Study On Extraction And Purification,Antioxidant Of Polysaccharide And Transcriptome Sequencing Analysis From Actinidia Arguta Fruit

Objectives: The polysaccharide from the fruit of Actinidia arguta was extracted using ultrasonic-assisted ionic liquid method,and the extraction process was optimized by response surface analysis.The obtained crude polysaccharide was further purified to study its antioxidant activity.The molecular regulation mechanism of A.arguta polysaccharide formation was explored by transcriptomic sequencing analysis.Methods: Ultrasound-assisted ionic liquid method was used to extract polysaccharide from A.arguta fruit.Firstly,the optimal ionic liquid and concentration were selected.Through single factor test and response surface analysis,the extraction process was optimized,and the regression model was established.The optimal extraction conditions of each factor were determined and verified by experiment.The crude polysaccharide of A.arguta fruit was deproteinized and depigmented,and further separated and purified by the ethanol fractionation method.The antioxidant activity of the polysaccharide components was determined.Illumina novaseq 6000 sequencing platform was used to sequence the transcriptome of A.arguta fruit in six periods including fruit setting stage,young fruit stage,green fruit stage,expansion stage,brittle ripening stage and initial ripening stage.The related genes of A.arguta fruit polysaccharide formation were screened by bioinformatics analysis.Results: After screening the ionic liquid with the best extraction effect was[BMIM]Br with a concentration of 0.5 mol/L.The optimal extraction conditions of polysaccharide from A.arguta fruit were determined by response surface method:ultrasonic time 70 min,ultrasonic power 350 W,and liquid-solid ratio 35:1 m L/g.Under this process,the yield of polysaccharide from A.arguta fruit was 3.52%.AB-8macroporous adsorption resin and 10% trichloroacetic acid were used to deproteinize and depigmentize the crude polysaccharide from A.arguta fruit,respectively,and then the antioxidant activities of the three kinds of polysaccharide components obtained by ethanol graded purification were detected in vitro.The DPPH free radical scavenging ability was as follows:SPS60>SPS30>SPS.The removal capacity of hydroxyl radical was as follows:SPS60>SPS30>SPS,and the total reduction capacity was as follows:SPS60>SPS30>SPS.Using the Illumina Novaseq 6000 sequencing technology platform,the transcriptome sequencing data of six periods including fruit setting stage,young fruit stage,green fruit stage,expansion stage,brittle ripening stage and initial ripening stage was obtained.The clean data of each sample exceeded 6.12 Gb,and the percentages of Q30 bases were all greater than 93.72%.The number of unigenes was115,508 with the average length of 752.28 bp,and the average length of N50 was 1229 bp.The number of transcript was 193264 with an average length of 944.32 bp,and an average length of N50 was 1560 bp.The unigenes obtained by transcriptome sequencing were successfully annotated to six major protein databases(KEGG,Pfam,Swiss-Prot,COG,GO,NR),with a number of 18453(15.98%),27503(23.81%),and 30896(26.75%),respectively.Taking | log2(foldchang)| > 1 and p-adjust < 0.001 as the screening conditions,22 differentially expressed genes were screened out in the pathways with significantly enriched polysaccharide-related differentially expressed genes,which were coding AXS、GAE、GALE、GME、RHM、SQD1、UER1、UXE and UXS1.Conclusions: Single factor test and response surface method were used to optimize the extraction process of polysaccharide from A.arguta fruit by the ultrasonic assisted ionic liquid,and the optimal extraction conditions were obtained.The three kinds of polysaccharides obtained by ethanol fractional purification were tested for antioxidant activity in vitro.Within the tested concentration range,there was a certain linearity relationship between the concentrations of the three polysaccharides and the antioxidant activity,among which sps60 had the best antioxidant activity and had great development potential.The transcriptome sequencing data of A.arguta fruit in six periods and the screened 22 differentially expressed genes lay a foundation for understanding the formation mechanism of polysaccharide from A.arguta fruit and cultivating A.arguta with high content of polysaccharide.