香蕉MaFLS1基因克隆、生物信息学分析及功能解析

Cloning,bioinformatics and functional analysis of MaFLS1 in banana

【目的】初步探讨黄酮醇合成酶FLS基因在香蕉果实中生物学功能。【方法】采用RT-PCR和PCR法克隆香蕉MaFLS1基因,对其进行生物信息学分析。运用qRT-PCR的方法研究其表达模式,同源重组构建MaFLS1基因的过表达载体,通过农杆菌介导的叶盘法转化Micro-Tom番茄,测定T1代果实中总黄酮含量。【结果】香蕉MaFLS1基因开放阅读框含有1080对碱基,编码359个氨基酸,理论等电点为5.41,预测分子质量为39 436.94 Da,是一种稳定的亲水酸性蛋白,属于α-酮戊二酸依赖性双加酶家族。通过分析香蕉MaFLS1的氨基酸序列,发现其不含信号肽和跨膜结构。系统进化树分析表明,FLS在不同物种间具有高度的氨基酸序列保守性。MaFLS1基因在香蕉果实发育成熟后期高度表达,前期基本不表达。通过测定转基因番茄中总黄酮的含量发现其总黄酮含量极显著高于野生型果实。【结论】MaFLS1在果实成熟后期高度表达,且能够显著增加果实中总黄酮的含量。

【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 homolo-gous 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.【Re-sults】We used the reverse transcribed Xiangfen 1 flesh cDNA as the template,primers were designed by 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 39436.94 Da.The unstable coeffi-cient 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://wolf-psort.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 on-line 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 ofα-helix(34.26%),irregular curls(42.9%),extended chains(16.71%)andβ-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 us-es dioxin to catalyze the oxidation of organic substrate,mainly by using ferrous as the active site cofac-tor 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 spe-cies,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 pro-tein 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 indi-cated 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 to-tal 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,indicat-ing 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.Simultane-ously,the gene can significantly increase the total flavonoid content in the tomato fruit by determining the content of total flavonoid.