| Strawberry(Fragaria×ananassa Duch.)is a perennial herb of the genus Fragaria in the Rosaceae.The fruit is bright in color,rich in in aroma and superb in texture,popular in consumers,with cultivars varieties and a wealth of germplasm resources.The quality of strawberry includes shape,size,color,flavor,sweetness,acidity and aroma.In particular,acidity,sweetness and color are important reference indexes for whether the fruit is highquality or not,and they are also one of the main factors to attract consumers to buy.Therefore,exploring the mechanism of strawberry fruit quality regulation has always been the focus of many researchers.In this paper,the transcriptome and metabolome data were detected from three strawberry varieties with typical differences in sweetness and acidity.Combined with the joint analysis of transcriptome,metabolome and gene expression profiles,two differential genes related to strawberry sugar and acid were screened.Cloning and Expression Analysis of the two genes have been completed.The purpose of this study was to explore the key genes that affect the difference of sugar and acid accumulation in strawberries of different varieties,and lay a foundation for further gene regulations at fruit quality.The main achievements are listed as follows:(1)In order to further explore the sugar and acid metabolism pathways and regulatory genes that affect strawberry fruit quality,Benihoppe(HY),Snow White(BX)and ToKun(TX)strawberry were used as test materials for joint analysis by transcriptome and metabolome sequencing.Combined with the determination and analysis of sugar and acid content in fruits of different strawberry varieties in the early stage of our laboratory,it was found that there was no significant difference in sugar content of the three strawberry varieties,but the sweetness in Snow White was significantly higher than that in the other two kinds of strawberries.The sugar acid ratio in ToKun which has the lowest total organic acid content is significantly higher than that in Snow White and Benihoppe.Based on metabolome data analysis,743 differential metabolites were obtained from the three comparison groups HY vs BX,TX vs BX and TX vs HY,150 metabolites were known,and 21 metabolites with significant content differences were screened out,which may be related to the differences of sugar and acid in the three strawberries.A total of 74.95Gb of Clean reads were obtained from transcriptome sequencing.A total of 74.95gb clean reads were obtained by transcriptome sequencing,and 77,213 genes were compared from strawberry genome Through the pairwise comparison of the differential genes of the three strawberries,it was found that there were 824 differentially expressed genes in three comparison groups.After correlation analysis of differential metabolites and differentially expressed genes,A module including SSADH-succinic acid-SDH2-2-Fumaric acid was found to be is significantly activated in the TCA cycle and GABA metabolic pathway and genes and metabolites in this modue were co-expressed.Finally,two candidate genes,FaSSADH and FaSDH2-2 were selected as two candidate genes which may be crucial regulatory genes in sugar and acid metabolism.(2)The FaSSADH gene which located at the lower end of the chromosome III in strawberry was cloned from the cDNA of ToKun.The coding sequence of FaSSADH gene is 1593 bp in length,encoding 530 amino acids.and the molecular weight of the protein was about 56.51KDA.Based on Sequence Alignmen,the conservative domain of of FaSSADH gene is relatively conserved and Subcellular localization predicted that FaSSADH protein was located in mitochondria without transmembrane structure and signal peptide.It was found that the expression levels of FaSSADH in Snow White and ToKun were significantly higher than that in Benihoppe through the relative expression of FaSSADH in three kinds of strawberries analyzed by qRT-PCR.Combined with the differences in the content of citric acid,malic acid and that of other organic acids,it is speculated that FaSSADH may negatively regulate the synthesis of organic acids in three varieties of Strawberry.The pNC-ET-28 vector of FaSSADH gene was constructed and transformed into the E.coli BL21 for prokaryotic expression successfully.The protein expression was obvious and existed in supernatant.The relative molecular weight of purified protein was consistent with the predicted value of 56.51KD.(3)The FaSDH2-2 gene which located at the upper end of the chromosome VI in strawberry was cloned from the cDNA of ToKun.The coding sequence of FaSDH2-2 gene is 861bp in length,encoding 286 amino acids,and the molecular weight of the protein was about 32.01KDA.Based on Sequence Alignmen,the conservative domain of of FaSDH2-2 gene is relatively conserved and Subcellular localization predicted that FaSDH2-2 protein was located in mitochondria without transmembrane structure and signal peptide.It was found that the expression levels of FASDH2-2 gene in Snow White and ToKun were significantly higher than that in Benihoppe through the relative expression of FASDH2-2 gene in three kinds of strawberries analyzed by qRT-PCR.Combined with the differences in the content of citric acid,malic acid and that of other organic acids,it is speculated that FASDH2-2 gene may also negatively regulate the synthesis of organic acids in three varieties of Strawberry.Most of expressed protein was reside in the sediment in the style of occlusion body through prokaryotic expression.The protein can be expressed when E.coli was shattered using ultrasonication and the relative molecular weight of the expressed protein was consistent with the theoretical value of 32.01KD... |
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