Agrobacterium tumefaciens the antagonistic fungi screening , fermentation and organotin compounds against TMV, anticancer mechanism
|Keywords||Agrobacterium tumefaciens Aspergillus niger Organotin Tobacco mosaic virus Tumor|
Plant diseases caused by the pathogenic Agrobacterium and plant viruses are located around the world, causing serious economic losses to agricultural production caused attaches great importance to the world. Root tumefaciens (Agrobacterium tumfaciens) the infected plant the rhizomes to form a crown gall tumor the susceptible plants growth retardation, Shushi weak, yield less short life, a direct impact on the production of fruit trees, thus affecting the agricultural economy development. Tobacco by tobacco mosaic virus (TMV) infection, its metabolic severely affected photosynthetic rate, the blades will be uneven chlorotic, yellow, plants dwarf, dry matter accumulation to reduce damage to the quality of tobacco, our current one of the most important agricultural diseases. This paper is divided into two parts. The first part is for crown gall biocontrol bacteria not drought disadvantage of short life, for the first time from the fruit tree roots screening antagonistic Agrobacterium tumefaciens biocontrol fungi in the soil, and to determine the biocontrol fungi fermentation conditions for plant development and application of crown gall biocontrol strains to expand organotin strain resources; and the other part is for general agents prevent TMV difficult in the case do not harm the host inhibit viral proliferation, Nankai University, Academician Li Zhengming, for the first time new compound 04-W0319 activity and mechanisms of resistance to TMV, and this on the basis of anti-tumor mechanism of the 04-W0319 system, organotin compounds for the development of low toxicity, high efficiency, expand the scope of its application reference. Soil samples collected from 12 Chinese provinces and cities in the first part of the study, 152 fungi were isolated and purified using the agar column screening, two anti Agrobacterium tumefaciens Agrobacterium tumefaciens T-37 (referred to as T-37) fungal strains through the filter paper method compared rescreening, confirmed that the antagonism of the two strains of Agrobacterium tumefaciens. According to Raper (1965) classification of Aspergillus, the main morphological characteristics of antagonistic fungal strains of Aspergillus niger (Aspergillus niger V.Tieghen 1867) he described standard form is basically the same, the identification of the strains belonging to the genus Aspergillus Aspergillus niger group an Aspergillus niger, denoted as Aspergillus niger xj (Aspergillus niger xj), and the strain was deposited in China Typical Culture Collection Center. According to the classification criteria of Brown Smith (1957), antagonistic strain identification Paecilomyces, Verticillium recorded as round sticks to Paecilomyces 49-01 (Paecilomyces verticillatus 49-01), as the new species. The xi strains separation monospores, eight single spore isolates, denoted xj-1, xj-2, xj-xj-xj-5 xj-6 xj-7, xj-8. Colony growth situation, sporulation, conidial germination rate that xj-5, T-37 inhibitory activity comprehensive comparison for the advantages of single spore isolates, their Following the the culture traits cultured and T -37 inhibitory activity were investigated xj-5 single spore isolates showed relatively stable genetic traits and the resulting inhibition zone diameter (20.13 ± 0.09 mm) than the control the xj strain (18.28 ± 0.21 mm). Xj-5 single spore strains as the original strain, its fermentation process conditions to explore. 2% maltose, glucose, starch, sucrose, lactose, glycerol, 6 kinds of carbon sources in the culture composition, and 3% of peptone, beef extract, yeast extract, ammonium nitrate, ammonium chloride, urea nitrogen sources for univariate screening choose maltose, sucrose as a carbon source, yeast extract, peptone as nitrogen source and to design L 9 (3 4 sup>) orthogonal experiment to optimize the C / N. Determining xj-5 strain fermentation medium composed as follows: maltose, 2%, sucrose, 2%, 3% of yeast extract, 1% peptone. Culture conditions in different inoculum size, medium volume, initial pH value of the culture temperature and fermentation medium were screened. Fermentation conditions determine the strain of the xj-5: inoculated with 15%, 10% of the amount of shake flask, incubation temperature 25 ° C, initial pH of the fermentation medium natural. According to filter out the above medium components and culturing conditions, the XJ-5 strain was inoculated shake flask culture and every 24h regular sampling and determination of the biomass and the diameter of the inhibition zone, dynamic curve drawn fermentation. The results show: xj-5 strain directly into the logarithmic growth phase, growth delay period, this may be because of the strong vitality of liquid added parent species, and differences in culture conditions, the strains were able to adapt quickly to the fermentation environment, direct access to the fast growing season, and the longer this time (1-8d), biomass in 8 days, reached maximum (161.24g / L), antibacterial ingredients are constantly secretion and accumulation. Subsequent delay period (9-12d) lasted a long time, and when in the first 10 days, the accumulation of metabolites peaked maximum inhibition zone diameter (54.48 mm). With the fermentation medium nutrient depletion, the emergence of the aging period (13-15d), the accumulation of toxins and bacterial autolysis led to a sharp decline in biomass, and the outflow of the cell contents and cell debris generated cause the pH of the fermentation medium value changes directly affect the activity of the antibacterial component xj strains, resulting in the inhibition zone diameter also fell sharply. Therefore, according to the xj-5 strain fermentation kinetics model, established xj-5 Strains the fermentation process of the active ingredient: Preparation maltose 2%, sucrose, 2%, 3% of yeast extract, peptone 1%, pH natural fermentation medium, the strain xj-5 inoculation amount of 15%, was inoculated into the liquid loading is 10% of liquid fermentation medium, 25 ° C, 120rpm, the shake flask culture 10d, to collect mycelia preparation fermentation concentrate, filter paper piece measured xj-5 strain inhibition zone diameter of 54.48 mm. The second part of the study, leaf smoke, organotin compounds 04-W0319 therapeutic effect of tobacco mosaic virus infection was 49.38%; passivation effect 73.23% 48.82%; protective effect. 500μg/mL 04-W0319 with the in vitro effects of TMV 30min, electron microscopy, virions breakage, grain structure has been destroyed; agarose gel electrophoresis showed 04-W0319 TMV-RNA in vitro degradation; TMV -CP the UV measurement results reflect a 04-W0319 inhibit tobacco mosaic virus in vitro polymerization process. Of ordinary cigarette K326 of PAL, POD and SOD activity were measured. PAL activity assay results show that: 500μg/mL 04-W0319 treatment of tobacco seedlings reach the enzyme activity peak value in the first seven days, nine days to a minimum, and tobacco in the 04-W0319 treatment and then inoculated with TMV increased activity can be . The POD activity test showed: Single 04-W0319 treatment of enzymatic activity in the seven days to reach the peak of enzymatic activity values, tobacco inoculated with TMV after 04-W0319 treatment also increased activity can be reached enzymatic activity in nine days The peak value, and is higher than the other processing. SOD activity measurement results show that: the control of enzyme activity and the incidence of 04-W0319 processing of TMV processing enzyme activity reached a peak in the first five days, followed by enzyme activity in nine days and reached another peak value, and then inoculated with TMV The same trend, used alone 04-W0319 processed tobacco seedlings enzyme activity reached a peak value in the first five days, and higher than the other treatment groups. The K326 the intercellular protein the discontinuous electrophoresis analysis by SDS-PAGE: compound 04-W0319 on K326 intercellular protein has little effect, and the induction of PR protein is not strong. 04-W0319 host chlorophyll content experimental results show: TMV infection greatly reduce ,04-W0319 processed tobacco leaf chlorophyll content increased 04-W0319 can reduce virus makes chlorophyll content of tobacco leaves on tobacco leaves The damaging effects of chloroplast chlorophyll content, thereby improving the disease resistance of the host; pharmaceutical processing tobacco leaf chlorophyll content is still lower than the healthy controls, which also shows the 04-W0319 not completely inhibit the destructive effects of the virus on chlorophyll. Therefore ,04-W0319 one hand in vitro with TMV role, reduce the TMV infection rate; the other hand, increase the activity of the host defense enzymes and increase the chlorophyll content, increase host resistance against TMV infection. The PC3, Bcap-37 and BGC823 tumor cell materials ,04-W0319 for 72h the the PC3 cell of IC 50 for 0.13μg/mL; role 48h cells Bcap-37 IC 50 0.18μg/mL; and at 48h BGC823 cells IC 50 for 0.17μg/mL. The cytotoxicity assays preliminary judge :04-W0319 3 induction of cell death is not primarily through cytotoxicity. Scratches notation experiments show that 04-W0319 can be reduced to the 3-cell movement ability to inhibit the growth of tumor cells. Morphological observation and cell culture supernatant LDH activity assay showed that: 72h apoptosis is always the apoptosis induced by 04-W0319 the PC3 and Bcap-37 cell death using the main ways; within 48 h is always 04-W0319 induced BGC823 cell death using the main ways. Flow cytometry cytometry results further reveal: the 0.15μg/mL 04-W0319 role in 48h, the of PC3 or Bcap-37 cells were arrested in G 2 / M phase and inhibits cell growth, reproduction; role of prolonged concentration increased role to induce apoptosis based; BGC823 cells, 0.15μg/mL 04-W0319 role 48h, cell cycle arrest in G 0 / G < sub> 1 period, and accompanied by the induction of apoptosis and inhibition of cell growth, reproduction; With the prolonged duration of action ,04-W0319 will BGC823 cell cycle block in G 2 ╱ M phase and associated with cell necrosis. Immunoblotting (Western blot, WB) The experimental results show :04-W0319 can stimulate PC3 Bcap-37 cells p21 protein expression, thus preventing the cells through the cell cycle G 2 ╱ M of the restriction point , inhibition of cell growth. BGC823 cells ,04-W0319 by expression of inhibit CyclinD proteins, hinder the cell through the cell cycle g 0 / G 1 of the restriction point, and inhibited cell growth. Overall ,04-W0319 the CDK 3 cells 2 and CDK , 4 no effect, suggesting that the role of 04-W0319 target relative specificity.