Screening Of High Starch Duckweed By Heavy-ion Irradiation And Its Molecular Mechanism

Due to its superior characteristics,such as high relative biological effectiveness,wide spectrum of mutation,high mutation efficiency and the lower recovery rate of DNA damages,induced heavy-ion irradiation mutagenesis has been utilized as an efficient method in plant breeding.It has become an important and effective technique for the improvement and breeding of new crop varieties.Besides of the generation of novel crop varieties,mutants generated by heavy-ion radiation can also be used for gene functional analysis in forward genetics studies,thus greatly promoting functional genomics studies in plants.Due to the characteristic of rapid growth and high starch content,duckweed has been considered as an ideal feedstock for bioethanol production.In this study,we employed the starchy energy plant duckweed in heavy ion radiation.The callus was irradiated with heavy-ion,and the half lethality rates and mutation rates were calculated,and the optimum irradiation conditions were determined.Based on the establishment of heavy-ion radiation mutant library of duckweed callus?more than 2000mutants?,we screened a high starch mutant.Furthermore,the mechanism of the high starch mutants was analyzed,and its growth photosynthesis and starch accumulation characteristics were measured.Finally,the effects and mechanism of heavy-ion mutation were analyzed by transcriptomics and other techniques,in order to lay the foundation for the breeding of duckweed by heavy-ion radiation mutagenesis,and provide new germplasm of high starch duckweed.The main results obtained are as follows:?1?Duckweed heavy-ion mutagenesis conditions was optimized.The results showed that with the increase of irradiation dose,the lethal rate of duckweed callus increased.The duckweed callus had a lower lethal rate under 20 Gy and its semi-lethal irradiation dose was about 50 Gy.In addition,we found that special phenotypes such as larger leaves,leaf folds,thicker roots and root entanglement appeared in regenerative plants of duckweed after heavy-ion radiation.?2?After large scale screening of high starch mutants by staining I₂-KI with 2 000duckweed regeneration plants,55 duckweed mutants were considered as candidate high starch mutants.Furthermore,the dry-to-wet weight ratio of the candidate high starch mutants was determined.The results showed that 15 mutants appeared with higher dry-to-wet weight ratio compared to the wide type.Subsequently,the starch content of15 duckweed mutants was determined and the starch content of 5 duckweed mutants was significantly higher than that of the wild type.Finally,the starch content and growth curve of five duckweed mutants were measured,and one high starch duckweed mutant?HS?was obtained with higher starch content and similar biomass accumulation compared to the wild type.?3?The growth curve of high starch duckweed mutant HS showed that the biomass of HS was not significantly different from that of the wild type,and the starch content of HS was significantly higher than that of the wild type,which was 2.57 times that of the wild type.Calculating starch yield by combining biomass and starch content,the results showed that the starch yield of HS was significantly higher than that of the wild type,which was twice that of the wild type.In addition,it was also found that the leaves of high starch duckweed HS were smaller than the wild type.?4?Nitrogen deficiency could induce starch accumulation in duckweed.The high starch mutant HS and the wild type strains were subjected to nitrogen deficiency treatment.The results showed that the starch content of the high starch mutant was significantly higher than that of the wild type after nitrogen deficiency treatment.Starch granules of the wild type strains and the high starch mutants after 0,1,3 and 5 days of nitrogen deficiency was observed by transmission electron microscope.The results showed that the high starch mutant HS and the wild type starch granules were larger than those of the untreated ones after nitrogen deficiency treatment,and the number of starch granules increased significantly with prolonged nitrogen deficiency.Further,at the same condition,the starch granules of the high starch mutant was larger than the wild type,and the number of starch granules was significantly more than that of the wild type.Further,we determined the maximum photochemical efficiency of PSII?Fv/Fm?and pigment content after nitrogen deficiency treatment.The results showed that the pigment content of the high starch mutants was higher than that of the wild type.The Fv/Fm of the wild type strains decreased significantly with the prolongation of nitrogen deficiency time while the high starch mutant maintained a high level.In addition,the Fv/Fm of the high starch mutant was lower than the wild type in the early stage of nitrogen deficiency,and higher than the wild type in the late stage during nitrogen deficiency.?5?To reveal the molecular mechanism of starch accumulation of the high starch mutant HS,we used next-generation sequencing technology to examine the transcriptome responses of HS and the wild type at three stages?0,1 and 5 days?after nitrogen deficiency.A total of 116.30 Gb of Clean Bases and a total of 47 616 Unigene were obtained.There were 8 437 differentially expressed genes,which were significantly enriched to 421 GO terms.In addition,9 Pathways were significantly enriched.In these differentially expressed genes,91 genes were involved in the starch metabolism of the high starch mutant HS.Differentially expressed genes enrichment analysis results showed that photosynthetic light-harvesting antenna-related proteins,photosynthesis,carbon fixation,monoterpenoid biosynthesis,phenylpropanin metabolism,biotin metabolism,plant-pathogen interaction,circadian rhythm,glyoxylate and dicarboxylate metabolism in high starch mutants significantly enrich.In order to verify the transcriptome sequencing results,we performed a Real-time PCR verification of genes that differ in photosynthesis and starch metabolism pathways.The results were consistent with the transcriptome sequencing.The photosynthetic light-harvesting protein and photosynthetic pathway related proteins of the high starch mutants were significantly different from the wild type strains.The starch metabolism pathway results showed that the high starch mutants had significant differences in the starch degradation pathway compared with the wild type strains.And?-amylase,?-amylase,starch phosphorylase and?-glucosidase gene expressions were significantly lower than the wild type.Finally,we measured the activity of?-amylase and?-amylase,and the results were consistent with the results of Real-time PCR and transcriptome sequencing.The activity of?-amylase and?-amylase in the high starch mutants was significantly lower than that of the wild type.