Application of the ZjUVI4 gene from jujube tree in rice grain shape improvement

Abstract

This invention discloses the application of the jujube ZjUVI4 gene in rice grain shape improvement, belonging to the field of plant genetic engineering technology. The invention cloned the coding region sequence of ZjUVI4 from the jujube variety 'Dongzao' using PCR. The full-length sequence is 774 bp, encoding 257 amino acids. The nucleotide sequence of the coding region of the ZjUVI4 gene is shown in SEQ ID NO:1. Then, an overexpression vector was constructed, and transgenic experiments were conducted using the rice variety 'Zhonghua 11' as the transformation recipient. The ZjUVI4 gene was heterologously overexpressed in rice, and its biological function in the positive regulation of rice grain shape was investigated. This invention reveals the biological function of the jujube ZjUVI4 gene in positively regulating seed size. By transforming the ZjUVI4 gene into rice, the transgenic rice lines showed a significant increase in grain length and thousand-grain weight, while grain width remained largely unchanged, providing a new gene resource for rice grain shape improvement and high-yield, high-quality breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and in particular to the application of the jujube ZjUVI4 gene in rice grain shape improvement. Background Technology

[0002] Rice is one of the world's most important food crops, and increasing rice yield is key to ensuring food security. Rice yield components include the number of effective panicles, the number of grains per panicle, and grain weight, with grain weight primarily determined by grain length, width, and thickness. Therefore, identifying and utilizing superior genes that regulate seed size (grain type), elucidating their molecular mechanisms, and conducting genetic improvement through modern breeding techniques are of great significance for improving rice yield and quality.

[0003] Fruit or seed size is closely related to cell division and cell expansion. Studies have shown that multiple pathways, including nuclear replication, plant hormone signaling, and the CLV-WUS signaling pathway, are involved in regulating plant organ size. Nuclear replication, in particular, refers to the multiple replication of DNA during mitosis without nuclear membrane division, leading to increased cell ploidy and thus affecting cell volume and organ size. In Arabidopsis and tomato, it has been shown to be closely related to leaf, flower, and fruit development. UVI4 (ULTRAVIOLET-B-INSENSITIVE4) is a negative regulator of the late cell cycle promoting complex / cyclic body (APC / C), regulating cell cycle progression by inhibiting APC / C activity. In Arabidopsis, tomato, and other plants, it has been shown to participate in leaf, flower, and fruit development.

[0004] Jujube (Ziziphus jujuba Mill.) is a perennial fruit tree belonging to the genus Ziziphus in the family Rhamnaceae. It is of significant economic, ecological, and medicinal value in my country. Originating in China, jujube has a cultivation history of over 7,000 years. Cultivated jujubes evolved from wild sour jujubes through long-term artificial selection and domestication. Fruit size is a very typical domestication trait—wild varieties have small fruits, while cultivated varieties have large fruits. Single fruit weight is not only an important domestication trait but also a crucial factor determining the yield per jujube tree, and a vital indicator for new variety breeding and jujube fruit quality grading. Therefore, identifying superior genes that regulate yield traits such as single fruit weight in jujube and elucidating their molecular regulatory mechanisms is of great significance for improving both yield and quality.

[0005] In the study of jujube fruit size, previous research has shown that ZjDA3 is a gene that regulates single fruit weight; transcription factors ZjWRKY23 and ZjWRKY40 positively regulate jujube fruit size by inhibiting the expression of ZjCKX5; and heterologous transgenic experiments have shown that ZjZOG may positively regulate jujube fruit size. However, the genetic transformation of the woody plant jujube is difficult, its growth cycle is long, and its genome has high heterozygosity, resulting in slow progress in verifying its gene functions. Currently, the function of the UVI4 homolog in jujube has not been reported, especially the effect of its heterologous overexpression on seed size in the monocotyledonous plant rice, which is a research gap. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the ZjUVI4 gene from jujube trees in rice grain shape improvement, which can effectively increase rice grain length and thousand-grain weight, providing a new gene resource for high-yield rice breeding.

[0007] This invention cloned the coding sequence of the ZjUVI4 gene from the jujube variety 'Dongzao'. The full-length sequence is 774 bp, encoding 257 amino acids. By constructing an overexpression vector and transforming it into rice 'Zhonghua 11', it was found that heterologous overexpression of ZjUVI4 significantly increased rice seed length and thousand-grain weight, while having no significant effect on grain width, thus achieving targeted improvement of rice grain shape.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of the jujube ZjUVI4 gene in rice grain shape improvement, wherein the coding region nucleotide sequence of the ZjUVI4 gene is shown in SEQ ID NO:1.

[0009] Secondly, the present invention provides a method for improving rice grain shape, comprising the following steps: Step 1: Using the cDNA of the jujube variety 'Winter Jujube' as a template, the coding region sequence of the ZjUVI4 gene is amplified using the primers shown in SEQ ID NO:3 and SEQ ID NO:4. The nucleotide sequence of the coding region of the ZjUVI4 gene is shown in SEQ ID NO:1. Step 2: The coding region sequence of the ZjUVI4 gene obtained in Step 1 is cloned into the plant expression vector pCAMBIA1300 to construct the recombinant overexpression vector pCAMBIA1300-ZjUVI4; Step 3: Transform the recombinant overexpression vector obtained in Step 2 into Agrobacterium, and then use the transformed Agrobacterium to transform rice to obtain positive rice plants that heterologously overexpress the ZjUVI4 gene.

[0010] Furthermore, the plant expression vector mentioned in step two is the pCAMBIA1300 vector, which contains a ZjUVI4 gene expression cassette driven by a strong promoter.

[0011] Furthermore, the rice variety mentioned in step three is 'Zhonghua 11'.

[0012] 1. This invention reveals the biological function of the ZjUVI4 gene in the woody plant jujube. Heterologous overexpression of the ZjUVI4 gene from jujube in the monocotyledonous plant rice yielded unexpected technical results. Experimental results showed that, compared with the wild type, the transgenic rice lines exhibited increased grain length of 13.08% and 15.49%, respectively, and increased thousand-grain weight of 7.06% and 9.92%, respectively, while grain width remained largely unchanged. This discovery provides a novel and efficient genetic resource for rice grain shape improvement and high-yield breeding.

[0013] 2. This invention further verifies the functional correlation between ZjUVI4 and its interacting protein ZjDA3. ZjDA3 has been reported as a negative regulator of jujube fruit size, while this invention confirms that ZjUVI4 is a positive regulator. The two have opposite functions, providing new genetic resources and improvement strategies for yield breeding of jujube and other fruit trees. Attached Figure Description

[0014] Figure 1 The relative expression levels of ZjUVI4 in transgenic lines (OE-1 and OE-2) are shown by qRT-PCR, where WT is wild-type and OE-1 and OE-2 are two transgenic lines.

[0015] Figure 2 Photographs comparing the grain length of rice seeds overexpressing ZjUVI4 with the control (scale bar = 1 cm), where A is before hulling and B is after hulling.

[0016] Figure 3 Photographs comparing the grain width of rice seeds overexpressing ZjUVI4 with the control (scale bar = 1 cm), where A is before hulling and B is after hulling.

[0017] Figure 4 The statistical results show the grain length, grain width, and thousand-grain weight of transgenic rice overexpressing ZjUVI4 compared to the control. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments. It should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0019] Given the difficulty and long cycle of genetic transformation in the woody plant jujube, making it difficult to verify gene function within the plant itself, this invention selects rice as a model crop as a heterologous expression receptor to explore the biological function of the ZjUVI4 gene in regulating seed size.

[0020] Example 1

[0021] Construction of ZjUVI4 gene overexpression vector.

[0022] Using the jujube variety 'Dongzao' as material, RNA was extracted and cDNA was obtained through reverse transcription. Specific primers were designed based on the coding region sequence of the ZjUVI4 gene, and 20 bp vector terminal homologous arms were added to the 5' ends of both the forward and reverse primers. The primer sequences are as follows: The upstream primer sequence is: ZjUVI4-F (GGTGTTACTTCTGTTGCAACATGCCGGAACCAAGAGATAGGCTTTC) (SEQ ID NO: 3) The downstream primer sequence is: ZjUVI4-R (TGAAGACAGAGCTAGTTACATCAACGCATTGACATCAAAGTGCGGAC) (SEQ ID NO: 4) Using 'winter jujube' cDNA as a template, the coding region sequence of the ZjUVI4 gene with a length of 774 bp was obtained by PCR amplification. Its nucleotide sequence is shown in SEQ ID NO: 1, encoding 257 amino acids. The amino acid sequence is shown in SEQ ID NO: 2.

[0023] Nucleotide sequence: ATGCCGGAACCAAGAGATAGGCTTTCAAGGCCAGTAGATGTTGCCGCTGTGTTCGTTCGGTGGCGGTCCCGCGGTGTAGTCAACGAGTCGGACGCTGCGTCGGACTTGTTTGGATCTCCGATGCGGCAGCAGACCACTGCGACGACGCCAATTGCTCGTAGAGTGACGGGTTTTGGGGCTACACGTGGTGGTGGGTTCGGAAGGGGCATTTTTGGAACCCCAAGAACTGTAAATCGGCGTGGCCGGAATCGTCCTGGATCTTCATCAGCGGCTAGAGAGAATACACCGGCGGGGACTGCCCGGCGGGGAAGGGGTCGTGTTACGAACAGTGTGTTACCTTCTTGGTACCCAAGGACCCCTCTCCGTGATATCACTGCGATAGTGACGGCTATTGAGAGGAGAAGAGCTCGCTTGAGAGGAGACGAAGGCCTACAAGTAGGGACTCCTATATACCAGGACCCAGGCGTTCTTGATCCTCCGGTGTCTGTTTCTCCTGCTCCACTCGAGCAGGATATCCCTATAGCATCTCCAAGTACAGCTGTCAGAAAGAAGGGTTGCCCACCTTCTGTTGGTAAAGTGCCTAAAATTCTGCTTGGAATCACTAATCAGACTACGGGAGAATCGGAGTGTCTCACACCCCAGAAGAAACTTTTGAACAACATTGACACAGTTGAGAAAGCAGTCAAGGAGGAATTGCAGAAACTGAAGAGGACCCCTAGTGCTAAGAAGGCAGAGAGAGAAAAAAGAGTCCGCACTTTGATGTCAATGCGTTGA Amino acid sequence: MPEPRDRLSRPVDVAAVFVRWRSRGVVNESDAASDLFGSPMRQQTTATTPIARRVTGFGATRGGGFGRGIFGTPRTVNRRGRNRPGSSSAARENTPAGTARRGRGRVTNSVLPSWYPRTPLRDIT AIVTAIERRRARLRGDEGLQVGTPIYQDPGVLDPPVSVSPAPLEQDIPIASPSTAVRKKGCPPSVGKVPKILLGITNQTTGESECLTPQKKLLNNIDTVEKAVKEELQKLKRTPSAKKAEREKRVRTLMSMR Simultaneously, the empty vector pCAMBIA1300 was double-digested with restriction endonucleases, separated by electrophoresis, and purified by gel extraction to obtain a linearized vector. A homologous recombination reaction system was prepared at a molar ratio of the target fragment to the linearized vector of 2–3:1 and incubated at 50°C for 15–30 min. The recombinant product was transformed into competent *E. coli* cells, subjected to ice bath, heat shock at 42°C, and then recovered. The cells were then plated on plates containing the appropriate antibiotics and incubated overnight at 37°C. Single colonies were picked for preliminary screening using colony-forming PCR. Positive clones were extracted, and plasmids were extracted and verified by restriction enzyme digestion. Finally, sequencing confirmed the sequence was correct. The correctly sequenced recombinant plasmid was named pCAMBIA1300-ZjUVI4. This recombinant vector contains a ZjUVI4 gene expression cassette driven by a strong promoter and is used for the efficient expression of the ZjUVI4 protein in transgenic plants.

[0024] Example 2

[0025] Genetic transformation of rice and acquisition of transgenic plants.

[0026] In this embodiment, the rice variety 'Zhonghua 11' was used as the recipient material, and genetic transformation was carried out using Agrobacterium-mediated transformation.

[0027] The culture medium formula used is as follows: N6 basal medium (1 L): KNO3 2830 mg, (NH4)2SO4 463 mg, KH2PO4 400 mg, MgSO4·7H2O 185 mg, CaCl2·2H2O 166 mg, MnSO4·4H2O 10.0 mg, H3BO3 1.6 mg, ZnSO4·7H2O 2.0 mg, Na2MoO4·2H2O 0.25 mg, CuSO4·5H2O 0.025 mg, CoCl2·6H2O 0.025 mg, FeSO4·7H2O 27.8 mg, Na2EDTA·2H2O 37.3 mg, Thiamine·HCl (B1) 1.0 mg, Pyridoxine·HCl (B6) 0.5 mg, Nicotinic acid 0.5 mg, Glycine 2.0 mg, 30 g of sucrose, 2.5–3 g of Phytagel, adjust pH to 5.8–5.9 with 1N KOH.

[0028] AB solid culture medium (1 L): 5×A stock solution (200 mL, containing (NH4)2SO4 2 g, Na2HPO4 6 g, KH2PO4 3 g, NaCl 3 g, sterilized) sterilized at 121℃ for 15 min; 1×B solid (800 mL, containing 16 g agar) sterilized at 121℃ for 15 min; After sterilization, mix under aseptic conditions and add 1 mL of filtered and sterilized 0.1 M CaCl2, 1 mL of 1.0 M MgCl2, 1 mL of 0.003 M FeCl3, and 10 mL of 20% glucose.

[0029] N6 / HC2 selection medium: N6 basal medium plus: Hygromycin B 2 mg / L and Cefotaxime 250-500 mg / L.

[0030] R / HC differentiation medium (1 L, pH 5.8): KNO3 2830 mg, (NH4)2SO4 463 mg, KH2PO4 400 mg, MgSO4·7H2O 185 mg, CaCl2·2H2O 166 mg, MnSO4·H2O 10.0 mg, H3BO3 3.0 mg, ZnSO4·7H2O 2.0 mg, Na2MoO4·2H2O 0.25 mg, CuSO4·5H2O 0.025 mg, CoCl2·6H2O 0.025 mg, FeSO4·7H2O 27.8 mg, Na2EDTA·2H2O 37.3 mg, Thiamine·HCl (B1) 10.0 mg, Pyridoxine·HCl (B6) 1.0 mg, Nicotinic acid 1.0 mg mg, Myoinositol 100mg, Sucrose 30 g, L-Proline 500 mg, Casein Hydrolysate (CH, hydrolyzed casein) 300 mg, 6-BA (6-benzyladenine) 3.0 mg / L, NAA (naphthaleneacetic acid) 0.5 mg / L, Hygromycin B 2mg / L, Cefotaxime 250-500 mg / L, Phytagel 3.0-4.0 g, adjust pH to 5.8 with 1NKOH.

[0031] HF / H rooting selection medium (1 L): KNO3 950 mg, NH4NO3 825 mg, KH2PO4 85 mg, MgSO4·7H2O 185 mg, CaCl2·2H2O 220 mg, MnSO4·H2O 8.45 mg, H3BO3 3.1 mg, ZnSO4·7H2O 5.2 mg, KI 0.415 mg, Na2MoO4·2H2O 0.125 mg, CuSO4·5H2O 0.0125 mg, CoCl2·6H2O 0.0125 mg, FeSO4·7H2O 13.9 mg, Na2EDTA·2H2O 18.65 mg, Thiamine·HCl (B1) 1.0 mg, Pyridoxine·HCl (B6) 0.5 mg, Nicotinic acid 0.5 mg, Glycine 2.0 mg, Myo-inositol 100 mg, Sucrose 10 g, Hygromycin B 30 mg / L, Cefotaxime 250 mg / L, Phytagel 2.5 g or Agar: 7-8 g, adjust pH to 5.8.

[0032] The specific steps are as follows: (1) Callus induction: Select plump, mature 'Zhonghua 11' seeds, remove the glumes, and place them in 50 mL centrifuge tubes (no more than 200 seeds at a time to prevent incomplete seed sterilization); add 10 mL of 70% ethanol, invert and mix well, and let stand for 1 min; discard the ethanol, add 30 mL of sterile deionized water and wash twice; discard the washing solution, add 30 mL of sodium hypochlorite with an effective chlorine concentration of 2%, and place on a shaker to shake at 80 rpm for 20 min for surface sterilization. In a clean bench, discard the disinfectant solution, wash three times with sterile water, then add 30 mL of sterile water and shake for 10 min; discard the washing solution, and wash once more with 30 mL of sterile water. Filter the seeds dry, spread them flat on N6 medium with the embryo side facing up, and incubate in the dark at 28℃ to induce callus formation.

[0033] (2) Callus subculture: After the seeds sown on N6 medium have grown for 10-13 days, the young shoots are removed, and the callus tissue is transferred to a new N6 medium and cultured for another 10-13 days. The differentiated small callus tissue is then transferred to a new N6 medium and cultured for another 10-13 days.

[0034] (3) Agrobacterium infection and co-culture: A small amount of Agrobacterium strain containing the recombinant plasmid pCAMBIA1300-ZjUVI4 was picked up with a sterile toothpick, dissolved in 100 μL of sterile water, mixed well, and spread on AB solid medium. It was then incubated in the dark at 25°C for 2 days. An appropriate amount of activated Agrobacterium was dissolved in a solution containing 40 μg / mL of... -1 Acetyleugenol in AAM staining solution. After immersing the pre-cultured callus tissue from step (2) in the staining solution for 2-5 min, blot dry on sterile filter paper, then spread it evenly on 2N6 / AS medium and incubate in the dark at 25°C for 3 days.

[0035] (4) Screening and differentiation culture: The above callus tissue was transferred to the selective medium N6 / HC2 and screened twice in a light incubator for 10 days each time. Then the resistant callus tissue was transferred to the differentiation medium R / HC and differentiated for three rounds of culture for 10 days each time until seedlings were differentiated.

[0036] (5) Rooting and transplanting: After the differentiated seedlings are transferred to HF / H medium and grown for 15 days, they are cultured in nutrient solution for 15 days; transplanted in greenhouse and cultivated and managed using conventional methods.

[0037] Using the above methods, multiple independent transgenic rice lines were obtained.

[0038] Example 3

[0039] qRT-PCR detection of ZjUVI4 expression level in transgenic rice.

[0040] 3.1 RNA extraction and cDNA synthesis Total RNA was extracted from wild-type 'Zhonghua 11' rice and various transgenic rice lines obtained in Example 2 using the TaKaRa MiniBEST RNA Extraction Kit (Cat. # 9769). The specific steps are as follows: (1) Quickly transfer fresh or cryopreserved plant tissue samples to a mortar pre-cooled with liquid nitrogen, and grind the tissue with a pestle, continuously adding liquid nitrogen until it is ground into powder (without obvious visible particles; incomplete grinding will affect the yield and quality of RNA). Add the ground sample (50-100 mg) to a 1.5 ml sterile centrifuge tube containing 450 μl Buffer RL (please confirm that 50×DTT Solution has been added before use), and repeatedly pipette until there is no obvious precipitate in the lysis buffer.

[0041] (2) Centrifuge the lysate at 12,000 rpm and 4 °C for 5 minutes.

[0042] (3) Carefully aspirate the supernatant into a new 1.5 ml sterile centrifuge tube.

[0043] (4) Add 1 / 2 volume of anhydrous ethanol to the supernatant or mixture from the sample lysis step (precipitation may occur at this time), and mix the solution evenly using a pipette.

[0044] (5) Immediately transfer the entire mixture (including the precipitate) into the RNA Spin Column (containing 2 ml CollectionTube). (If the volume of the mixture is greater than 600 μl, add it in batches, and each batch should not exceed 600 μl.)

[0045] (6) Centrifuge at 12,000 rpm for 1 minute and discard the filtrate. Transfer the RNA Spin Column back into the 2 ml Collection Tube.

[0046] (7) Add 500 μl of Buffer RWA to the RNA Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0047] (8) Add 600 μl of Buffer RWB to the RNA Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0048] (9) DNase I digestion (optional).

[0049] (10) Repeat step (8).

[0050] (11) Replace the RNA Spin Column onto a 2 ml Collection Tube and centrifuge at 12,000 rpm for 2 minutes.

[0051] (12) Place the RNA Spin Column on a 1.5 ml RNase Free Collection Tube, add 50-200 μl of RNase Free dH2O or 0.1% DEPC treated water to the center of the RNA Spin Column membrane, and let it stand at room temperature for 5 minutes.

[0052] (13) Elute RNA by centrifuging at 12,000 rpm for 2 minutes.

[0053] Using TaKaRa's PrimeScrip reverse transcription kit TMII. 1st Strand cDNA Synthesis Kit (Cat.# 6210A) for reverse transcription synthesis of first-strand cDNA. Specific steps are as follows: Take 1 μg of total RNA, add 1.0 μL of Oligo(dT)15 primer and 1.0 μL of 10 mM dNTP, and add RNase-free H2O to a final volume of 10 μL. Incubate at 65°C for 5 min, then quickly cool on ice. Add 4.0 μL of 5× reverse transcription buffer, 1 μL of AMV reverse transcriptase, and 0.5 μL of RNasin ribonuclease inhibitor to the above system, and add RNase-free H2O to a final volume of 20 μL. Incubate the reaction system at 42°C for 1 hour, then heat at 70°C for 15 min, and finally incubate at 0-5°C for 5 min. Store the synthesized cDNA at -20°C for later use.

[0054] 3.2 Real-time quantitative PCR detection Using cDNA obtained through reverse transcription as a template and the rice OsActin gene as an internal control, qRT-PCR was performed on a Bio-Rad CFX96 instrument using SYBR Green I dye. The reaction program was: 94℃ pre-denaturation for 300 s; then 94℃ denaturation for 5 s, 60℃ annealing for 15 s, and 72℃ extension for 10 s, for a total of 40 cycles. Each sample was tested in triplicate.

[0055] The primer sequences used for detection are as follows: ZjUVI4(RT)-F:CATCAGCGGCTAGAGAGAATAC (SEQ ID NO: 5) ZjUVI4(RT)-R:CTTGGGTACCAAGAAGGTAACA (SEQ ID NO: 6) OsActin-F: TGACGGAGCGTGGTTACTCATTCA (SEQ ID NO: 7) OsActin-R:TCTTGGCAGTCTCCATTTCCTGGT (SEQ ID NO: 8) Based on the qRT-PCR results, two transgenic lines with significantly higher ZjUVI4 expression levels than the wild type were screened from the multiple transgenic lines obtained in Example 2, and named OE-1 and OE-2, respectively. These two lines were used for subsequent phenotypic identification. The results are as follows: Figure 1 As shown, the expression level of ZjUVI4 in the OE-1 and OE-2 lines was significantly higher than that in the wild type.

[0056] Example 4

[0057] Phenotypic analysis of transgenic rice seeds.

[0058] Wild-type 'Zhonghua 11' rice and the OE-1 and OE-2 transgenic rice obtained in Example 2 were grown in a greenhouse and managed under the same conditions. After the seeds matured, they were harvested and dried. Ten seeds were randomly selected from each material, and the seed length and width were measured using calipers. Simultaneously, seeds from each material were randomly selected, and their thousand-seed weight was measured, repeated three times.

[0059] Measurement results as follows Figures 2 to 4 Table 1 shows the results. The results indicate that, in terms of grain length, the OE-1 and OE-2 transgenic lines increased by 13.08% and 15.49% compared to the wild type, respectively; in terms of grain width, there was no significant difference between the two transgenic lines and the wild type; and in terms of thousand-grain weight, the OE-1 and OE-2 transgenic lines increased by 7.06% and 9.92% compared to the wild type, respectively. These results demonstrate that heterologous overexpression of ZjUVI4 can significantly increase the grain length and thousand-grain weight of rice seeds.

[0060] Table 1. Statistical results of wild-type and transgenic rice seed phenotypes It should be noted that the above embodiments are only used to illustrate the present invention, but the present invention is not limited to the above embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The application of the jujube ZjUVI4 gene in rice grain shape improvement, characterized by: The coding region nucleotide sequence of the ZjUVI4 gene is shown in SEQ ID NO:

1.

2. A method for improving rice grain shape, characterized in that: Includes the following steps: Step 1: Using the cDNA of the jujube variety 'Winter Jujube' as a template, the coding region sequence of the ZjUVI4 gene is amplified using the primers shown in SEQ ID NO:3 and SEQ ID NO:

4. The nucleotide sequence of the coding region of the ZjUVI4 gene is shown in SEQ ID NO:

1. Step 2: The coding region sequence of the ZjUVI4 gene obtained in Step 1 is cloned into the plant expression vector pCAMBIA1300 to construct the recombinant overexpression vector pCAMBIA1300-ZjUVI4; Step 3: Transform the recombinant overexpression vector obtained in Step 2 into Agrobacterium, and then use the transformed Agrobacterium to transform rice to obtain positive rice plants that heterologously overexpress the ZjUVI4 gene.

3. The method according to claim 2, characterized in that: The plant expression vector mentioned in step two is the pCAMBIA1300 vector, which contains a ZjUVI4 gene expression cassette driven by a strong promoter.

4. The method according to claim 2, characterized in that: The rice variety mentioned in step three is 'Zhonghua 11'.

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