| Aralia elata Seem.(Miq.),is a kind of common wild vegetable from Araliaceae.It possess high value of nutrition and health care.In recent years,due to the increase of social demand and the continuous decrease of wild resources,artificial cultivation is still in the in itial stage,thus the yield can not meet the market demand.In addition,because of the slow natural variation,Aralia elata Seem.(Miq.)cultivars are relatively single,and germplasm resources shortage;Furthermore,the Aralia elata Seem.(Miq.)has many spines,which bring great difficulties for the artificial collection of it.Therefore,carrying out innovative research on germplasm resources,cultivating new varieties with high yield and high quality,researching and establishing cocmercialized breeding base and technical system of Aralia elata Seem.(Miq.)have become urgent problems to be solved in the development and utilization of Aralia elata Seem.(Miq.),which is of great significance for the further development and utilization of Aralia elata Seem.(Miq.)resources.In this study,the germplasm resources of Aralia elata Seem.(Miq.)were created with the method of artificial mutagenesis,so as to establish the technical system of Aralia elata Seem.(Miq.)mutagenesis,construct the mutant pool of Aralia elata Seem.(Miq.),screen out the mutant with high yield and high quality or some special quality,and provide the excellent germplasm resources for the breeding of wild vegetables.At the same time,the study on the molecular mechanism of acquiring mutants by artificial mutagenesis is helpful to provide theoretical basis for the innovation of germplasm resources of Aralia elata Seem.(Miq.),and to provide reference for related studies on artificial mutagenesis.The main research results are as follows:Sodium azide,EMS and 60Co-gamma rays were used to treat Aralia elata Seem.(Miq.),and mutant libraries were established.A total of 147 mutants of various types were obtained.Albino,yellowing and purple leaf mutants were obtained in Aralia elata Seem.(Miq.)treated with sodium azide and EMS,with mutation rates of 1.1% and 1.6% respectively,and yellowing and purple leaf mutants were obtained by 60Co-gamma ray treatment with mutation rates of 10%.Mutants with spiny and sparse spines were obtained by sodium azide,EMS and 60Co-gamma ray treatments.The mutation rates of sodium azide and EMS treatments were lower,about 1.1%,and those of 60Co-gamma ray treatments were higher,about 6%.Purple stem mutants were obtained by sodium azide,EMS and 60Co-gamma ray treatment.The mutation rates of sodium azide and EMS treatment were 0.6% and 0.9%,respectively.The mutation rates of 60Co-gamma ray treatment were 2%.Mutants with fast growth rate and slow growth rate were obtained by sodium azide and EMS,with an average mutation rate of 1.33%,and mutants with slow growth rate were obtained by 60Co-gamma ray treatment,with mutation rate of 8%.Different concentrations of sodium azide have different effects on Aralia elata Seem.(Miq.)mutants.When the concentration was 0.05%,no leaf mutant,thorn mutant and purple stem mutant appeared,but the mutant with fast growth rate appeared.Leaf mutants,s pine number mutants and purple stem mutants all appeared when the concentration was above 0.2%;Leaf Mutants and less spine mutants had higher mutation rate when the concentration was 0.2%;purple stem mutants and growth rate mutants had higher mutation rate when the concentration was 0.8%.Different concentrations of EMS treatment had different effects on Aralia elata Seem.(Miq.)mutants.At the concentration of 0.05%,there were no leaf mutants,thorn number mutants and purple stem mutants,but there were growth rate mutants.When the concentration was above 0.1%,leaf mutant,thorn mutant,purple stem mutant and growth rate mutant appeared,and the higher the concentration,the higher the mutation rate.There were no leaf mutants,thorn mutants and purple stem mutants in Aralia elata Seem.(Miq.)treated with sodium azide on seeds,but there were growth rate mutants.There were leaf mutants,thorn mutants,purple stem mutants and growth rate mutants after treatment of tender buds,young roots and whole pla nt,and there was no significant difference in mutation rate among them.There were no leaf mutants,spine mutants and purple stem mutants in Aralia elata Seem.(Miq.)treated with EMS on seeds,but there were growth rate mutants.The mutation rates of leaf mutants and spine mutants obtained from young roots and whole plants were higher,while there was no significant difference between purple stem mutants and growth rate mutants.There were 147 surviving Aralia elata Seem.(Miq.)mutants were obtained by sodium azide,EMS and 60Co-gamma ray treatment.The nutrient content of tender buds of 11 high-quality mutants with fast growth,large leaves and few thorns was analyzed.It was found that the mutants with the highest saponin content were T4,T6 and T1;the mutants with the highest flavonoid content were control(wild type),T4 and T5;the polysaccharide content was T6,T7 and T10;and the soluble sugar content was T2,T5,T6 and T8,respectively.The ones with more pr otein are T4,T5 and T6.T4 is the most abundant with saponin and flavone.T4 and T6 are the most abundant with saponin and protein,and T5 mutant is the least prickle.Spinose Aralia elata Seem.(Miq.)(group C)was as the control group and less prickles o f Aralia elata Seem.(Miq.)(TW)as the experimental group for transcriptome analysis,in the experimental group,there were 3353 genes were up-regulated expressions and 2,187 down-regulated.According to KEGG analysis,among the top 20 pathways with the most enrichment of differentially expressed genes,phenylpropane biosynthesis,plant signal transduction and protein processing had the most gene enrichment,followed by synaptic vesicle circulation,antigen processing,calcium signal and other genes.In ad dition,genes involved in the biosynthesis of terpenoids,alkaloids and flavonoids were also enriched.There were 35 differentially expressed genes involved in terpenoid metabolism,21 were down-regulated and 14 were up-regulated in the less spines group.Some were only expressed in group C(Spinose),such as citrate synthase,sesquiterpene synthase and terpene synthase.There were 7 differentially expressed genes involved in lignin biosynthesis,and these 7 differentially expressed genes were down-regulated in the less prickles group(TW).There were 4 differentially expressed genes related to cell wall,two of which were cell wall receptor kinases and the other two were cellulose synthases.All the four genes are down-regulated compared to the spinose Aralia elata Seem.(Miq.)group.There were 9 differentially expressed genes involved in flavonoid metabolism,all of which were down-regulated in the TW group.The flavonol 3-sulfonyl transferase gene was only expressed in group C.There were 5 differentially expressed genes involved in phenylalanine metabolism,3 of which were up-regulated,encoding branching acid mutase and branching acid synthase.Two of then were down-regulated,encoding(3S,6E)-enol synthase and 5-enol-pyruvyl shikimate-phosphate synthase.And one of the chorismate mutase and chorismate synthase were only expressed in the TW group.Among the 21 differentially expressed genes encoding cytochrome P450,6 were down-regulated and 15 were up-regulated.The up-regulated expression was far more than the down-regulated expression.There were 11 differentially expressed genes involved in photosynthesis,among which 2 were down-regulated and 9 were up-regulated.There were 7 differentially expressed genes involved in ethylene biosynthesis,3 were down-regulated and 4 were up-regulated.There were 8 differentially expressed genes involved in ethylene signal transduction,all of which were up-regulated.There were 12 differentially expressed genes involved in auxin signal transduction pathway,of whi ch 1 was down-regulated and 11 were up-regulated.Two differentially expressed genes involved in gibberellin biosynthesis and its regulation were up-regulated.The differentially expressed genes involved in the biosynthesis of jasmonic acid and the biodegradation of abscisic acid were 1 only,and their expressions were up-regulated.A total of 154,803 second-order mass spectra were obtained by proteomics analysis.A total of 2013 proteins were identified,and 352 differentially expressed proteins were found,.Among them,217 were up-regulated and 135 were down-regulated in the TW group(less spines)compared with the control group(C,more spines).Most of the differentially expressed proteins were concentrated in the metabolic pathway,followed by biosynthesis of secondary metabolites.Proteins involved in photosynthesis,carbon metabolism,amino acid biosynthesis,phenylpropane metabolism and terpene metabolism were also significantly enriched.Among these differentially expressed proteins,77 differentially expressed proteins were involved in the primary metabolism of Aralia elata Seem.(Miq.),including 11 proteins involved in amino acid metabolism,52 proteins involved in photosynthesis,10 proteins involved in sugar metabolism,and 4 proteins involved in lipid metabolism.Among the 11 differentially expressed amino acid metabolic proteins,9 were up-regulated and 2 were down-regulated.There were 52 proteins were involved in photosynthesis,only one(subunit of rubisco)was down-regulated,while the other 51 were up-regulated.There were 50% of the differentially expressed proteins involved in glucose metabolism were up-regulated,while the differentially expressed proteins involved in fat metabolism were up-regulated.Participating in the difference of secondary metabolism proteins are mainly involved in secondary metabolites biosynthesis and degradation,such as phenolic compounds,nucleoside substance,flavonoids,lignin,alkaloids,saponins,and terpenoid substances.All kinds of expression level of substance metabolism is not the same,which involved in the biosynthesis of terpenoids substances are mostly lower expression of genes.The differentially expressed proteins involved in the regulation of metabolic processes include proteins with functions suc h as antioxidant enzymes,auxin binding proteins and cell wall construction,all of which are up-regulated.The association analysis of transcriptomics and proteomics showed that 19 of them were down-regulated and 37 of them were up-regulated at both the transcription level and the protein expression level.Among them,photosynthesis-related genes(proteins)were significantly up-regulated.In terms of secondary metabolites,the expressions of sandalwood terpene synthase and berberine pontase were significantly up-regulated in the presence of different levels of terpenoids and alkaloid genes(proteins).The expression of beta-amyrin 28-oxidase-like and Furostanol glycoside 26-O-beta-glucosidase were both down-regulated.SNP analysis of genes involved in spine formation,secondary metabolite biosynthesis and gene expression regulation showed that 625 sites of 52 genes had base mutations.The types of mutations were G-A,C-T,T-C,A-G,T-A,G,G-A,C and 17 forms,of which C-T mutations accounted for 17.28% of the total sites,followed by A-G and G-A mutations,15.52% and 14.72%.The number of mutation sites in each gene is different,the least is 1,such as 4-coumaric acid Co A ligase(TRINITY_DN22459_c0_g1)and flavonol 3-sulfosyltransferase(flavonol 3-sulfosyltransferase);the most is WRKY transcription factor 16(TRINITY_DN22768_c1_g1),with 82 mutation sites;the second is 3-hydroxy-3-methylglutaric coenzyme A synthase(TRINITY_DN23524_c0_g1).44 mutation sites,but the relationship between base mutation and gene function needs further study.In conclusion,the leaf mutants of albino,yellowing and purple leaves,the number mutants of spines with more spines and less spines,the purple stem mutants and the growth rate mutants of Aralia elata Seem.(Miq.)with 60Co-gamma rays can be induced.The mutation rate of sodium azide and EMS is low,about 1%-2%,and that of 60Co-gamma ray treatment is high,reaching 10%.Four mutant libraries including leaf mutant,thorn mutant,stem mutant and growth rate mutant were established.A total of 147 mutants were found.Ten mutants with fast growth,high nutrient content and few thorns were screened out.Transcriptional combinational proteomics analysis of the mutant with less terpenoids showed that the up-regulated expression of genes involved in the metabolism of terpenoids was more than that of the mutant with less terpenoids.The genes involved in lignin biosynthesis and cell wall were down-regulated.The genes involved in flavonoid metabolism were downregulated.The genes involved in auxin signal transduction and ethylene signal transduction were up-regulated.Genes involved in photosynthesis are basically up-regulated.The molecular mechanism of the less spines mutant and nutrient content change were preliminarily clarified,which is of great significance to the innovation and development of Aralia elata Seem.(Miq.)germplasm resources in eastern Liaoning. |
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