Optimization Of Earthworm Peptide Extraction Process And Improvement Of Cold Tolerance Of Cucumber Seedlings

Low temperature is the main environmental factor limiting the geographical distribution and growth and development of plants.Plants often experience low temperature stress in autumn,winter and early spring,resulting in stunted growth and even wilting and death during the growth cycle,which seriously affects yield and quality and causes huge economic losses.As another new type of plant growth regulating substance in addition to the five major plant hormones,exogenous peptides play an important role in promoting plant growth,increasing crop yield,improving fruit quality and enhancing crop resistance.Therefore,it is important to study the effect of earthworm peptides on the cold resistance of plants and apply them to agricultural production.In this study,the extraction process of earthworm peptides was optimised by using the yield of earthworm peptides as an indicator,and their antioxidant activity and stability were measured.The main findings of the study are as follows:1.Using orthogonal tests to optimize the combination of four levels of temperature,pH,feedto-liquid ratio and enzymatic digestion time,the best extraction process of earthworm peptides was obtained:pH 7,feed-to-liquid ratio 1:1,enzymatic digestion time 5h and temperature 35℃.The highest yield of earthworm peptide was 4.01%,and the scavenging rate of DPPH·,·OH and O2-· radicals was determined by the determination of DPPH· and ·OH radicals,which showed a positive correlation with the concentration of earthworm peptide.The scavenging rate of O2-· showed a trend of increasing and then decreasing,with the highest value occurring at a peptide concentration of 0.4 mg/mL and a scavenging rate of 12.56%.Finally,the exclusion chromatograms and molecular mass distribution of earthworm peptides at different temperatures,pH values and UV lamp irradiation times were measured,and it was found that earthworm peptides showed good stability at different factor levels.2.The resistance indexes of cucumber seedlings were measured by spraying earthworm polypeptide at different temperatures and concentrations,and it can be concluded that under different temperature conditions,earthworm polypeptide can increase the content of photosynthetic colour,proline and soluble sugar in cucumber seedlings,thus improving the cold resistance of cucumber seedlings.The earthworm peptide reduced the malondialdehyde content in cucumber seedlings and alleviated the accumulation of malondialdehyde content in cucumber seedlings caused by low temperature stress,and also increased the activities of antioxidant enzymes(SOD,POD and CAT).3.The resistance indexes of cucumber seedlings were measured by low temperature(5℃)stress for 8h.It was found that earthworm peptide could alleviate the accumulation of MDA content in cucumber seedlings and significantly increase the content of photosynthetic pigments,SOD,POD and CAT.The GO and KEGG enrichment analysis of DEGs by transcriptome sequencing revealed that earthworm peptide induced mainly the enrichment of glutathione metabolic pathway.The genes related to glutathione were also found to be up-regulated under low temperature stress;transcription factors were screened by analysing the DNA binding domain information of the genes.By performing functional annotation,we obtained a family of transcription factors induced by earthworm peptides,mainly WRKY family,NAC family and MYB family,which are mainly associated with low temperature stress.Then,by protein interaction network(PPI)analysis,two key genes,F0F1-ATP and psaA,induced by earthworm peptides were derived,indicating that F0F1-ATP and psaA play a decisive role in association with other proteins.Finally,by qRT-PCR analysis,the trend was found to be consistent with the transcriptome sequencing trend results,which proved that the transcriptome sequencing results were reliable.