Mutagenesis,screening,Physiological And Biochemical Characteristics And Omics Analysis Of Pyropia Yezoensis Mutant

Pyropia yezoensis grow in the intertidal zone,which are mainly distributed in the western North Pacific Ocean.They are important economic macroalgae.Py.yezoensis industry has high economic benefits and broad market prospects.However,there are some restrictive factors,such as long growth cycle,high cost,germplasm degradation and frequent occurrence of diseases.These factors have impeded the development of Py.yezoensis industry.The effective method of solving these problems is breeding,which is screening new varieties with excellent characteristics.In this paper,a mutant strain of E was obtained by chemical mutagenesis of free living conchocelis using pure line NA of Py.yezoensis as an initial strain.The physiological and biochemical characteristics,omics of mutant strain E in different generations including conchocelis and thalli were studied.Besides,breeding and cultivation were conducted in nori field.The main research results were as follows:?1?The physiological and biochemical characteristics of mutant E and initial strain NA filaments.In morphology,there was no significant differences between mutant E and NA filaments under the microscope.But at the same temperature,especially at18?,the filaments of mutant E were easier to develop into conchosporangial branches than the initial strain NA.The conchosporangial branches formed at 18?were stimulated by lowering temperature and shortening light time to release conchospores.Results showed that the conchosporangial branches released conchospores,and the release lasted over 9 days.Besides,the releasing peak appeared on the 5th⁷th d.Among them,there were more conchospores at 16?,up to?3.15±0.10?×10⁷ g^-1.In addition,the drilling rate of E?21.33%±5.33%?was lower than that of NA?60.44%±3.08%?.This indicated that it was more difficult for E filaments to drill into shells than NA.The culture conditions of mutant strain E filaments were investigated using the proportion of conchosporangial branches in total filaments as the parameter.Results showed that the optimum culture conditions of mutant E filaments were 16?,20?mol photons m^-2s^-1 white light and 25‰³0‰salinity.?2?Comparative analysis of transcriptome between the mutant strain E and initial strain NA conchocelis.A total of 26,949 differentially expressed genes were obtained,including 11,948 up-regulated and 15,001 down-regulated genes.Results showed that respiration increased in the mutant strain E,which might provide energy for the development of filaments.The signal transduction mediated by the second messenger decreased,but the hormone signal transduction mediated by ubiquitin-proteasome pathway increased.This suggested that hormones might play an important role in conchosporangial branches formation of strain E.Other up-regulated proteins,such as cytoskeleton,transcriptional related proteins and amino acid synthesis related proteins,might provide support for the development of mutant strain E filaments?the formation of conchosporangial branches?.?3?The physiological and biochemical characteristics of the mutant E and initial strain NA blades.Under the microscope,there was no significant differences between the young blades of E and NA??10 mm?.The growth rate of young blades of mutant E(0.79±0.05 mm d^-1)was higher than that of the initial strain NA(0.26±0.06 mm d^-1).At the same time,the increment of the young E blades?62.50±12.11?was greater than that of strain NA?8.00±5.15?,which indicated that the mutant E could release more monospores under the same conditions.Taking photosynthetic activity as a parameter,the culture conditions of young blades of mutant E were studied.The optimum culture conditions of mutant E young blades were 16?,60⁸0?mol photons m^-2s^-1 white light,30‰of salinity and 3:1 of C/N ratio.Then the young blades?¹0 mm?were transferred to 10?.After 4 wks,they grew into large blades.The blades were slender,and there was no significant differences between the two strains both in surface and longitudinal view.Compared with NA,mutant E had the advantage of growth.After 4 wks,the length of E blade was 3.5 times that of NA,and the weight of E blade was about 3 times that of NA.In addition,the results showed that the photosynthetic activity and some pigments?including?-carotene,?-carotene,phycoerythrin and phycocyanin?contents of E blades were higher than those of NA,but the cell respiration rate of E was lower than that of NA?p<0.05?.During the period of dehydration,Fv/Fm,Y?I?andY?II?of blades decreased.When absolute water content fell to 9%,the value of Y?II?was close to zero.After rehydration for 15 min,Y?I?increased rapidly,Fv/Fm and Y?II?recovered over half.After rehydration for 1 h,the activity of photosynthesis almost recovered.In the process,although some values of parameters of E were higher than NA,the trends were similar.?4?Comparative analysis of transcriptome and proteome were conducted between the mutant strain E and initial strain NA blades.The transcriptomic analysis obtained1,549 differentially expressed genes,including 657 up-regulated genes and 892 down-regulated genes.Proteomic analysis identified 41 differentially expressed proteins,including 22 up-regulated proteins and 19 down-regulated proteins.The integrated analysis of differentially expressed genes and proteins showed the early phase of carbohydrate catabolism enhanced in mutant strain E,such as glycolysis,pentose phosphate pathway and pyruvate degradation process.That suggested the content of acetyl coenzyme A increased.The electron transporter decreased in the respiratory chain,and the respiration rate decreased in mutant strain E.This suggested the respiration decreased in mutant strain E.The decrease of respiration indicated the increased acetyl coenzyme A did not enter the three carboxylic acid cycle for further decomposition.The possible reason was that acetyl coenzyme A participated into the synthesis of fatty acid as a precursor.In addition,some genes and proteins related to anabolism including photosynthesis and protein synthesis were up-regulated.Besides,values of photosynthesis parameters and some pigments contents increased.That suggested that anabolism such as photosynthesis and protein synthesis enhanced in mutant strain E.The rapid growth of mutant E blades might be due to the decrease of catabolism and the increase of anabolism.Moreover,the increased expression of proteins associated with resistance to stress,such as heat shock protein and antioxidants,indicated that the E blades might have the potential of high stress resistance.?5?The free living conchocelis of mutant strain E and initial strain NA were cut and sprinkled on surface of shells,and the density was 600 segments/cm².At the early stage of growth,the culture conditions were 18.5²0?,the highest light intensity was less than 4000 lx,salinity 24.8²6.5‰.Then the culture temperature increased to23²6?,the highest light intensity was less than 2500 lx,and the salinity was24.8²6.5‰.In the process,the filaments developed into sporangial branches,which released conchospores after maturation.Results showed that the total amount of conchospores released by mutant strain E was larger than that of NA,and the maximum amount could reach 2.40±0.21 million per shell.?6?The conchospores of mutant strain E and NA were collected by splashing water on the net,and two nets were used for each sample.The nets with conchospores were cultured both in Wendeng,Weihai?36°47'54''N 121°58'03''E?and in the intertidal zone of Lanshan Port,Rizhao?35°5'24''N 119°18'00''E?.Both strains grew well in Weihai,The mean length and width of E blades and NA blades had no significant differences.The results of photosynthetic activity showed that the Fv/Fm,Y?I?,Y?II?and contents of most photosynthetic pigments?including zeaxanthin,chlorophyll a,beta-carotene,phycoerythrin and phycocyanin?in the mutant E blades were higher than those of NA?p<0.05?.Besides,the density of the experimental blades was higher than the others in nori field,because the experimental strains could release more monospores.The results of nutrient analysis showed that the blades of Py.yezoensis contained higher content of carbohydrates?40.8-41.0 g/100 g DW?and proteins?36.8-37.1 g/100 g DW?,followed by ash?18.3-19.3 g/100 g DW?,and a small amount of crude fiber?3.5 g/100 g DW?and fat?1.2-1.6 g/100 g DW?.Compared with the initial strain NA,the content of carbohydrate,protein and fat in the blades of mutant E were higher,while the ash content was lower.Compared with three representative cereals?wheat,rice and soybean?,Py.yezoensis was a kind of green healthy food with higher protein and lower fat.In the blades,18 kinds of amino acids,including 8 kinds of essential amino acids and 10 kinds of nonessential amino acids,were determined by amino acid composition analysis.Methionine and cystine were the limiting amino acids.Except methionine and isoleucine,the contents of other amino acids?including 9 kinds of flavor amino acids?in the E blades were higher than those of NA.Among the flavor amino acids,the total amount of sweet and umami amino acids account for over 50%of the total amino acids detected.Fatty acid analysis showed that 14 kinds of fatty acids were detected,including 3 kinds of saturated fatty acids,6 kinds of monounsaturated fatty acids and 5kinds of polyunsaturated fatty acids.The contents of total fatty acids and mono-fatty acids in the mutant E blades were higher than those of NA.In Rizhao,the density,length,photosynthetic activity and the pigments content of the mutant E blades were higher than that of NA?p<0.05?.Water quality detection found that there was a lack of nitrogen and phosphorus in Rizhao filed,and a lack of nitrogen in Weihai filed.Under the nitrogen deficiency,the growth of Py.yezoensis cultivated in the two fileds were significantly different,which was possibly affected by different environment and culture methods.In the later stage of cultivation,the initial strain NA and other blades in nori filed appeared decay,while the mutant E had better growth,darker color and stronger tenacity.This indicated that mutant E had stronger ability to resist nutrition deficiency.In this study,the mutant strain E was selected by the mutagenesis of free living conchocelis.The conchocelis of mutant strain E easily developed into conchosporangial branches,and the blades of mutant strain E had higher growth rate than the initial strain NA.In addition,the changes of omics were analyzed between mutant E and initial strain NA both in conchocelies and blades.In mutant strain E conchocelis,the signal transduction mediated by the second messenger decreased,but the hormone signal transduction mediated by ubiquitin-proteasome pathway increased.This suggested that hormones might play an important role in conchosporangial branches formation of mutant strain E.In mutant strain E blades,the increase of anabolism and the decrease of the catabolism might provide support for the rapid growth of the mutant strain E blades.The omics data could provide theoretical basis for Py.yezoensis molecular breeding.In addition,the breeding and trial cultivation of mutant strain E were carried out in nori field,which accumulated practical experience for the artificial cultivation in the future.