Metabolome And Transcriptome Analysis Of Three Successive Generations Of A.tuberosum Rottl.Ex Spreng.under Cutting Stress

In the natural environment,plants as sessile organisms can not avoid all kinds of adverse growth factors.In order to cope with changes and challenges,plants have evolved complex adaptive mechanisms.The study on the mechanism of adaptation to adversity is a hot spot in the current plant research,which has strong scientific value and practical significance.However,most of the researches on plant stress focus on single factor or single effect,there has not been much discussion of complex factors or repetitive stresses that are more complex and relevant.In this study,the response mechanism of three successive generations of stress treatment was analyzed by using metabonomics method,so as to accumulate experience for the following research.ObjectiveThe mechanism of three successive generations of A.tuberosum Rottl.ex Spreng.cutting stress response was studied from the point of view of metabolism,transcription and their joint analysis.Methods1.Conventional planting of A.tuberosum Rottl.and obtaining of stress materials.The pot-picking planting method was used to establish a stable and repeatable planting system of A.tuberosum Rottl.under the laboratory conditions,which could make A.tuberosum Rottl.grow well and obtain enough materials The stress degree,stress time,time point of clearing cutting,watering and fertilizing measures under three stress treatments of cutting,flooding+cutting,low temperature+flooding+cutting were investigated,the new long leaves of A.tuberosum Rottl.showed stable growth after several cycles of stress treatment,and three successive generations of A.tuberosum Rottl.stress materials were obtained for subsequent analysis.2.Analysis of amino acid metabolite of three successive generations of A.tuberosum Rottl.under different stress treatments.Metabonomics of three generations of CK Group(cutting Stress),group A(flooding+cutting Stress)and Group B(low temperature+flooding+cutting Stress)were analyzed,The amino acids were determined by OPA-FMO pre column derivatization HPLC,Then,the differential amino acids were screened and the trend of differential amino acids was analyzed to obtain the optimal stress treatment methods,and the stress materials were obtained for metabolomic and transcriptome analysis.3.Metabolite analysis of three successive generations of A.tuberosum Rottl.under cutting stressIn this study,three successive generations of A.tuberosum Rottl.under cutting stress samples and control samples were used as experimental materials,and the metabolites were detected and identified by ultra-High-performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)using a wide target metabonomics technique,to screen significant difference metabolites,significant difference metabolic pathway,to analyze the trend of the difference metabolites.4.Transcriptome analysis of three successive generations of A.tuberosum Rottl.under cutting stressThree successive generations of cutting stress samples and control samples of A.tuberosum Rottl.were used as experimental materials,and the data obtained from three successive generations of cutting stress samples of A.tuberosum Rottl.were analyzed by transcriptome sequencing technique and Illumina HiSeqTM 2500 sequencing platform,the differentially expressed genes of three successive generations of A.tuberosum Rottl.under cutting stress were screened and analyzed by trend analysis,GO analysis and typical KEGG pathway.5.The combined analysis of metabolism group and transcription group under three successive cutting stress in A.tuberosum Rottl.Based on the data of differential metabolite abundance and differential gene expression,a correlation coefficient model was established to calculate the Pearson correlation coefficient between gene expression and metabolite abundance,the correlation between differential metabolites and differential genes in three successive generations of A.tuberosum Rottl.under cutting stress was studied.Results1.Analysis of amino acid metabolite of three successive generations of A.tuberosum Rottl.under different stress treatments.The results showed that 14,13 and 16 amino acids were detected in three successive generations of CK Group(cutting stress),a group(flooding+cutting stress)and B group(low temperature+flooding+cutting stress),respectively.Aim at the comparison of CK,A and B groups of A.tuberosum Rotlt.,six different amino acids of three successive generations were screened out.The stress effect of cutting stress and(flooding+cutting stress)mainly caused the down-regulated expression of different amino acids,the stress(low temperature+flooding+cutting stress)mainly caused the up-regulation of differential amino acids.The contents of different amino acids in CK Group,a group and B group were 3,3 and 4 species,respectively,and the changing trend was 1,2 and 4 species.Glycine(Gly)is a common amino acid in CK,A and B,and its content tends to decrease.In view of the fact.that the variation of different amino acids in CK Group was single and regular,which was helpful to explore the more complicated mechanism of plant multi-generation stress,the stubble stress was selected as the sample treatment method for the follow-up metabolome and transcriptome analysis.2.Metabolite analysis of three successive generations of A.tuberosum Rottl.under cutting stressUHPLC-MS/MS method was used to detect 729 metabolites in three successive generations of cutting-stressed A.tuberosum Rottl.samples and control samples(S1,S2,S3,CK),75 differential metabolites were obtained.There were 8 change trends of different metabolites in three successive generations of A.tuberosum Rottl.cutting stress,among which there was a significant trend,the trend was 0(continuous decline),The metabolism of amino acids and their derivatives,organic acids and other kinds of differential metabolites were greatly affected by cutting stress,among which 11 differential metabolites were found in amino acids and their derivatives,eight of the three successive generations showed a continuous downward trend,two showed a first-and then-constant trend,one showed a first-and then-downward trend,and there were six different metabolites in organic acids,and four showed a continuous downward trend in the three successive generations,there were 2 different metabolites in the alkaloids,which showed a continuous downward trend in the three successive generations.The results showed that the accumulation of amino acids,organic acids and alkaloids was inhibited and the content of total alkaloids decreased under the stress of three successive generations of stubble.Piperidinic acid,1-lysine and 1-arginine,which were closely related to plant stress,decreased continuously in three successive generations of A.tuberosum Rottl.cutting.3.Transcriptome analysis of three successive generations of A.tuberosum Rortl.under cutting stressThree successive cutting-stressed A.tuberosum Rottl.samples and control samples(S1,S2,S3,CK)were sequenced using Hiseq2500.There were 3 biological repeats and 12 samples in each sample.103892 genes were obtained and 1491 DEGs were screened.There were 8 expression trends of DEGs,among which trend 3(first unchanged and then down),trend 0(continuous down)and trend 7(continuous up)were statistically significant.The results of KEGG analysis showed that most DEGs involved in amino acid-related anabolism pathway,protein processing pathway in endoplasmic reticulum,carbon metabolism and plant hormone signal transduction pathway decreased continuously in three successive generations of A.tuberosum Rottl.cutting.4.Combined analysis of metabolism group and transcription group under cutting stress in three successive generations of A.tuberosum Rottl.A correlation coefficient model was established by combining analysis of metabolism and transcriptome of three successive generations of A,tuberosum Rottl.cutting stress.The results showed that there were significant differences and close relationships between different genes and different metabolites in three successive generations of A.tuberosum Rottl.cutting stress,piperidinic acid and 1-lysine,which are closely related to plant stress,were regulated by many different genes and showed a continuous decline trend in the third generation,so it should be considered as a marker of cutting stress in follow-up research.ConclusionThis study revealed the molecular response mechanism of A.tuberosum Rottl.under the stress of three successive generations of cutting,and from the perspective of metabolism,transcription and joint analysis,we explored the trends and related pathways of differential metabolites,differentially expressed genes,some important clues related to the stress of three successive generations of A.tuberosum Rottl.cutting were screened out.The study accumulated experience for the study of multi-generation stress in plants.