Cloning, bioinformatics and functional analysis of MaFLS1 in banana

Abstract: 【Objective】Banana is an important tropical fruit crop, and it contains abundant flavonoids. Flavonoids are the most important and widely involved in plant growth and development, which play an important role in plant stress resistance. Although the flavonol synthase gene has been studied in other plants, its function has not been reported in banana fruit. In this paper, the function of MaFLS1 gene in banana pulp was preliminarily identified by carrying out transgenic heterologous functional verification on micro Tom tomato.【Methods】The open reading frame of the MdFLS1 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR). The sequences were obtained and analyzed by various bioinformatics methods, i.e. TMHMM online software, SignalP, SOPMA, MEGA7.0 software and so on. The expression pattern of MaFLS1 was studied by qRT-PCR during banana fruit ripening. The over-expression vector of MaFLS1 gene was constructed by homologous recombination and transformed as heterologous gene into Solanum lycopersicum L. (micro-Tom) by bladed disc conversion method with recombinant agrobacterium transformants, and the phenotypes of T1 generation transgenic positive plants and wild-type plants were observed, such as plant height, leaf color, fruit size and fruit color, and the total flavonoids were detected by Spectrophotometer.【Results】We used the reverse transcribed Xiangfen 1 flesh cDNA as the template, primers were designedby Primer Premier 5.0, and the PCR amplification was performed. The amplification results showed that the size of the amplified band was consistent with the reference sequence. Sequence analysis showed that the length of open reading box of the MaFLS1 contained 1080 bp bases and encoded 359 amino acids, the theoretical PI was 5.41, and the molecular mass was 39 436.94 Da. The unstable coefficient of the MaFLS1 protein was 37.35, and the total average value of hydrophilicity was -0.178, which belonged to be a stable hydrophilic acidic protein. The position of the MaFLS1 protein in banana was predicted, which was localized in the cytoplasm by using subcellular localization website (https://wolfpsort.hgc.jp/). The results of TMHMM online software analysis indicated that the MaFLS1 protein had no transmembrane domain, and there was also no signal peptide by using SignalP software. SOPMA online software (http://web. expasy. org/) was applied to predict the secondary structure of the MaFLS1 protein, and the results showed that the protein was mainly composed of α- helix (34.26% ), irregular curls (42.9%), extended chains (16.71%) and β-corner (6.13%). The prediction of the tertiary structure of this protein was carried out by SWISS- MODEL online software (http://swissmodel.expasy.org/). When the sequence of a protein was highly similar to a known structural protein sequence, the structure of the protein can be modeled. The conserved domain of MaFLS1 was predicted through the Protein Conservative Domain Prediction website (SMART), and the results showed that the MaFLS1 protein contained two functional domains, one was the highly conserved N-terminal region of a protein with 2- oxoglutarate/Fe (Ⅱ) dependent dioxide, located in the 49th to 160th amino acid positions. In plants, Fe (Ⅱ) 2OG dioxygenase domain enzymes catalyze the formation of plant hormones such as ethylene, gibberellin, pigments and flavonoids. The other was an enzyme structure with a Fe2+ and 2-ketoglutarate (2OG) dependent dioxygenase domain, located in the 206th-306th amino acids. The enzyme usually uses dioxin to catalyze the oxidation of organic substrate, mainly by using ferrous as the active site cofactor and 2OG as the co substrate, and decarboxylates to form succinate and CO2. DNAMAN software analysis showed that the amino acid similarity was about 51% between MaFLS1 protein and other species, and it was showed that FLS genes had a highly conserved amino acid sequence between different species by using MEGA7.0 software, and it was showed the genetic relationship between MaFLS1 protein and MaFLS3 and MaFLS2 was relatively close, it may be because banana belongs to herbaceous plants, and its hereditary distance is far away from the other species. The result of real-time PCR indicated that the expression level of the MaFLS1 gene was high in the later stage of fruit ripening, but it was almost not expressed in the early stage, and the results showed that after 65 d after cutting off buds, the relative expression increased sharply, indicating that the gene was expressed in the later stage of fruit development, which may participate in the maturity process of banana fruits. By measuring the total flavonoid content in the transgenic tomato, it was found that the total flavonoid content in wild-type fruits was 0.46 mg·g^-1 , which was significantly lower than the 0.58 mg·g^-1 in transgenic fruits, indicating that the MaFLS1 gene can indeed increase the total flavonoid content in fruits, and it was found that the transgenic fruits were smaller and lighter in color than the wild-type fruits, which may be possibly due to increased synthesis of flavonol branches and reduced anthocyanin content.【Conclusion】By comparing the expression of the MaFLS1 gene in the process of fruit maturity, the results showed that the expression of the gene increased significantly in the later stage of banana fruit maturity. Simultaneously, the gene can significantly increase the total flavonoid content in the tomato fruit by determining the content of total flavonoid.