MaDGAT3 gene and application thereof in increasing flavonoid content of banana fruits

Abstract

The invention discloses a MaDGAT3 gene and application thereof in increasing the flavonoid content of banana fruits. The nucleotide sequence of the MaDGAT3 gene is as shown in SEQ ID NO: 1. According to the application disclosed by the invention, a high-flavonoid-content banana resource'fragrant powder No.3 'is taken as a research object, through transcriptome and metabolome combined analysis, diacylglycerol acyltransferase 3 is found to be a key gene influencing flavonoid synthesis, and after the gene is silenced in banana fruits, the flavonoid content is obviously reduced; the MaDGAT3 plays an important role in positively regulating and controlling the flavonoid content of the banana fruits, the discovery of the gene provides an important target spot for creating the banana fruits with high flavonoid content through a genetic engineering means in the later period, and healthy and long-term development of the banana industry is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and specifically relates to MaDGAT3 Genes and their application in increasing the flavonoid content of banana fruit. Background Art

[0002] Bananas are an important fruit widely grown in tropical and subtropical regions. Flavonoids are a class of secondary metabolites found throughout plants, exhibiting a variety of important biological activities. In plants, flavonoids not only participate in plant growth and development, and in resisting pests and diseases, but also act as pigments in the formation of flower color. Flavonoids possess numerous benefits in the human body, including antioxidant, anti-inflammatory, hypoglycemic, anti-allergic, cardiovascular, and antibacterial and antiviral properties, demonstrating broad application prospects in medicine and food. Bananas are also rich in flavonoids, making them an important dietary source for the human body. Flavonoids in bananas primarily include quercetin, kaempferol, and isoquercetin. These flavonoids impart antioxidant and health-promoting properties to bananas, contributing to their nutritional and economic value.

[0003] Currently, research on the regulation of flavonoid biosynthesis is limited, and the relevant functional genes remain unclear. Despite the important role of flavonoids in plant and human health, and the crucial impact of flavonoid content in banana fruit on its quality and value, relatively little research has been conducted on the regulation of flavonoid biosynthesis in banana fruit. Existing research has primarily focused on the extraction, isolation, identification, and bioactivity analysis of flavonoids, while the molecular regulatory mechanisms underlying flavonoid biosynthesis remain relatively understudied. In bananas in particular, the functional genes involved in regulating flavonoid biosynthesis remain largely unknown, significantly limiting the research and application of genetic engineering approaches to precisely manipulate flavonoid content in banana fruit and, consequently, improve banana quality. In-depth research on the regulatory mechanisms of flavonoid biosynthesis in banana fruit and the identification of key functional genes has important theoretical and practical implications for enhancing the nutritional value and quality of banana fruit, as well as promoting the sustainable development of the banana industry. Summary of the Invention

[0004] The object of the present invention is to provide MaDGAT3 Genes and their application in increasing the flavonoid content of banana fruit.

[0005] MaDGAT3 gene, the MaDGAT3 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0006] MaDGAT3 protein, the amino acid sequence of the MaDGAT3 protein is shown in SEQ ID NO: 2.

[0007] Contains the MaDGAT3 Gene carrier.

[0008] Contains the MaDGAT3 Engineered bacteria that carry gene vectors.

[0009] Detection MaDGAT3 Primers for any fragment of the gene.

[0010] described MaDGAT3 Application of genes in increasing flavonoid content in banana fruit.

[0011] A method for increasing the flavonoid content of banana fruit, comprising overexpressing the MaDGAT3 Gene.

[0012] Beneficial effects of the present invention: The present invention uses the high flavonoid content banana resource "Xiangfen No. 3" as the research object, and through the combined analysis of transcriptome and metabolome, it is found that diacylglycerol acyltransferase 3 is a key gene affecting flavonoid synthesis. After silencing this gene in banana fruits, the flavonoid content is significantly reduced, indicating that MaDGAT3 It plays an important role in positively regulating the flavonoid content of banana fruits. The discovery of this gene provides an important target for the subsequent creation of banana fruits with high flavonoid content through genetic engineering, which is conducive to the healthy and long-term development of the banana industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This study analyzed the content of key metabolites in the fruit of "Xiangfen No. 3" at different developmental stages.

[0014] Figure 2 Analysis of the main components and DEG genes of the transcriptome of "Xiangfen No. 3" fruits at different developmental stages.

[0015] Figure 3 KEGG and GO enrichment analysis of differentially expressed genes in the transcriptome of "Xiangfen No. 3" fruits at different developmental stages.

[0016] Figure 4 This study analyzed the main components and differential metabolites of the fruit metabolome of "Xiangfen No. 3" at different developmental stages.

[0017] Figure 5 KEGG enrichment analysis of differential metabolites in the fruit metabolome of "Xiangfen No. 3" at different developmental stages.

[0018] Figure 6 The results of the 9-quadrant joint analysis of the transcriptome and metabolome of the fruit of "Xiangfen No. 3" at different developmental stages were analyzed; The figure is divided into quadrants 1-9 from left to right and from top to bottom. Quadrants 1 and 9 represent genes and metabolites with opposite differential expression patterns and inconsistent regulatory trends. Metabolite expression changes may be due to negative regulation of genes. Quadrants 3 and 7 represent genes and metabolites with consistent differential expression patterns and positive correlations. Metabolite expression changes may be due to positive regulation of genes. Other quadrants represent non-differential expression of genes and metabolites in the differential group.

[0019] Figure 7 Heat map of key DEGs from the joint analysis of transcriptome and metabolome of fruits of “Xiangfen 3” at different developmental stages.

[0020] Figure 8 The key DEGs cis-acting elements were analyzed by joint analysis of the transcriptome and metabolome of the fruit of "Xiangfen No. 3" at different developmental stages.

[0021] Figure 9 Functional analysis of MaDGAT3 regulating banana flavonoid biosynthesis. DETAILED DESCRIPTION

[0022] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] Example 1 Analysis of key metabolite contents in different developmental stages of “Xiangfen No. 3” fruit High performance liquid chromatography was used to determine the total phenols, tannins, carotenoids, and total flavonoids in banana fruits of "Xiangfen No. 3" at 15, 25, 45, 85, and 88 days after bud breaking. The results showed that 45, 85, and 88 days after bud breaking were the key periods for changes in the above metabolites. Figure 1 ).

[0024] Example 2 Analysis of the main components and DEG genes of the transcriptome of “Xiangfen No. 3” fruits at different developmental stages Transcriptome sequencing analysis was performed on 45d, 85d and 88d old "Xiangfen No. 3" banana fruits. The results of principal component analysis and differentially expressed gene (DEG) analysis showed that ( Figure 2 ), the reproducibility of fruit samples at each time point was good, and the number of differentially expressed genes between the 45d vs 88d and 85d vs 88d comparison groups was significantly higher than that between the 45d vs 85d comparison group, indicating that 88d may be a critical period for banana fruit development.

[0025] Example 3 KEGG and GO enrichment analysis of differentially expressed genes in the transcriptome of “Xiangfen No. 3” fruit at different developmental stages Further KEGG and GO enrichment analysis was performed on the transcriptomes of the above groups. The results showed that ( Figure 3 ), 45d vs 88d, 85d vs 88d, and 45d vs 85d comparison groups were mainly enriched in flavonoid biosynthesis, phenylpropanoid biosynthesis, sugar and fatty acid metabolism pathways, indicating that these pathways may be the key factors in dominating banana nutrient metabolism.

[0026] Example 4 Analysis of the main components and differential metabolites of the metabolome of the fruit of “Xiangfen No. 3” at different developmental stages In order to better understand the dynamic changes of nutritional metabolites during the development of "Xiangfen No. 3" banana fruits, metabolome analysis was further conducted on banana fruits at three different developmental stages: 45 days, 85 days, and 88 days. The results also showed that ( Figure 4 ), the number of differential metabolites between the 45d vs 88d and 85d vs 88d comparison groups was significantly higher than that between the 45d vs 85d comparison group, further indicating that the transition process from 85d to 88d may be a key time point for changes in nutrients in banana fruit.

[0027] Example 5 KEGG enrichment analysis of differential metabolites in the metabolome of fruits of “Xiangfen No. 3” at different developmental stages Further analysis showed that ( Figure 5 ), metabolic pathways such as terpenes, tannins, steroids, phenolic acids, organic acids, nucleotides and their derivatives, lipids, lignin, flavonoids, alkaloids, amino acids and their derivatives showed significant differences in the three comparison groups of 45d vs 88d, 85d vs 88d, and 45d vs 85d, and flavonoids, phenolic acids, and lipids may be the main factors causing the dynamic changes of nutritional metabolites during the development of "Xiangfen No. 3" fruit.

[0028] Example 6 KEGG enrichment analysis of differential metabolites in the metabolome of fruits of “Xiangfen No. 3” at different developmental stages In order to analyze the correlation between the changes in metabolites and gene expression in the fruits of Xiangfen No. 3, a 9-quadrant analysis of transcriptome and metabolome was further performed. The results showed that ( Figure 6 ), most of the genes are involved in the regulation of changes in the content of metabolites in the fruits of "Xiangfen No. 3".

[0029] Example 7 Joint analysis of transcriptome and metabolome of fruits of “Xiangfen No. 3” at different developmental stages Heat map of key DEGs and analysis of cis-acting elements Heat map and gene cis-acting element analysis were performed on the common genes of the three comparison groups of 45d vs 88d, 85d vs 88d and 45d vs 85d in the above 9-quadrant analysis results. The results showed that ( Figure 7-8 ), compared with 88 days, most of the above-mentioned common genes were up-regulated at 45 days and 85 days. Further analysis of their cis-acting elements showed that the MaDGAT3 gene had a flavonoid biosynthesis response element, indicating that this gene may be a key factor affecting the changes in metabolites during the development of "Xiangfen No. 3" fruit.

[0030] Example 8 Functional analysis of MaDGAT3 regulating banana flavonoid synthesis To further verify the above MaDGAT3 The function of genes in regulating the synthesis of banana flavonoids was constructed. MaDGAT3 The VIGS transient gene silencing vector of the gene was used and the "Xiangfen No. 3" banana fruit was infected by Agrobacterium-mediated method. The results showed that ( Figure 9 Middle A), compared with the control, in the corresponding VIGS silenced plants MaPDS (indicator gene, fruit turns green after silencing) and MaDGAT3 The expression levels of the genes were significantly decreased, and after 11 days of VIGS silencing treatment, MaPDS The peel of the silent banana showed a significant chlorosis. MaDGAT3 Physiological and biochemical analysis of banana fruits after gene modification showed that ( Figure 9 Middle B), and MaPDS Compared to the instant silence fruit, MaDGAT3 The contents of chlorophyll a and b in the instantaneous silent fruit were both high, while the total flavonoids content decreased significantly by about 30%. There was no significant difference in total phenols and anthocyanins between the two, indicating that MaDGAT3 It plays an important role in positively regulating the total flavonoids content in bananas.

[0031] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. MaDGAT3 A gene characterized by described MaDGAT3 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. MaDGAT3 protein, characterized in that The amino acid sequence of the MaDGAT3 protein is shown in SEQ ID NO:

2.

3. Containing the claim 1 MaDGAT3 Gene carrier.

4. Containing the claim 3 MaDGAT3 Engineered bacteria that carry gene vectors.

5. Detection of claim 1 MaDGAT3 Primers for any fragment of the gene.

6. The method according to claim 1 MaDGAT3 Application of genes in increasing flavonoid content in banana fruit.

7. A method for increasing the flavonoid content of banana fruit, characterized in that: Overexpression of the compound of claim 1 in banana MaDGAT3 Gene.

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