| Machilus rehderi(M.rehderi)is an evergreen small tree belonging to the genus Machilus of Lauraceae.It is rich in wild resources.Polysaccharide is one of the main chemical components in the leaves of M.rehderi,which has the effects of anti-oxidation,anti-cancer and hypoglycemic.However,at present,the research on the polysaccharides in the leaves of M.rehderi focuses on the determination of the content.Therefore,it is of great significance to explore the extraction process,functional activity and biosynthetic pathway of polysaccharides for the further exploration and utilization of the germplasm resources of this tree species.In this study,the tender leaves(Y),mature leaves I(M)and mature leaves II(O)of M.rehderi were used as research materials.The optimum extraction process of polysaccharides was obtained by response surface design method,and the physicochemical properties and antioxidant activity of polysaccharides were analyzed.Then,high-throughput sequencing technology was used to sequence the transcriptome of the leaves of M.rehderi in three periods,and the biosynthetic pathway and key enzyme genes of polysaccharides in the leaves were explored.The main results and conclusions are as follows:(1)On the basis of single factor experiment,according to the response surface(Box-Behnken)experimental design method,the optimum extraction process of polysaccharides from M.rehderi leaves in three periods was determined as follows Y period: solid-liquid ratio 30: 1 m L/g,ultrasonic temperature 60 °C,ultrasonic time 60min;M period: solid-liquid ratio 30: 1 m L/g,ultrasonic temperature 55 °C,ultrasonic time 60 min;O period: solid-liquid ratio 30: 1 m L/g,ultrasonic temperature 60 °C,ultrasonic time 60 min.The order of polysaccharide yield was M(13.18 %)> O(11.98 %)> Y(11.95 %);(2)The polysaccharides in the leaves of M.rehderi in the three periods were mainly composed of protein,neutral sugar and uronic acid,and the content of uronic acid was the highest(all> 40%).For protein,the content of Y period was the highest(5.09%),and the content of the three periods was significantly different;for neutral sugar,the content of M.rehderi was the highest(14.76%),but there was no significant difference among the three periods.For uronic acid,the content(68.01%)was the highest in the M.rehderi,and the content of the three periods was significantly different,and the molecular weight reached 104 orders of magnitude.The monosaccharide components of the leaves in the three periods were glucose,rhamnose,galactose,mannose,glucuronic acid and fucose,and the glucose content was the highest,especially in the M.rehderi;(3)The in vitro antioxidant activity of polysaccharides from M.rehderi leaves at three stages showed that the antioxidant capacity of polysaccharide solution increased with the increase of concentration within a certain range,and showed a dose-dependent relationship.The scavenging ability of DPPH was the highest in Y and M periods,which was close to that of VC.At the concentration of 80 ug/m L,the scavenging of ABTS free radical by leaf polysaccharide in O period was close to that of VC,and higher than that in Y and M periods.The scavenging rate of hydroxyl radicals by leaf polysaccharides in three periods was less than 50 %.At the concentration of 400 ug/m L,the scavenging ability of leaf polysaccharides in Y period to hydroxyl radicals was significantly higher than that in M and O periods.Leaf polysaccharides in the three periods had strong antioxidant activity,but they were much lower than VC.(4)Based on Illumina high-throughput sequencing technology,a total of219,970,953 raw reads were obtained from the leaves of the three periods,with Q30 greater than 93.94% and GC content of 46.56%.Based on the clean reads after quality control,477,585 longest transcripts(Unigenes)were spliced using Trinity software according to the method of no-reference transcriptome.Unigenes were annotated to the NCBI da Tablease.Among them,the NR da Tablease annotated the most Unigenes,with180,081(37.71%);the number of Unigenes annotated in the remaining da Tableases gradually decreased,and the NT da Tablease was annotated to 101,687(21.29%).The Swiss-Prot da Tablease annotated 99,500(20.83%);The KOG da Tablease annotated70,268(14.71%);the Pfam da Tablease annotated 54,920(11.50%);The KEGG da Tablease annotated 31,736(6.65%);The GO da Tablease annotated 30,567(6.40%).A total of 7,316 differentially expressed genes were identified in Y and M stages,13,752 differentially expressed genes were identified in Y and O stages,and 22 differentially expressed genes were identified in M and O stages.A total of 394 Unigenes were involved in four polysaccharide biosynthesis pathways of M.rehderi,involving a total of 11 key enzyme genes(Mr GER1,Mr GMD,Mr GMPP,Mr HK,Mr MPI,Mr PMM,Mr RHM,Mr SUS,Mr UER1,Mr UGDH,Mr UGE).The expression of Mr GMPP in the polysaccharide synthesis pathway of M.rehderi leaves at three stages was relatively high.In summary,there was no significant difference in in vitro antioxidant activity between the three periods.Considering the factors such as polysaccharide extraction rate,chemical composition,monosaccharide composition and leaf yield,it was considered that the leaves of M.rehderi,M period were the best picking period,and Its leaves are more suitable for the development of polysaccharide products of M.rehderi than Y and O periods. |
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