon is propably related to it's promoter elements.An experiment about cytopathic effect of the chicken embryo fibroblasts had been undertaken using total crude protein extract from T0 leaves of the transgenic ChIFN-γtobacco to determine the ChIFN-γbioactivity. Activate the passage monolayer chicken fibroblast cell when the cells culture 24 hours was infected with TCID50 of vesicular stomatitis virus (VSV). The ChIFN-γprotein titer per milligram transgenic tobacco was 5.12×103 U as calculated by morphological observation on cells at 22h and 42 h.Using mechanical friction method, the phenotype susceptive Tobacco Mosaic Virus (TMV) was inoculated. During the initial six days, the inhibiting rate of transgenic tobacco lines to virus was 100%. Unfortunately, this rate dropped gradually, and the inhibiting rate got stable at 32% after 16 day's treatment. Moreover, significant inhibited effect to this virus had been investigated in the transgenic lines. After one week, diverse mosaic and necrotic lesion were obtained from the control group, and the, infection rates reached 95%. All stated above had proved that the transgenic tobacco enhanced the inhibiting effects to TMV.With rapid seeding on floating plates, a large number of leaves were obtained from the transgenic plants . Basis on the data that per gram feedstuff contains 50U ChIFN-γprotein and one chick feeds on 20 g feedstuff every day, the leaf homogenate of transgenic tobacco was then added to the feedstuff. Subsequently, animal feeding experiments were proceed with five groups, in whichs 10 chickens were used for each one. The tested chickens were subjected to virulent strain Newcastle Disease Virus (NDV) for 1-10 days, and then killed after 10 days. The results showed that feeding with ChIFN-γprotein group had higher weight growth rate than that of the control (feeding-free). Therefore, ChIFN-γprotein demonstrated the ability to add up weight of chicken. Meanwhile, the results of serum agglutination proved that NDV antibody titer of feeding ChIFN-γgroups was higher than that of the control; suggestting ChIFN-γprotein could improve the ability of antivirus in chicken. In addition, the immunoprotection ratio to virus infection reached to 80% by feeding 300U ChIFN-γprotein, thus this protein might significantly increase chicken anti-viral capacity via oral feeding mode.Natural inoculation of tobacco aphids as well as feeding wild perfect insects of Spodoptera litura or tobacco budworm with in vitro transgenic tobacco leaves sustantially verified that the, transgenic tobacco leaves showed significantly a positive effects on the resistance to insect. In tobaccos, the nicotine content, self-protease activity, volatile substances were quantified, and plant tissue was further investigated by means of slice staining. Compared with the control, no significant difference in nicotine content was obtained from the transgenic lines, and insertean protease activity was lower by 5.4% in the feeding transgenic group after 12 hours. The leaf thickness, cell density, cell wall thickening, glandular hairs increase demonstrated a positive trend toward the insect resistance in transgenic tobacco. In addition,Some differences in volatile components were investigated from the transgenic tobacco, e.g. the contents duvatriendiol and 4,8,13-Duvatriene-1,3-diol, which are closely related to the insect resistance increased directly. ChIFN-γgene activation of several transcription factors may be binding the cis-regulatory elements of trichome development-related genes, increases the expression of genes of the trichome development, leading to increase in the number of trichomes, the secretion of terpenoid, which may be linked to the main mechanisms of the transgenic ChIFN-γgene tobaccos.In conclusion, we substantially created the transgenic lines expressing active ChIFN-γprotein. Transgenic ChIFN-γgene plants may have a certain role in enhancement the resistance to animal and plant virus, as well as to insect. Our findings may provide a foundation for the application of plant bioreactor in ChIFN-γprotein production. Rather, it also demonstrates a new clue to prevent or/and recover avian disease via direct oral feeding with transgenic plants.
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