Study On Biological Effects And Mutagenesis Breeding Of Salvia Miltiorrhiza SP₁-1Induced By Spaceflight

In this paper, Space Danshen SP1-1was bred through the space inducing mutation via the Chinese seven manned spacecraft "Shen-Zhuo7". In order to investigate the mutagenic effects of space environment on Danshen, the comparative research of the botanical characteristics, resistance, DNA molecular marker, physiology and biochemistry, photo synthetic effect and yield, inherent quality and pharmacodynamics were studied between CK and SP1-1, which was lay the foundation for selecting and breeding high yield, superior quality and a strong resistance Danshen germplasm resources.The main results as follows:1. Study on botanical characteristics of SP1-1The botanical characteristics of root, stem, leaf and flower were observed between CK and SP1-1in this study, the main character of SP1-1is included dwarf aerial stem and thicker, stem section space shorter, leaf blade thickness, leaf surface collapse, leaf larger, dark green, root epidermal reddish-brown, the botanical characteristics differences of CK and SP1-1was more obvious. Common disease of root surveying showed that a lower incidence of root rot disease and root knot nematode disease were observed in SP1-1, the results showed that SP1-1had excellent botanical characters and stronger disease resistance to root disease, which was belong to an excellent Danshen germplasm resource.2. Study on root exudates between SP1-1and CKThe rhizosphere soil of CK and SP1-1were extracted with different solvents i.e. Hexane and ethyl acetate, the GC/MS technique was applied to analyze the root exudates, the results showed that the kinds and contents of root exudates in CK were significantly higher than SP1-1. The constituents of root exudates could inhibit the growth in continuous cropping obstacles and reduce the disease resistance of crops. Therefore, the difference root exudates between CK and SP1-1must affect the growth and disease resistance of Danshen, further research would study to investigate which components of root exudates played an important role to affect the the growth and disease resistance in the process of Danshen planting.3. Study on cytological effects and molecular marker of SP1-1The chromosome configuration of CK and SP1-1root tips were observed, as the results showed that the chromosome number was2n=16, Chromosome type was similar, but there were difference between the morphology and arm ratio.RAPD molecular marker technology was used to analyse the genetic diversity of CK and SP1-1, random RAPD primers A1,A13,A15amplified stable and reproducible differential fragments A1-450,A1-900,A13-700and A15-1100. The four differential fragments were cloned and sequenced, the sequence data has been submitted to GenBank to compare with BLAST tools, the BLAST analysis showed that partial sequence of A1-450and Al-900amplified from SP1-1has a certain similarity with SMCCR,SMCCR and SMGGPPS genes, which was the regulatory key enzyme genes of tanshinone and salvianolic acid biosynthesis pathway in Salvia miltiorrhiza. Partial sequence of A13-700and A15-1100has higher similarity with Boea hyqrometrica and Arabidopsis thaliana gene respectively.4. Analysis of soluble protein, CAT, POD and SOD isozyme of SP1-1Polyacryamide gel electrophoresis (PAGE) was used for analyzing the soluble protein, CAT, POD and SOD isozyme of CK and SP1-1. The result showed that, the number, Rf and expression level of soluble protein bands had difference comparing to CK, there are14bands in SP1-1and7bands in CK about soluble protein. there are2bands of CAT in SP1-1and CK, but expression level of No.2band in SP1-1was increased than CK; the number and expression level of POD isozyme in CK and SP1-1showed obvious differences, the band of Rf (0.325) in SP1-1and the band of Rf (0.075,0.542) in CK were lacked, the expression level of No.5,6,7bands in SP1-1were higher than CK.5. Study on the Correlation between photosynthetic characteristics and yield of SP1-1Study on photosynthetic showed that the total content of chlorophyll a,b, chl(a+b)and carotenoids of SP1-1was higher than CK in different growth stage. The content of chla in SP1-1was higher than that of CK in flowering and late-flowering stage significantly(P<0.05). The ratio of chla/b of SP1-1was lower than CK by increasing the content of chlorophyll b in SP1-1.The diurnal change of net photosynthetic rate (Pn) of SP1-1and CK showed a typical double peaked curves and significant midday depression of photosynthesis. The net photosynthetic rate (Pn) of SP1-1was higher than CK. For the intracellular CO2(Ci), their daily variation curves were shapes like letter "U". Responses of photosynthetic rate to light showed that the light compensation point (LCP,65.72μmol·m·s-1) and the light saturation point (LSP,754.13μmol·m-2·s-1) of SP1-1was higher than CK (LCP,51.36μmol·m-2·s-1; LSP,643.58μmol·m-2·s-1). The yield survey of showed that the yield of SP1-1increased about14%than CK, the yield increase is not significant difference between CK and SP1-1, however, SP1-1has a certain advantages and potential to increase yield.6. Study on evaluation of comprehensive quality of SP1-1In order to compare the difference of drug quality between CK and SP1-1, the content of salvianolic acid B, dihydrotanshinone I, tanshinone II A, petroleum extraction, polysaccharide and trace elements were measured in this study. The results showed that the contents of salvianolic acid B, tanshinone ⅡA and polysaccharide in SP1-1was increased by13%,50%and16%respectively compared with CK. The kinds of petroleum extraction were similar, but the relative content has obvious difference. Comprehensive analysis showed that the inherent quality of the SP1-1was superior to CK. In this study, HPLC fingerprint technology was developed to detect13batches Danshen samples from different places. The fingerprint data were managed by related statistical softwares. SP1-1had relatively distant relationship from CK groups, the result showed that the characteristics of chemical composition of SP1-1showed obvious differences compared to CK.7. Pharmacodynamic study of SP1-1in blood stasis ratsIn this study, the urgent blood stasis syndrome model of rats by injecting adrenaline was duplicated. The model groups all displayed status of concentration, viscosity, aggregation and coagulability. Compared with the model group, the decoction of CK and SP1-1could reduce plasma viscosity, whole blood middle, high shear viscosity(50/s,100/s,200/s), blood reduced whole blood low and high shear viscosity, index of erythrocyte rigidly significantly(P<0.05). At the same time, the decoction of CK and SP1-1could reduce the parameters such as whole blood viscosity (low sheers), index of erythrocyte aggregation, index of erythrocyte deformabilty, and there was no significant difference when they compared with the model group. Compared with CK group, SP1-1could reduce plasma viscosity, whole blood (middle, low) shear viscosity, the relative index of whole blood high and low shear, erythrocyte aggregation and erythrocyte rigidly in different degree.