| Oligosaccharides,as small molecule carbohydrate polymers,are widely used in nutritional food and medicine industries due to their unique biological probiotic properties and biological activities.At present,studies have identified the immune activity of Lycium barbarum oligosaccharides produced by enzymatic hydrolysis and acid hydrolysis of Lycium barbarum polysaccharides,which laid a theoretical foundation for the further study of the active fragments of Lycium barbarum polysaccharides.Most of the studies focused on the functional activity,probiotic properties,extraction methods and structural characteristics of oligosaccharides,but the mechanism of oligosaccharide accumulation in Lycium barbarum fruit has not been reported.Therefore,in this study,the oligosaccharide content,structural characteristics and anabolic enzyme activities of Lycium barbarum fruits with different growth and development were studied,and the mechanism of oligosaccharide anabolism in Lycium barbarum fruits was explored by proteomics method.The key proteins related to oligosaccharide anabolism in Lycium barbarum fruits were verified by real-time fluorescent quantitative PCR technology The results are as follows:(1)The crude oligosaccharides of Lycium barbarum were extracted by alcohol solution method.The free protein was removed by savage method,and the content of oligosaccharides and protein in oligosaccharides were determined.The results showed that the content of oligosaccharides in Lycium barbarum fruit changed continuously during the whole growth stage,and the oligosaccharides accumulated in the green fruit stage(14 d)and the late yellowing stage(28 d)43%and 22.33%respectively.The content of oligosaccharide was significantly different from the other three stages(p<0.05).The average content of soluble protein was 0.59%.The molecular weight at different developmental stages was mainly distributed in 0.9?1.2 KDa by HPLC.With the development of Lycium barbarum,the molecular weight first increased and then decreased.The maximum molecular weight was 1.18 KDa at 35 days,the results of GC-MS showed that the oligosaccharides were composed of arabinose,rhamnose,xylose,galactose and mannose.Infrared spectra showed that the characteristic peaks of sugars appeared at 3000?2800 cm-1 and 1400?1200 cm-1 in the five growth stages.The absorption peak at about 1640 cm-1 was sugar COOH stretching vibration absorption peak.The conformation of sugar ring was mainly in the form of ?-pyran ring.X-ray analysis showed that there was an extra sharp narrow diffraction peak at 29° in the XRD spectrum of LBO2 in five periods.It can be concluded that the structure of LBO2 in five periods was semi crystalline.The results of thermal property analysis showed that there were two main stages in the thermal decomposition process.The first stage was the removal of crystal water,the second stage was the oxidative decomposition,the molecular skeleton of oligosaccharides was broken,and the thermal stability of Lycium oligosaccharides in air was good.The morphology of LB02 in the five stages was similar to sponge like arrangement,and a single oligosaccharide molecule was similar to zigzag.(2)During the fruit development of Lycium barbarum,the activities of sucrose phosphate synthase and sucrose synthase increased significantly from 7 to 28 days after flowering,which were 1449.17(IU/g)and1.354(U/g),respectively.The activities of sucrose phosphate synthase and sucrose synthase decreased from 28 days after flowering.The activity of acid invertase increased to 0.52(U/g)on the 21st day and decreased slightly on the 35th day.The activity of fructokinase increased significantly from 7 to 14 days,reached the maximum of 0.315(U/g)on 28 days,and decreased on 28 to 35 days.The activity of ?amylase increased from 7 to 28 days.The maximum activity was 0.904(U/g)on 28 days,and decreased on 35 days.The endoglucanase activity increased significantly from 7 d to 28 d,reached the highest value of 4.046(U/g)at 28 d,and began to decline after 28 d.(3)A total of 1799 differentially expressed proteins were screened during the whole growth and development of Lycium barbarum.133 differentially expressed proteins related to oligosaccharide anabolism were screened according to go database.According to the analysis of KEGG pathway enrichment and differential protein expression,there were 22 differential proteins in 14 d/7 d,which were mainly acid invertase,?-amylase and sucrose synthase involved in starch and sucrose metabolism pathway.It showed that Lycium barbarum polysaccharide accumulation was mainly realized in green fruit stage,and oligosaccharides were produced in this process.21 d/7 d.a total of 60 differential proteins were screened.Fructose diphosphate aldolase and phosphoglycerate kinase were up-regulated in the early stage of yellowing.Other aldehyde dehydrogenase,aldose 1-epimerase,endoglucanase and glucose-6phosphate isomerase were involved in the process of glucuronic acid metabolism and glucuronic acid metabolism in the early stage of yellowing,LBP decomposes to form oligosaccharide accumulation,which may provide energy to cells after further decomposition.106 and 113 key differential proteins were screened at 28 d/7 d and 35 d/7 d,respectively.They mainly regulate oligosaccharide anabolism by participating in fructose and mannose metabolism,starch and sucrose conversion,glucose production,pentose and nucleotide sugar metabolism.(4)The results showed that the agarose electrophoresis bands of RNA purified from five samples of Lycium barbarum in different periods were clear and complete,the results of dissolution curve showed that there was only one smooth single peak in the dissolution curve of 10 genes including EF1a.The expression level of sucrose phosphate synthase(A0A059T2S5)increased first and then decreased during the whole growth process,and the highest expression level was 0.155 at 21d.The expression patterns of hexosyltransferase(A0A0V0IJ15)and sucrose synthase(M0ZT40)were similar during the growth and development of Lycium barbarum.The expression levels increased from 7 days to 14 days and then decreased rapidly to 0.084 and 0.003 on 28 days,respectively.The maximum expression levels were 0.225 and 0.338 on 35 days,respectively.The expression levels of P37829 and M0ZVJ3 were similar,showing a trend of first decreasing and then increasing,with the lowest values of 0.261 and 0.005 on the 14th day and gradually increasing values of 0.177 and 0.004 on the 14th-35th day.The expression level of raffinose synthase(A0A0V0ITL0)increased to 0.011 on the 35th day.The expression of ?fructofuranosidase(G9IHI6)increased continuously from 7 to 28 days,and the highest expression was 2.782 on 28 days,and decreased on 28 to 35 days.The expression levels of rhamnose 1(A0A0V0HSE1)and ?-mannosidase(A0A0V0IX45)in Lycium barbarum during the whole development process showed a trend of first decreasing and then increasing,gradually decreasing at 7-21 days,and significantly increasing at 21 days(0.037 and 0.003,respectively).The expression of(Q9ZSP9)increased from 14 to 28 days,and reached the highest level at 28 days.This is consistent with the results of proteomics analysis,which provides a reference for the study of oligosaccharide anabolism. |
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