Impact Of Carbon Mon Irradiation On Isatis Indigotica And Mechanism Research On An Indirubin Increasing Mutant

Heavy ion irradiation is one of new mutagenesis approaches for biological organisms,which can generate new phenotypes due to its higher linear energy transfer(LET)and relative biological effectiveness(RBE)compared to other ionizing radiations.Isatis indigotica is a commonly used traditional Chinese herb,whose leaves(da qing ye)and roots(ban lan gen)have antiviral and anticancer potencies,so it is significant to cultivate a new variety that has a high content of active ingredients.In order to evaluate the impact of heavy ion irradiation on physiological index,biochemical index,potency and molecular level of Isatis indigotica,gradient doses of carbon ions(irradiation doses:30Gy,60Gy,90Gy and 120Gy)were used to irradiate it,genetic stable materials after three generations were used to test the indexs,and an indirubin increasing mutant was successfully isolated.Indirubin,as a main effective ingredient of I.indigotica,was widely reported for its clinical application on anticancer activity.My dissertation also foused on studying the mechanistic details of this mutant on its evevated capability to synthesize indirubin.The major results can be summarized as follows:1.The impact of different irradiation dosages on physiological index,biochemical index and potency.My results showed that the seedling surviving rate and root fresh weight of I.indigotica were gradually decreased with enhanced irradiation intensity.But SOD,POD,MDA and free proline were altered differently.Compared to other dosages,30Gy exhibited the minimum inhibition of growth and maximum increase of main effective ingredients 4(3H)quinoxalinol and indirubin in roots,by 2.2 and 2.3 fold respectively.Therefore,30Gy is the best carbon ion irradiation dosage for I.indigotica mutation breeding.2.The impact of different irradiation dosages on DNA and total protein polymorphism,posttranslational modifications and stress-related pathways were also comprehensively evaluated.SRAP analysis results indicated that the degree of genome variation was increased with enhanced irradiation intensity.Compared to wide type,mutants recoved after using 30Gy had 33.59%polymorphic variations for DNA.SDS-PAGE gels stained by CBB,Pro-Q and Western blotted by ubiquitin antibody showed that total protein,phosphoprotein and ubiquitinated protein had been changed but they were not positively correlated with the irradiation intensity,which indicates that plants may had a compensatory mechanism for the damage caused by heavy ion irradiations.Before and after salt stress treatments,the expression levels of a stress response gene liLEA in the mutants were both up-regulated compared to WT.liSDD1,however,showed less change under cold stress.3.SSH was used to identify differentially expressed genes between WT and indirubin increasing mutant.Among many up-regualted genes,TSAI is closely related to indirubin biosynthesis.TSAI is a subunit of tryptophan synthase,which regulates the biosynthesis of indole derivative indirubin and plant hormone IAA.The full-length cDNA sequences of TSA1 and IAA19 were cloned by RACE.The phylogenetic analysis indicated that TSA1 from Isatis indigotica is most closely related to the one from Isatis tinctoria(98%identity at the amino acid level).Whereas IAA19 is most closely related to the one from Arabidopsis thaliana(94%).The TSA1 and IAA19 were also sequenced in the mutant,it showed no difference to WT.4.The auxin response gene IAA19 and IAA6 showed reduced sensitivity to IAA treatment in the mutant compared to WT,suggesting up-regulation of TSA1 may have caused the accumulation of both auxin and indirubin,both of which use tryptophan as their precursors.In order to verify this result,an I.indigotica regeneration system was established.Over-expression of TSA1 in WT I.indigotica indeed resulted in an increased indirubin production.