Functional Analysis Of VcCOMT40 And VcCCOAOMT20 Genes In Blueberry (Vaccinium Corymbosum L.)

Anthocyanins are the main bioactive substances in blueberry(Vaccinium corymbosum L.)for the prevention and treatment of various human diseases.The different content and proportion of anthocyanins not only affect the appearance quality of blueberry fruit,but also affect its nutritional quality and processing quality.Because of the high content of malvidin glycosides,blueberry has a significant hypoglycemic effect.Malvidin glycosides is a class of methylated modified anthocyanins in the fruits of Vaccinium spp.plants,but its mechanism of methylation in blueberry fruit remains unclear.Therefore,based on the genomic and transcriptome data of V.corymbosum'Draper',the O-methyltransferase(OMT)related gene families were analyzed by bioinformatics.In this study,the functions of VcCOMT40 and VcCCoAOMT20 genes with high expression content in highbush blueberry ‘Northland' fruit color transformation stage(S4)were analyzed by enzyme activity test in vitro,transient overexpression test and VIGS test in blueberry fruit development stage,stable overexpression test in tobacco.The main research results are as follows:1.The fruit development stage of ‘Northland' was divided into six stages,and the anthocyanin contents in different stages of S1-S6 were analyzed by HPLC.It was found that cyanidin glycosides,delphinin glycosides and malvacorin glycosides were the main components in S4(color changing stage),S5(pink fruit stage)and S6(blue fruit stage),respectively.Acylated anthocyanins appeared in the fruits of S5 and S6.Methylated anthocyanins were produced in large quantities from S5 stage.2.92 COMT genes and 20 VcCCoAOMT genes were identified from the genome of blueberry ‘Darper'.According to the q RT-PCR of fruit development stage,VcCOMT40 with the highest expression level at S4 stage were selected.Based on the transcriptome data analysis of fruit development stage,the relative expression of VcCCoAOMT 20 was also the highest at S4 stage.The subcellular localization of these two genes showed that they were located in the cytoplasm.3.In vitro enzyme activity test of VcCOMT40 showed that it can catalyze the methylation of caffeic acid and 5-OH ferulic acid,which are the precursor compounds in pathway of lignin synthesis.It can't catalyze flavone and flavonol.After overexpression of VcCOMT40 gene in ‘Notrhland' fruit,the fruit became hard,the number of stone cells in pulp increased,the number of seeds increased,the cells in ventricles arranged closely,and the cell walls became thicker.The results showed that the content of total anthocyanins did not change significantly;according to the types of glycosyl,the content of galactosyl anthocyanins decreased,the content of acetylated anthocyanins decreased significantly,while the content of glucosyl anthocyanins increased,and the content of arabinoyl anthocyanins did not change significantly.According to the classification of anthocyanin aglycones,the contents of cyanidin glycosides and peonidin glycosides in the injected fruits increased,while the contents of delphinidin glycosides,petunidin glycosides and malvidin glycosides decreased compared with the control.VIGS test showed that the VcCOMT40 gene silencing in‘Northland' fruit showed that the degree of fruit lignification.When VcCOMT40 was transferred into tobacco expression system and overexpressed in it.The results showed that the vascular bundle cells became thicker and more densely arranged.The the ratio of G-type lignin to S-type lignin(G / S)changed significantly by GC-MS.It is concluded that VcCOMT40 is a functional gene in lignin synthesis.4.The activity of VcCCoAOMT20 in vitro showed that the substrates of VcCCoAOMT20 were caffeic acid,5-OH ferulic acid in lignin synthesis pathway and delphinin 3-O-glucoside and cyanidin 3-O-glucoside in anthocyanin synthesis pathway.The overexpression of VcCCoAOMT20 in ‘Northland' fruit showed that the structure of blueberry fruit changed.The number of stone cells did not increase significantly,but the pericarp sclerenchyma cells increased.The formation of anthocyanins in the fruit was changed and the methylated anthocyanins increased.The VIGS experiment of VcCCoAOMT20 in ‘Northland' fruit showed that there was no seed.And the pulp color turned red.Silencing the gene resulted in a significantly lower degree of fruit lignification.Overexpression of VcCCoAOMT20 gene in tobacco system can obviously observe that the cortical cells are flat near the vascular bundle cells.The vascular bundle cells walls are thickened and arranged more densely,tightly wrapping the pulp cells.The content of lignin monomer in transgenic tobacco was determined by GC-MS.The content of G-type lignin increased significantly and the content of S-type lignin also increased.The G/S ratio of transgenic tobacco changed significantly.It is proved that this gene is not only a functional gene in lignin synthesis pathway,but also a functional gene in anthocyanin synthesis pathway.Conclusions: VcCOMT40 and VcCCoAOMT20 genes were obtained from the fruits of 'Northland' blueberry.VcCOMT40 can catalyze the methylation of caffeic acid and 5-OH ferulic acid,which are precursors of lignin synthesis pathway,and change the fruit structure of 'Northland'.Besides the above functions,VcCCoAOMT20 can also catalyze delphinin 3-O-glucoside and cyanidin 3-O-glucoside,which change the composition and content of anthocyanins in 'Northland' fruit.