Application of PaBBX32-1 gene in promoting formation of plant leaf epidermal hair

Abstract

The invention provides an application of a gene PaBBX32-1 in promoting formation of epidermal hair of plant leaves. According to the invention, a PaBBX32-1 gene is separated and cloned from platanus acerifolia, an overexpression vector is constructed by using the PaBBX32-1 gene, and the overexpression vector is introduced into cells or tissues of a required plant through agrobacterium-mediated genetic transformation. The overexpression of the PaBBX32-1 gene can promote the epidermal hair development of plant leaves.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the PaBBX32-1 gene in promoting the formation of epidermal hairs in plant leaves. Background Technology

[0002] The London plane tree (scientific name: *Platanus × acerifolia* (Aiton) Willd.) is an important deciduous tree belonging to the genus *Platanus* in the family Platanaceae. Due to its rapid growth, beautiful tree shape, and strong resistance, it is widely used in urban landscaping. The densely covered leaf surface with trichomes not only enhances its ornamental value but also plays a vital role in ecological functions, such as absorbing airborne particulate matter, reducing noise pollution, and mitigating the urban heat island effect. It is a typical resilient urban greening tree species.

[0003] Epidermal trichomes are structures formed by the specialized differentiation of epidermal cells. They are widely distributed throughout plants and possess various physiological functions, including regulating transpiration, reflecting ultraviolet radiation, defending against pests and diseases, and resisting environmental stresses. In urban ecosystems, epidermal trichome density is closely related to a plant's ability to absorb pollutants and its resistance to external stresses. Therefore, understanding the molecular regulatory mechanisms of plant epidermal trichome development is of significant practical importance for improving the stress resistance and ecological adaptability of greening plants.

[0004] Previous studies have shown that the formation and development of epidermal hairs are regulated by a series of transcription factors. However, current research on epidermal hair formation in Platanus orientalis mainly focuses on negative regulatory factors (such as pathways that inhibit epidermal hair formation), and there is a lack of in-depth exploration of the positive regulatory mechanisms that promote epidermal hair formation. The identification of related functional genes and regulatory networks remain unclear. Summary of the Invention

[0005] In view of this, the present invention provides an application of the PaBBX32-1 gene in promoting the formation of epidermal hairs on plant leaves. Overexpression of the PaBBX32-1 gene can promote the development of epidermal hairs on plant leaves.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an application of the PaBBX32-1 gene in promoting the development of epidermal hairs on plant leaves.

[0007] Preferably, the specific steps of the application are as follows: the PaBBX32-1 gene is isolated and cloned from the sycamore tree, an overexpression vector is constructed using the PaBBX32-1 gene, and the vector is introduced into the cells or tissues of the desired plant.

[0008] Preferably, the overexpression vector is introduced into the desired plant by introducing the overexpression vector into the cells of the desired plant through Agrobacterium-mediated genetic transformation.

[0009] Preferably, the required plants include Arabidopsis thaliana and Platanus orientalis.

[0010] Preferably, the nucleotide sequence of the PaBBX32-1 gene is shown in SEQ ID NO:1.

[0011] Preferably, the amino acid sequence encoded by the PaBBX32-1 gene is shown in SEQ ID NO:2.

[0012] Secondly, the present invention provides an overexpression vector containing the nucleotide sequence of the PaBBX32-1 gene.

[0013] Thirdly, the present invention provides an engineered bacterium containing the overexpression vector described above.

[0014] Preferably, the engineered bacteria is Agrobacterium GV3101.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention isolates and clones the PaBBX32-1 gene from *Platanus × acerifolia*, constructs an overexpression vector using the PaBBX32-1 gene, and introduces the overexpression vector into the cells or tissues of the desired plant through Agrobacterium-mediated genetic transformation. Overexpression of the PaBBX32-1 gene can promote the development of epidermal hairs on plant leaves.

[0016] This invention systematically verifies the functions of BBX family members such as PaBBX32-1, clarifying their mechanism of action in promoting epidermal hair formation. This will provide a theoretical basis and technical path for molecular breeding of superior sycamore germplasm, and is expected to cultivate urban greening tree species with stronger resistance and better ecological functions, which has important scientific significance and practical application prospects. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the original intermediate carrier pMD®18 structure provided in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the pMD®18-PaBBX32-1 recombinant plasmid structure provided in Example 1 of the present invention; Figure 3 This is a schematic diagram of the structure of the vector pRI101-EGFP provided in Embodiment 2 of the present invention; Figure 4 This is a schematic diagram of the pRI101-PaBBX32-1 recombinant plasmid structure provided in Embodiment 2 of the present invention; Figure 5 This is a graph showing the expression level of the PaBBX32-1 gene in the transgenic line provided in Example 3 of the present invention; Figure 6 This is a comparison image of the epidermal hairs of control Arabidopsis thaliana and transgenic Arabidopsis thaliana PaBBX32-1 provided in Example 3 of the present invention; Figure 7 This is a comparison of the number of epidermal hairs between the CK plant (plant transformed with empty vector) and the PaBBX32-1 transgenic line at 15 days, as provided in Example 3 of this invention. Figure 8 This is a graph showing the expression levels of genes that intrinsically regulate the number of epidermal hairs in Arabidopsis thaliana in the CK and PaBBX32-1 transgenic lines provided in Example 4 of this invention. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0019] Epidermal trichomes are structures formed by the specialized differentiation of epidermal cells. They are widely distributed throughout plants and possess various physiological functions, including regulating transpiration, reflecting ultraviolet radiation, defending against pests and diseases, and resisting environmental stresses. In urban ecosystems, epidermal trichome density is closely related to a plant's ability to absorb pollutants and its resistance to external stresses. Therefore, understanding the molecular regulatory mechanisms of plant epidermal trichome development is of significant practical importance for improving the stress resistance and ecological adaptability of greening plants.

[0020] Previous studies have shown that the formation and development of epidermal hairs are regulated by a series of transcription factors. However, current research on epidermal hair formation in Platanus orientalis mainly focuses on negative regulatory factors (such as pathways that inhibit epidermal hair formation), and there is a lack of in-depth exploration of the positive regulatory mechanisms that promote epidermal hair formation. The identification of related functional genes and regulatory networks remain unclear.

[0021] To address the aforementioned technical problems, this invention provides an application of the PaBBX32-1 gene in promoting the development of epidermal hairs on plant leaves.

[0022] Further, the specific steps of the application are as follows: the PaBBX32-1 gene is isolated and cloned from the sycamore tree, an overexpression vector is constructed using the PaBBX32-1 gene, and the vector is introduced into the cells or tissues of the desired plant.

[0023] Furthermore, the overexpression vector is introduced into the desired plant specifically by introducing the overexpression vector into the cells of the desired plant through Agrobacterium-mediated genetic transformation.

[0024] Furthermore, the required plants include Arabidopsis thaliana and Platanus orientalis.

[0025] Furthermore, the nucleotide sequence of the PaBBX32-1 gene is shown in SEQ ID NO:1, with a full length of 825 bp.

[0026] Furthermore, the amino acid sequence encoded by the PaBBX32-1 gene is shown in SEQ ID NO:2, and the sequence shown does not include the terminator sequence.

[0027] Secondly, the present invention provides an overexpression vector containing the nucleotide sequence of the PaBBX32-1 gene.

[0028] Thirdly, the present invention provides an engineered bacterium containing the overexpression vector described above.

[0029] Fourthly, the present invention provides an engineered bacterium, wherein the engineered bacterium is Agrobacterium GV3101.

[0030] Example 1: Isolation and Cloning of the PaBBX32-1 Gene This embodiment aims to illustrate how to isolate and clone the PaBBX32-1 gene from Platanus orientalis and verify its nucleotide sequence.

[0031] The specific steps are as follows: 1. Mixed tissue samples from different parts of the Platanus orientalis (buds, petioles, young leaves, etc.) were selected, and total RNA was extracted using the CTAB method. The CTAB buffer formulation was as follows: NaCl 1.4 mol / L; EDTA (ethylenediaminetetraacetic acid) 20 mmol / L; Tris·Cl 100 mmol / L; 2% (w / v) polyvinylpyrrolidone K30 (pvp) and 2% β-mercaptoethanol.

[0032] The extraction steps are as follows: 1) After grinding the sample with liquid nitrogen, add 4 mL of preheated CTAB extraction solution and incubate in a water bath at 65°C for 5 minutes.

[0033] 2) Add an equal volume of chloroform:isoamyl alcohol (volume ratio 24:1), invert to mix, let stand for 5 min, and centrifuge at 10000 rpm / min for 10 min at 4℃.

[0034] 3) Take 3 mL of the supernatant and repeat step 2).

[0035] 4) Take the supernatant, add 1 / 3 of the supernatant volume of LiCl, precipitate at -20℃ for 12 hours, centrifuge at 10000rpm / min at 4℃ for 10min, discard the supernatant, wash the precipitate twice with 75% ethanol, and dissolve it in an appropriate amount of DEPC (diethyl pyrocarbonate) to treat the water for later use.

[0036] 2. Using the extracted total RNA as a template, the first strand of cDNA was synthesized using a commercial reverse transcription kit (ClonExpress II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd.). The reaction conditions were: 42℃ for 2 min, 37℃ for 15 min, and 85℃ for 5 s.

[0037] 3. The ORF region of the PaBBX32-1 gene was obtained from the transcriptome data, and the PaBBX28 / 29-1 gene was amplified from *Platanus orientalis* using specific primers P1 (forward primer) + P2 (reverse primer). The PCR reaction system was as follows: 94℃ pre-denaturation for 4 min, 94℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, 35 cycles, and extension at 72℃ for 10 min. It should be noted that the primer pairs were designed based on the nucleotide sequence of the PaBBX32-1 gene, and a mixed sample of different parts of *Platanus orientalis* was used as a template for PCR amplification. The DNA sequences of the primer pairs for amplifying PaBBX32-1 are shown below: P1 forward primer (F): 5'-GATGAAATACCGGGTTTGCGAGC-3', P2 reverse primer (R): 5'-ATCCCCTCAAACCACACCATTAG-3'.

[0038] The amplified products were purified after electrophoresis and then ligated into the pMD®18-T vector ( Figure 1-2 (As shown). The positive clone was verified by sequencing, and the target sequence was obtained and named PaBBX32-1-T. Its complete sequence is SEQ ID NO:1, and the protein sequence encoded by this sequence is shown in SEQ ID NO:2.

[0039] SEQ ID NO:1:ATGAAATACCGGGTTTGCGAGCTGTGCAATGGAGAAGCTACGGTCTACTGCGAGTCGGATTCAGCGTTTCTGTGTTGGAAGTGTGATGCTAAGGTTCACGAGGCAAATTTTCTAGTTGCTCGTCATGTCCGCCGGACTGTCTGTTCCAAATGTAAAGGATTCGATGGGAATCATATCTCCGGCGTCGGATTCAGGCCGCGATCGATCTGCCGATCTTGCTTGCCGGAGTCGGGCTGTGATGACTTGGATTATTCTCGGTCATCATCTTCTTCTAGTGCGGAATCCTTCGCTGCTGCTCCTTGGAAAATCGATTTTGATCAACAACGACTGGAAAAAAAAAACTTGTTCGACTTCTGAAACTGAGGTTTCCAGCAATGAATCAAGTTTTCCAACCAAGTTTCCTGGCGACGA TGATGATTCAACGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGACGCTGCTGAGATCGCCACCAGGTCCGAAGAAGGTGGACTGAAGACTGAGGGCATTCTTGTAAATTGGTGCAGAAAGCTGGGCCTAAAGAATGGCTGCTATGTGCCCCTTGCCTCCACCGCCTTGGGTGTGTGTGTTTGCGTAAACTGACGGTTTTTGCCCTT TAGGGTCTCCCTCGCTTCGTCGCTTTGGTTATCGTGGAAACTTTGCGTGGGGAGGTTCAGCATCCACGTGTCAGACCTAAAGCGGCTTGAAGAGATCTCTGGCGTGCCAGCTAAGCTGATCTTATTGGCTGAGTCGAAGCTCTCACGTTTTCTAAAGATGGAGAAATCTCAGCTTCATCAAGAAGGTTGGGCTGAATGCTCTGTTTTGA SEQ ID NO:2:MKYRVCELCNGEATVYCESDSAFLCWKCDAKVHEANFLVARHVRRTVCSKCKGFDGNHISGVGFRPRSICRSCLPESGCDDLDYSRSSSSSAESFAAAPWKIDFDQQRLEKKTCSTSETEVSSNESSFPTKFP GDDDDSTKKKKKKKKKKKTLLRSPPGPKKVDVKTEGILVNWCRKLGLKNGCYVPLASTALGVCLRKLTVLPFRVSLASSLWLSWKLCVGSSASTCQNLKRLEEISGVPAKLILLAESKLSRFLKMEKSQLHQEGWAECSV Example 2: Construction and transformation of the PaBBX32-1 gene overexpression vector into Arabidopsis thaliana To clarify the biological function of the PaBBX32-1 gene in plants, this embodiment constructs a plant overexpression vector for the gene and introduces it into Arabidopsis thaliana to observe phenotypic changes in transgenic plants.

[0040] 1. Using the PaBBX32-1-T plasmid obtained in Example 1 as a template, homologous recombination primers containing KpnI restriction sites were designed and synthesized for PCR amplification of the target gene fragment. The amplification product was purified and recovered by agarose gel electrophoresis and then digested with KpnI.

[0041] Simultaneously, the plant expression vector pRI101-EGFP was linearized by KpnI single enzyme digestion. Subsequently, the digested PaBBX32-1 fragment was ligated to the linearized pRI101-EGFP vector using the ClonExpress II One Step Cloning Kit and transformed into competent E. coli DH5α. It should be noted that primer pairs were designed for the construction of the pRI101-EGFP expression vector. The primer pairs were designed based on the nucleotide sequences of the PaBBX32-1 gene and the pRI101-EGFP vector. The DNA sequences of the primer pairs are shown below, with lowercase letters representing sequences on the pRI101-EGFP vector: P5 forward primer (F): 5'-cccgtcgaccccgggggtaccATGAAATACCGGGTTTGCGA-3', P6 reverse primer (R): 5'-catgaattcggatccggtaccAACAGAGCATTCAGCCCAACC-3'.

[0042] After antibiotic resistance screening and sequencing to identify positive clones, the successfully constructed expression vector was named pRI101-PaBBX32-1 (see...). Figure 3-4 The recombinant plasmid was then introduced into the Agrobacterium tumefaciens GV3101 strain via electroporation for subsequent Arabidopsis transformation.

[0043] 2. This invention employs the floral dip method for genetic transformation of Arabidopsis thaliana. The specific steps are as follows: 1) Incubate Agrobacterium GV3101 containing the target vector on solid LB medium containing the corresponding antibiotic resistance (kanamycin) at 28°C for 2-3 days. Pick positive single colonies and inoculate them into liquid selective LB medium containing the corresponding antibiotic resistance (kanamycin). Incubate overnight at 28°C with a shaker at 200 rpm until the bacterial concentration reaches OD500. 600 The value is approximately 0.6-0.8. It should be noted that the promoter can be a 35S promoter or other enhanced promoters, and the expression vector should contain a suitable selection marker gene (such as a kanamycin resistance gene) to facilitate the identification and screening of transgenic plant cells or plants.

[0044] The LB medium formula is 10 g / L peptone + 5 g / L yeast extract + 10 g / L NaCl + 100 mg / L Kan, and the solid medium is supplemented with 1.5 g / L agar.

[0045] 2) Centrifuge the shaken bacterial solution at 5000 rpm for 5 min, and resuspend it in 5% sucrose solution (with 40 μL / 100 mL of Arabidopsis thaliana conversion surfactant). Soak the Arabidopsis thaliana inflorescence in the resuspended solution for about 45 s, and keep it in the dark and moist for 24 h.

[0046] 3) Disinfect the harvested Arabidopsis thaliana transgenic positive seedlings with 75% alcohol for 10 min, then disinfect twice with 100% alcohol for 2 min each time. Finally, rinse 5-6 times with sterile water and spread them evenly on MS medium for screening. Perform PCR detection on the selected positive plants, observe the phenotype in the field, and collect seeds for sowing until homozygous T3 generation seeds are obtained. It should be noted that the primer sequences used for detection are as follows. The primers are designed based on the PaBBX32-1 gene nucleic acid sequence, and RT-PCR amplification is performed using cDNA from transgenic plant samples as templates. The DNA sequences of the primer pairs are as follows: P9 forward primer (F): 5'-TCAAACAGAGCATTCAGCCCAA-3', P10 reverse primer (R): 5'-ATAACCAAAGCGACGAAGCGAG-3'.

[0047] The MS medium formula is 4.4 g / L MS powder + 30 g / L sucrose + 7.5 g / L agar + 50 mg / L kanamycin + 50 mg / L cephalosporin.

[0048] Example 3: Phenotypic observation of PaBBX32-1 transgenic Arabidopsis thaliana T3 generation transgenic Arabidopsis seeds were directly sown in flowerpots along with the unloaded control (CK) and cultivated under long-day conditions (16h light / 8h darkness). The epidermal trichome phenotype was observed. Results showed that the number of epidermal trichomes on the leaves of the PaBBX32-1 transgenic lines (OE1, OE4, OE11) was significantly higher than that of the control group: the average number of epidermal trichomes on the leaves of the control plants was 30.4 days; the average number of epidermal trichomes on the leaves of the PaBBX32-1 transgenic lines OE1, 4, and 11 were 45.6 days, 45.4 days, and 38.8 days, respectively. Figure 5-7 The above results indicate that overexpression of the PaBBX32-1 gene significantly enhances the development of epidermal hairs in Arabidopsis leaves.

[0049] Example 4: Transcriptome Data Analysis of PaBBX32-1 Transgenic Arabidopsis thaliana Transcriptome sequencing was performed on 10-day-old WT and PaBBX32-1 transgenic lines, and the expression levels of genes related to epidermal hair development were analyzed. Figure 8 As shown, the expression of genes negatively regulating epidermal trichome development, namely JAZ1, TRICHOMELESS1 (TCL1), TCL2, ENHANCER OF TRY AND CPC 3 (ETC3), and TRIPTYCHON (TRY), in the PaBBX32-1 transgenic line was significantly lower than that in the control plant. However, the expression of genes positively regulating epidermal trichome development, namely GLABRA1 (GL1) and GL3, was slightly higher in the PaBBX32-1 transgenic line than in the control plant. This indicates that PaBBX32-1 regulates the development of epidermal trichomes in Arabidopsis rosette leaves mainly by downregulating the negative regulators of epidermal trichome development.

[0050] In conclusion, PaBBX32-1 may promote the formation of epidermal trichomes in Arabidopsis thaliana by inhibiting the expression of negative regulators of epidermal trichome development, thus confirming its positive regulatory role in epidermal trichome development.

[0051] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0052] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of a PaBBX32-1 gene in promoting the development of epidermal hairs in plant leaves.

2. The application according to claim 1, characterized in that, The specific steps of the application are as follows: the PaBBX32-1 gene is isolated and cloned from the sycamore tree, an overexpression vector is constructed using the PaBBX32-1 gene, and the vector is introduced into the cells or tissues of the desired plant.

3. The application according to claim 2, characterized in that, The overexpression vector is introduced into the desired plant cells through Agrobacterium-mediated genetic transformation.

4. The application according to claim 2, characterized in that, The required plants include Arabidopsis thaliana and Platanus orientalis.

5. The application according to claim 1, characterized in that, The nucleotide sequence of the PaBBX32-1 gene is shown in SEQ ID NO:

1.

6. The application according to claim 1, characterized in that, The amino acid sequence encoded by the PaBBX32-1 gene is shown in SEQ ID NO:

2.

7. An overexpression vector containing the nucleotide sequence of the PaBBX32-1 gene as described in claim 5.

8. Engineered bacteria containing the overexpression vector of claim 7.

9. The engineered bacteria according to claim 8, characterized in that, The engineered bacteria is Agrobacterium GV3101.

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