Cloning And Expression Analysis Of Anthocyanin Synthesis Hydroxylase Gene And Promoter In Vitis Amurensis

The peel of Vitis amurensis is rich in many natural anthocyanins.in the process of anthocyanin biosynthesis,Phenylalanine is catalyzed by a series of enzymes to produce trihydroxyflavanone,and then under the action of F3H to produce dihydro kaempferol.The products undergo the action of F3’H and F3’5’H to produce anthocyanins.Therefore,F3H,F3’H and F3’5’H are the key enzymes for anthocyanin and anthocyanin synthesis,which are very important in the formation of anthocyanins.The present study is of Vitis amurensis "Shuanghong"cultivars as the experimental material.According to the important role of F3H,F3’H and F3’5’H in the formation of anthocyanins.Cloning the open reading frame(ORF)and promoter sequence based on Vitis amurensis F3H,F3’H and F3’5’H gene sequences from NCBI databas,and an expression vector was constructed for the prokaryotic expression of the gene and the transient expression of the promoter-transformed tobacco leaves.The main research results are as follows:1、The F3H,F3’H and F3’5’H gene sequences of Vitis amurensis "Shuanghong" were cloned to 1091 bp,1530 bp and 1528 bp,respectively.The physical and chemical properties of isoelectric point,molecular weight and secondary structure of "Shuangfeng" comparison with"Shuanghong"is different.2、F3H,F3’H and F3’5’Hgenes were used to construct prokaryotic expression vectors in BL21 strain of E.coli for prokaryotic expression.F3H,F3’H and F3’5’H genes were at 25℃、30℃ and 25℃,the training time is 7 h,11 h,and 11 h,and the IPTG concentration is 0.8 mmol/L,the target protein has a high-concentration expression band in the supernatant sample that is consistent with the predicted molecular weight of the recombinant protein.3、The promoters of the F3H,F3’H and F3’5’H genes were cloned by Tail-PCR,and the promoter sequence information was analyzed by bioinformatics software.The length of cloned promoters of F3H,F3’H and F3’5’H genes were 14.54 bp,1051 bp and 1270 bp.Predictive analysis by online analysis software such as BLASTn,BDGP,PlantCARE,and PLACE It were found that all three promoters contained the basic elements CAAT-box and TATA-box.In addition to having light-responsive elements that have an important effect on the anthocyanin formation of Vitis amurensis,it also contains a series of hormone-responsive elements,such as salicylic acid-responsive elements,abscisic acid-responsive elements,auxin-responsive elements,etc.4、Analysis of different promoter deletion constructs activity in tobacco leaves were transformed and transiently expressed.According to the measurement results,it is found that these three promoters have weak activity compared to CaMV 35S promoter.In F3H promoter,Gus enzyme activity first decreased and then increased with the increase of the length of promoter deletion segment;in F3’H promoter,Gus enzyme activity increased with the increase of the length of promoter deletion segment;in F3’5’H promoter,Gus enzyme activity first increased and then decreased with the increase of the length of promoter deletion segment.