Effects Of StCYS1 Gene On Enzymatic Browning And Salt Tolerance Of Potato

Protease inhibitors?proteinaseinhibitors,PIs?are widely found in animals,plants and microorganisms.Cystatin?CYS?is mainly involved in seed germination,plant growth and development,programmed cell death and response to biotic and abiotic stresses.CYS of Potato?Solanum tuberosum L.?is a small molecular weight soluble protein existing in its tubers and plants.at present,its research is mainly focused on the response and regulation of potato stress.In the early stage of the laboratory,through RNA-Seq analysis,it was found that CYS may be involved in the regulation of enzymatic browning of fresh-cut potatoes,but the transgenic function has not been verified;the expression of CYS in E.coli?Escherichiacoli,E.coli?can improve the salt tolerance of bacteria.In this experiment,diploid potato"MDS"overexpressing CYS gene StCYS1 was used to verify its effect of inhibiting browning and salt tolerance,and to explore the mechanism of inhibiting tuber browning and improving salt tolerance.In this study,using tetraploid potato"Desiree"as material,the gene editing vector of StCYS1 was successfully constructed by CRISPR/Cas9 technique.The purpose of this study is to explore whether it has the characteristic of enhanced browning by obtaining potato StCYS1 mutant plants,in order to lay a foundation for further research.The main results are as follows:?1?Effect of overexpression of StCYS1 on enzymatic browning of diploid potato tubers.The browning degree of potato tuber slurry of three lines OE1,OE2 and OE3 was significantly lower than that of wild type WT at 0 h,1 h,2 h and 3 h at room temperature.The contents of PPO activity,ABTS,DPPH free radical scavenging ability,iron reduction ability of FRAP,and 16 kinds of single free amino acids in wild type and overexpression lines were determined respectively.The results showed that overexpression StCYS1 significantly increased PPO activity,indicating that PPO activity was not the main reason for reducing browning of overexpression lines,but overexpression StCYS1 significantly improved the total antioxidant capacity of potato.Overexpression of StCYS1 significantly decreased the content of total free amino acids and tyrosine,the main substrate of potato tuber browning.The results showed that overexpression of StCYS1 could reduce browning by increasing antioxidant capacity and reducing total free amino acid content and tyrosine content of potato.?2?Effect of overexpression of StCYS1 on salt tolerance of diploid potato plants.The potted seedlings of WT and overexpression lines grew for 50 days.Under the condition of continuous treatment with 1%NaCl for 7 days,the leaves of WT lines showed yellowing,water loss and wilting from the 5th day,and OE1-3 lines could still maintain a good growth state.The chlorophyll SPAD value of 0,1,3,5 and 7 days of treatment and the chlorophyll content of 7 days were determined.The results showed that the chlorophyll degradation of OE1-3 strain was significantly lower than that of wild type.The results of determination of H₂O₂,MDA,relative conductivity and proline content in plant leaves showed that the accumulation of H₂O₂ and MDA of OE1-3 strain was significantly lower than that of wild type,and the relative conductivity was significantly lower than that of wild type,while the accumulation of proline was significantly higher than that of wild type.The results showed that the overexpression of StCYS1 could improve the salt resistance of plants by slowing down the chlorophyll decomposition,maintaining a high chlorophyll level,and improving the ability of plants to resist oxidative stress and osmotic stress.?3?The single target gene editing vector of StCYS1 was constructed by CRISPR/Cas9technique.Three first generation heterozygous mutant positive plants were obtained by infecting potato leaves and stems by Agrobacterium tumefaciens transformation.The mutation types were single base deletion?target sequence 14 base C?and single base substitution?target sequence 13 base mutation from C to T?,homozygous mutant was not found.The results showed that the CRISPR system was suitable for potato gene editing and could lead to deletion mutation of potato genome.The detection of StCYS1 gene expression of3 positive plants showed that the expression of StCYS1 was not significantly affected,and there was no significant difference in tuber browning between 3 positive plants and WT.The results showed that the single base deletion and heterozygous mutation caused by single base substitution of two alleles in StCYS1 had no significant effect on potato chip browning.