Cloning,expression Characterization Of Flavanone-3’,5’-hydroxylase Gene From Ribes Nigrum L.

The black currant(Ribes nigrum L.)is one of the characteristic berries of the cold region,which is rich in anthocyanins and has various health functions such as improving vision,reducing blood pressure,slowing down inflammation,and inhibiting cancer cells.Flavonoids-3’ 5’-hydroxylase(F3’5’H)is a key enzyme for the synthesis of delphinidin,which is one of anthocyanin.To systematically elucidate the role of the F3’5’H gene in anthocyanin biosynthesis of black currant and its expression pattern,this study used the samples of black currant(’Brodforp’)were collected at the horticultural experiment station,which belongs to the Northeast Agricultural University in the Harbin(44°04′0″N,125°42′0″E)region of China.F3’5’H gene was cloned from black currant and used qPCR to study F3’5’H gene in different development stages of different tissues and organs such as leaves,flowers,and buds were analyzed.HPLC-MS/MS techniques were used to analyze the anthocyanins content of black currant at different stages and organs.The prokaryotic expression systemof F3’5’H was constructed.The results are as follows:(1)The F3’5’H gene was successfully cloned from the flower of black currant using PCR technique.The open reading frame sequence was 1530 bp,which encoded 509 amino acids.The bioinformatics method was used to predict the relative molecular mass of F3’5’H is 57.11 kDa,the theoretical isoelectric point is 9.26,is a hydrophilic protein,no signal peptide,and a transmembrane Region.Sequence alignment results showed that the F3’5’H had three CYP450-specific conserved sequences(FGAGRRICAG,PPGP,AGTDT),belongs to CYP450 family.The amino acid sequence of the F3’5’H has higher similarity with other plants F3’5’H,such as grapevine,arabidopsis thaliana,wild soybean,Cyclamen sinensis,Rumornaceae,Petunia,etc.The sequence similarity is in the range of 75%-80%,with the closest relatives to the grape.(2)On the basis of cloning,the expression level of F3’5’H gene in different tissues and organs of black currant and fruit development period was detected and compared by real-time fluorescence quantitative PCR.The results showed that the mRNA transcript were expressed in different tissue,the lowest expression level was in flowers,and the mainly expressed was in the skin of berry.The expression analysis showed that F3’5’H gene expression was tissue specific,very high in the skin of berry.During fruit development,the expression of F3’5’H gene gradually increased along with the development of fruit,reached the highest level in the late fruit development and then decreased.It is speculated that it may play a role in the formation of anthocyanins in the berry.(3)The content of anthocyanins in different tissue and development stages of ’Broud’ black currants were analyzed by HPLC-MS/MS method.The results showed that 17 kinds of anthocyanins were detected.The total anthocyanin content was higher in the skin than in other tissue parts,and the total content of anthocyanin gradually increased with the development of the fruit until the fruit reached full maturity,reaching a maximum of 37.25 mg/g,while in the roots,stems,leaves,flowers,buds,etc.was less than 2.00 mg/g;And found that delphinidin anthocyanins accounted for more than half of the total anthocyanin content,followed by anthocyanins,accounting for about a quarter.The relative expression of F3’5’H gene and the total anthocyanin content were similar.The relative expression of F3’5’H gene was also higher in organs with higher content of anthocyanins,indicating the accumulation of anthocyanins and F3’5’H gene expression is positively correlated.(4)Therecombinant vectors of F3’5’H from black currant were constructed for its expression in E.coli.F3’5’H was cloned into prokaryotic exoression vector pET28 b and expressed in three different E.coli(E.Coil strain NO.1,E.Coil strain NO.2,E.Coil strain NO.3).By the optimization of the induction temperature and time,the recombinant protein was successfully expressed in E.coil strain NO.3,the IPTG concentration was 1.0 mM,the optimal conditions for induction were 16°C for 16 h.SDS-PAGE results revealed that recombinant protein molecular weight consisted with the prediction of protein molecular weight.The recombinant protein was purified and present in the form of an inclusion body with a concentration of 0.41 mg/mL.The specific protein with a purity of 60% indicates that it provides the basis for the verification of the function of the F3’5’H gene of black currant and the study on the activity of its encoded enzyme.