Application of ArMYB15 gene in regulating formation of red leaves of poplar

Abstract

The application of ArMYB15 gene in regulating formation of red leaves of poplar belongs to the field of biotechnology and gene engineering, and specifically relates to ArMYB15 gene, amino acid coded by the ArMYB15 gene and application in regulating formation of red leaves. The nucleotide sequence of the ArMYB15 gene is shown as SEQ ID No. 1. In the application, ArMYB15 gene is screened, gene cloning and vector construction are carried out, pCAMBIA1300-ArMYB15-EGFP is transformed into leaf of Populus alba var. pyramidalis Bunge 84K by means of agrobacterium mediation, and after co-cultivation and selection culture, resistant bud points are obtained, and after rooting culture, resistant plants are obtained. The R2R3-MYB transcription factor coded by ArMYB15 gene promotes accumulation of anthocyanin and its derivatives, causes leaves to be red, and new germplasm of red leaf forest trees with stable leaf color and excellent ornamental traits is obtained.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic engineering, specifically relating to the ArMYB15 gene and its encoded amino acids and their application in regulating red leaf formation. Background Technology

[0002] Colorful-leaved trees possess unique ornamental value due to their beautiful tree shapes and vibrant foliage, playing an irreplaceable role in urban forestry development. In my country, colorful-leaved tree species are mainly created through traditional breeding techniques such as mutation breeding or hybridization breeding. Obtaining stable colorful-leaved varieties takes a long time, and the leaves frequently revert to their original green color during cultivation, severely reducing their landscape value. Therefore, genetic engineering techniques for the transformation of leaf color-regulating genes are an effective means of creating stable colorful-leaved tree species.

[0003] The leaf color of forest trees is mainly determined by the relative content of three major pigments: chlorophyll, carotenoids, and anthocyanins. Among them, anthocyanins can make plants appear red or purple. The biosynthetic pathway of anthocyanins in plants has been largely elucidated. The structural genes involved in this pathway are directly regulated by MYB transcription factors or regulated by the MYB-bHLH-WD40 (MBW) transcriptional complex. Environmental factors such as light and temperature can induce the expression of MYB transcription factors, thereby promoting the accumulation of anthocyanins.

[0004] The genome sequence of the American red maple (Acer rubrum), also known as the red-flowered maple, has been published, but the gene related to leaf color formation has not been identified, and the function of ArMYB15 remains unclear. Therefore, cloning the ArMYB15 gene and conducting genetic transformation in the model plant *Populus simonii* 84K can provide important genetic resources for the creation of colorful-leaved tree species. Summary of the Invention

[0005] The present invention relates to the application of the ArMYB15 gene in regulating the formation of red leaves in poplar trees. The nucleotide sequence of the ArMYB15 gene is shown in SEQ ID No. 1.

[0006] Furthermore, the poplar tree mentioned is *Populus simonii* 84K.

[0007] Furthermore, the application of the ArMYB15 gene in regulating the formation of red leaves in poplar trees is that the ArMYB15 gene promotes the accumulation of anthocyanins and their derivatives in the leaves of transgenic lines, thus turning the leaves red.

[0008] Furthermore, the ArMYB15 gene regulates the formation of red leaves in poplar trees through overexpression.

[0009] Furthermore, the amino acid sequence of the protein encoded by the ArMYB15 gene is shown in SEQ ID No. 2.

[0010] The beneficial effects of this invention are as follows:

[0011] This invention screens for the ArMYB15 gene based on transcriptome data from both red and green leaves of the same American red maple plant. The expression profiles of differentially expressed MYB family genes are analyzed, and the MYB transcription factor ArMYB15, which is highly expressed in red leaves but not in green leaves, is further screened. Then, using red leaf cDNA as a template, the coding region sequence of the ArMYB15 gene is amplified. Primers containing homologous arms are designed for vector construction, and the ArMYB15 gene is introduced into the pCAMBIA1300-EGFP vector via homologous recombination, achieving gene cloning and vector construction. Finally, the pCAMBIA1300-ArMYB15-EGFP is transformed into leaves of *Populus simonii* 84K using Agrobacterium-mediated leaf disc transformation. After co-culture and selection, resistant buds are obtained, and resistant plants are obtained after rooting culture. This invention uses qRT-PCR technology to detect the expression of ArMYB15 in Populus aurea 84K, as well as phenotypic observation of transgenic plants and anthocyanin-targeted metabolome determination. It demonstrates that the ArMYB15 gene encodes the R2R3-MYB transcription factor, which promotes the accumulation of anthocyanins and their derivatives, resulting in red leaves. This yields a new red-leaved forest tree germplasm with stable leaf color and excellent ornamental traits. Attached Figure Description

[0012] Figure 1 A differential expression map of ArMYBs genes in red and green leaves of the same American red maple.

[0013] Figure 2 Electrophoresis image of the ArMYB15 gene clone;

[0014] Figure 3 A diagram illustrating the Agrobacterium-mediated genetic transformation process of *Populus alba* 84K.

[0015] Figure 4 Figure 1 shows the molecular detection results of DNA and RNA levels in ArMYB15 transgenic plants.

[0016] Figure 5 Image showing the color changes of leaves of ArMYB15 transgenic plants and wild-type plants under strong light.

[0017] Figure 6 The graph shows the changes in anthocyanin and its derivative content in ArMYB15 transgenic plants and wild-type plants. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0019] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0020] Example 1:

[0021] The nucleotide sequence of the ArMYB15 gene is shown in SEQ ID No. 1, as follows:

[0022] ATGGTGAGAGCTCCTTGTTGTGAGAAGATGGGATTGAAGAGGGGTCCATGGACTCCTGAAGAAGATCGAATTTTAATCTCCTACATCCACAAACACGGCCATGCAAACTGGCGCGCTCTACCAAAACAAGCAGGTCTGTTAAGATGTGGGAAGAGTTGCAGACTTCGCTGGATAAACTACTTGAGGCCTGATATTAAGAGAGGAAACTTCACTAAAGAAGAAGAAGAAGCCATCATCAATTTGCATCAAATGTTGGGCAACAGGTGGTCAGCTATCGCAGCAAAATTACCAGGAAGGACAGACAATGAGATAAAAAATGTGTGGCACACCCACTTGAAGAAGAGACTCATCAAACAAAACCAGGCCAATTCGGACACCAAAAAACACTCAAAGACTACAATAACCACAAAATCCGAGTCAAGCCAATCAGAGTCAGAACTCACAAATCCTGCAAGTAATAGCCTTCCAATGTCGCCGCAACAGTCTTCTAGCGATGTCTCCTCAGTCACAGCTCTCACAACTGGAGAAACAAATGATCACATGGAGGAGGAGAACAAAGGAGAACATGAAGTAGTTGATATTGACTCGTTTGATATTAGTTTCCCAGCAATCGATGAGAGTTTCTGGTCAGATGCATTATCATCAGATAACAACTCAAGCTTAAGTACATTAGATCTTGGACAAGGGATGATCAATAACGATGATCAGTTTCAAATTGACGAGTCTCTATTTTCTTCAGTGGATTTCATGGGGTATGATTATGATGCAAAATCTGAACATGATGGCATGGACTTCTGGTACAACATTTTGCTTACAACTGAGGGTGAATCAATGGTACCCCTTTTCTGA

[0023] The amino acid sequence encoded by the ArMYB15 gene is shown in SEQ ID No.2, as follows:

[0024] MVRAPCCEKMGLKRGPWTPEEDRILISYIHKHGHANWRALPKQAGLLRCGKSCRLRWINYLRPDIKRGNFTKEEEEAIINLHQMLGNRWSAIAAKLPGRTDNEIKNVWHTHLKKRLIKQNQANSDTKKHSKTTITTTKSESS QSESELTNPASNSLPMSPQQSSSDVSSVTALTTGETNDHMEEENKGEHEVVDIDSFDISFPAIDESFWSDALSSDNNSSSLSTLDLGQGMINNDDQFQIDESLFSSVDFMGYDYDAKSEHDGMDFWYNILLTTEGESMVPLF

[0025] 1. Cloning of the ArMYB15 gene and construction of an overexpression vector

[0026] (1) Screening of the ArMYB15 gene

[0027] Based on transcriptome data from the red and green leaves of the same American red maple, the expression profiles of differentially expressed ArMYBs genes in the red and green leaves were analyzed and further visualized using the heatmap function of the Maiwei Cloud platform.

[0028] In this embodiment, a total of 28 differentially expressed ArMYBs genes were screened, and 20 of the ArMYBs genes were highly expressed in red leaves. Figure 1 This image shows the differential expression of ArMYB genes in the red and green leaves of the same American red maple. The gene marked in red, evm.TU.Contig311.18, is the ArMYB15 gene, with a mean expression level of 1.19 in the red leaves and 0 in the green leaves.

[0029] (2) Cloning of the ArMYB15 gene

[0030] Using cDNA from red leaves of American red maple (Acer rubrum) as a template, the ArMYB15 gene was amplified. The amplification primers are shown in Table 1.

[0031] The PCR reaction system is as follows: 12.5 μL of 2 × PrimeSTAR Max Premix, 1 μL each of forward and reverse primers, 2 μL of cDNA template, and ddH2O to a final volume of 25 μL.

[0032] The PCR reaction conditions were as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 56℃ annealing for 5 s, 72℃ extension for 1 min 10 s, 35 cycles; 72℃ final extension for 5 min. The PCR products were recovered and ligated into the pEASY-T1 vector, transformed into E. coli TOP10 competent cells, and positive clones were screened for sequencing verification.

[0033] In this embodiment, the ArMYB15 gene was cloned. Its coding region contains 849 nucleotide sequences and encodes 282 amino acid sequences. The nucleotide sequence of the ArMYB15 gene is shown in SEQ ID No. 1, and the amino acid sequence encoded by the ArMYB15 gene is shown in SEQ ID No. 2. Figure 2 The image shows an electrophoresis diagram of the ArMYB15 gene clone; where M is the DL 2000 DNA Marker, and lanes 1-5 are the ArMYB15 amplification products.

[0034] Table 1 Primers for ArMYB15 gene cloning

[0035]

[0036] (3) Construction of plant overexpression vector for ArMYB15 gene

[0037] The plant overexpression vector plasmid pCAMBIA1300-EGFP was digested with the restriction endonuclease BamHI. Simultaneously, primers for amplifying the ArMYB15 gene were constructed using a vector with introduced homologous arms (primers are shown in Table 2). After purification and recovery, the plasmid was ligated to a linear vector using homologous recombination. The ligation system consisted of: 5 μL of 2 × Spark HiFi Seamless Cloning Mix, 1 μL of the linear pCAMBIA1300-EGFP vector, 3 μL of the recovered ArMYB15 product, and ddH2O to a final volume of 10 μL. The reaction was carried out at 50°C for 15 min. The ligation product was transformed into E. coli competent cells TOP10 and cultured overnight at 37°C on kanamycin (50 mg / L) resistant plates. Positive single clones were picked for sequencing verification, and the plasmid was extracted for later use.

[0038] Table 2 Primers for constructing plant expression vectors

[0039]

[0040] (4) Obtaining the 35S::ArMYB15 engineered bacteria

[0041] The recombinant plasmid pCAMBIA1300-ArMYB15-EGFP was used to screen for resistance in *Agrobacterium* competent cells GV3101 on plates containing kanamycin (50 mg / L) and rifampin (50 mg / L). The cells were incubated upside down at 28°C for 2–3 days. Single *Agrobacterium* clones were picked, plasmids were extracted, and plasmid PCR was performed for verification. Positive bacterial cultures were then stored for later use.

[0042] 2. Obtaining the ArMYB15 transgenic line and identifying its leaf color phenotype.

[0043] (1) Genetic transformation of *Populus silveraefolius* 84K

[0044] Activation of 35S::ArMYB15 engineered bacteria, OD of the bacterial culture solution 600 When the concentration is around 0.5, it is ready for use. Select healthy *Populus simonii* 84K, cut 3-5 leaves, make wounds, and transfer them into activated bacterial solution. Shake slowly for 10 min to infect, wipe off any residual bacterial solution, and transfer to meristem culture medium. After dark culture for 2-3 days, wash 5-7 times with cephalosporin solution (50 mg / L), then wash 5-7 times with clean water. After drying the leaves, transfer them to selection medium for further culture. The resistant buds that grow normally and survive on the selection medium are then transferred to stem-elongation medium. After the buds elongate, rooting culture is performed. The culture media used in the experiment are shown in Table 3.

[0045] Table 3 Culture medium formulation

[0046]

[0047] In this embodiment, resistant buds were obtained through co-culture and selective culture, and resistant strains were obtained after rooting culture. Figure 3 This diagram illustrates the genetic transformation process of *Populus alba* 84K mediated by *Agrobacterium*. A represents the co-culture stage, B the selection culture stage, C the selected resistant shoots, and D the resistant plants after rooting culture.

[0048] (2) Molecular detection of ArMYB15 transgenic silver poplar 84K

[0049] Leaves from ArMYB15 transgenic plants and wild-type *Populus simonii* 84K plants were collected and ground with liquid nitrogen. Genomic DNA was extracted from the leaves using a DNA extraction kit. PCR detection of the transgenic lines was performed using the DNA as a template. PCR primers are shown in Table 4. The PCR reaction system consisted of: 12.5 μL of 2 × Rapid Taq Master Mix, 1 μL each of forward and reverse primers, 2 μL of cDNA template, and ddH2O to a final volume of 25 μL. The PCR conditions were as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min 10 s, 30 cycles; and a final extension at 72℃ for 5 min.

[0050] Table 4 Primers for PCR detection of transgenic lines

[0051]

[0052] Total RNA was extracted from plants using an RNA extraction kit, and 1 μg was reverse transcribed into cDNA. Using cDNA as a template, qRT-PCR was performed on the target genes of the transgenic lines. The qRT-PCR primers are shown in Table 5. The qRT-PCR reaction system was as follows: 12.5 μL of 2 × TB Green Premin Ex Taq II Fast qPCR, 1 μL each of forward and reverse primers, 2 μL of cDNA template, and ddH2O to a final volume of 25 μL. The qRT-PCR reaction conditions were as follows: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 40 cycles; 95℃ for 10 s, 65℃ for 5 s, 95℃ for 5 s. A 2 -ΔΔCT The relative expression level of the ArMYB15 gene was calculated using this method.

[0053] Table 5 Primers for qRT-PCR detection of transgenic lines

[0054]

[0055] This embodiment uses PCR to detect the insertion of exogenous ArMYB15 in transgenic lines; Figure 4 Figure 1 shows the molecular detection results of DNA and RNA levels in ArMYB15 transgenic plants; Figure A shows the PCR amplification results of pCAMBIA1300-ArMYB15-EGFP positive plasmid, wild-type WT, negative control water, and 7 transgenic plants (OE15-1~OE15-7); Figure B shows the relative expression level of ArMYB15 in transgenic plants compared with wild-type WT; Figure C shows the relative expression level of ArMYB15 in transgenic plants compared with low-expression OE15-5 plants. It was found that all 7 lines could amplify the exogenous ArMYB15 gene (…). Figure 4A); The relative expression levels of ArMYB15 were measured, and it was found that the expression levels of OE15-1 to OE15-7 were all higher than those of wild-type WT (A). Figure 4 B), further analysis of the relative expression levels of each transgenic line compared to the lowest expression level of OE15-5 revealed that the expression levels of OE15-1, OE15-2, and OE15-4 were significantly higher than those of the OE15-5 line (B). Figure 4 C).

[0056] (3) Observation of leaf color of ArMYB15 transgenic silver poplar 84K

[0057] Transformers with relatively high expression levels and healthy growth, such as OE15-4, were selected for mass propagation, with wild-type *Populus simonii* 84K serving as a control. After 30 days of rooting culture, the seedlings were transplanted into soil containing a substrate of nutrient soil, vermiculite, and perlite (volume ratio 3:2:1) under a light intensity of approximately 5000 Lux. After 20 days, seedlings with similar growth were transplanted to a light intensity of approximately 16000 Lux and cultured for 15 days. Leaf color changes were then observed.

[0058] (4) Anthocyanin-targeted metabolome assay of ArMYB15 transgenic *Populus alba* 84K.

[0059] Leaves (1-4 leaves) from wild-type and OE15-4 strains without mechanical damage were collected, flash-frozen in liquid nitrogen, and then subjected to anthocyanin-targeted metabolomics analysis. Each sample was ground, and 50 mg of powder was weighed and dissolved in 500 μL of extraction buffer (50% methanol aqueous solution containing 0.1% hydrochloric acid). The mixture was vortexed for 5 min and sonicated for 5 min. Centrifuged at 12000 rpm for 3 min at 4℃, and the supernatant was collected and the process was repeated once. The two supernatants were combined, filtered through a 0.22 μm microporous membrane, and LC-MS / MS was used to analyze anthocyanin biosynthesis pathways and metabolites. Differential metabolites were screened based on a fold change ≥ 2 or a fold change ≤ 0.5.

[0060] In this embodiment, it was found that after strong light treatment, the leaves of the ArmyB15 overexpressing line OE15-4 turned red. Figure 5 ). Figure 6 The graph shows the changes in anthocyanin and its derivative content in the ArMYB15 transgenic plant (OE15-4) and the wild type (WT). Compared with the wild type WT, except for proanthocyanidin B4, delphinidin-3-O-moribiglycoside and pelargonidin-3-O-p-coumaryl-5-O-galactoside, the remaining 18 anthocyanins and their derivatives accumulated in large quantities in OE15-4.

Claims

1. The application of the ArMYB15 gene in regulating the formation of red leaves in poplar trees, characterized by, The nucleotide sequence of the ArMYB15 gene is shown in SEQ ID No.

1.

2. The application of the ArMYB15 gene in regulating the formation of red leaves in poplar trees according to claim 1, characterized in that, The poplar tree mentioned is the silver gland poplar 84K.

3. The application of the ArMYB15 gene according to claim 1 in regulating the formation of red leaves in poplar trees, characterized in that, The application of the ArMYB15 gene in regulating the formation of red leaves in poplar trees is that the ArMYB15 gene promotes the accumulation of anthocyanins and their derivatives in the leaves of transgenic lines, thus turning the leaves red.

4. The application of the ArMYB15 gene according to any one of claims 1 to 3 in regulating the formation of red leaves in poplar trees, characterized in that, The ArMYB15 gene regulates the formation of red leaves in poplar trees through overexpression.

5. The application of the ArMYB15 gene according to claim 1 in regulating the formation of red leaves in poplar trees, characterized in that, The amino acid sequence of the protein encoded by the ArMYB15 gene is shown in SEQ ID No. 2.

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