Gene bpmixta01 for regulating initiation of epidermal hair development of morus alba and expressed protein and application thereof

Abstract

The application discloses a gene BpMIXTA01 for regulating and controlling initial development of Broussonetia papyrifera epidermal hair as well as an expression protein and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2. The gene belongs to the ninth sub-group MIXTA gene of the R2R3 MYB transcription factor family. Function verification shows that overexpression of BpMIXTA01 in Arabidopsis thaliana can significantly increase the number of epidermal hairs, and can restore the epidermal hair loss phenotype of the Arabidopsis thaliana hairless mutant, proving that BpMIXTA01 is a positive regulation factor of epidermal hair formation. The application first discloses a key regulation gene for initial development of Broussonetia papyrifera epidermal hair cells, provides an important gene resource for analyzing the molecular mechanism of epidermal hair development of woody plants, and can cultivate a hairless forage Broussonetia papyrifera new variety through genetic engineering means, and has important theoretical value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the gene BpMIXTA01 that regulates the initiation of hair development in the epidermis of paper mulberry, its expressed protein, and its applications. Background Technology

[0002] Paper mulberry (Broussonetia papyrifera) is a deciduous tree belonging to the genus Broussonetia in the family Moraceae. It is naturally distributed in East and Southeast Asia, possessing characteristics of both a native species and a pioneer plant. This species has high crude protein content in its leaves, reaching 20-30% in the wild. It also exhibits strong resistance to adverse conditions and rapid growth, making it widely used in feed processing, papermaking, and ecological restoration, thus demonstrating significant economic value.

[0003] Despite abundant wild paper mulberry resources, its densely hairy leaf epidermis results in poor palatability and low protein utilization, hindering its large-scale promotion in livestock farming. While improved varieties obtained through hybridization have partially alleviated the shortage of feed protein, the diverse types of paper mulberry epidermis and complex genetic background make it difficult to achieve targeted breeding of completely hairless superior germplasm using traditional breeding methods. Therefore, creating new hairless forage paper mulberry varieties using modern biotechnology has become an important direction for promoting the upgrading of the paper mulberry industry.

[0004] Plant epidermal trichomes are unique structures formed by the differentiation of epidermal cells. Based on cell number and physiological function, they can be classified into unicellular and multicellular types, and glandular and non-glandular trichomes. The epidermal trichomes of Broussonetia papyrifera leaves have a more complex composition, containing both unicellular glandular and non-glandular trichomes. Their developmental regulatory mechanisms differ from those of previously reported model plants, making them uniquely valuable for research. Existing studies have shown that members of the ninth subgroup of the R2R3 MYB transcription factor family play a crucial regulatory role in epidermal cell differentiation, and these functions have been validated in species such as Arabidopsis thaliana, tomato, cotton, and poplar.

[0005] Currently, research on paper mulberry mainly focuses on assessing its feed value, analyzing fiber characteristics, and optimizing pulping and papermaking processes. Basic biological research is relatively weak, especially regarding the molecular regulatory mechanisms of epidermal hair development, which have not yet been systematically elucidated. With the completion of the paper mulberry whole-genome sequencing and breakthroughs in genetic transformation technology, a technological foundation has been laid for analyzing the functions of key genes in epidermal hair development at the molecular level and creating new hairless germplasm.

[0006] It is worth noting that epidermal hairs serve as an important structural barrier for plants to resist external stresses, and a moderate increase in their density can help improve the plant's tolerance to adverse conditions such as drought and pests. Therefore, under different breeding objectives, regulating epidermal hair density can serve both the breeding of hairless forage varieties and the application in stress-resistance breeding scenarios to enhance plant environmental adaptability, demonstrating dual application potential. 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 the gene BpMIXTA01, which regulates the initiation of trichome development in mulberry trees. Another technical problem to be solved by the present invention is to provide the application of the gene BpMIXTA01, which regulates the initiation of trichome development in mulberry trees, in transgenic mulberry trees.

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

[0009] A gene, BpMIXTA01, that regulates the initiation of hair development in the epidermis of paper mulberry trees has the nucleotide sequence shown in SEQ ID NO.1.

[0010] The protein encoded by the gene BpMIXTA01, which regulates the initiation of hair development in the epidermis of the paper mulberry tree, has the amino acid sequence shown in SEQ ID NO.2.

[0011] The recombinant expression vector, expression cassette, recombinant bacteria, or host cell of the gene BpMIXTA01, which regulates the initiation of hair development in the epidermis of the paper mulberry tree.

[0012] The application of the gene BpMIXTA01, which regulates the initiation of epidermal hair development in Broussonetia papyrifera, in promoting the formation of epidermal hairs in plants, wherein the plant is Arabidopsis thaliana or Broussonetia papyrifera.

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

[0014] (1) Construct a plant overexpression vector containing the gene BpMIXTA01 that regulates the initiation of hair development in the epidermis of Broussonetia papyrifera;

[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-BpMIXTA01.

[0018] The application of the gene BpMIXTA01, which regulates the initiation of epidermal hair development in Broussonetia papyrifera, in restoring epidermal hair formation in Arabidopsis thaliana hairless mutants, specifically the gl1 mutant.

[0019] The application of the gene BpMIXTA01, which regulates the initiation of epidermal hair development in Broussonetia papyrifera, 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 some embodiments, the plant is Arabidopsis thaliana or paper mulberry.

[0021] Beneficial effects: Compared with the prior art, the technical advantages of this invention are as follows:

[0022] This invention, through stereomicroscopy and scanning electron microscopy, observed the epidermal hairs on the leaves of the paper mulberry tree. The results showed that epidermal cells begin to develop protrusions on the fourth day after seed coat rupture, subsequently swelling and extending to develop into epidermal hairs. Combining gene sequence structural characteristics and evolutionary analysis, three MIXTA genes were identified in the paper mulberry genome. Through gene expression profile analysis at different time points before and after the initiation of epidermal hair development, and verification through genetic transformation in Arabidopsis thaliana, the BpMIXTA01 gene was determined to be a key gene regulating the initiation of epidermal cell development in paper mulberry. The results showed that, during gene sequence evolution analysis, this gene clustered with known genes regulating the initiation of epidermal cell development in poplar and cotton within the same MIXTA subcategory. Gene expression pattern analysis revealed that this gene was specifically highly expressed on the day epidermal cell development began in mulberry leaves, exhibiting an expression pattern of first increasing and then decreasing during epidermal cell development, highly overlapping with the dynamic developmental process of epidermal cells. Functional validation results showed that overexpression of BpMIXTA01 in wild-type Arabidopsis significantly increased the number of epidermal hairs. Overexpression of this gene in the hairless Arabidopsis mutant (gl1) completely restored its epidermal hair phenotype to the wild-type level, indicating that BpMIXTA01 can effectively promote epidermal cell protrusion and differentiation into epidermal hairs. In summary, this verifies that the BpMIXTA01 gene is a key gene regulating the initiation of epidermal cell development in mulberry trees. It can be expected that genetic manipulation such as gene silencing or gene editing of this gene can yield new hairless mulberry tree varieties. Attached Figure Description

[0023] Figure 1 This describes the dynamic development process of the epidermal hairs in the paper mulberry and the plant's growth status.

[0024] Figure 2 A scanning electron microscope image showing the dynamic development process of paper mulberry epidermal cells;

[0025] Figure 3 This is a graph showing the results of cluster evolution analysis of the MIXTA gene sequences.

[0026] Figure 4 This is a multiple sequence alignment diagram of the MIXTA gene;

[0027] Figure 5 This is a graph showing the qRT-PCR expression pattern analysis of the BpMIXTA01 gene. The bar numbers represent the average of three biological replicates, and the error bars represent the standard error of three biological replicates.

[0028] Figure 6 This is a schematic diagram of the overexpression vector structure of the BpMIXTA01 gene;

[0029] Figure 7 Figure showing the functional validation results of the BpMIXTA01 gene from Broussonetia papyrifera transformed into wild-type Arabidopsis thaliana.

[0030] Figure 8 The figure shows the functional verification results of the BpMIXTA01 gene of Broussonetia papyrifera transformed into the hairless mutant (gl1) of Arabidopsis thaliana. Detailed Implementation

[0031] 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.

[0032] Example 1:

[0033] (1) Observation of the dynamic process of differentiation and development of hairs on the epidermis of paper mulberry.

[0034] Preliminary observations of the aboveground parts of the paper mulberry plant showed that the epidermal hairs had fully developed along with the true leaves, even in tissues almost invisible to the naked eye. This made it difficult to identify the initiation point of epidermal hair development and obtain a continuous developmental process. However, in the cotyledons of the paper mulberry, the epidermal hairs develop synchronously with the cotyledons, overcoming the difficulty of identifying the initiation point of epidermal hair development in true leaves. Therefore, this study used the epidermal hairs in the cotyledons of the paper mulberry as the research object to obtain their dynamic developmental process. Mature paper mulberry seeds were collected, dried, and inducing vernalization at low temperature before being sown on MS medium. Phenotypic observations were conducted on paper mulberry leaves at different developmental stages after seed coat rupture. Sampling was performed from the day of seed coat rupture to 20 days after seed coat rupture. Samples were taken every 24 hours in the early stage, and every 72 hours after the first true leaf emerged, for a total of 12 stages. The collected samples were preserved in fixative, and the initiation process of epidermal hair development was observed using scanning electron microscopy.

[0035] Morphological observation reveals that the surface of the cotyledons of the paper mulberry is smooth 1-3 days after seed coat rupture, with almost no epidermal hairs visible to the naked eye. However, by the 4th day of development, transparent, glassy epidermal hairs that are almost integrated with the leaf can be observed. By the 5th day, the epidermal hairs become clearly visible, and subsequently, as the leaf develops, the epidermal hairs become increasingly obvious and visible. Figure 1 The dynamic development process of epidermal hairs observed by scanning electron microscopy almost perfectly matches the development process observed in previous morphological observations. It can be seen that the epidermal cells did not show obvious protrusions on day 3, but showed obvious protrusions on day 4, indicating that the epidermal cells had begun to develop at this stage, and subsequently gradually enlarged and extended to develop into epidermal hairs. Figure 2 ).

[0036] (2) Identification and sequence evolution analysis of the MIXTA gene

[0037] MIXTA genes belong to the ninth subclass of R2R3-MYB genes. Their sequence characteristics include two conserved MYB domain units and AQWESAR. AE RL Motif sequences of RES were obtained. Based on MIXTA sequence characteristics, three MIXTA genes were identified in *Broussonetia papyrifera*. Members of the MIXTA gene family in the following species were also identified: 8 in *Populus deltoides*, 10 in *Gossypium hirsutum*, 3 in *Arabidopsis thaliana*, 7 in tomato (*Solanum lycopersicum*), 3 in cucumber (*Cucumissativus*), 5 in rice (*Oryza sativ*), 7 in maize (*Zea mays*), 8 in willow (*Salix purpurea*), 11 in tobacco (*Nicotiana tabacum* L.), and 4 in snapdragon (*Antirrhinum majus*). A phylogenetic tree was constructed using MEGA 5.0 software based on the Maximum Likelihood Tree method. The results showed that the BpMIXTA01 gene clustered in the same branch with key genes regulating poplar fluff development and key genes regulating cotton fluff initiation development. Figure 3 It is speculated that it may play a regulatory role in the initial development of hairs on the epidermis of paper mulberry.

[0038] (3) Cloning of the BpMIXTA01 gene of Broussonetia papyrifera and analysis of its expression pattern in the dynamic process of epidermal hair differentiation and development.

[0039] Primers BpMIXTA01F (5'-TTCTACAGCGTTAGGTTTG-3') and BpMIXTA01R (5'-AGACGTGACAGGGAAAGA-3') were designed based on the full-length sequence of the BpMIXTA01 gene identified in the Broussonetia papyrifera genome. Using cDNA from leaves on the day of Broussonetia papyrifera epidermal cell protrusion as a template, BpMIXTA01 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 BpMIXTA01 gene is structurally identical to the PdeMIXTA02 gene, which regulates poplar fluff development, and the GhMML4 gene, which regulates cotton fluff initiation. Both possess a typical R2R3 MYB domain and contain a motif sequence unique to the ninth subgroup. Figure 4 Leaves were collected from *Broussonetia papyrifera* at 1 to 20 days after seed coat rupture. RNA was extracted from these samples for cDNA reverse transcription. Quantitative primers for the BpMIXTA01 gene were designed: C0004F: 5'-ACGGGACTAGATCAACACGA-3'; C0004R: 5'-CGACGGAGTTGTAGACGAAC-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 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 BpMIXTA01 gene during the dynamic development of paper mulberry epidermal cells was analyzed using a T-test. The results showed that the BpMIXTA01 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 development process of epidermal cells. Figure 5 It is speculated that it plays a key regulatory role in the initial differentiation of paper mulberry epidermal cells.

[0040] (4) Construction of BpMIXTA01 gene overexpression vector

[0041] Using pCAMBIA1301 as the backbone vector, the full-length BpMIXTA01 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-BpMIXTA01. Figure 6 ).

[0042] (5) Functional verification of BpMIXTA01 gene transfected into Arabidopsis thaliana.

[0043] 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).

[0044] The constructed overexpression vector pCAMBIA1301-BpMIXTA01 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 seconds, then cultured in the dark in a growth chamber for 24 hours. Normal growth conditions were then restored until maturity, at which point the plants were divided and seeds were harvested (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 the pCAMBIA1301-BpMIXTA01 plasmid was used as the positive control PCR amplification template. Results showed that the amplification band of the positive transgenic plants was the same size as the amplification band of the pCAMBIA1301-BpMIXTA01 plasmid positive control, while no band was detected in the corresponding lane of the negative control. Figure 7 A). After the seedlings had grown in the soil for 10 days, the number of epidermal trichomes in a designated 1.5 mm area of ​​the first true leaf was observed under a microscope. The results showed that overexpression of BpMIXTA01 altered the phenotypic phenotype of Arabidopsis leaves, specifically, the epidermal trichome density of transgenic plants was significantly greater than that of wild-type Arabidopsis leaves. Figure 7 B). 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 7 B). To verify the reliability of this result, five lines of BpMIXTA01 transgenic Arabidopsis were selected, and 10 positive plants from each of their T2 generations were selected. The number of epidermal hairs was counted in the same area of ​​the first true leaf. The results showed that the BpMIXTA01 gene can significantly increase the number of epidermal hairs in Arabidopsis (B). Figure 7 C).

[0045] 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 BpMIXTA01 could restore the hairless phenotype of gl1 leaves, we transformed gl1 mutants with BpMIXTA01 and observed and photographed seedlings grown in nutrient pots for two weeks under a stereomicroscope. PCR detection was performed on transgenic plants selected for hygromycin resistance. Figure 8 A) found that in 50 T1-positive transgenic lines, 15 heterologously expressed BpMIXTA01 lines exhibited a wild-type phenotype, with complete restoration of trichome development on rosette leaves, while trichomes were not observed in untransformed gl1 lines. Figure 8 B). Compared to the gl1 mutant, transgenic plants overexpressing the BpMIXTA01 gene showed a highly significant increase in the number of epidermal hairs. Figure 8 C). Transformation experiments in Arabidopsis thaliana confirmed that the BpMIXTA01 gene has the potential to promote the differentiation of epidermal cells into protruding cells, thus confirming the regulatory role of this gene in the initiation of epidermal cell development in Broussonetia papyrifera. Therefore, genetic transformation technology can be used to knock out or silence the BpMIXTA01 gene in Broussonetia papyrifera to breed new glabrous Broussonetia papyrifera varieties.

[0046] 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. A gene BpMIXTA01 that regulates the initiation of hair development in the epidermis of paper mulberry, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The protein encoded by the gene BpMIXTA01, which regulates the initiation of hair development in the epidermis of paper mulberry, 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 gene BpMIXTA01, which regulates the initiation of hair development in the epidermis of the paper mulberry tree as described in claim 1.

4. The application of the gene BpMIXTA01, which regulates the initiation of epidermal hair development in Broussonetia papyrifera as described in claim 1, in promoting the formation of epidermal hairs in plants.

5. The application according to claim 4, characterized in that, The plant in question is 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 gene BpMIXTA01, which regulates the initiation of hair development in the epidermis of Broussonetia papyrifera 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-BpMIXTA01.

8. The application of the gene BpMIXTA01, which regulates the initiation of epidermal hair development in Broussonetia papyrifera as described in claim 1, in restoring epidermal hair formation in Arabidopsis thaliana hairless mutants.

9. The application of the gene BpMIXTA01, which regulates the initiation of epidermal hair development in Broussonetia papyrifera as described in claim 1, the protein described in claim 2, or the recombinant expression vector, expression cassette, recombinant bacteria, or host cell 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 Arabidopsis thaliana or Broussonetia papyrifera.

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