Identification And Proteomics Analysis Of Salt-tolerant Mutants Of Raspberry

Raspberry is the "third-generation" fruit trees in China,it is characterized by rich nutrition and high economic value.The black raspberry "Kaiou" has features with high yield and big fruit size,but not suited to saline-alkali areas.In order to improve the ability to adapt to saline soil and expand the planting area,the raspberry loose callus has been subjected to chemical mutagenesis(0.15% EMS),and use high Na Cl(2%)condition to screened cells which preform salt-tolerance.Eight salt-tolerant mutants were obtained by regeneration of somatic embryos.The growth indexes of 8 salt-tolerant mutant plants of raspberry and control group were measured.It was found that the leaf area of the No.9 mutant(2.93cm2)was highly significantly smaller than the control group(9.6cm2),and the petiole length(2.22 cm)was also significantly shorter than that of the control(3.00 cm).There were no significant differences in other groups.Electron microscopic observation of pollen grains no changes in shape between mutant plants with control group(No.9,10,11,12).Through the RAPD polymorphism analysis,we found that all of the mutant plants exhibit genetically altered.Control group used 13 pairs of effective primers amplified 122 bands in total.In contrast,No.1 mutant added 21 bands,reduced 14;No.2 added 26,reduced 9;No.8 added 23,reduced 14;No.9 added 11,reduced 13;No.11 added 10,reduced 20;No.12 added 15,reduced 28;No.13 added 18,reduced 13.A two-dimensional electrophoresis system with good reproducibility was established,and the optimization of the raspberry leaf tissue was carried out on the focusing time,focusing voltage,salt bridge(filter paper)and loading amount in IEF program.The final procedure is: 10000 V,8h,replace the salt bridge,the amount of 0.5mg.In order to analyze the proteomics changes of salt-tolerant plants,leaf tissus was used to compared the differences in proteomics between the No.9 mutant and the control.The results showed that:1.Find a total of 30 protein spots,including 20 upregulated spots and 10 downregulated spots.And 26 protein spots were successfully identified by mass spectrometry,16 spots were photosynthetic related proteins(diphosphate ribulose carboxylase,diphosphate ribulose carboxylase activating enzyme,phosphoketanokinase,light system II protein complex,light-harvesting pigment a / b binding Protein);3 spots were energy-producing protein(ATP synthase);2 spots were nitrogen metabolism-related protein(glutamine synthetase);and the growth and shape control proteins(actin),plant allergic reactions(2-cys peroxidoxin),enzyme activating factor(14-3-3 protein),molecular chaperone protein(Cpn7 protein)were all one spots.2.26 spots were mapped to Arabidopsis protein database.21 spots were successfully mapped,including 14 upregulated spots and 7 downregulated spots.These spots are annotated by Gene Ontology.The results showed that the upregulated protein was mainly existed in the chloroplast thylakoid and involved in the biological processes such as photosynthesis,pathogen invasion and cold response,and had ATP binding activity,RNA polyuridine binding activity,chlorophyll binding activity.Downregulated protein existed in chloroplast matrix,involved in cytokinin response and calcium ion response,with protein binding activity and calcium ion binding activity.KEGG pathway analysis showed that the upregulated protein was involved in the polysaccharide degradation pathway,and the downregulated protein was mainly involved in the metabolic pathway of glycerophosphate.3.Find the genes corresponding to 21 Arabidopsis mapping proteins.The protein-related genes were PSBO2,CAB3,RCA,LHCB6,PSBP-1,CYB561,FIB4,2-Cys Prx B and psb C.The down-regulated genes were PRK,CPN20,GS2 and ACT7.In summary,it is concluded that the response of plant No.9 to salt stress is to increase the stability of photosynthetic system,enhance the osmotic adjustment ability,combine with pathogen invasion,cell signal transduction and the change of two kinds of multi-modulating factors.These process are associated with the up-expression of RCA,CAB3,PSBP-1,2-Cys Prx B and other genes that can respond to pathogen invasion and cold,and the down-expression of PRK and GS2 genes in response to cytokinin.