Waxberry MrMYB6 gene and application thereof

Abstract

The invention discloses a myrica rubra MrMYB6 gene and application thereof. The sequence of the MrMYB6 gene is as shown in SEQ ID No. 1. Based on RNA-Seq, transcriptome analysis of bioinformatics and gene regulatory network analysis, the invention discovers the MrMYB6 gene capable of promoting accumulation of soluble sugar, salicylic acid and jasmonic acid in waxberry fruits. The gene is transferred into arabidopsis thaliana, and it is found that the gene can promote growth of arabidopsis thaliana by increasing the content of soluble sugar, salicylic acid and jasmonic acid. Therefore, the gene can be used for genetic improvement of plants.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a bayberry MrMYB6 gene and an application thereof. Background Art

[0002] Bayberry (Myriea rubra Sieb.Et Zucc) is a fruit native to the subtropical region of southern China. Bayberry fruit is sweet and sour, rich in nutritional value, rich in vitamin C, flavonoids, polyphenols, organic acids and other functional ingredients, deeply loved by consumers, and has high commercial value.

[0003] Fruit development is not only a stage in the life cycle of plants, but also an essential process for seed reproduction. Existing studies have shown that as the fruit grows, its cells undergo a series of changes, such as cell division, cell expansion, and an increase in primary and secondary metabolites. The fruit development process undergoes a series of biochemical reactions, which vary from species to species. In general, these biochemical reactions lead to corresponding biochemical changes, such as sugar metabolism, organic acids, pigment accumulation, fruit softening, and changes in flavor and aroma volatile content.

[0004] Studying the growth and development of bayberry fruit and exploring its molecular mechanisms will help improve the quality of bayberry fruit and create greater commercial value. Summary of the invention

[0005] In view of the above problems, based on RNA-Seq, transcriptome analysis and bioinformatics methods, this application conducted a comparative study on the bayberry fruits of two cultivars at different developmental stages (10 days, 20 days, 30 days, 4 days, and 50 days after pollination), and found that the two bayberries had the same regulatory modules and different regulatory modules on the gene regulatory network. In the further research process, the MrMYB6 gene related to the synthesis of soluble sugars, salicylic acid (JA) and jasmonic acid (SA) was found. Then, a transcription vector containing the MrMYB6 gene was constructed, and transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation, and Arabidopsis thaliana plants with MrMYB6 overexpression were constructed, further proving that the MrMYB6 gene has the effect of increasing the content of soluble sugars, JA and SA and promoting plant growth.

[0006] In one aspect of the present application, a Myrica rubra MrMYB6 gene and a protein encoded by the gene are provided, wherein the sequence of the MrMYB6 gene is shown in SEQ ID NO. 1. The amino acid sequence of the protein encoded by the Myrica rubra MrMYB6 gene is shown in SEQ ID NO. 2.

[0007] The second aspect of the present application provides the use of the bayberry MrMYB6 gene in preparing an agent for regulating plant growth or fruit development.

[0008] In some embodiments, the MrMYB6 gene promotes plant growth or fruit development by promoting the synthesis of soluble sugars and organic acids.

[0009] In some embodiments, the organic acid includes salicylic acid and jasmonic acid.

[0010] In some embodiments, the plant is Arabidopsis thaliana or Bayberry.

[0011] In some embodiments, the fruit is bayberry fruit.

[0012] The third aspect of the present application provides an expression vector containing the MrMYB6 gene.

[0013] The fourth aspect of the present application provides a host cell containing the MrMYB6 gene.

[0014] Compared with the prior art, the present invention has the following advantages: the present invention discovered a new gene, MrMYB6 gene, from bayberry, which regulates the content of soluble sugar, JA and SA in bayberry fruit. In addition, the MrMYB6 gene was transferred into Arabidopsis thaliana and found that the MrMYB6 gene can promote the growth of Arabidopsis thaliana and increase the content of soluble sugar, JA and SA in its plants. Therefore, the MrMYB6 gene can be used to regulate plant growth or fruit development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Bayberry fruit conditions at 10, 20, 30, 40, and 50 days after pollination, A: fruit appearance, B: dynamic changes in fruit drop rate during fruit development, C: dynamic changes in fruit weight during fruit development;

[0016] Figure 2 Soluble sugar and organic acid contents of bayberry fruit at 10, 20, 30, 40, and 50 days after pollination;

[0017] Figure 3 The number of differentially expressed genes (DEGs) in bayberry fruit at 20, 30, 40, and 50 days compared with 10 days after pollination;

[0018] Figure 4 Expression of genes involved in sugar metabolism pathways at different developmental stages of bayberry fruit. A: Expression patterns of sugar metabolism-related enzyme genes in two cultivated bayberry varieties. Red indicates high expression levels, blue indicates low expression levels, and expression levels are standardized by row. B: MrSPS1 gene expression in ZJ, C: MrSPS1 gene expression in DA, D: MrFK7 gene expression in ZJ, E: MrFK7 gene expression in DA.

[0019] Figure 5 Transcription patterns of genes involved in plant hormone-related pathways, A: Expression patterns of hormone-related pathway genes in two cultivated bayberry varieties, expression levels were normalized by row, B: MrPP2C gene expression in ZJ, C: MrPP2C gene expression in DA, D: MrAXR1 gene expression in ZJ, E: MrAXR1 gene expression in DA, F: MrARF17 gene expression in ZJ, G: MrARF17 gene expression in DA;

[0020] Figure 6 Cluster analysis results of DA gene expression, A: Gene expression patterns in clusters, C1-C13 are clusters, red represents high expression, blue represents low expression, cluster colors are listed next to the heat map, which are the same as the colors of the expression dynamics of the five important clusters listed on the right side of the heat map; B: Gene ontology enrichment analysis results of DA varieties, red represents significant enrichment in the corresponding pathway;

[0021] Figure 7 Cluster analysis results of gene expression in ZJ, A: Gene expression patterns in clusters, C1-C12 are clusters, red represents high expression, blue represents low expression, cluster colors are listed next to the heat map, which are the same as the colors of the expression dynamics of the five important clusters listed on the right side of the heat map; B: Gene ontology enrichment analysis results of ZJ variety, red represents significant enrichment in the corresponding pathway;

[0022] Figure 8 Changes of transcriptome over time during the development of ZJ and DA fruits. A: Changes of the 10 largest gene clusters (G1-G10) over time. B: GO terms of genes enriched in the G1-G10 clusters.

[0023] Fig. 9 Key transcription factors in fruit development, A: Differentially expressed transcription factors in DA fruit development, B: Overlap of differentially expressed transcription factors in DA at different developmental stages, C: Transcription factor families detected in two bayberry cultivars, D: Phylogenetic analysis of MYB transcription factors, the outer blue dots refer to the MYBTFs detected in this study, the orange and red dots refer to the differentially expressed TFs (transcription factors) in ZJ and DA, respectively, E: Heat map showing the expression patterns of key transcription factors in two bayberry cultivars;

[0024] Fig.10 Expression profiles of candidate genes in Bayberry, heat map of tissue-specific expression;

[0025] Fig.11MrMYB6 positively regulates leaf growth and SA synthesis, A: Phenotypes of MrMYB6 overexpressing Arabidopsis (OE), RNAi Arabidopsis (RNAi) and wild-type Arabidopsis (WT) 21 days after germination, B: Germination rate of WT, OE and RNAi at 7 days, C: Leaf area of ​​WT, OE and RNAi, D: Leaf fresh weight of WT, OE and RNAi, E: Leaf dry weight of WT, OE and RNAi, F: Stem cross section of WT, OE and RNAi 21 days after germination, electron microscope scale = 100 μm, ph refers to phloem, xy refers to xylem; ep refers to epidermis, G: Epidermal cell number of WT, OE and RNAi 21 days after germination, H: SA content of WT, OE and RNAi 28 and 35 days after germination;

[0026] In the figure, ZJ refers to the bayberry cultivar “Zaojia”, DA refers to the bayberry cultivar “Dingao”; SA refers to jasmonic acid, JA refers to salicylic acid (JA); T10, T20, T30, T40, and T50 refer to bayberry fruits at 10, 20, 30, 40, and 50 days of pollination, respectively. DETAILED DESCRIPTION

[0027] In order to describe the present invention more specifically, the technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and specific implementation methods. It should be pointed out that the embodiments are only a detailed description of the present invention and cannot be used to limit the protection scope of the present invention. All features disclosed in the embodiments of the present invention, or steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way and are within the protection scope of the present invention. Technologies not covered by the present invention can be implemented by existing technologies.

[0028] In order to explore the development characteristics of bayberry fruit and the possible common biological regulation mechanisms, this application first selected two types of bayberry for research, compared the fruit development of the two types of bayberry at different developmental stages, and found that the fruit size, soluble sugar and organic acid content of the two types of bayberry had a significant correlation with the change trend of the development period, indicating that the two types of bayberry have similar regular biological network regulation mechanisms. These two types of bayberry are Dingao Bayberry and Zaojia Bayberry. Among them, Dingao Bayberry is one of the "four major bayberry" varieties in Zhejiang Province, and the picking period is early June; Zaojia is a new variety of bayberry recognized by the Zhejiang Provincial Forestry Variety Approval Committee, and the picking period is also early June. Both types of bayberry are early-maturing bayberries and are representatives of traditional fine cultivars and emerging fine cultivars, respectively, and can effectively represent early-maturing bayberries.

[0029] Furthermore, the present application adopts a temporal transcriptome analysis method, sets different developmental stages of the fruit, obtains transcriptome data at different developmental stages through RNA-seq and performs comparative analysis, uses bioinformatics technology to find differentially expressed genes (DEGs), performs enrichment analysis on DEGs, constructs a gene regulatory network (GRN), and combines the fruit development to find the biological regulatory network and key regulatory genes that regulate the growth and development of bayberry fruit.

[0030] Through research, the present invention discovered the MrMYB6 gene, whose sequence is shown in SEQ ID NO.1, and the amino acid sequence of the MrMYB6 gene is shown in SEQ ID NO.2.

[0031] Furthermore, the present invention constructed a vector containing the MrMYB6 gene and introduced it into host cells through Agrobacterium transformation to obtain MrMYB6 gene overexpression plants, confirming that the MrMYB6 gene has the effect of promoting fruit development and increasing the content of soluble sugar and organic acid in the fruit.

[0032] Example 1 Bayberry Fruit Development

[0033] Fruits of two bayberry cultivars Zaojia (ZJ) and Dingao (DA) at different developmental stages (10, 20, 30, 40, and 50 days after pollination) Figure 1 A shows the results of fruit weight and fruit drop rate. Figure 1 B and Figure 1 C (For details of the experimental materials and methods, see "1. Experimental materials" and "10. Extraction and determination of endogenous JA, SA and soluble sugars" below). The results in the figure show that there is no significant difference in the weight of single fruit between the two cultivars ZJ and DA; the fruit drop rate of DA is significantly higher than that of ZJ; between 40 and 50 days after pollination, the fruit drop rate of DA increases particularly significantly, while the fruit drop rate of ZJ does not change much; in addition, there is a significant correlation between the fruit weight and fruit drop rate of DA and ZJ (P<0.05).

[0034] The contents of soluble sugar and organic acid (salicylic acid and jasmonic acid) in bayberry fruits at different development stages are as follows: Figure 2 As shown. It can be seen that the soluble sugar and organic acid contents of DA were significantly higher than those of ZJ; the soluble sugar contents of DA and ZJ remained at a low level during fruit development, and increased significantly during 20-40 days and reached the highest level at 40 days; the salicylic acid (JA) and jasmonic acid (SA) contents of the two showed a rapid growth trend from 20 to 50 days, and the growth trend of JA content during 20-30 days was faster than that during 30-40 days; there was a significant correlation between the soluble sugar and organic acid contents of DA and ZJ (P<0.05).

[0035] The above results indicate that the fruit development of DA and ZJ bayberry, including fruit size, fruit drop rate, soluble sugar and organic acid content, has similar regular biological regulatory networks.

[0036] Example 2 Identification of candidate differentially expressed genes (DEGs) related to fruit development

[0037] RNA-seq analysis was performed on ZJ and DA fruit samples at different developmental stages. Compared with T10, the number of DEGs in other developmental stages was Figure 3 As shown (the experimental methods are detailed in "2. RNA extraction, transcriptome library construction and RNA-seq data processing" below). In general, we obtained 13515 and 13733 expressed genes (FPKM>1) in ZJ and DA cultivars, respectively. Compared with T10, there were 5168 DEGs in ZJ late fruits (T20, T30, T40, T50) and 4105 DEGs in DA late fruits (difference fold>2, FDR<0.01). Among the DEGs of ZJ, those related to plant-type cell wall and beta-glucosidase activity accounted for a higher proportion; those of DA were concentrated in pathways related to organic acid signaling pathway, regulation of hormone levels, cell wall organization, and cytoskeletal protein binding, indicating that the expression patterns of the differentially expressed genes detected in the two cultivars showed high dynamics at different developmental stages of the fruit.

[0038] Compared with T10, we detected 451 DEGs (186 up-regulated and 265 down-regulated) in T20 of ZJ, and these genes were mainly enriched in the process of nucleosome organization. However, the DEGs in T20 of DA were concentrated in the organic acid signaling pathway, regulation of developmental growth, and intrinsic component of membrane.

[0039] Compared with T10, we detected 2764 DEGs (1577 up-regulated and 1187 down-regulated) in T30 of ZJ, which were mainly enriched in organic acid signaling pathway, anthocyanin-containing compound biosynthetic process, response to hormone, and plant-type cell wall (Table S4). For DA, 1518 DEGs were detected in T30, which were mainly enriched in plant-type secondary cell wall biogenesis, sugar-metabolism signaling pathway, and cell wall polysaccharide biosynthetic process. In summary, sugar metabolism and organic acid signaling pathways, cell wall biosynthesis, and secondary metabolite accumulation pathways were detected in both cultivars, which is consistent with the above-mentioned study on fruit development.

[0040] Example 3 Transcription of DEGs in the Sugar Metabolism Signaling Pathway

[0041] Important genes related to sugar metabolism have a considerable impact on all stages of fruit development. During fruit development, carbohydrates not only provide energy for fruit development, but also contribute to the accumulation of sugars and improve fruit quality. Soluble sugars, such as glucose, sucrose, and fructose, are involved in the genetic regulation of plant metabolism and defense responses, which is beneficial to plant growth and development. Research on sucrose metabolism has mostly focused on sugar metabolic enzymes, such as sucrose synthase (SS), sucrose phosphate synthase (SPS), fructokinase (FK), hexokinase (HK), etc. The expression of sugar metabolism-related genes in two bayberry cultivars at different developmental stages (10, 20, 30, 40, and 50 days after pollination) is shown below. Figure 4(AE) (For details of the experimental method, see "4. Reverse transcription quantitative PCR of DEGs" below). It is worth noting that the expression of MrSS2 (GENE_013917) and MrSS (GENE_025677) underwent an obvious dynamic change during the development stage. During fruit development, the expression levels of MrSS in ZJ and DA remained at a high level. The expression of MrSS2 in ZJ decreased slightly at T40 and then increased sharply at T50. In contrast, for DA, the expression level of MrSS2 began to increase at T30. In addition, throughout the fruit development process, the expression levels of MrSPS1 in the two cultivars gradually increased, showing more obvious dynamic changes in the cultivar DA ( Figure 4 BC). MrSPS2 showed similar expression patterns in both cultivars ( Figure 4 A).

[0042] Fructose kinase (FK) helps phosphorylate fructose to form fructose 6-phosphate (F6P) and plays a role in various stages of plant development. The expression of MrFK7 gradually increased in both cultivars ( Figure 4 DE). In contrast, MrFK2 had higher expression levels and greater fluctuations in both cultivars ( Figure 4 A), the expression of MrFK2 decreased from T10 to T30 in DA, while that of MrFK2 decreased from T20 to T40 in ZJ. In addition, the expression of MrHK2 increased during fruit development, however, a decrease in expression was observed at T50 in ZJ and T30 in DA, respectively. In general, the sugar metabolism-related enzymes of the two cultivars shared common characteristics, however, sucrose synthases showed different expression patterns in the two cultivars.

[0043] Example 4 Transcription of DEGs in the organic acid signaling pathway

[0044] Organic acids are thought to play a key role in forming sour taste and lowering pH. A total of 67 genes were mapped to the organic acid pathway, of which 34 genes were expressed in ZJ and 32 in DA. Several genes were found to have significant expression changes. For example, K + It is a key activator in the organic acid signaling pathway. + Outflow Reactor 6(K +Homologous genes of effluxantiporter 6 (KEA6), including GENE_022715 and GENE_025628, showed a decrease during the fruit development stage in the DA cultivar, whereas a short-term increase was detected at the T20 stage in ZJ. The transcript levels of O-acyltransferase WSD1 (MrWSD1) (GENE_021286, GENE_002136) showed a continuous increasing trend at all stages in both cultivars, however, a decrease in the expression of GENE_002136 at T40 was detected in DA. MrTIFY6B showed a significant decrease at the T40 stage in both cultivars, and the overall expression level of MrTIFY6B in ZJ was higher than that in DA.

[0045] In particular, SA and JA are important organic acids that promote fruit ripening and affect fruit quality. The experimental results show that 42 genes of SA-related signaling pathways and 37 genes of JA-related signaling pathways are expressed in the two cultivars, respectively. For example, the expression of the homologous gene MsPHS1 (GENE_011967) of the dual-specific protein phosphodiester continued to decrease in cultivar DA. Differently, in cultivar ZJ, its expression was upregulated at T20 and then decreased. MrSAD2 (GENE_017042) may be involved in the nuclear transport of SA signals. Its transcription showed an increasing trend in each developmental stage of DA, and its expression in ZJ decreased at T30. The SA signaling protein MrPP2C (GENE_020213, a homologous gene of PP2V, and the expression of PP2C has been found to be induced by ethylene) was also upregulated during the development of cultivar DA, but was significantly downregulated at T50 in cultivar ZJ ( Figure 5 AC). It is noteworthy that MrSGT1 (GENE_009680) and MrAXR1 (GENE_012252) of both cultivars were classified into clusters enriched with JA signaling pathway genes (cluster 8 for ZJ and cluster 2 for DA), and their expression levels were significantly upregulated in both cultivars at different developmental stages, but slightly decreased in T40 ( Figure 5 DE). Therefore, we suspected that the distinct differences in the expression of JA- and SA-related genes might be significantly associated with the fruit development of the two cultivars.

[0046] Example 5 Conserved and divergent dynamic transcriptional signatures between two cultivars

[0047] To gain insight into transcriptome dynamics across developmental stages, we then performed hierarchical clustering ( Figure 6 ; Figure 7; the optimal K for ZJ / DA was 12 / 13) analysis. For ZJ and DA cultivars, the hierarchical clustering network consisted of 13,515 and 13,733 differentially expressed genes and had 12 and 13 clusters, respectively. For both cultivars, genes with similar expression patterns were enriched in the same biological pathways. For example, cluster 8 of cultivar ZJ contained 1,477 (10.9%) genes with low expression in the early stages and high expression in the later stages. Notably, SA-mediated signaling pathways and JA-mediated signaling pathways were overrepresented in the clusters. In cultivar DA, we observed similar expression patterns of genes involved in SA- and JA-mediated pathways (Cluster 2 in DA; Figure 6 A). In addition, genes in cluster 5 of ZJ were closely related to sugar metabolism, of which 1064 (7.9%) genes were involved in cellular carbohydrate metabolism, glucan metabolism, and polysaccharide metabolism. The expression of genes in cluster 5 (C5) peaked at T20 and T50 and remained at low levels at other developmental stages. In C5, GENE_012076 encoding alpha-amylase 3 (which has been found to act on glucan substrates to release maltooligosaccharides) and GENE_002496 encoding isoamylase 3 are known to be involved in the degradation of starch to sucrose, and these enzymes may play an important role in carbohydrate metabolic pathways. For cultivar DA, genes related to sugar metabolism were classified into groups 11 (C11) and 13 (C13), which had similar expression patterns to those of C5 of ZJ. In summary, the results indicate that the SA-mediated pathway is a similar regulatory module present between the two cultivars.

[0048] Several genes in the same pathway had different expression patterns in the two cultivars. The transcription of 754 (5.6%) genes in cluster 10 (C10) of cultivar ZJ increased continuously from T10 to T30 and then decreased to a stable level. The genes in this cluster were enriched in the regulation of olefin metabolic process and the regulation of ethylene biosynthetic process. In addition, in ZJ, the genes in cluster 6 (C6) (750 out of 13,515) were enriched in cellulose-related pathways and sugar metabolic pathways and were highly expressed at T20. In addition, the genes in cluster 9 (C9) (1,071 out of 13,515) were highly expressed at T30. These genes were enriched in the lignin biosynthesis process and the xylan metabolic and biosynthetic pathways. Therefore, the genes in C6 and C9 are related to plant-type primary and secondary cell wall biogeneration, respectively. C6 and C9 contained genes annotated as cellulose synthase A catalytic subunits, which are involved in the formation of the primary cell wall. In contrast, in cultivar DA, genes related to the cellulose, lignin, and xylan pathways were enriched in cluster 7 (C7) and their expression levels continued to decrease during the developmental stages. Overall, this result further confirmed the existence of differences in regulatory modules between the two cultivars.

[0049] To further understand the common and unique transcriptome dynamics of the two bayberries, K-means clustering was used to obtain 50 dynamic clusters of DEGs according to their expression patterns. Figure 8 A), we found 7 similar clusters (G1-G7) and 3 special clusters (G8-G10). The genes in the G2 cluster showed a continuous upward expression trend in both bayberries, especially in the later stages of fruit development. The expression of genes in the G8 cluster of DA was similar to that of the G2 cluster. These similar gene expression dynamics may make the two bayberries have similar biological processes. Different gene ontology (GO) categories, including cell wall biosynthesis, hormone regulation process, and stress response process, were enriched in the dynamic clusters of the two bayberries at multiple time periods ( Figure 8B), however, some GO terms were specific to one bayberry species. In both bayberries, the G2 cluster (which showed a significant increase in gene expression at later stages of development) was enriched in stress response, JA-, and SA-mediated pathways, and the G8 cluster with similar gene expression trends in DA had similar pathways. Genes in cluster 8 of ZJ and cluster 2 of DA for JA- and SA-related pathways were overexpressed, and similarly, genes in G2 of both bayberries and G8 of DA had similar expression patterns, suggesting that JA and SA play similar roles in fruit development in both bayberries. Notably, GO terms associated with JA and SA were related to stress response. This similarity in biological function suggests that JA and SA-responsive genes maintain similar expression patterns.

[0050] Example 6 Transcriptional regulatory network related to fruit development and ripening

[0051] Transcription factors (TFs) were more concentrated in the network than other genes and also had more sample-specific expression profiles. In our study, 710 TFs and 730 TFs were found in ZJ and DA, respectively, at different developmental stages. These TFs had more sample-specific expression profiles, and the number of differentially expressed TFs in different samples increased ( Fig. 9 AB). In particular, transcription factor families including MYB, basic helix-loop-helix (bHLH), ethylene responsive factor (ERF), MADS-Box, WRKY, and basic leucine zipper (bZIP) showed the highest enrichment in ZJ and DA ( Fig. 9 C). For example, WRKY proteins constitute a large family of plant-specific transcription factors that are crucial in plant defense, biotic and abiotic stresses. Interestingly, in both cultivars, more than 30% of MrWRKYs expression was grouped in clusters associated with the SA and JA pathways, which are expressed later in development (cluster 8 for ZJ and cluster 2 for DA, Figure 6 A; Figure 7 A). In cultivar DA, the transcription of MrWRKY18, MrWRKY31, and MrWRKY75 increased significantly. In contrast, the expression levels of MrWRKYs in cultivar ZJ were lower, showing different expression patterns between the two cultivars ( Fig. 9E). In our study, MADS-Box proteins such as MrJOINTLESS, MrAGL8, and MrMADS6 were significantly upregulated during the developmental stages of both cultivars. The JOINTLESS gene, encoding a MADS-Box transcription factor, is known to promote abscission zone development in tomato. During fruit development, the transcription of MrAGL8 (AGL8) was upregulated and showed significantly high expression levels in both cultivars ( Fig. 9 E), indicating that MrAGL8 plays a key role in regulating fruit development. MADS-Box transcription factor 6 was upregulated during this process and has been reported to play a role in floral organ identity and floral meristem determination.

[0052] Notably, MYB TFs have been reported to be involved in leaf and root development. In our study, the expression of MrMYB86 (GENE_020272) and MrMYB6 (GENE_000172) in both cultivars increased consistently during the developmental stages ( Fig. 9 E). Interestingly, the transcription of MrMYB108 and MrMYB6 showed a continuous increase in DA and had different expression patterns in the two cultivars, suggesting that these MrMYB TFs may be required for sustained plant growth, development, and resistance to hypoxia. To further explore the gene regulatory network (GRN) of fruit development, we constructed a multi-layered hierarchical GRN by combining all the differentially expressed TFs and potential target genes (PTGs) involved in sugar metabolism and organic acid signaling pathways mentioned above. As a result, a total of 21 TF-PTG pairs were identified as being located at the bottom, middle, and top levels of the network. Therefore, we identified five key TFs upstream of fruit genes, including MYB, NAC, GRF, and WRKY genes (P<0.05). Notably, MrMYB6, a member of the MYB family, regulated 105 downstream soluble sugar, SA, and JA signaling pathway genes (P<0.05). We randomly selected 15 genes from these 105 genes and detected the presence of MrMYB6 binding motif (GGTAG) in their proximal regions, which further illustrates this GRN in fruit development. The gene sequence of MrMYB6 and the amino acid sequence of the encoded protein are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0053] Example 7 MrMYB6 positively regulates leaf growth and the synthesis of soluble sugars, JA and SA

[0054] Tissue-specific analysis showed that MrMYB6 was most highly expressed in fruit tissues compared with the other seven tissues ( Fig.10), indicating that MrMYB6 plays a key role in fruit development.

[0055] To prove the function of MrMYB6, molecular experiments were further conducted using Arabidopsis thaliana, where plant MrMYB6 overexpression vectors and RNAi vectors were constructed and transformed into A. thaliana. Three independent overexpression (MrMYB6 overexpression) and RNAi Arabidopsis lines (MrMYB6 gene silencing) were obtained for further testing (for details of the experimental methods, see "8. Plasmid construction and plant gene transformation" and "9. Measurement of physiological characteristics of A. thaliana" below). At 35 days after germination, the MrMYB6 overexpression lines had larger leaves (the size of the eight leaves was calculated from the bottom leaf) and earlier seed germination than the WT (wild type), while the RNAi lines showed smaller leaves and later seed germination than the WT ( Fig.11 AE). We further examined the cell morphology and number and found that MrMYB6 overexpression plants had an increase of 11.24% and 60.86% compared with WT and RNAi lines, respectively ( Fig.11 FG), indicating that MrMYB6 is a major positive transcription factor in plant growth and development.

[0056] Soluble sugars and organic acids (including JA and SA) play fundamental roles in various developmental processes and stress responses. To explore whether MrMYB6 affects the synthesis of organic acids and soluble sugars, we measured the contents of soluble sugars, JA, and SA at different developmental stages using HPLC. In our study, at 28 and 35 days, the accumulation of soluble sugars, SA, and JA in the MrMYB6 overexpression line was greater than that in the WT (increases of 16.3%, 37.4%, and 28.1%, respectively), while the accumulation in the RNAi line was less than that in the WT ( Fig.11 H). Therefore, increased expression of MrMYB6 resulted in increased soluble sugar, SA, and JA content in both cultivars during fruit development. Overall, the accumulation of soluble sugars and organic acids was highly correlated with MrMYB6 transcription, suggesting that MrMYB6 plays an important role in regulating fruit development and plant growth by coordinating the expression of genes in the organic acid and soluble sugar pathways.

[0057] The following are the experimental materials and methods used in the examples.

[0058] 1. Test materials

[0059] 15-year-old 'Zaojia' and 'Dingao' bayberries were obtained from Lanxi, Zhejiang Province, China (29.22°N, 119.45°E) and cultivated under 15.0 h of light, 22-25°C, and 75% humidity. They were watered with 1 L Hoagland nutrient solution every 2 weeks. Fruits and pedicels of bayberries were collected 10, 20, 30, 40, and 50 days after pollination. Fruits from three trees of the same cultivar constituted one replicate, and three replicates were sampled at each time point. Fruits, mature leaves, immature leaves, roots, xylem, phloem, bark, and petioles were taken as samples for RNA-seq, with three biological replicates in each group.

[0060] 2. RNA extraction, transcriptome library construction and RNA-seq data processing

[0061] Total RNA from fruit samples of different time periods and tissues was extracted using RNA Easy Kit (Qiagen), immediately frozen in liquid nitrogen, and stored at -80°C until use. According to the instructions for use of the kit, after purification and digestion of DNA with RNase-free DNase (Qiagen), RNA samples (5 μg per sample) were analyzed using Agilent 2100 Bioanalyzer (28S / 18S=1.8-2.0) and NanoDrop ND-2000 (A 260 / A 280=1.9-2.1). Then, sequencing was performed using the Illumina Hiseq4000 platform according to the index code and instructions. Paired-end sequencing and library construction were performed by Biomarker Technologies. The processing of transcriptome data was the same as Quan M, Liu X, Xiao L, et al. (2020). Transcriptome analysis and association mapping reveal the genetic regulatory network response to cadmium stress in Populus tomentosa [J]. Journal of Experimental Botany and Xu W, Zhao Y, Chen S, et al. (2020). Evolution and Functional Divergence of the Fructokinase Gene Family in Populus [J]. Frontiers in Plant Science. A total of 211.56 GB of clean data was obtained for the total sample, with an average Q30 value of > 86.69%. In short, the clean transcriptome data obtained after quality control was then compared with the reference genome data of Bayberry (http: / / www.bayberrybase.cn / ) using TopHat (v.2.0.0) with default parameters. Transcript levels were normalized according to FPKM (fragments per kilobase of transcript per million fragments) using Cufflinks (v.2.1.1) with default options. Genes with a change greater than 2-fold and P < 0.05 (adjusted for false discovery rate, Q < 0.05) were considered to be significantly differentially expressed genes.

[0062] 3. Measurement of tissue specificity

[0063] The calculation method of tissue-specific score is as described previously (Yang, YR, Zhang, JY, Li, YQ, and Xia, N. (2006). Evaluation of detection of circulating antibody, circulating antigen and circulating immune complexes in human hydatidosis. [J] Molecular Biology & Evolution 23 (3), 530.). In order to further quantify the tissue specificity of gene expression, the tissue-specific score of gene i (T i ) is calculated as follows:

[0064]

[0065] Where n is the total number of tissue samples, and aij is the average expression of gene i in tissue j.

[0066] 4. Reverse transcription quantitative PCR of DEGs

[0067] To evaluate the expression of DEGs, we extracted total RNA using Qiagen RNeasy Kit (Qiagen, Shanghai, China) according to the instructions and purified it using RNase-Free DNase Set (Qiagen). According to the instructions, mRNA was reverse transcribed to obtain cDNA using a reverse transcription system (Promega, Madison, WI, USA). Gene-specific primers were used for qRT-PCR using SYBR Premix Ex Taq (TaKaRa) on a 7500 Fast Real-Time PCR System. Actin (retrieval number: EF145577) was used as an internal reference and repeated three times. The PCR program was as follows: initial denaturation at 94°C for 5 minutes; 40 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds; and 70°C to 95°C to obtain the final melting curve. The sequences of the primers are as follows:

[0068] Table 1 Summary of primer sequences

[0069]

[0070]

[0071] 5. Functional enrichment analysis and visualization

[0072] GO annotations were extracted from the M. rubra database (http: / / www.bayberrybase.cn / ) (Ren et al., 2019), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http: / / www.genome.jp / kegg) (Hiroyuki et al., 2012) was used to obtain annotations. The results of GO enrichment analysis were input into the OmicShare platform (https: / / www.omicshare.com / tools) together with the P value corrected using the Benjamini and Hochberg (1995) FDR method, which was used to build a visual dendrogram of the GO analysis results. GO terms with a corrected P < 0.05 were considered significantly enriched.

[0073] 6. Identification of homologous genes, amino acid sequence alignment and phylogenetic analysis

[0074] Homologous genes of the model plant A. thaliana from the PlantTFDB database (http: / / planttfdb.gao-lab.org / ) were used as potential reference sequences, and candidate homologous genes were identified using BLASTp searches (adjusted E value < 0.01) (Xu et al., 2020). To identify the conserved domains of candidate genes, the PFAM database (http: / / pfam.xfam.org / ) was used, and the potential homologous protein sequences were aligned using MEGA 11 software (Kumar et al., 2018). A phylogenetic tree of protein sequences was constructed using MEGA 11 with the neighbor joining (NJ) method and 1000 bootstrap replicates (Kumar et al., 2018). In our analysis, we removed all positions with less than 90% site coverage; the total missing data, alignment gaps, and ambiguous bases at any position were less than 10%.

[0075] 7. Constructing a multi-level gene regulatory network using the backward elimination random forest method

[0076] The differentially expressed TF genes were evaluated using PlantPAN v.2.0 to identify cis-regulatory elements in their 2 kb promoter regions (Chow et al., 2016). These motifs were predicted as targets of 28 TFs based on 80% confidence. Then, the ion transporter genes were used together with the 129 genes and 28 TFs to construct a multi-layer hierarchical gene regulatory network (ML-hGRN) by adopting the backward elimination random forest (BWERF) algorithm (Deng et al., 2017).

[0077] 8. Plasmid construction and plant gene transformation

[0078] Cloning, expression analysis, and genetic transformation are the three approaches we utilized on A. thaliana plants to develop overexpression and RNAi lines. Seedlings were grown under long-day conditions with 16 h of light and 8 h of darkness. The full-length coding region of MrMYB6 was cloned from the M. rubra cloning template cDNA using gene-specific primers (Table 1). During the cloning of the full-length gene, the coding region of MrMYB6 gene was cloned into the pDONR222 vector. Next, the coding region was recombined with the destination vector pGWB405 (Nakagawa et al., 2007) using LR reaction according to Quan et al. (2020) and verified by PCR sequencing. All binary vectors were introduced into Agrobacterium tumefaciens GV3101 and then transformed into Nicotiana benthamiana via Agrobacterium-mediated transformation along with the gene silencing suppressor P19. The Agrobacterium-mediated floral dip method was used to create transgenic A. thaliana seedlings (Clough and Bent, 1998). Transgenic plants were confirmed by PCR analysis using vector and gene-specific primers. The forward and reverse primers for MrMYB6 gene were as follows:

[0079] SEQ ID NO.15 (forward primer 3′-5′): gacacagccagcagcaAGGCAGGCGAGCGAGGCGACGG;

[0080] SEQ ID NO. 16 (reverse primer 5′-3′): catgattacgaattcTGGCGAGAGCGAGCGACGCCAAG.

[0081] 9. Measurement of physiological characteristics of A. thaliana

[0082] In the presence of 1% sucrose and 1 / 2×MS medium (Na+: 0.1 mM CaCl2, 0.1 mM MgSO4 -, 0.5 mM NaCl and 0.3 mM ZnSO4) plates. The number of germinations was counted until 5 days (d) after stratification (almost emerged embryos). The germination rate (GR) was calculated as the ratio of germinated seeds to the total number of seeds of WT A.thaliana and transgenic plants. Mature leaves of transgenic and WT A.thaliana plants were measured at 28 days (DAG) and 35 days (DAG) after germination. The seedling length of A.thaliana was calculated using ImageJ software (https: / / imagej.en.softonic.com / ). Stem tissues of 28-day-old WT A.thaliana and transgenic plants were treated with 10 μ / mL of propidium iodide (C27H34I2N4) solution at 30°C in the dark for 1 min.

[0083] 10. Extraction and determination of endogenous JA, SA and soluble sugars

[0084] The content of plant JA, SA, and soluble sugars in A. thaliana was quantified and extracted as described previously (Huang et al., 2022). Nearly 0.2 g of tissue from WT plants and transgenic plants was frozen in liquid nitrogen and quickly ground into powder. 2 mL of isopropanol-HCl buffer was quickly added to the powder and shaken at 4 °C for 30 min. After shaking for 35 min, 4 mL of CH2Cl2 was added and centrifuged at 11000g for 6 min. The lower organic phase after centrifugation was immediately frozen in liquid nitrogen. All samples were kept in a dark environment and dissolved in 150 μL of methanol at 30 °C for 5 min. For HPLC-MS / MS analysis, all solutions were quickly filtered with a 0.45 μm filter according to the manufacturer's instructions (Huang et al., 2022). A hybrid triplet / quadrupole ion trap mass spectrometer (SCIEX 6500QTrap, Applied Biosystems, Foster City, CA) was used according to Huang et al. (2017). Each sample had three biological replicates.

[0085] 11. Data Analysis

[0086] For data analysis, Microsoft Excel 2010 (Microsoft Corp., Redmond, WA) and SPSS (v.17.0; IBM, Chicago, IL, USA) were used for data analysis. One-way and two-way ANOVA were used to examine the significance of treatment differences. P values ​​were calculated using Student's t-test (*P < 0.05, **P < 0.01). All samples were normally distributed in terms of variance homogeneity after data normalization.

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Claims

1. A Myrica rubra MrMYB6 gene, characterized in that: The sequence of the MrMYB6 gene is shown in SEQ ID NO.

1.

2. A protein, characterized in that The protein is the protein encoded by the bayberry MrMYB6 gene according to claim 1, and its amino acid sequence is shown in SEQ ID No.

2.

3. Use of the MrMYB6 gene as claimed in claim 1 in preparing an agent for regulating plant growth or fruit development.

4. The use according to claim 3, characterized in that The MrMYB6 gene promotes plant growth or fruit development by promoting the synthesis of soluble sugars and organic acids.

5. The use according to claim 4, characterized in that The organic acids include salicylic acid and jasmonic acid.

6. The use according to claim 3, characterized in that The plant is Arabidopsis thaliana or Myrica rubra.

7. The use according to claim 3, characterized in that The fruit is bayberry fruit.

8. An expression vector containing the MrMYB6 gene according to claim 1.

9. A host cell for the MrMYB6 gene according to claim 1.

Images