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Screening And Functional Verification Of Synthetic Gene Of Grape Stilbene Compound Trans-scirpusin A Under UV-B Stress

Posted on:2020-07-03Degree:MasterType:Thesis
Country:ChinaCandidate:X YuFull Text:PDF
GTID:2433330602952691Subject:Food Science
Abstract/Summary:PDF Full Text Request
UV-B(Ultraviolet Radiation B)is one of the most important factors affecting plant growth and development.As a common environmental factor,UV-B not only affects plant morphological structure and physiological metabolism,but also affects plant fruit quality.Grapes are rich in anthocyanins,soluble sugars and organic acids,as well as natural plant polyphenols.Stilbenes are common plant polyphenols with good biological activity.Trans-scirpusin A is a kind of stilbenes with little research but high attention.As a quality functional factor of grapes,its pharmacological activity has attracted much attention,but its synthetic pathway has rarely been reported.In this paper,the effects of UV-B radiation on grape physiology were studied.Based on the biosynthetic mechanism of trans-scirpusin A,a key biosynthetic gene(SAH)of trans-scirpusin A was screened by transcriptome sequencing technology,and the key genes were cloned,expressed and subcellular localized.The main results were as follows:(1)The effects of UV-B radiation on grape physiology are various.After UV-B radiation stress,the activities of SOD and POD in grapes increased with the increase of stress time.The range of POD activity was 35.63-66.99 U.g-l.min-1.Although the trend of SOD activity affected by UV-B radiation was the same as POD,the range of change was lower than POD,and the range of change was 40.96-63.82 U.g-l.min-1.CAT activity reached the maximum value of 23.09 U.g-1.min-1 at 60 min of UV-B radiation.The change trend of CAT activity increased first and then decreased.The change range of CAT activity was least affected by UV-B radiation.With the increase of UV-B radiation time,the contents of glucose,fructose and sucrose in glucose increased in varying degrees.The effect of UV-B radiation on soluble glucose was glucose>fructose>sucrose>sorbitol.After UV-B radiation,the content of organic acids in grapes will also change.The effects of UV-B radiation on different organic acids are different,and the proportion of single organic acids in total organic acids will be significantly affected.The content of stilbenes in grapes was positively correlated with the time of UV-B radiation.Different stilbenes were affected by UV-B radiation.Resveratrol monomer content had the greatest response to UV-B radiation,followed by trans-e-viniferins and trans-scirpusin A.UV-B radiation had the least effect on trans-?-viniferins and pterostilbene.(2)Based on transcriptome sequencing results,the effects of 30 minutes of UV-B radiation on grape were analyzed at the level of gene expression difference.The results showed that there were 13906 differentially expressed genes after 30 minutes of UV-B radiation compared with untreated ones.According to GO annotation classification,5560 differentially expressed genes belonged to biological process classification,5024 differentially expressed genes were classified into cell composition classification,and 3322 differentially expressed genes were classified.Allo-expressed genes are classified into molecular functional categories.At the same time,differentially expressed genes were classified and enriched in KEGG metabolic pathways.Results A total of 4891 differentially expressed genes were classified into five major KEGG metabolic pathways,of which 208 differentially expressed genes were classified into cell processes,259 differentially expressed genes were related to environmental information processing,1017 differentially expressed genes were related to genetic information processing,and 2988 differentially expressed genes were related to genetic information processing.394 differentially expressed genes are related to the biological system.Differentially expressed genes are mainly concentrated in three regions:ribosome,plant pathogen interaction and flavonoid biosynthesis.Three possible Trans-scirpusin A biosynthetic genes were screened and verified by RT-qPCR using GO,KEGG annotation and HsCYPIB1 comparison results.(3)Based on the analysis of gene differential expression multiple and gene homology,the most probable SAH gene was selected for subsequent analysis.The open reading frame(ORF)of the SAH gene was 837 BP long.It encoded 278 amino acids.The calculated isoelectric point of the protein was 6.63 and its molecular weight was 31.8 Kda.With the help of DNA man analysis software and BLAST function of NCBI website,the target gene was compared with four other CYP genes with hydroxylation function.The homology of the target gene with ginseng,lotus root,reference gene and grape was 68.19%,70.95%,39.98%and 51.94%,respectively.Cloning,expression and subcellular localization of the key trans-scirpusin A synthesized by screening.SDS-PAGE and Western blot results showed that the target fragment was successfully transformed into Pichia pastoris,and the target protein could be expressed stably.The molecular weight of the protein was 31.8 KDa,which was consistent with the expectation.The results of subcellular localization analysis showed that the target protein was mainly expressed in the nucleus and a small amount in the cell membrane,which was consistent with the predicted results of subcellular localization.
Keywords/Search Tags:UV-B, grape, stilbenes, western blot, subcellular localization
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