Analysis Of Metabolites In Wheat Germination Process Based On UPLC-QTOF/MS Method

Seed germination is an orderly physiological growth process in which seeds begin from swelling and is the beginning of the seed growth process.Seed germination could be devided into three stages of swelling,water absorption,slow water absorption and growth and water absorption.This process involves metabolic activities such as glycolysis,amino acid metabolism,and lipid metabolism,which not only providing the energy needed for wheat germination,but also providing a material basis for wheat growth.Seed germination is a critical period of concern for plant growth.In this study,a non-target metabolomics analysis method based on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)was used to study the wheat germination process,and the following results were obtained.1.Xianmai 13 and Zhoumai 26 were germinated and the morphological changes during wheat germination were observed.In order to explore the effects of different extraction methods on metabolites from germinated wheat,a UPLC-QTOF/MS-based pretreatment method and analysis method for germinated wheat metabolomics samples were established.The germination was carried out for 2 days.Three factors of solvent,extraction method and extraction time were tested at three levels of orthogonal test to determine the best combination of extraction: 80% acetonitrile(0.1% formic acid)was shaken for 30 min.2.Dynamic analysis of non-target metabolites at ten different time points of germination of Xianmai 13 and 113 metabolites identified by positive and negative ion mode.OPLS-DA analysis and fold change analysis were combined to screen 16 differential metabolites.It includes one amine,four amino acids,one azole,one saccharide,two nucleic acids,three organic acids,one pteridine,one pyrazine,one steroid and one vitamin.The contents of amino acid metabolites,melibiose,dihydrofolate,2,5-dimethylpyrazine and vitamin B6 gradually increased with the germination time,betaine,desulfurization biotin,4-hydroxyphenylpyruvate,the content of GGPP gradually increases with the germination time.3.UPLC-QTOF/MS non-target metabolomics method was used to identify different wheat varieties.In this part,the samples of Xianmai 13 and Zhoumai 26 germination at 0,24 and 48 h were used as research objects.The differences of metabolites in different germination stages of different varieties of wheat and different varieties of wheat were analyzed,v 130 kinds of metabolites patterns were identified by positive and negative ion,OPLS-DA analysis and fold change analysis screened 25 differential metabolites.The two wheat varieties can be distinguished by metabolomics.Compared with the Xianmai 13,the germinated Zhoumai 26 has higher levels of amino acid,lipid and organic acid metabolites,especially the healthy unsaturated fatty acids oleic acid and gamma-linolenic acid.Based on the results,the metabolomic analysis method of germinated wheat was established by UPLC-QTOF/MS technology.The dynamic changes of metabolites in different germination stages of Xianmai 13 were studied.The method was applied to the identification of different wheat varieties.This study provides technical support for the multi-faceted study of wheat metabolomics.