A drought-tolerant functional gene, MaPUB21, in the banana plant and its application in improving plant drought tolerance.

Abstract

This invention discloses a drought-resistant functional gene, MaPUB21, in the banana plant and its application in improving plant drought tolerance. The nucleotide sequence of this gene is shown in SEQ ID NO.2. This invention constructs a silencing vector (MaPUB21-pTRV) targeting the MaPUB21 gene and uses Agrobacterium-mediated transformation to transiently silence it in banana plants. Drought tolerance analysis shows that after drought stress treatment, compared with the wild type, the MaPUB21-silenced lines exhibit significantly lower levels of malondialdehyde, superoxide anion, and hydrogen peroxide, while significantly higher levels of proline and peroxidase activity. The results indicate that silencing the MaPUB21 gene effectively improves plant drought tolerance. The gene of this invention provides a new resource for molecular breeding of plant stress resistance and is of great significance for cultivating drought-resistant plants and improving the utilization rate of arid land.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving a functional gene MaPUB21 isolated and cloned from banana plant, and the application of this gene in the genetic improvement of drought resistance in plants. The gene was transiently transformed and expressed in banana plant and it was found that the drought resistance of the silent line was significantly stronger than that of the wild type of banana plant. Background Technology

[0002] Bananas are evergreen, perennial, large monocotyledonous herbaceous plants belonging to the genus *Musa* in the family Musaceae. They are one of the world's oldest cultivated fruit trees, exhibiting rich genetic diversity (Liu et al., 2019; Zhou et al., 2023). Most modern cultivated bananas evolved from two wild species, *Musa acuminata* (chromosome type AA) and *Musa balbisiana* (chromosome type BB), through intraspecific or interspecific hybridization (Simmonds, 1962). Internationally common cultivated types include Cavendish (AAA), Plantain (AAB), and Pisang awak (ABB). Bananas have a wide geographical distribution, which means they are frequently affected by environmental factors during their growth and development, such as drought, low temperatures, and salinity. Therefore, the development of superior, stress-resistant new varieties has become a key factor in the development of the banana industry. In recent years, with the continuous development of molecular biology technology, the discovery of resistance genes to quickly breed new stress-resistant banana varieties has become an indispensable technology.

[0003] Drought stress is a type of water stress, primarily caused by the inability of soil or air to provide effective water supply to plants, thus affecting their normal growth. To adapt to drought stress, plants have evolved a series of complex adaptive mechanisms to ensure their survival and reproduction. At the physiological level, drought signals trigger the plant's antioxidant system to produce peroxidases to maintain redox homeostasis, prevent cell damage by scavenging free radicals, reduce membrane peroxidation, maintain membrane integrity, and promote the accumulation of metabolites such as proline and betaine to enhance drought resistance. At the molecular level, the activation of drought-induced signaling pathways and metabolic and physiological responses is crucial for plants to cope with drought.

[0004] Ubiquitination is an important post-translational modification. The 26S-proteasome degradation system (UPS) is a complex, precise, and reversible protein post-translational modification mechanism, primarily responsible for degrading a large number of enzymes, structural proteins, and regulatory proteins. In this system, ubiquitin acts as a tag molecule, precisely labeling substrate proteins and selectively degrading them via the 26S proteasome, thus determining their fate and location. Furthermore, it influences the function of intracellular proteins by regulating their activity and stability, thereby modulating the transmission of numerous intracellular signals. The UPS consists of ubiquitin (UB), ubiquitin activator (E1), ubiquitin conjugate (E2), ubiquitin ligase (E3), and the 26S proteasome. The ubiquitination process, through binding to specific proteins and following an E1-E2-E3 multi-enzyme cascade, plays a crucial role in the degradation of target proteins. The ubiquitin ligase E3, due to its ability to specifically recognize and bind to target proteins and determine protein degradation, is considered the most critical component of this system. It mainly consists of three types: HECT (Homologous to the E6-AP carboxyl terminus), RING (Really interesting new gene) / U-box, and CRLs (Cullin-RING ligases).

[0005] U-boxes are variants of RING-type E3 ligases, widely distributed in eukaryotes. This conserved domain consists of 70 amino acid residues and was first identified in yeast from ubiquitin fusion degradation protein 2 (UFD2). The U-box gene family has been identified in various plants, with 62 identified in tomato, 125 in soybean, and 69 in apple. Multiple studies have shown the important roles of plant U-box proteins (PUBs) in drought stress, ABA signaling, and the regulation of vesicle and membrane protein transport. In Arabidopsis thaliana, AtPUB18 and AtPUB19 negatively regulate ABA signaling by inducing an ABA hypersensitive response, thereby responding to drought stress. AtPUB46 and AtPUB48 play key roles in drought and oxidative stress, and heat tolerance in Arabidopsis thaliana, respectively. In rice, OsPUB15 is involved in the positive regulation of plant tolerance to salt and drought stress. In soybean, GmPUB8 negatively regulates the plant's response to drought stress.

[0006] The Banana 'Cinnamomum campestris' is a widely cultivated variety in the banana industry and an ideal material for studying banana drought resistance and cloning drought-resistant genes. Therefore, identifying members of the U-box gene family in Banana 'Cinnamomum campestris' and analyzing their drought-resistant functions is of great significance for banana stress-resistance breeding. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the functional gene MaPUB21 in improving the drought resistance of plants. This invention isolates and clones a functional gene from the banana plant, which the applicant names MaPUB21. Transient transformation and expression of the functional gene MaPUB21 in banana plants revealed a significant increase in drought resistance in the silenced lines, indicating that the functional gene MaPUB21 described in this invention is related to the drought resistance of plants and has the function of regulating plant drought resistance.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention seeks protection for the use of the functional gene MaPUB21, whose nucleotide sequence is shown in SEQ ID NO:2, in at least one of the following (a1)-(a3):

[0010] (a1) Improve the drought resistance of plants;

[0011] (a2) Prepare products to improve the drought resistance of plants;

[0012] (a3) Cultivate new plant germplasm with improved drought resistance.

[0013] Secondly, the present invention seeks protection for the use of biological materials associated with the functional gene MaPUB21, whose nucleotide sequence is shown in SEQ ID NO:2, in at least one of the following (a1)-(a3):

[0014] (a1) Improve the drought resistance of plants;

[0015] (a2) Prepare products to improve the drought resistance of plants;

[0016] (a3) Cultivate new plant germplasm with improved drought resistance;

[0017] The biomaterial associated with the functional gene MaPUB21 is at least one of the following (b1)-(b2):

[0018] (b1) The protein encoded by the functional gene MaPUB21;

[0019] (b2) Biological materials used to silence, suppress or interfere with the expression of the functional gene MaPUB21;

[0020] The biological material used to silence, inhibit, or interfere with the expression of the functional gene MaPUB21 is at least one of (c1) to (c10):

[0021] (c1) Nucleic acid molecules used to silence, suppress, interfere with, or gene edit the expression of the functional gene MaPUB21;

[0022] (c2) An expression cassette containing the nucleic acid molecule described in (c1);

[0023] (c3) A recombinant vector containing the nucleic acid molecule described in (c1) or a recombinant vector containing the expression cassette described in (c2);

[0024] (c4) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3);

[0025] (c5) A transgenic plant cell line containing the nucleic acid molecule described in (c1), or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3);

[0026] (c6) A transgenic plant tissue containing the nucleic acid molecule described in (c1), or a transgenic plant tissue containing the expression cassette described in (c2), or a transgenic plant tissue containing the recombinant vector described in (c3);

[0027] (c7) A transgenic plant organ containing the nucleic acid molecule described in (c1), or a transgenic plant organ containing the expression cassette described in (c2), or a transgenic plant organ containing the recombinant vector described in (c3);

[0028] (c8) A transgenic plant containing the nucleic acid molecule described in (c1), or a transgenic plant containing the expression cassette described in (c2), or a transgenic plant containing the recombinant vector described in (c3);

[0029] (c9) Regenerative cells, tissue cultures or protoplasts derived therefrom of the transgenic plant described in (c8);

[0030] (c10) Propagation material of the transgenic plant as described in (c8).

[0031] Furthermore, the above-mentioned application is characterized in that, using the functional gene MaPUB21 as the target gene, through genetic engineering methods, the expression of the functional gene MaPUB21 is silenced, inhibited, or interfered with, or the activity or content of the protein encoded by the functional gene MaPUB21 is reduced in the target plant, so as to improve the drought resistance of the plant, or to cultivate new plant germplasm with improved drought resistance and apply it in production.

[0032] Thirdly, the present invention seeks to protect a method for improving the drought resistance of plants, using the functional gene MaPUB21 with the nucleotide sequence shown in SEQ ID NO: 2 as the target gene, and through genetic engineering methods, silencing, inhibiting or interfering with the expression of the functional gene MaPUB21 or reducing the activity or content of the protein encoded by the functional gene MaPUB21 in the target plant, thereby improving the drought resistance of the plant.

[0033] Fourthly, this invention seeks to protect a method for cultivating new plant germplasm with improved drought resistance. Using the functional gene MaPUB21, whose nucleotide sequence is shown in SEQ ID NO: 2, as the target gene, transgenic plants are obtained by silencing, inhibiting, or interfering with the expression of the functional gene MaPUB21 in the target plant through genetic engineering methods. The transgenic plants are then cultivated to obtain new plant germplasm with improved drought resistance for production application.

[0034] In the technical solution of this invention, the protein encoded by the functional gene MaPUB21 is the protein shown in (d1) or (d2) below:

[0035] (d1) A protein having the amino acid sequence shown in SEQ ID NO: 1;

[0036] (d2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (d1).

[0037] In the technical solution of this invention, the plant is Arabidopsis thaliana or banana, and the banana is Banana buds, but is not limited to these.

[0038] In a specific embodiment of the present invention, the primer pair used to clone the functional gene MaPUB21 is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0039] In a specific embodiment of the present invention, the primer pair used to amplify (c1) the nucleic acid molecule for silencing the functional gene MaPUB21 is shown in SEQ ID NO:13 and SEQ ID NO:14.

[0040] The applicant isolated and cloned a functional gene from the banana plant and named it MaPUB21. Its nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:1.

[0041] This invention involves sampling *Calabash merbau* seedlings under dehydration-simulated drought stress at corresponding time points and analyzing the relative expression level of the functional gene MaPUB21 described in this invention using qRT-PCR. The results showed that the relative expression level of the functional gene MaPUB21 gradually increased with the extension of drought treatment time, indicating that the functional gene MaPUB21 has the function of regulating plant drought resistance in response to drought stress treatment.

[0042] During the research, the inventors overexpressed the functional gene MaPUB21 from the banana plant in Arabidopsis thaliana, and the resulting Arabidopsis thaliana exhibited significantly weaker drought resistance than the wild-type Arabidopsis thaliana. Silencing the functional gene MaPUB21 from the banana plant in the plant resulted in plants with significantly stronger drought resistance than the wild-type banana plant.

[0043] The beneficial effects of this invention are:

[0044] This invention provides the application of the functional gene MaPUB21 in *Musa javanica* in improving plant drought tolerance. Overexpression and silencing vectors were constructed and stably transformed into *Arabidopsis thaliana* and transiently transformed into *Musa javanica*, obtaining overexpressing lines in *Arabidopsis thaliana* and gene-silenced lines in *Musa javanica*, which were then subjected to drought treatment. Results showed that the malondialdehyde (MDA) content in the overexpressing lines was significantly higher than that in the wild type, and the contents of superoxide anion and hydrogen peroxide were also higher, indicating more residual reactive oxygen species and greater cell damage. Compared with the wild type, the overexpressing lines contained lower levels of proline and peroxidase. Conversely, the VIGS-mediated MaPUB21-silenced *Musa javanica* lines showed significantly lower MDA content, and lower superoxide anion and hydrogen peroxide contents, with other physiological and biochemical results being the opposite of the overexpressing *Arabidopsis thaliana* lines. These results indicate that interfering with or silencing the expression of the MaPUB21 gene enhances the plant's reactive oxygen species scavenging capacity, thereby improving drought tolerance. This research is of great significance for banana stress resistance breeding. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the technical process of the present invention.

[0046] Figure 2 This is a diagram illustrating the temporal expression pattern of the functional gene MaPUB21 under dehydration-simulated drought stress.

[0047] Figure 3 Subcellular localization map of the protein encoded by the functional gene MaPUB21; imaging of MaPUB21 in GFP field, DAPI field, bright field, and mixed field.

[0048] Figure 4 This is a schematic diagram illustrating the positive identification of Arabidopsis thaliana transgenic MaPUB21 according to the present invention.

[0049] Figure 5This study measured the phenotypic and physiological parameters of the MaPUB21 gene overexpression lines and wild-type (WT) before and after dehydration-simulated drought treatment. Specifically, A represents the phenotype of 45-day-old Arabidopsis seedlings before and after 15 days of drought treatment; B represents the proline content of 45-day-old Arabidopsis seedlings after 15 days of drought treatment; C represents the malondialdehyde content of 45-day-old Arabidopsis seedlings after 15 days of drought treatment; D represents the superoxide anion content of 45-day-old Arabidopsis seedlings after 15 days of drought treatment; E represents the hydrogen peroxide content of 45-day-old Arabidopsis seedlings after 15 days of drought treatment; and F represents the peroxidase content of 45-day-old Arabidopsis seedlings after 15 days of drought treatment.

[0050] Figure 6 This is a schematic diagram of the quantitative analysis of VIGS-mediated gene silencing expression in this invention.

[0051] Figure 7 This diagram illustrates the drought resistance analysis of the VIGS-mediated functional gene MaPUB21 silencing line in banana plants according to the present invention. In the diagram, A represents the phenotype of 45-day-old banana seedlings before and after 15 days of drought treatment; B represents the proline content of 45-day-old banana seedlings after 15 days of drought treatment; C represents the malondialdehyde content of 45-day-old banana seedlings after 15 days of drought treatment; D represents the superoxide anion content of 45-day-old banana seedlings after 15 days of drought treatment; E represents the hydrogen peroxide content of 45-day-old banana seedlings after 15 days of drought treatment; and F represents the peroxidase content of 45-day-old banana seedlings after 15 days of drought treatment. Detailed Implementation

[0052] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0053] This invention provides the functional gene MaPUB21 of the banana plant, the nucleotide sequence of which is shown in SEQ ID NO:2. The amino acid sequence of the protein MaPUB21 encoded by this functional gene is shown in SEQ ID NO:1, encoding 409 amino acids. Research results show that overexpression of the functional gene MaPUB21 can reduce the content of proline and peroxidase, and simultaneously reduce the plant's ability to scavenge reactive oxygen species (ROS), indicating that overexpression of this functional gene can reduce the plant's drought tolerance. Conversely, silencing the functional gene MaPUB21 can increase the content of proline and peroxidase, and simultaneously improve the plant's ability to scavenge reactive oxygen species (ROS), indicating that silencing this functional gene can improve the plant's drought tolerance. In this invention, the MaPUB21 gene is constructed using homologous recombination technology. Its recombination expression method can be implemented using conventional techniques in the art, without special limitations.

[0054] This invention designs primer pairs (as shown in SEQ ID NO:3 and SEQ ID NO:4) for cloning the cDNA sequence of the functional gene MaPUB21. The source of the primer pairs is not particularly limited; they can be synthesized by a biosynthetic company well-known in the art. In this embodiment, the primer pairs were synthesized by Shanghai Biotech Co., Ltd.

[0055] The functional gene MaPUB21 was overexpressed in plants using the P1300-MaPUB21 recombinant vector, and the pTRV-PUB21 recombinant vector was silenced in banana plants. This invention does not impose any particular limitations on the construction methods of the P1300-MaPUB21 and pTRV-PUB21 recombinant vectors; any construction method well-known in the art can be used.

[0056] The P1300-MaPUB21 and pTRV-PUB21 recombinant vectors were transformed into target plants using Agrobacterium-mediated genetic transformation. This invention does not impose any particular limitation on the Agrobacterium-mediated genetic transformation method; conventional methods well-known in the art can be used. The target plants preferably include Arabidopsis thaliana or Musa balsamina.

[0057] This invention uses PCR amplification to screen positive overexpression plants. The primers used for PCR amplification to screen positive overexpression plants are shown in SEQ ID NO:11 and SEQ ID NO:12. After PCR amplification, if the plant line to be tested can amplify a fragment of the expected size (500bp), it indicates that they are positive overexpression lines.

[0058] Positive silencing lines were screened from the positive transgenic lines using qRT-PCR. qRT-PCR detection revealed that the expression level of the target gene (functional gene MaPUB21) in the tested plants was significantly lower than that in the wild type, thus classifying them as positive silencing lines. The qRT-PCR primer sequences for the functional gene MaPUB21 are shown in SEQ ID NO:5 and SEQ ID NO:6, and the primer sequences for the internal reference gene MaACT1 are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0059] Finally, the drought resistance of overexpressing and gene-silenced plants was tested: the obtained overexpressing and gene-silenced lines were subjected to drought treatment, and the phenotypes, physiological indicators and reactive oxygen species of the overexpressing and gene-silenced lines were measured to verify their drought resistance. (1) Detection of reactive oxygen species: Anti-O in leaves was determined using conventional methods. 2- And H2O2 accumulation. The results showed that overexpressing Anti-O in plants 2- The levels of both reactive oxygen species (ROS) and H2O2 were significantly higher in the overexpressing lines compared to the wild type, indicating that the drought tolerance of the overexpressing lines was weaker and lower than that of the wild type. In contrast, the levels of both ROS in the silent lines were significantly lower than in the wild type, indicating enhanced drought tolerance.

[0060] (2) Malondialdehyde (MDA) content determination: Standard methods revealed that the MDA content in the overexpression lines was significantly higher than that in the wild type, indicating that the cell damage in the overexpression lines was stronger than that in the wild type, meaning that the drought resistance of the overexpression lines was weaker than that of the wild type. The MDA content in the silent lines was significantly lower than that in the wild type, indicating that their cell membrane damage was less severe and their drought resistance was stronger.

[0061] (3) Determination of proline content and peroxidase activity: Conventional methods showed that the proline content and peroxidase activity of the overexpressing plants were significantly lower than those of the wild type, indicating that the drought resistance of the overexpressing plants was weaker than that of the wild type. The proline accumulation and peroxidase activity of the silent lines were significantly higher than those of the wild type, further confirming their improved drought resistance.

[0062] The above results indicate that overexpression of the banana functional gene MaPUB21 in plants can significantly reduce the drought tolerance of the plants (see...). Figure 5 Specifically, this manifests as increased accumulation of reactive oxygen species, enhanced cell damage, and decreased activity of osmotic regulators and antioxidant enzymes. Silencing the functional gene MaPUB21 in *Musa acutissima* via VIGS-mediated induction significantly enhanced drought tolerance in the resulting plant lines (see...). Figure 7 Specifically, this manifests as a reduction in reactive oxygen species accumulation, a decrease in cell damage, and an increase in the activity of osmotic regulators and antioxidant enzymes.

[0063] The following examples illustrate the MaPUB21 gene in the banana plant provided by this invention and its application in improving plant drought resistance. However, these examples should not be construed as limiting the scope of protection of this invention.

[0064] Example 1: Cloning of the full-length cDNA of the MaPUB21 gene from the banana plant.

[0065] Our research group previously used a highly efficient yeast expression system to screen a functional gene, MaPUB21, from banana plants. Primers were designed based on the MaPUB21 gene sequence, and its full-length form was amplified from banana plants using RT-PCR. The detailed steps are as follows:

[0066] (1) RNA extraction and cDNA synthesis: 1 μg of banana RNA was treated with 1 U of DNase I at 37°C for 30 min and immediately placed on ice. 1 μL of 50 mM EDTA was added and treated at 65°C for 10 min and immediately placed on ice. First-strand cDNA was synthesized according to the reverse transcription kit instructions.

[0067] (2) Gene cloning and amplification: The obtained first-strand cDNA was used for PCR amplification of the MaPUB21 gene. The cloning primers are as follows:

[0068] Forward primer: 5'-ATGGAAGCTCCATTCTTGTTCC-3' (SEQ ID NO:3)

[0069] Reverse primer: 5'-ATGATTCAATCTCAAGAAGTCTTT-3' (SEQ ID NO:4).

[0070] The PCR reaction procedure and PCR reaction system for cloning the functional gene MaPUB21 are shown in Tables 1 and 2:

[0071] Table 1 PCR reaction procedure

[0072]

[0073] Table 2 PCR reaction system

[0074]

[0075] After amplification, a single-band PCR product is generated. After electrophoresis on a 1% agarose gel, the specific target band is recovered using a gel extraction kit following the instructions.

[0076] (3) Vector construction and transformation: The purified solution was ligated with the Peasy-Blut vector. Ligation system (10µL): 1 µL of PCR purified product, 3 µL of Peasy-Blut vector, 4 µL of ligase and 2 µL of sterile water. Ligation was carried out at 37℃ for 15 min, and the mixture was transformed into E. coli competent cells DH5α by heat shock method. PCR verification and sequencing were performed using primers for the target gene sequence (SEQ ID NO:3 and SEQ ID NO:4) (completed by Shanghai Sangon Biotech Co., Ltd.).

[0077] (4) Sequence analysis: Bioinformatics analysis of the cDNA sequence showed that the full length of the MaPUB21 gene is 1227 bp (as shown in SEQ ID NO:2), and the open reading frame encodes 408 amino acids (as shown in SEQ ID NO:1).

[0078] Example 2: qRT-PCR analysis of the MaPUB21 gene under different stress conditions

[0079] To analyze the response pattern of the MaPUB21 gene in banana plants to drought, qRT-PCR was used to analyze the expression pattern of the MaPUB21 gene under different abiotic conditions.

[0080] Total RNA was extracted from banana plants using the CTAB method, and first-strand cDNA was synthesized according to the TOYOBO reverse transcription kit instructions. The qRT-PCR reaction system (20 μL) consisted of: 10 μL 2×Mix, 0.1 μL cDNA, 5 μL primers (including MaPUB21 gene detection primers and internal control primers), and 4.9 μL water. The primer sequences are shown below:

[0081] (1) Primers for qRT-PCR detection of the MaPUB21 gene:

[0082] Forward primer: 5'-ATAATGCGCGTGTCGGAGAT-3' (SEQ ID NO:5)

[0083] Reverse primer: 5'-GAGCTTCCTCACCGTTCCAA-3' (SEQ ID NO:6)

[0084] (2) Internal reference primer:

[0085] Forward primer: 5'-ATTGTGCTTGATTCTGGTGATG-3' (SEQ ID NO:7)

[0086] Reverse primer: 5'-TTCAGCAGTGGTAGTGAAGGAA-3' (SEQ ID NO:8)

[0087] MaACT1 was used as an internal reference gene, and the amplified fragment length was 208.

[0088] The reaction procedure and reaction system for qRT-PCR are shown in Tables 3 and 4.

[0089] Table 3 qRT-PCR reaction procedure

[0090]

[0091] Table 4 qRT-PCR reaction system

[0092]

[0093] Samples were taken from non-transgenic banana seedlings under dehydration-simulated drought stress at corresponding time points, and the relative expression level of the MaPUB21 gene was analyzed by qRT-PCR. Figure 2 As shown, the expression level of the encoding gene increased over 12 hours with prolonged treatment time. This indicates that the MaPUB21 gene responds to drought stress.

[0094] Example 3: Subcellular localization of the protein encoded by MaPUB21

[0095] Based on the MaPUB21 nucleotide sequence and the 35S-GFP vector diagram (Zhang F, Pan ZJ, Han CY, DongHZ. et al. Pyrus betulaefolia ERF3 interacts with HsfC1a to coordinatelyregulate aquaporin PIP1;4 and NCED4 for drought tolerance. Hortic Res. 2024;11.uhae090.), XbaI and BamHI restriction sites were added before and after the gene sequence, respectively. Plasmids extracted from the correctly sequenced target gene were used as templates and amplified using specific primers containing XbaI / BamHI restriction sites (SEQ ID NO:9 and SEQ ID NO:10). The specific primers containing XbaI / BamHI restriction sites are as follows:

[0096] Forward primer: 5'-gagaacacgggggactctagaATGGAAGCTCCATTCTTGTTCC-3' (SEQ ID NO:9)

[0097] Reverse primer: 5'-gcccttgctcaccatggatccATGATTCAATCTCAAGAAGTCTTT-3' (SEQ ID NO: 10).

[0098] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 60 s, 35 cycles, followed by a 72℃ extension for 5 min after each cycle. The stop codon TAG was removed from the 3′ gene to allow for gene fusion with GFP. The PCR product was subjected to 1% agarose gel electrophoresis, and the target band was recovered using a gel electrophoresis kit. The 35S-GFP vector plasmid was digested with XbaI and BamHI restriction endonucleases at 37℃ for 3 hours, and then purified and recovered. The digested 35S-GFP vector and the gel-recovered MaPUB21 fragment were ligated using recombinant ligase at 37℃ for 30 min, and then transformed into competent E. coli DH5α cells. The transformed bacterial culture was tested by PCR. Cultures with positive PCR results were sent to the company for sequencing. The plasmid from the culture with correct sequencing results was extracted, and the resulting recombinant vector was named MaPUB21-GFP. The recombinant MaPUB21-GFP vector plasmid was transformed into Agrobacterium competent cells GV3101.

[0099] Agrobacterium-mediated transient transformation of tobacco: Agrobacterium single clones (containing MaPUB21-GFP, GFP empty vector, and P19 plasmid) were picked and activated in LB medium at 28°C with shaking at 220 rpm overnight. 10 μL of the overnight culture was transferred to 5 mL of LB-MES medium, and 2 μL of 100 mM acetylsylgenone was added. The culture was incubated at 28°C with shaking at 220 rpm for 16 h. The bacterial suspension was collected by centrifugation and resuspended in 10 mM MgCl2 solution. The OD600 value of the resuspended target gene bacterial suspensions (MaPUB21-GFP and GFP) was adjusted to 1.0; the OD600 value of the resuspended P19 bacterial suspension was adjusted to 0.7. The target gene bacterial suspension and P19 bacterial suspension were mixed in equal volumes. 100 mM acetylsylgenone was added to the mixed bacterial suspension at a 1:500 volume ratio, and the mixture was incubated at room temperature in the dark for 3 h. Select leaves with good growth and flat surfaces for injection. After injection, protect them from light for 12 hours and then allow them to grow in a normal environment. Observe and verify the results after 2-3 days, ensuring adequate water supply during this period.

[0100] See results Figure 3 . Figure 3 The subcellular localization of the MaPUB21-GFP encoded protein is shown in the imaging of the MaPUB21-GFP encoded protein in bright field, GFP field, Mcherry field, and mixed field. Based on the subcellular localization, it can be known that the protein encoded by the MaPUB21 gene is located in the cell membrane.

[0101] Example 4 Genetic transformation of Arabidopsis thaliana

[0102] 1. Construction of plant transformation vectors

[0103] Based on the multiple cloning site of the PCMBIA1300 vector and the coding region sequence of the MaPUB21 gene, upstream and downstream PCR primers (SEQ ID NO:9 and SEQ ID NO:10) were designed with XbaI and BamHI restriction sites. PCR amplification was performed using the MaPUB21 gene plasmid as a template. The PCR reaction system and amplification procedure were the same as those used for the MaPUB21 gene cloning. After amplification, the amplified product was purified and recovered using gel electrophoresis.

[0104] The PCMBIA1300 vector double digestion reaction volume was 40 µl, containing: 10 µl of the PCMBIA1300 vector plasmid, 4 µl of 10×M buffer, 1 µl each of XbaI and BamHI, and 24 µl of double-distilled water. After digestion at 37℃ for 3-4 h, the product was purified and recovered. In the ligation reaction system, the molar ratio of MaPUB21 gene to PCMBIA1300 vector was 2:1, with a total reaction volume of 10 µl. This system contained: 1 µl of 10× buffer, 1 µl of DNA recombinase, 4 µl of the double-digested MaPUB21 gene, 2 µl of the double-digested PCMBIA1300 vector product, and 2 µl of double-distilled water. The reaction was carried out at 37℃ for 30 min to obtain the ligation product. The ligation product was transformed into *E. coli* DH5α and cultured on LB agar plates containing 50 mg / L kanamycin for 16 h. After selecting positive clones, the cells were shaken to extract plasmids for PCR identification. Sequencing confirmed the absence of coding frame mutations, and a recombinant clone containing the inserted target fragment was obtained, which was named P1300-MaPUB21.

[0105] 2. Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0106] (1) Agrobacterium culture: Take Agrobacterium tumefaciens bacterial culture stored in ultra-low temperature freezer, streak it on LB agar plates with 50 mg / L kanamycin and 50 mg / L rifampin, and incubate at 28°C for 36-48 hours. Scrape off the streaks and add them to liquid MS medium (2.37 g / L MS + 50 g / L sucrose + 0.1 mg / L LIBA, pH=5.8). Incubate at 28°C for 30 min with shaking. When the bacterial concentration reaches OD600=0.8-1.0, add 200 μl / L of surfactant sweet77 for inoculation.

[0107] (2) Infection: Select robust wild-type Arabidopsis thaliana plants with a seedling age of about 30 days, main inflorescence with pods, secondary inflorescences about 2-10 cm long, a small number of flowers, and good growth for genetic transformation. Before transformation, cut off the flowering inflorescences, then place them upside down in a glass bottle containing the prepared Agrobacterium tumefaciens bacterial solution, vacuum the bottle, maintain a pressure of 0.05 MPa for 5 minutes, and place them on their side in the dark for 24 hours.

[0108] (3) Cultivation: Cultivate the plants according to conventional methods until they bear fruit and harvest mature seeds (T0 generation).

[0109] 3. Screening of transgenic positive seedlings

[0110] The above method was used to obtain Arabidopsis thaliana T0 generation seeds transgenic with the MaPUB21 gene. The surface of the T0 generation seeds was sterilized and evenly distributed on MS selective medium containing 50 mg / L hygromycin and 50 mg / L termethin. The seeds were cultured at 22℃ under light for 16 h / d. After one week of growth, plants with fast growth and long roots were selected and transplanted into sterilized nutrient soil for a period of time.

[0111] 3.1 DNA extraction from transgenic Arabidopsis thaliana

[0112] Following the above method, transgenic Arabidopsis thaliana with the MaPUB21 gene was obtained. DNA was extracted from each Arabidopsis thaliana plant, and gene primers were designed for PCR amplification to identify positive seedlings.

[0113] (1) Take 0.1g of Arabidopsis thaliana leaves and grind them thoroughly in a 2mL centrifuge tube with liquid nitrogen. Then add 500µL of CTAB solution (preheated at 65℃).

[0114] (2) 65℃ constant temperature water bath for 30 minutes, and invert it once every 10 minutes.

[0115] (3) After the water bath, cool to room temperature, add 500 µL of chloroform in a fume hood, mix by inverting, and centrifuge at 12000 rpm for 10 min at room temperature.

[0116] (4) After centrifugation, transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol solution, mix by inverting the tube, and centrifuge at 12000 rpm for 10 min at room temperature.

[0117] (5) After centrifugation, a white flocculent precipitate appears at the bottom of the tube. Carefully discard the supernatant, add 1 mL of 75% anhydrous ethanol to wash twice, resuspend the precipitate, and centrifuge at 12000 rpm for 1 min.

[0118] (6) Carefully discard the supernatant. After the alcohol has completely evaporated, add 50µL ddH2O and place in a constant temperature oven at 65℃ for 30 minutes until the precipitate is completely dissolved.

[0119] 3.2 Detection of positive transgenic plants

[0120] PCR amplification was performed using gene-specific primers. PCR amplification was performed using the gene upstream primer and the vector downstream primer (SEQ ID NO: 11 and SEQ ID NO: 12). The PCR amplification procedure and system are shown in Tables 1 and 2. After PCR amplification, the selected transgenic lines were able to amplify a fragment of the expected size (500 bp), indicating that they were positive transgenic lines (e.g., [examples would be inserted here]). Figure 4 (As shown).

[0121] Forward primer: 5'-atcgccctcgccctcgccgga-3' (SEQ ID NO:11)

[0122] Reverse primer: 5'-ATCGAAGCTGGTGCCGTCTG-3' (SEQ ID NO:12)

[0123] MaPUB21 was expressed in Arabidopsis thaliana using Agrobacterium-mediated transformation. Molecular genetic analysis identified transgenic Arabidopsis with a single-copy homozygous insertion, stably expressing MaPUB21 from generation T1 to T2, thus demonstrating stable heritability of the phenotypic trait. Insertion site analysis confirmed that the phenotypic changes in the MaPUB21 transgenic materials were not caused by the transgenic manipulation affecting other genes. Therefore, this transgenic material provides a material basis for this research project. Two overexpression lines, MaPUB21-1 and MaPUB21-2, were ultimately obtained.

[0124] Example 5: Drought Resistance Analysis of MaPUB21-GFP Transgenic Plants

[0125] To further verify the function of MaPUB21 in improving plant drought tolerance, wild-type and overexpression lines were subjected to drought treatment for a certain period of time. Wild-type and overexpression-positive Arabidopsis seedlings (MaPUB21-1 and MaPUB21-2) of uniform age and good growth were treated with dehydration to simulate drought stress for 15 days under the same culture conditions. The phenotypes after treatment were observed, and conductivity, proline, and other parameters were measured.

[0126] Figure 5 Phenotypic and physiological parameters of MaPUB21-GFP gene overexpression lines and wild-type lines before and after drought treatment were determined. Figure 5 In the figure, A represents the phenotype of Arabidopsis thaliana seedlings at 15 days old before and after drought treatment 15 days later; Figure 5 In this context, B represents the proline content of Arabidopsis thaliana seedlings that were 15 days old after 15 days of drought treatment. Figure 5 C in the figure represents the malondialdehyde content of Arabidopsis thaliana seedlings at 15 days of age after 15 days of drought treatment; Figure 5 D in the figure represents the superoxide anion content of Arabidopsis thaliana seedlings aged 15 days after 15 days of drought treatment; Figure 5 E in the figure represents the hydrogen peroxide content of Arabidopsis thaliana seedlings at 15 days of age after 15 days of drought treatment; Figure 5 F in the figure represents the peroxidase content of Arabidopsis thaliana seedlings that are 15 days old after 15 days of drought treatment.

[0127] The above indicators are important measures for evaluating drought resistance. The results show that the overexpression line is weaker in growth than the wild type, with higher levels of malondialdehyde, superoxide anion and hydrogen peroxide, and lower levels of proline and peroxidase. Figure 5 shows that overexpression of MaPUB21 reduces the drought resistance of plants.

[0128] Example 6 Transient transformation of banana seedlings

[0129] 1. Construction of virus-induced gene silencing vector

[0130] The viral silencing vector was constructed according to the method in Example 4. The viral silencing vector pTRV2 has XbaI and SacI double restriction sites. Upstream and downstream primers (SEQ ID NO:13 and SEQ ID NO:14) were designed according to primer design principles to amplify the MaPUB21 gene and insert it between the two restriction sites on the vector, resulting in the recombinant vector pTRV-MaPUB21, which was then transformed into Agrobacterium GV3101 competent cells. The sequences of the gene silencing primers are as follows:

[0131] Forward primer: 5'-gtgagtaaggttaccgaattcATGGAAGCTCCATTCTTGTTCC-3' (SEQ IDNO: 13)

[0132] Reverse primer: 5'-cgtgagctcggtaccggatccTTCTCCATGACGACATGAGACATAA-3' (SEQ ID NO: 14).

[0133] 2. Virus-induced gene silencing in banana seedlings

[0134] (1) Agrobacterium culture: Agrobacterium tumefaciens culture stored in an ultra-low temperature freezer was cultured in LB liquid medium supplemented with kanamycin 50 mg / L and rifampin 50 mg / L at 28°C and 220 rpm for 12 h. The cultured bacterial culture was centrifuged at 6000g for 10 min to collect the bacterial cells. The precipitate was resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 mM acetylsyl syringone, pH 5.6) until the concentration reached OD 600 =0.8-1.0;

[0135] (2) Induction of bacterial culture: Place the bacterial culture with the adjusted OD value in the dark and induce it at room temperature for 4 hours at 100 rpm;

[0136] (3) Injection of banana seedlings: pTRV1 and pTRV2 bacterial solutions were mixed in a 1:1 volume ratio as the control group, and pTRV1 and pTRV-MaPUB21 bacterial solutions were mixed in a 1:1 volume ratio as the experimental group. The seedlings were injected with banana seedlings that were 45 days old, had the same growth status and good health.

[0137] 3. VIGS Material Identification

[0138] After injection, banana seedlings were treated in the dark at room temperature for 12 hours, followed by normal culture for 3 days. RNA was extracted from seedlings of each strain from both the control and experimental groups. Gel electrophoresis was used to confirm the integrity of the RNA structure. The concentration was determined using Nanodrop (200-1000 ng / μL). The total RNA amount was adjusted to 3 μg, and then reverse transcribed into cDNA. Primers were designed using banana MaACT1 as an internal reference gene for amplification. The nucleotide sequence of the internal reference primers is as follows:

[0139] MaACT1 forward primer: 5'-ATTGTGCTTGATTCTGGTGATG -3' (SEQ ID NO:7)

[0140] MaACT1 reverse primer: 5'-TTCAGCAGTGGTAGTGAAGGAA-3' (SEQ ID NO:8)

[0141] The bands amplified using MaACT1 showed consistent brightness, indicating that the reverse-transcribed cDNA concentration was the same. Then, qRT-PCR was performed using MaPUB21-specific primers and the banana internal control primer MaACT1 to analyze the expression levels of the tested strains. The nucleotide sequence of the MaPUB21-specific primers is as follows:

[0142] Forward primer: 5'-ATAATGCGCGTGTCGGAGAT-3' (SEQ ID NO:5)

[0143] Reverse primer: 5'-GAGCTTCCTCACCGTTCCAA-3' (SEQ ID NO:6).

[0144] Based on the expression level of the MaPUB21 gene, two plants with lower expression levels were selected as virus-silencing positive lines and named MaPUB21-1-pTRV1 and MaPUB21-1-pTRV2.

[0145] See results Figure 6 As shown. Figure 6The gene expression level in gene-silenced positive plants was detected by qRT-PCR using gene-specific primers and the internal control primer MaACT. This indicates that the MaPUB21 gene in the virus-silenced banana seedling positive lines was silenced.

[0146] Example 7: Drought Resistance Analysis of pTRV-MaPUB21 Transgenic Plants

[0147] To further verify the function of the MaPUB21 functional gene in plant drought tolerance, wild-type and silent lines were subjected to drought treatment for a certain period of time. Wild-type and virus-silenced positive banana seedlings (TRV1 and TRV2) of uniform age and good growth were treated with dehydration to simulate drought stress for 15 days under the same culture conditions. The phenotypes after treatment were observed, and malondialdehyde (MDA) and other parameters were measured.

[0148] Figure 7 Phenotypic and physiological parameters of pTRV-MaPUB21 gene-silenced lines and wild-type lines before and after drought treatment were determined. Figure 7 In the figure, A represents the phenotype of banana seedlings aged 45 days before and after 15 days of drought treatment; Figure 7 In this context, B represents the proline content of banana seedlings aged 45 days after 15 days of drought treatment. Figure 7 The C in the figure represents the malondialdehyde content of banana seedlings aged 45 days after 15 days of drought treatment. Figure 7 D in the figure represents the superoxide anion content of banana seedlings aged 45 days after 15 days of drought treatment. Figure 7 E in the figure represents the hydrogen peroxide content of banana seedlings aged 45 days after 15 days of drought treatment. Figure 7 F in the figure represents the peroxidase content of banana seedlings aged 45 days after 15 days of drought treatment.

[0149] The above-mentioned indicators are all important measures for evaluating drought resistance. The results show that the gene-silenced lines have stronger growth than the wild-type lines, with lower levels of malondialdehyde, superoxide anion, and hydrogen peroxide, while having higher levels of proline and peroxidase. Figure 7 It can be demonstrated that silencing the MaPUB21 functional gene can enhance the drought resistance of plants.

[0150] sequence list

[0151] SEQ ID NO:1 amino acid sequence

[0152] MEAPFLFQCPISLELMEDPVTIATGVTYERKNIEKWLFTYKKLTCPATMQRLESFDLTPNHTVKRLISSFLEHAADEEPVTSPPCSYDAVDHDKLVSLLKSIQTGPFKASGLRKLKVLVEKNDELQKDLIRSGGIEVLGCVMSHVVVENSDFTAFRACEEALGVLSLLPLSDDATVELILKPDSLKPMMVIIQRGSAEARVHAMSILMKISKISNEWITKMVTDQDVDIVKSLLDLLSDEISTKLSSSSLDVLLEIVATSNKNRLKAIEAGAVCILLELLPDASRHNCEKVLLLLKRLCECAEGRSAFADHGLGVAAVSKKIMRVSEMATKLGVQIMWLMSSFHPREKLLEEMMVFGTVRKLLALLHIDGRSSSTNEKAIKMMKLHGAVWRQYHCFPSELKDFLRLNH

[0153] SEQ ID NO:2 Nucleotide sequence

[0154]

Claims

1. Silencing, inhibiting, or interfering with functional genes whose nucleotide sequences are as shown in SEQ ID NO.

2. MaPUB21 In at least one of the following applications (a1)-(a3): (a1) Improve the drought resistance of plants; (a2) Prepare products to improve the drought resistance of plants; (a3) Cultivate new plant germplasm with improved drought resistance; The plant in question is a banana.

2. Functional genes with nucleotide sequences as shown in SEQ ID NO.2 MaPUB21 The relevant biomaterials are used in at least one of the following (a1)-(a3): (a1) Improve the drought resistance of plants; (a2) Prepare products to improve the drought resistance of plants; (a3) Cultivate new plant germplasm with improved drought resistance; With the functional genes MaPUB21 The relevant biological materials are for silencing, inhibiting, or interfering with the functional genes. MaPUB21 Expression of biological materials; The gene used to silence, suppress, or interfere with the functional gene MaPUB21 The expressed biological material is at least one of (c1) to (c10): (c1) Used to silence, suppress, interfere with, or gene edit the functional gene. MaPUB21 Expressed nucleic acid molecules; (c2) An expression cassette containing the nucleic acid molecule described in (c1); (c3) A recombinant vector containing the nucleic acid molecule described in (c1) or a recombinant vector containing the expression cassette described in (c2); (c4) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3); (c5) A transgenic plant cell line containing the nucleic acid molecule described in (c1), or a transgenic plant cell line containing the expression cassette described in (c2), or a transgenic plant cell line containing the recombinant vector described in (c3); (c6) A transgenic plant tissue containing the nucleic acid molecule described in (c1), or a transgenic plant tissue containing the expression cassette described in (c2), or a transgenic plant tissue containing the recombinant vector described in (c3); (c7) A transgenic plant organ containing the nucleic acid molecule described in (c1), or a transgenic plant organ containing the expression cassette described in (c2), or a transgenic plant organ containing the recombinant vector described in (c3); (c8) A transgenic plant containing the nucleic acid molecule described in (c1), or a transgenic plant containing the expression cassette described in (c2), or a transgenic plant containing the recombinant vector described in (c3); (c9) Regenerative cells, tissue cultures or protoplasts derived therefrom of the transgenic plant described in (c8); (c10) Propagation material of the transgenic plant as described in (c8); The plant in question is a banana.

3. The application according to claim 1 or 2, characterized in that, With the aforementioned functional genes MaPUB21 Using genetic engineering methods, the functional genes are silenced, suppressed, or interfered with in target plants. MaPUB21 The expression or reduction of the functional genes MaPUB21 The activity or content of encoded proteins can be used to improve the drought resistance of plants, or to cultivate new plant germplasm with improved drought resistance for production applications.

Images