Molecular Mechanism Of GST Regulating Anthocyanin Accumulation In Strawberry Fruit

Anthocyanin are the main pigments that make up the fruit color of strawberry.Anthocyanin,also known as anthocyanidin,are flavonoid polyphenolic compounds.The popular strawberry fruit contains a lot of flavonoids,which determines the color of angiosperms,fruits,seed coats,stems,leaves and roots.In addition,it is applied as a natural pigment and anti-aging additive to food dyeing,cosmetics and health care products and other aspects.Strawberry is one of the seven largest fruits in the world.It is known as the ’Queen of Fruits’ and has important nutritional,medicinal and economic values.Strawberry has been shown to have high antioxidant activity,which is related to the content of polyphenolic compounds in the fruit,especially anthocyanin,which is the important constituent of phenolic compounds in mature strawberry fruits.Therefore,elucidating the molecular mechanism of anthocyanin accumulation in strawberry fruit is essential for strawberry production.In this paper,the Benihoppe strawberry fruit was used as a test material.Two GSTs genes were cloned and named as FaGST173 and FaGST660 and based on the results of the integration analysis of the previous transcriptome and metabolites.In this paper,two GSTs transporter proteins were transferred to bioinformatics and functional verification analysis,and real-time quantitative PCR was used to analyze the expression levels of FaGST173 and FaGST660.The over-expression vectors pBI121-FaGST173 and pBI121-FaGST660 were constructed,and transformed into Arabidopsis Col-0,and several transgenic plants were identified.The interference expression vectors pHellsgate2-FaGST173 and TRV2-FaGST660 were constructed and the strawberry fruit was injected for sexual function verification.Construction of the GFP fusion proteins pRI101-GFP-FaGST173 and pRI101-GFP-FaGST660 for subcellular localization of the genes.The molecular mechanism of GSTs gene during anthocyanin synthesis and transport was verified by the above experiments.The experimental study achieved the following main results:1.Two GST genes FaGST173 and FaGST660 were cloned from the red strawberry fruit.The results of phylogenetic tree indicated that FaGST173 and FaGST660 had the highest homology with ZmBz2,and also had high homology with the already published AtTT19 and PhAN9 with the function of chlorophyll transporter.The results showed that the homology of FaGST173/FaGST660 to ZmBz2 reached 57.36%,it is speculated that FaGST173/FaGST660 is also involved in the process of transporting anthocyanin.2.Real-time quantitative PCR analysis of the spatio-temporal expression patterns of FaGST173 and FaGST660 genes showed that FaGST173 gene had the highest relative expression in flowers,followed by young leaves and lowest in fruits.The expression analysis of fruit in six different developmental stages of strawberry showed that FaGST173 expression was significantly up-regulated,the highest expression in red fruit stage and slightly down-regulated in mature stage,indicating that this gene may be associated with anthocyanin accumulation.The relative expression of FaGST660 gene in stems was found to be the highest,and the expression in other parts was relatively low.The expression analysis of fruit in six different developmental stages of strawberry showed that the expression of FaGST660 was significantly up-regulated,indicating that this gene may be associated with anthocyanin accumulation.3.The results of GST activity assay in six different developmental stages of strawberry showed that the overall trend of the enzyme activity in the green and white phase was higher and then decreased,and the gene expression and anthocyanin of FaGST173 and FaGST660 genes.The accumulation pattern was similar,indicating that this FaGST173 and FaGST660 may be associated with anthocyanin transport.The expression pattern of anthocyanin functional gene showed that the expression of CHS1/3,F3H1,DFR2,ANS1 and UFGT1 genes was highly correlated with anthocyanin accumulation,and played a key role in the biosynthesis pathway of anthocyanins.4.The expression patterns of FaGST173 and FaGST660 genes in red strawberry and its white meat mutant were slightly higher than that in the white pulp and peel of strawberry,which was consistent with the anthocyanin content pattern;however,the expression of FaGST660 gene was in red The grass berry meat is lower than the white,indicating that the FaGST173 gene plays a key role in the accumulation process of anthocyanins in strawberry fruits.At the same time,the expression patterns of anthocyanin synthesis and regulation related genes in red and white flesh and fruit were detected.It was found that the expression levels of ANS1 and UFGT1 in red peel were significantly higher than that in white,while only anthocyanin regulatory gene was in bHLH3.The expression level in the red peel is higher than that in the white,indicating that these genes are the key synthetic and regulatory genes for the anthocyanin accumulation in strawberry fruit.5.Response patterns of FaGST173 and FaGST660 genes to ABA treatment:The results showed that the accumulation of anthocyanins in the control group was very low,and ABA treatment could significantly promote the accumulation of anthocyanins,and the anthocyanin content was positively correlated with the treatment concentration.The ABA concentration was 50 mL L-1,the anthocyanin content was 45 m Lg-1,and the concentration of 100 mg L-1 anthocyanin was 74 mg L-1.The expression of FaGST173 and FaGST660 genes was also induced by ABA treatment.Compared with the control,the expression level was increased by 200 times when the ABA concentration was 100 mg L-1,while the expression level of FaGST660 was decreased at the concentration of 100 mg L-1.6.Subcellular localization:In order to determine the subcellular localization of FaGST173 and FaGST660 genes,FaGST173-GFP and FaGST660-GFP fusion expression vectors were constructed and transformed into N.benthamiana epidermis by injection.The results showed that both FaGST173 and FaGST660 were localized to the cytoplasm.7.FaGST173 protease activity assay:The prokaryotic expression vector pMAL-C5X-FaGST173 was constructed,and the protein was induced and purified for enzyme activity assay.The protein was found to have GST transport protease activity,and the optimal temperature of protein activity was 25℃-30℃.8.Genetic transformation and gene function verification:Plant overexpression vectors PBI121-FaGST173 and PB1121-FaGST660 were constructed,and the heterologous plant Arabidopsis thaliana was successfully transformed,and finally several transgenic plants were obtained.The plant interference vectors pHellsgate2-FaGST173 and TRV2-FaGST660 were constructed and the octoploid strawberry fruit was successfully injected.It was found that FaGST173 played a key role in the strawberry fruit coloring process.RT-PCR results showed that FaGST173 gene was significantly inhibited in the injection area,and anthocyanin accumulation was reduced by 780%.In addition,the expression of anthocyanin biosynthesis genes CHS1/3,F3H1,DFR2,ANS1 and UGT1 and the regulatory gene MYB10 were also significantly observed.inhibition.