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| Cloning and Expression Analysis of F3'H Gene and Promoter from Vitis amurensis |
| ZHANG Yu1,2, XU Zhi-Hui1, REN Shao-Qi1, YANG Ming-Hui1, CHEN Meng1, LIU Hai-Feng1,* |
1 College of Agriculture, Yanbian University, Yanji 133002, China;
2 Jilin Academy of Agricultural Sciences, Changchun 130033, China |
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Abstract Flavonoid-3'-hydroxylase (F3'H) is the key enzyme in the second stage of the anthocyanin synthesis pathway, it is very important in the anthocyanin synthesis pathway. In order to explore the formation mechanism of F3'H in the formation of anthocyanins in Vitis amurensis, the F3'H gene and its promoter sequence were cloned from the 'shuanghong' variety of V. amurensis, and the bioinformatics and prokaryotic expression analysis were performed on it, and the different length deletion fragment promoters of F3'H gene were transformed into tobacco (Nicotiana tabacum) leaves for transient expression. The results showed that the ORF of the F3'H gene was 1 530 bp in length and the promoter sequence was 1 051 bp in length. Bioinformatics analysis showed that the composition of the 'Shuanghong' variety F3'H was different from that of the 'Shuangfeng' variety in isoelectric point, molecular weight and secondary structure. The prediction of promoter cis-acting elements showed that in addition to the core promoter elements CAAT-box and TATA-box, there were a series of light-responsive elements, stress elements, hormone response elements in the promoter sequence. The transient expression results of different length deletion fragment promoters transformed into tobacco leaves showed that as the length of the deletion fragment promoter increases, the GUS enzyme activity of the F3'H promoter showed an upward trend. Through the construction of prokaryotic expression vector, it was found that the F3'H recombinant protein had a relative molecular weight of the predicted recombinant protein in the supernatant sample under the conditions of 30 ℃, culture time 11 h, and isopropyl β-D-thiogalactoside (IPTG) concentration 0.8 mmol/L, expression bands with consistent mass and high concentration. This study provides basic data for further study on the regulation mechanism of Vitis amurensis F3'H gene in the process of anthocyanin synthesis and the transcription mechanism of promoter.
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Received: 17 March 2021
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Corresponding Authors:
* Liufenge_1989@163.com
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