BpMYB090, a gene regulating the development of epidermal hairs on mulberry leaves, its expressed protein, and its applications.

Abstract

This invention discloses a regulatory gene BpMYB090 for the development of epidermal hairs on paper mulberry leaves, its expressed protein, and its applications, belonging to the field of plant genetic engineering technology. The nucleotide sequence of this gene, BpMYB090, is shown in SEQ ID NO.1. This gene belongs to the ninth subgroup of the R2R3 MYB transcription factor family, MIXTA class, and its expression level is significantly positively correlated with the density of epidermal hairs on paper mulberry leaves. Overexpression of BpMYB090 in Arabidopsis thaliana significantly increases the number of epidermal hairs and restores the epidermal hair-deficient phenotype of the Arabidopsis thaliana mutant gl1, confirming that BpMYB090 is a positive regulator of epidermal hair formation. This invention reveals for the first time a key regulatory gene for the development of epidermal hairs on paper mulberry leaves. Furthermore, based on the positive regulatory function of this gene, new hairless forage paper mulberry varieties can be bred through gene silencing or editing techniques, possessing significant theoretical value and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, it relates to the gene BpMYB090 that regulates the development of epidermal hairs on mulberry leaves, its expression protein, and its applications. Background Technology

[0002] Paper mulberry (Broussonetia papyrifera) is a deciduous tree belonging to the genus Broussonetia in the family Moraceae. Naturally distributed in East and Southeast Asia, it is a typical native tree species and pioneer plant. Due to its high crude protein content in its leaves (wild paper mulberry leaves can contain 20-30% crude protein), strong resistance to adverse conditions, and rapid growth, it is widely used in animal feed, papermaking, and vegetation restoration, making it a woody forage crop with significant economic value.

[0003] However, the extremely high density of hairs on the bark of wild paper mulberry leaves not only affects protein utilization but also results in poor palatability, limiting its large-scale utilization. While the breeding of hybrid paper mulberry has alleviated the shortage of feed protein to some extent, as a woody plant with complex bark hair types, it is difficult to obtain completely hairless superior germplasm through traditional hybridization breeding. Therefore, the use of modern biotechnology to cultivate new hairless feed paper mulberry varieties has become an urgent need for industrial development.

[0004] Epidermal trichomes are specialized structures formed by the differentiation of plant epidermal cells. Based on cell number, they can be classified into unicellular and multicellular epidermal trichomes; based on secretory capacity, they can be classified into glandular trichomes and non-glandular trichomes. The leaf epidermal trichomes of the paper mulberry (Broussonetia papyrifera) exhibit a rich variety of types, including unicellular glandular trichomes and non-glandular trichomes, differing from model plants and possessing unique characteristics. Epidermal trichome development is regulated by transcription factors, among which genes in the ninth subgroup of the R2R3MYB transcription factor family play a crucial role in regulating epidermal cell differentiation. Their function has been confirmed in multiple species, including Arabidopsis thaliana, tomato, cotton, and poplar.

[0005] Currently, research on paper mulberry mainly focuses on its feed value and pulping / papermaking, lacking systematic research on its basic biology. Furthermore, research on the molecular mechanisms of paper mulberry epidermal hair development remains lacking. With the completion of paper mulberry whole-genome sequencing and the establishment of a genetic transformation system, a technical foundation has been provided for elucidating the regulatory mechanisms of paper mulberry epidermal hair development at the molecular level and creating new hairless paper mulberry germplasm.

[0006] Furthermore, epidermal hairs, as the first barrier between plants and their external environment, play a crucial role in plant stress resistance. High-density epidermal hairs can enhance a plant's adaptability to stresses such as drought and pests; therefore, increasing epidermal hair density is also of significant application value in breeding programs aimed at enhancing plant environmental adaptability. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a regulatory gene BpMYB090 for the development of epidermal hairs on paper mulberry leaves. Another technical problem to be solved by the present invention is to provide the application of the regulatory gene BpMYB090 for the development of epidermal hairs on paper mulberry leaves in transgenic paper mulberry.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A gene BpMYB090 that regulates the development of hairs on the leaf epidermis of Broussonetia papyrifera is shown in SEQ ID NO.1.

[0010] The protein encoded by the gene BpMYB090, which regulates the development of epidermal hairs on the leaves of the paper mulberry tree, has the amino acid sequence shown in SEQ ID NO.2.

[0011] A recombinant expression vector, expression cassette, recombinant bacteria, or host cell containing the gene BpMYB090, which regulates the development of the epidermal hairs of the Broussonetia papyrifera leaves.

[0012] The application of the gene BpMYB090, which regulates the development of epidermal hairs on the leaves of the paper mulberry tree, in promoting the formation of epidermal hairs in plants, wherein the plant is Arabidopsis thaliana or paper mulberry.

[0013] In some embodiments, the application includes the following steps:

[0014] (1) Construct a plant overexpression vector containing the BpMYB090 gene, which regulates the development of leaf epidermal hairs;

[0015] (2) Transform the plant overexpression vector into the target plant;

[0016] (3) Transgenic plants with increased number of epidermal hairs were obtained through cultivation and screening.

[0017] In some embodiments, the plant overexpression vector is pCAMBIA1301-BpMYB090.

[0018] The application of the gene BpMYB090, which regulates the development of epidermal hairs on the leaves of Broussonetia papyrifera, in restoring epidermal hair formation in the hairless mutant of Arabidopsis thaliana, specifically the gl1 mutant.

[0019] The application of the BpMYB090 gene regulating the development of leaf epidermal hairs, the protein, or the recombinant expression vector, expression cassette, recombinant bacteria, or host cell in the study of the molecular mechanism of plant epidermal hair development.

[0020] In the aforementioned application, the plant is Arabidopsis thaliana or Broussonetia papyrifera.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention, by comparing the significant differences in epidermal hair density between wild-type and hybrid Broussonetia papyrifera leaves, combined with gene sequence evolutionary analysis, expression profile analysis, and functional verification through Arabidopsis thaliana genetic transformation, has for the first time cloned the key gene BpMYB090, which regulates epidermal hair development in Broussonetia papyrifera. This gene belongs to the ninth subgroup of the R2R3 MYB transcription factor family, specifically the MIXTA class, and clusters with known genes regulating epidermal cell development in Arabidopsis thaliana within the same evolutionary branch. Expression pattern analysis showed that BpMYB090 was most highly expressed in the terminal leaves of wild-type Broussonetia papyrifera, where epidermal hair density was highest. Its expression gradually decreased as the leaf unfolded and epidermal hair density decreased, while it was almost not expressed in the leaves of the less hairy hybrid Broussonetia papyrifera at all developmental stages, indicating a significant positive correlation between gene expression level and leaf epidermal hair density. Functional verification results showed that overexpression of BpMYB090 in wild-type Arabidopsis thaliana significantly increased the number of epidermal hairs, and overexpression of this gene in the hairless mutant (gl1) of Arabidopsis thaliana completely restored its epidermal hair phenotype to the wild-type level. Therefore, this invention reveals for the first time that BpMYB090 is a key transcription factor regulating the development of pubescent hairs in Broussonetia papyrifera, providing a core gene target for elucidating the molecular regulatory mechanism of pubescent hair development in Broussonetia papyrifera, and filling the research gap in the cloning and functional verification of key genes for pubescent hair development in woody plants. At the same time, it is expected that by genetically manipulating this gene, new hairless Broussonetia papyrifera germplasm can be obtained, effectively solving the industrial problem of poor palatability and low protein utilization caused by dense pubescent hairs in wild Broussonetia papyrifera, and providing important gene resources and feasible technical paths for breeding high-quality forage Broussonetia papyrifera varieties and alleviating the shortage of forage protein. Attached Figure Description

[0023] Figure 1 The images show the microscopic observation results of the epidermal hair density of wild-type and hybrid mulberry leaves. In the images, AD represents the microscopic observation results, A and B are the front and back of the wild-type mulberry leaf, respectively; C and D are the front and back of the hybrid mulberry leaf, respectively; EH represents the scanning electron microscopic observation results, E and F are the front and back of the wild-type mulberry leaf, and G and H are the front and back of the hybrid mulberry leaf.

[0024] Figure 2 This is a comparison chart of the epidermal hair density of leaves at different developmental stages of wild-type and hybrid paper mulberry. AF represents leaves of wild-type paper mulberry, where A and B are the front and back faces of the terminal leaf, C and D are the front and back faces of the unfolded leaf, and E and F are the front and back faces of the mature leaf; GL represents leaves of hybrid paper mulberry, where G and H are the front and back faces of the terminal leaf, I and J are the front and back faces of the unfolded leaf, and K and L are the front and back faces of the mature leaf.

[0025] Figure 3The image shows the multiple sequence alignment results of the BpMYB090 gene. The amino acid sequence encoding the cloned BpMYB090 gene was compared with the Arabidopsis thaliana AtMYB16, AtMYB17, and AtMYB107. The results show that the gene has a typical R2R3 MYB domain and contains a conserved motif unique to the ninth subgroup, indicating that the sequence structure is highly conserved.

[0026] Figure 4 This figure shows the expression pattern of the BpMYB090 gene in leaves of *Broussonetia papyrifera* at different developmental stages. The relative expression levels of the BpMYB090 gene in the terminal leaves, unfolded leaves, and mature leaves of wild-type and hybrid *Broussonetia papyrifera* were detected using qRT-PCR. The bar chart data represent the average of three biological replicates, and the error bars represent standard errors. The results showed that the expression level of the BpMYB090 gene was highest in the terminal leaves of wild-type *Broussonetia papyrifera*, gradually decreasing as the leaves unfolded, while it was almost not expressed in the leaves of hybrid *Broussonetia papyrifera* at any developmental stage.

[0027] Figure 5 This is a schematic diagram of the BpMYB090 gene overexpression vector. This vector will be used for subsequent Arabidopsis thaliana genetic transformation functional verification experiments.

[0028] Figure 6 The figure shows the functional validation results of the BpMYB090 gene transformation in wild-type Arabidopsis thaliana. A is the electrophoresis image of the transgenic lines detected by PCR; B is the relative expression level of the BpMYB090 gene in the transgenic lines and wild-type Arabidopsis thaliana detected by qRT-PCR; C is the phenotypic observation of leaf epidermal trichomes in the transgenic lines and wild-type Arabidopsis thaliana; D is the statistical result of the number of epidermal trichomes in the same leaf area of ​​the first true leaf in the transgenic lines and wild-type Arabidopsis thaliana. The bar chart values ​​are the average values ​​of 10 plants, and the error bars represent standard errors. The results indicate that overexpression of the BpMYB090 gene can significantly increase the number of leaf epidermal trichomes in Arabidopsis thaliana.

[0029] Figure 7 The figure shows the functional validation results of the BpMYB090 gene transformation of the Arabidopsis thaliana hairless mutant (gl1). A is the PCR electrophoresis image of the transgenic lines; B is the relative expression level of the BpMYB090 gene in the transgenic lines and the gl1 mutant detected by qRT-PCR; C is the observation of the epidermal trichome phenotype of the leaves in the transgenic lines and the gl1 mutant; D is the statistical result of the number of epidermal trichomes in the same leaf area of ​​the first true leaf in the transgenic lines and the gl1 mutant. The bar chart values ​​are the average values ​​of 10 plants, and the error bars represent the standard errors. The results indicate that overexpression of the BpMYB090 gene can completely restore the epidermal trichome-deficient phenotype of the gl1 mutant to the wild-type level. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] Example 1:

[0032] (1) Microscopic observation and comparative analysis of the density of epidermal hairs on paper mulberry leaves

[0033] The test materials were wild-type paper mulberry and a hybrid paper mulberry with less hair. Healthy, disease-free plants were selected, and mature leaves were collected from the outer center of the canopy. The fresh leaves were washed with clean water to remove surface dust and blotted dry with filter paper. Small pieces measuring 0.5 cm × 0.5 cm were cut from the center of each leaf (avoiding the midrib). These leaf pieces were placed on slides and photographed using an optical microscope. For scanning electron microscopy (SEM), leaf tissue from the same location as observed in the optical microscope was selected and cut into 3 mm × 5 mm pieces. The samples were first fixed with 2.5% glutaraldehyde phosphate buffer (pH 7.2) at 4℃ for 4–6 hours. After fixation, they were rinsed three times with 0.1 mol / L phosphate buffer for 15 minutes each time. Subsequently, they were dehydrated using a gradient of ethanol concentrations (30%, 50%, 70%, 80%, 90%, 100%) for 15 minutes each time. The dehydrated samples were then subjected to critical point drying (using a CO2 critical point dryer). The dried sample was adhered to the sample stage with conductive adhesive and then subjected to ion sputtering gold coating (coating thickness approximately 10-20 nm). The treated sample was observed under a scanning electron microscope with an accelerating voltage of 5-10 kV. The morphology, surface microstructure, and density of the epidermal hairs were observed at both low and high magnification, and representative fields of view were photographed.

[0034] Under an optical microscope, the morphology and distribution density of the leaf epidermal hairs can be clearly observed, and scanning electron microscopy further reveals the ultrastructure and density differences of the epidermal hairs. The results show that the epidermal hairs on the upper surface of wild-type mulberry leaves are more densely distributed, with trichomes covering the epidermal cells. Figure 1 A, E); the density of epidermal hairs on the underside of the leaf is significantly higher than that on the upper side, and the epidermal hairs intertwine to form a dense pubescence (A, E). Figure 1 B, F). The upper surface of the leaves of the hybrid mulberry tree has sparse epidermal hairs, which contrasts sharply with the density of the wild type, but epidermal hairs are still distributed, and most of the visible epidermal hairs are bristle-type (B, F). Figure 1 C, G); The density of epidermal hairs on the underside of the leaves of the hybrid mulberry was also significantly lower than that of the wild type, with epidermal cells and some stomata clearly visible, and no continuous hairy covering layer formed. Figure 1 (D, H). A combination of optical and scanning electron microscopy revealed a significant difference in leaf epidermal hair density between wild-type and hybrid paper mulberry. While hybridization significantly reduced the overall epidermal hair density of paper mulberry leaves, as one of the woody plants with relatively complex epidermal hair types, it is difficult to select completely hairless superior paper mulberry varieties through hybridization.

[0035] (2) Observation of epidermal hair density of mulberry leaves at different developmental stages

[0036] Healthy, disease-free wild-type and less-hairy hybrid paper mulberry plants were selected and sampled during the growing season (June). Based on the leaf's position on the branch and its degree of unfolding, the tested leaves were divided into three developmental stages: terminal leaves: tender leaves taken from the tip of the branch that were not yet fully unfolded; the leaves were soft in texture (…). Figure 2 A, B, G, H); Expanded leaves: taken from the 2nd-4th leaves below the top leaf, the leaves are basically expanded, the leaf color is light green, and they are in the functional establishment period ( Figure 2 C, D, I, J); Mature leaves: taken from the 5th-8th leaves in the middle of the branch, the leaves are fully unfolded, dark green, highly leathery, and have the most vigorous physiological functions. Figure 2 E, F, K, L). Three plants were selected from each strain, and five leaves were collected from each plant at different developmental stages for observation under an optical microscope. The results showed that the epidermal hairs on the terminal leaves of wild-type mulberry leaves were dense, almost covering the entire epidermal surface, with a large number of interwoven trichomes visible. The epidermal hair density of mature leaves decreased significantly, and the epidermal hairs were sparsely distributed in the field of view. This indicates that the epidermal hair density of wild-type mulberry leaves shows a significant decreasing trend with leaf development, with the highest epidermal hair density in the terminal leaves and the lowest in mature leaves. Figure 2 AF). The epidermal hair density of hybrid paper mulberry remained at a low level at different developmental stages, without exhibiting the high-density hairs found in wild-type paper mulberry. Furthermore, the epidermal hair density showed minimal variation with leaf development, remaining generally stable. Figure 2 GL).

[0037] (3) Evolutionary analysis of the R2R3 MYB gene family

[0038] From the genomic protein sequences of *Broussonetia papyrifera*, candidate proteins containing the MYB domain were searched using HMMER 3.0 software. The Hidden Markov Model (HMM) used was the MYB domain model (PF00249) from the Pfam database. The candidate protein sequences obtained from the initial screening were then submitted to the Pfam (http: / / pfam.xfam.org / ) and SMART (http: / / smart.embl-heidelberg.de / ) databases for domain confirmation. Sequences lacking complete R2 and R3 repeats were removed, ultimately identifying members of the *Broussonetia papyrifera* R2R3 MYB transcription factor family. ClustalW software was used to perform multiple sequence alignment of the identified *Broussonetia papyrifera* R2R3 MYB transcription factors with known R2R3 MYB transcription factor amino acid sequences in *Arabidopsis thaliana*. The alignment results were imported into MEGA 11.0 software, and a phylogenetic tree was constructed using the neighbor-joining method. The bootstrap value was set to 1000 repetitions to assess the reliability of each branch. Phylogenetic analysis clearly divides these transcription factors into different evolutionary branches.

[0039] (4) Cloning and expression pattern analysis of the BpMYB090 gene in Broussonetia papyrifera

[0040] Primers BpMYB090F (5'-TTGCTTGCTAGAAACTTTGA-3') and BpMYB090R (5'-CACAGGTAAATTTGAGGGTT-3') were designed based on the full-length sequence of the BpMYB090 gene identified in the Broussonetia papyrifera genome. Using cDNA from leaves on the day of Broussonetia papyrifera epidermal cell protrusion as a template, BpMYB090 was homologously cloned. The target fragment was ligated into the Blunt vector and sequenced. Its nucleotide sequence is shown in SEQ ID NO. 1, and its expressed protein amino acid sequence is shown in SEQ ID NO. 2. Based on the structural characteristics of the MIXTA gene, multiple sequence alignment revealed that the BpMYB090 gene is structurally identical to the AtMYB16, AtMYB17, and AtMYB107 genes in Arabidopsis thaliana that regulate epidermal hair development. All of them possess a typical R2R3 MYB domain and contain a motif sequence specific to the ninth subgroup. Figure 3Top leaves, expanded leaves, and mature leaves of wild-type and hybrid paper mulberry trees were collected, and RNA was extracted from the collected samples for cDNA reverse transcription. Quantitative primers for the BpMYB090 gene were designed: 0103F: 5'-ACGGCTTGGCTACTTTCCC-3'; 0103R: 5'-ATCCTCAATCCACCGCTCT-3'. The BpACT gene was used as an internal reference gene, with primer sequences BpACTF: 5'-AATGGTGAAGGCTGGGTT-3'; BpACTR: 5'-ACCGTGCTCAATGGGATA-3'. The real-time quantitative PCR experimental procedure was as follows: a 20 μL reaction system was prepared including 100 ng of cDNA, 4 pmol of forward and reverse primers, 10 μL of AceQ qPCR SYBR Green Master Mix, and sterile ultrapure water was added to a final volume of 20 μL. The reaction procedure first involves denaturation at 95°C for 3 minutes, followed by 40 reaction cycles: denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds. Gene expression calculations were performed using 2- ΔCT The method, namely ΔCT=CT 目的基因 -CT 内参基因 The relative expression pattern of the BpMYB090 gene in leaves of Broussonetia papyrifera at different developmental stages was analyzed using a T-test. The results showed that the BpMYB090 gene was specifically highly expressed on the day epidermal cell development began, and its expression pattern initially increased and then decreased during epidermal cell development, highly overlapping with the dynamic developmental process of epidermal cells. Figure 4 It is speculated that it plays a key regulatory role in the initial differentiation of paper mulberry epidermal cells.

[0041] (5) Construction of BpMYB090 gene overexpression vector

[0042] Using pCAMBIA1301 as the backbone vector, the full-length BpMYB090 gene was recombined into the multiple cloning site between the KpnI and XbaI restriction endonucleases via homologous recombination. CaMV35S was used as the promoter of the target gene. Double digestion with KpnI and XbaI was performed for verification, yielding clear bands on gel electrophoresis, confirming the successful vector construction. Sequencing confirmed the vector sequence. This vector was named pCAMBIA1301-BpMYB090. Figure 5 ).

[0043] (6) Functional verification of the BpMYB090 gene transfected into Arabidopsis thaliana.

[0044] Colombian wild-type Arabidopsis thaliana (col-0) used for plant transformation was obtained from laboratory preservation. Arabidopsis seeds were surface-sterilized in 75% ethanol for 30 seconds, then washed three times with sterile water, followed by surface sterilization in 10% NaClO (v / v) for 10 minutes, and then washed six times again with sterile water. The seeds were then evenly sown in 1 / 2 MS medium containing 3% sucrose and 0.8% agar at pH 5.8. Seeds were first vernalized by incubation in the dark at 4°C for 3 days, then transferred to a growth chamber (22-23°C, 16 h light / 8 h dark) for germination. After approximately two weeks of growth, the Arabidopsis seedlings were transplanted into nursery pots with a soil:black soil:perlite:vermiculite ratio of 3:3:1:1 and grown under the same conditions (22-23°C, 16 h light / 8 h dark).

[0045] The constructed overexpression vector pCAMBIA1301-BpMYB090 was transformed into Agrobacterium tumefaciens GV3101 (pMP90). Agrobacterium culture was expanded, and the bacterial cells were collected. The concentration was adjusted to OD=0.8 with a suspension (1 / 2 MS + 0.5% sucrose) for Arabidopsis transformation experiments. Wild-type Arabidopsis transformation was performed using the floral organ immersion method. During the peak flowering period of wild-type Arabidopsis (approximately 4 weeks of growth), the inflorescences were immersed in the prepared Agrobacterium suspension for 30 s, cultured in the dark in a growth chamber for 24 h, and then normal growth conditions were restored until maturity. Seeds were then harvested by division (T1). T1 generation transgenic resistant plants were screened using MS medium containing 30 mg / L hygromycin. The selected resistant plants were transplanted into soil and placed in a growth chamber (22-23℃, 16 h light / 8 h dark) for normal management. Specific primers were designed for PCR detection of resistant plants selected under hygromycin resistance. Wild-type Arabidopsis thaliana (WT) genomic DNA was used as the negative control PCR amplification template, and pCAMBIA1301-BpMYB090 plasmid was used as the positive control PCR amplification template. Results showed that the amplification band of the positive transgenic plants was consistent in size with the amplification band of the pCAMBIA1301-BpMYB090 plasmid positive control, while no band was detected in the corresponding lane of the negative control. Figure 6 A). To further clarify the relationship between the expression level of the BpMYB090 gene and phenotypic changes, the transcription level of BpMYB090 in leaves of transgenic positive lines and wild-type Arabidopsis thaliana was detected using qRT-PCR. The results showed that the BpMYB090 gene was highly expressed in both transgenic lines, and its expression level was significantly higher than that in the wild-type control. Figure 6B) indicates that the exogenous gene has been successfully integrated and transcribed. After the seedlings have grown in the soil for 10 days, the number of epidermal trichomes in a designated 1.5 mm region of the first true leaf is observed under a microscope. The results show that overexpression of BpMYB090 alters the phenotypic phenotype of Arabidopsis leaves, specifically, the epidermal trichome density of transgenic plants is significantly greater than that of wild-type Arabidopsis leaves. Figure 6 C). Simultaneously, overall observation of the transgenic plants revealed that this change did not only occur in the first true leaf of Arabidopsis thaliana; the density of epidermal hairs on the entire leaf of Arabidopsis thaliana was significantly increased compared to the wild type. Figure 6 C). To verify the reliability of this result, five transgenic Arabidopsis thaliana lines using the BpMYB090 gene were selected, and 10 positive plants from each of the T2 generations were chosen. The number of epidermal hairs was counted in the same area of ​​the first true leaf. The results showed that the BpMYB090 gene can significantly increase the number of epidermal hairs in Arabidopsis thaliana (C). Figure 6 D).

[0046] The AtGL1 gene in Arabidopsis thaliana is a core gene in the GL1-GL3-TTG1 (MBW) complex, which regulates the development of leaf epidermal hairs. Mutation of AtGL1 directly leads to the hairless phenotype in the gl1 mutant. To investigate whether overexpression of BpMYB090 can restore the hairless phenotype of gl1 leaves, BpMYB090 was transformed into the gl1 mutant. Seedlings grown in nutrient pots for two weeks were observed and photographed under a stereomicroscope. PCR detection was performed on transgenic plants selected for hygromycin resistance. The results showed that the amplification band of the positive transgenic plants was consistent in size with the amplification band of the positive control pCAMBIA1301-BpMYB090 plasmid, while no band was detected in the corresponding lane of the negative control. Figure 7 A). To further clarify the relationship between the expression level of the BpMYB090 gene and phenotypic changes, qRT-PCR was used to detect the transcription level of BpMYB090 in the leaves of transgenic positive lines and gl1. The results showed that the BpMYB090 gene was highly expressed in all transgenic lines, and its expression level was significantly higher than that of the gl1 control (A). Figure 7 B), indicating that the exogenous gene has been successfully integrated and transcribed. In 50 T1-positive transgenic lines, 27 heterologously expressed BpMYB090 lines exhibited a wild-type phenotype, with complete restoration of trichome development on rosette leaves, while trichomes were not observed in the untransformed gl1 lines. Figure 7 C). Compared to the gl1 mutant, transgenic plants overexpressing the BpMYB090 gene showed a highly significant increase in the number of epidermal hairs. Figure 7D). Transformation experiments in Arabidopsis thaliana confirmed that the BpMYB090 gene has the potential to promote the differentiation of epidermal cells into epidermal hairs, thus confirming the regulatory role of this gene in the development of epidermal hairs in Broussonetia papyrifera. Therefore, genetic transformation technology can be used to knock out or silence the BpMYB090 gene in Broussonetia papyrifera to breed new hairless Broussonetia papyrifera varieties.

[0047] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. The regulatory gene BpMYB090 for the development of hairs on the leaf epidermis of Broussonetia papyrifera leaves has the nucleotide sequence shown in SEQ ID NO.

1.

2. The protein encoded by the gene BpMYB090, which regulates the development of leaf epidermal hairs as described in claim 1, has the amino acid sequence shown in SEQ ID NO.

2.

3. A recombinant expression vector, expression cassette, recombinant bacteria, or host cell containing the regulatory gene BpMYB090 for the development of leaf epidermal hairs as described in claim 1.

4. The application of the BpMYB090 gene regulating the development of leaf epidermal hairs as described in claim 1 in promoting the formation of plant epidermal hairs.

5. The application according to claim 4, characterized in that, The plant in question is either Arabidopsis thaliana or Broussonetia papyrifera.

6. The application according to claim 4, characterized in that, Includes the following steps: (1) Construct a plant overexpression vector containing the regulatory gene BpMYB090 for the development of leaf epidermal hairs as described in claim 1; (2) Transform the plant overexpression vector into the target plant; (3) Transgenic plants with increased number of epidermal hairs were obtained through cultivation and screening.

7. The application according to claim 6, characterized in that, The plant overexpression vector was pCAMBIA1301-BpMYB090.

8. The application of the gene BpMYB090, which regulates the development of epidermal hairs on Broussonetia papyrifera leaves as described in claim 1, in restoring epidermal hair formation in Arabidopsis thaliana hairless mutants.

9. The application of the regulatory gene BpMYB090 for the development of epidermal hairs of Broussonetia papyrifera leaves as described in claim 1, the protein as described in claim 2, or the recombinant expression vector, expression cassette, recombinant bacteria, or host cell as described in claim 3 in the study of the molecular mechanism of plant epidermal hair development.

10. The application according to claim 9, characterized in that, The plant in question is either Arabidopsis thaliana or Broussonetia papyrifera.

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