Studies On The Establishment Of Suspension Cell Lines And Salt-tolerance Metabolic Mechanisms Of Nitraria Tangutorum Bobr.

Nitraria tangutorum Bobr., a shrub belonging to the Zygophyllaceae family, is one of e ndemic species in China, which has superior resistance to severe drought and salt stress,and is an important constructive or dominant species in deserts, semi-deserts and saltin g lands. Therefore the study on its salt tolerance mechanism is of great significance to the cultivation of new salt-tolerant plant species and finding of new ways to improve their salt-tolerance. In this research, stems and leaves of wild N.tangutorum have been used as explants to induce callus, the loose type callus were screened and built a stabl e and rapid growth suspension cells, with emphasis on the study of its growth characte ristics. Our study has further focused on its growth rate, osmoregulation substance cont ent, the degree of membrane damage and the changes of antioxidant enzyme activity of the cells under salt stress; analysis of the differences of the cells metabolite profiling under different salt stress, selection of salt-tolerant biomarker metabolites; construction o f major metabolic pathways of Nitraria tangutorum cells under salt-stress; These studies are important to explain the metabolic mechanism of the cells salt tolerance. The mai n results are summarized as follows:1. Stem segments are better than leaves in inducing callus. The optimum combination to induce callus and establish N.tangutorum suspension cell lines is the MS medium containing 2,4-D 1.5 mg?L-1, NAA 0.2 mg?L-1 and 6-BA 0.4 mg?L-1 were chosen as the optimum combination to induce callus and establish N.tangutorum suspension cell lines. The best inoculation way is as follows: Add 7.5 mL dosage of mother liquor to a 100 mL conical flask with 30 mL MS; have it cultured on an orbital shaker(120 r?min-1) at 26~28 °C in the dark. The best time for the culture of the second generation cells is the 9th day upon inoculation. On the 3rd day of inoculation, the cells are most vigorous in metabolism when their light absorption value reaches 0.69. On the 7th day, their mitotic index reaches its maximum 5.1%, indicating that they are quite active in splitting. The survival rate in the lag phase and stationary phase decreases slightly faster than logarithmic phase, which, however, still remains between 84%~93%.2. Salt treatment can affect the growth of suspension cells. The low salt concentration(100 mmol?L-1) can accelerate their growth. They can reach their medium phase of growth at the fastest speed, and the maximum value of growth limitation(0.937 9 mg?mL-1). However, high salt concentration(≥150 mmol?L-1) will inhibit the cells’ growth, the time to reach the medium phase of its fast growth will be delayed, and the growth limit value decreased. It can be seen from the cells growth critical concentration(C50) that the cell salt tolerance value of N.tangutorum suspension cells an reach as high as 227 mmol?L-1, indicating they have relatively high salt tolerance.3. N.tangutorum suspension cells can adapt to the salt stress environment by changing the content of osmoregulation substances and the activity of antioxidant enzymes. At low salt concentration(100 mmol?L-1), the content of soluble sugar, soluble protein and proline was higher than that in the control group, and show increase with the increase of the incubation time. While at the high salt concentration(≥150 mmol?L-1), the content will first show the tendency of increase and then decrease. Moreover, the higher of the salt concentration, the earlier can they reach the maximum value, the soluble sugar in particular. In 100 mmol?L-1 treatment group, the content of MDA shows an increasing trend.While in 150、200、250 mmol?L-1 treatment group, the contents show the increasing trend first and then gradually tend to balance. Meanwhile, the tendency of the activity of SOD and POD is basically the same as that of MDA, that is, antioxidase repair rate keeps side by side with membrance oxisidase rate. The activity of SOD reaches its maximum value earlier than that of POD, which is the first defense line for antioxidase regulation. It is obvious from the correlation analysis of physiological indexes under salt stress that the correlation between the fresh quality of cells and contents of soluble sugar and MDA are highly negative significant(P < 0.01) and negative significant(P < 0.05) respectively; while the correlation between the content of MDA and the activity of POD and SOD is highly positive significant(P<0.01);. the contents of soluble sugar, soluble protein and proline is a highly positive significant(P<0.01); the correlation between the content of osmotic adjustment substances and enzymes activity is a positive significant(P<0.01). All of them indicate that the N.tangutorum suspension cells can alleviate the osmotic stress and improve the activity of antioxidant enzymes through increasing soluble sugar to scavenge scavenge the grease of protection membrane of superoxide free radical. Moreover, the osmotic regulation and antioxidant regulation are being regulation carried out simultaneously and correlatively.4. Analysis of changes of N.tangutorum cells’ metabolic profiling under salt stress. By means of gas chromatography/time-of-flight mass spectrometry, a totals of 373 and 629 substance peaks have been detected from the aqueous phase and organic phase, respectively. Through construction of partial least squares–discriminant analysis model, statistics analysis and database search, a total of 25 metabolites bearing potential biomarkers have been selected and identified, including Fructose, Galactose, Glucose, Xylopyranose, Mannose, Proline, Alanine, Valine, Malic acid, Succinic acid, Benzoic acid, Ethanedioic acid, Benzoic acid, Benzenedicarboxylic acid, Butanoic acid, Azelaic acid, Butanediamine, Acetamide, Hexanediamine, Octadecadienoic acid, Octadecanoic acid, Hexadecanoic acid, Linolenic acid, Eicosane, Dodecane and Sitosterol.5. Based on the analysis of N.tangutorum cells major metabolite composition and relative content under salt stress, the main metabolic pathways of their responding the salt-stress have been constructed, including carbohydrate metabolism, amino acid metabolism, fatty acid metabolism, tricarboxylic acid cycle, and so on. Carbohydrate metabolism is active under stress and the glucose, galactose, and mannose tend to rise while those of fructose and the xylopyranose tend to fall. Amino acid metabolism tends to be active and the content of proline, alanine and valine tends to rise, the proline in particular. With 250 mmol?L-1 treatment, the content of proline is about 100 times than that of control group. Fatty acid metabolism tends to speed up with the rise of the contents of octadecadienoic acid, octadecanoic acid, hexadecanoic acid, which can reach the maximum value under 100 mmol?L-1 treatment. The organic acids metabolism tends to be relatively stable, the relative contents of malic acid, succinic acid, benzoic acid, ethanedioic acid, benzenedicarboxylic acid remain almost unchanged or little rise. Therefore, N.tangutorum cells enhance the salt resistance ability by regulating the metabolism of carbohydrate, amino acids, organic acids, fatty acids and other secondary metabolites synergistically.In conclusion, this study has established the N.tangutorum suspension cell system, analyzed the growth and physiological indicators of the cells under salt stress, and thus determined the cell’ salt tolerance value; a total of twenty-five N.tangutorum cells’ potential salt-tolerant biomarker metabolites have been selected, and thus explained the metabolic mechanism of the N.tangutorum cells for their adaptation to salt stress. The results of our research will provide excellent materials and theoretical foundation for further study on the mechanism of Nitraria sal-tolerance, selection of new salt-tolerant plant species as well as exploring new salt-tolerant of approaches.