Pear PyYABBY gene and application

Abstract

The invention discloses a PyYABBY gene of a pear and application of the PyYABBY gene. The PyYABBY gene separated from Dangshan pear and capable of promoting pear leaf enlargement has a nucleotide sequence as shown in SEQ ID No.1, and an amino acid sequence coded by the PyYABBY gene is as shown in SEQ ID No.2 in a sequence table. In order to verify the function of the PyYABBY gene, a yabby mutant arabidopsis thaliana (lacking the YABBY gene) is adopted, and a supplementation experiment is carried out. The results show that the leaf of the PyYABBY back-complemented strain is obviously larger than that of the yabby mutant (without the YABBY gene) and that of the wild-type arabidopsis thaliana, and further confirms that the PyYABBY gene plays a positive regulation role in leaf size development and can promote leaf enlargement. A new gene resource is provided for molecular mechanism research of pear tree leaf development, and meanwhile, a potential application target is provided for pear tree variety improvement and molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. It describes the isolation and cloning of a PyYABBY gene related to pear leaf size from 'Dangshan Crisp Pear', as well as the development and application of molecular markers. Background Technology

[0002] Pear (Pyrus) is a perennial woody plant belonging to the Rosaceae family. It is the third most cultivated fruit tree species in my country, with at least 22 different species worldwide and over 5,000 recorded or preserved varieties (Potter et al., 2007; Li et al., 2022). 'Dangshan Crisp Pear' (P. bretschneideri Rehd.), also known as the Chinese White Pear (Asian Pear), has round fruit characterized by its crispness, juiciness, high sugar content, low acidity, delicate aroma, and sweet flavor. European pear (P. communis) is another important economic variety widely cultivated in Western countries. It possesses unique phenotypes and fruit quality characteristics distinct from Asian pears. Its fruit is typically pear-shaped, with soft, delicate flesh, few stone cells, and a rich aromatic flavor (Wu et al., 2018). However, it is noteworthy that while there is considerable research on pear fruit traits, systematic studies on the differences in leaf development and morphology between Eastern and Western pear species are scarce.

[0003] Leaves are crucial sites for nutrient production and photosynthesis, providing energy for fruit growth and development. Leaves are one of the main characteristic organs of pear plants, representing their genetic traits. Different pear varieties exhibit significant differences in fruit morphology, flavor, storability, and adaptability, which are closely related to their genetic background and leaf development characteristics. Leaves are essential for photosynthesis and the production of organic matter, playing a vital regulatory role in physiological activities such as photosynthesis, respiration, transpiration, and reproduction. Currently, research on various indicators of plant leaf growth processes, leaf morphology, and single-plant leaf area is widespread both domestically and internationally, and these indicators can effectively reflect plant growth levels (Chen et al., 2013; Guo Rui, 2022). The leaf margins of European pears are mostly blunt serrated or entire, while those of Oriental pears show greater differentiation (Teng Yuanwen, 2017). However, research on the molecular mechanisms of pear leaf development remains limited, especially regarding the genetic basis of differences in leaf development among different pear varieties.

[0004] In the early stages, SNP testing was conducted on 22 Oriental pear and 24 Western pear germplasm resources to identify the differentiation intervals of the two pear species, and the PyYABBY gene was found to be within the differentiation interval (Wu et al., 2018). The YABBY family is a class of transcription factors unique to plants, belonging to a subfamily of the zinc finger protein superfamily (Hussain et al., 2021), and plays an important role in plant growth, development, and stress response. This family contains two typical domains: the C-terminal YABBY domain (with a helical-loop-helical structure) and the N-terminal C2C2 type zinc finger domain (Baxvanis & Landsman 1995; Kanaya et al., 2002; Sawa et al., 1999). Currently, members of the YABBY family have been systematically identified and studied in various plants, including Arabidopsis thaliana (Bowman, 2000), rice (Yamaguchi et al., 2004), wheat (Zhao et al., 2006), tomato (Li et al., 2018), and grape (Zhang et al., 2019). The YABBY gene plays a crucial role in the formation of lateral organs during plant growth and development (Tanaka et al., 2017), such as the formation of front-back polarity (Sawa et al., 1999), abaxial cell differentiation (Bowman 2000), leaf expansion, and floral organ development (Golz & Hudson, 1999). In Arabidopsis thaliana, there are six YABBY members: CRABS CLAW (CRC), YABBY1 / FILAMENTOUS FLOWER (FIL), YAB-BY2, YABBY3, YABBY5, and INNERNO OUTER (INO). FL / YAB1, YAB2, YAB3, and YAB5 are specifically expressed in the abaxial region of lateral organs such as cotyledons, leaves, and flowers, participating in leaf development and floral organ formation (Stahleet et al., 2009); CRC is involved in the development of carpels and nectaries (Bowman & Smvth., 1999); and INO mainly plays a role in regulating ovule development (Villanueva et al., 1999). A comparative analysis of the leaf phenotypes of the Oriental pear variety 'Dangshan Su Li' and the European pear variety 'Zao Hong Komis' revealed a significant difference in leaf area, with 'Dangshan Su Li' having a significantly larger leaf area than 'Zao Hong Komis'. However, whether the PyYABBY gene mediates leaf type differentiation between pear varieties in the East and West by regulating leaf area development-related pathways has not yet been reported in relevant studies. Summary of the Invention

[0005] The purpose of this invention is to provide a pear leaf development-related gene, PyYABBY, which has the function of promoting leaf enlargement.

[0006] Another object of the present invention is to provide the application of this gene.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A PyYABBY gene that affects leaf size was isolated from 'Dangshan Crisp Pear'. Its nucleotide sequence is shown in SEQ ID No. 1, containing a 657 bp open reading frame and encoding 219 amino acids. The encoded amino acid sequence is shown in SEQ ID No. 2 of the sequence listing.

[0009] A recombinant expression vector containing the PyYABBY gene described in this invention.

[0010] The recombinant expression vector uses pCAMBIA1301 as the starting vector, and the insertion site of the PyYABBY gene is between Xba I and BamHI.

[0011] Genetically engineered bacteria containing the PyYABBY gene described in this invention.

[0012] The primer pair for cloning the cDNA sequence of the PyYABBY gene described in this invention is shown in SEQ ID No. 3 for the upstream primer PyYABBY-F1 and in SEQ ID No. 4 for the downstream primer PyYABBY-R1.

[0013] The application of the PyYABBY gene, recombinant expression vector, and genetically engineered bacteria described in this invention in promoting leaf enlargement.

[0014] Beneficial effects

[0015] Compared with the prior art, the present invention has the following advantages and effects:

[0016] 1. The discovery of the PyYABBY gene provides a new target for molecular breeding of pear trees. This gene can significantly increase leaf area by regulating leaf development, thereby reducing agricultural costs and achieving environmental friendliness.

[0017] 2. The PyYABBY gene was functionally verified in Arabidopsis thaliana using Agrobacterium-mediated genetic transformation. The results showed that the PyYABBY gene cloned in this invention has the function of promoting leaf enlargement, providing a more efficient approach for molecular breeding. Attached Figure Description

[0018] Figure 1 Statistical analysis of leaf phenotype (a) and leaf area size (b) for Oriental pear 'Dangshan Crisp Pear' and Western pear 'Early Red Comice';

[0019] Figure 2 Transcriptional expression level (a) and quantitative fluorescence expression level (b) of PyYABBY in Oriental pear 'Dangshan Crisp Pear' and Western pear 'Zaohong Comis'.

[0020] Figure 3 This is a schematic diagram of the carrier in Embodiment 2 of the present invention.

[0021] Figure 4 Functional analysis of Arabidopsis thaliana transformed with the PyYABBY gene in this invention.

[0022] Among them, (a) shows the leaf phenotype of wild-type Arabidopsis thaliana, yabby mutant Arabidopsis thaliana (YABBY gene missing), and PyYABBY-replenished Arabidopsis thaliana; (b) shows the expression level of YABBY gene in wild-type Arabidopsis thaliana, yabby mutant Arabidopsis thaliana (YABBY gene missing), and PyYABBY-replenished Arabidopsis thaliana; and (c) shows the leaf area statistics of wild-type Arabidopsis thaliana, yabby mutant Arabidopsis thaliana (YABBY gene missing), and PyYABBY-replenished Arabidopsis thaliana. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention.

[0024] Example 1: Expression pattern analysis of PyYABBY in pear leaves from both East and West

[0025] Based on a comparative analysis of genomic data from pear populations in the East and West (Wu et al., 2018), we located the PyYABBY gene within the identified speciation interval. To investigate the expression characteristics of this gene, we selected representative Eastern pear varieties 'Dangshan Su Li' and Western pear varieties 'Zao Hong Komis' as experimental materials. Fresh young leaf tissues from both pear varieties were collected at the Feicheng Experimental Base of Shandong Agricultural University, and gene expression profiles were obtained using high-throughput transcriptome sequencing (RNA-seq). The gene expression level was quantitatively analyzed using the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method. The results showed that the PyYABBY gene exhibited significantly high expression in the leaves of the Eastern pear 'Dangshan Su Li', while showing relatively low expression in the leaves of the Western pear 'Zao Hong Komis'. Figure 2a). This expression difference may be related to the speciation characteristics of pears in the East and West. Meanwhile, qRT-PCR experiments confirmed that the expression level of PyYABBY in the leaves of 'Dangshan Crisp Pear' was higher than that in 'Early Red Comice'. Figure 2 b).

[0026] Example 2: Isolation, cloning, and construction of the PyYABBY gene overexpression vector

[0027] 1 μg of RNA from 'Dangshan Crisp Pear' leaves was used for reverse transcription using a one-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Primers SEQ ID No. 3 and SEQ ID No. 4 were designed using SnapGene software according to general primer design principles. The 50 μL reaction mixture included 200 ng cDNA, 1× buffer (TransStart FastPfu Buffer), 10 mM dNTPs, 1 U Taq polymerase (TransStart FastPfu DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from TRANS), and 500 nM of the primers. The PCR reaction was performed on an Eppendorf amplification system according to the following program: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 1 minute, 35 thermal cycles; 72℃ extension for 5 minutes; and storage at 4℃. A single PCR band was generated. After recovery and purification, the PCR product was used to insert the PyYABBY sequence (with the stop codon removed) into the pCAMBIA1301 vector using homologous recombination technology. The vector was then transformed into *E. coli* DH5α using the heat shock method and administered in a 100 μg / mL solution. -1 K + The plasmid was cultured in LB solid medium, positive clones were screened, amplified, and sequenced. Sequencing results showed that the full-length PyYABBY gene is 657 bp, with its nucleotide sequence shown in SEQ ID No. 1, encoding a protein of 219 amino acid residues, with the sequence shown in SEQ ID No. 2. The correctly sequenced plasmid was transformed into Agrobacterium strain GV3101 using a freeze-thaw method, and then cultured in 50 μg / mL medium. -1 K + 100μg mL -1 Rifampin + The culture was carried out in LB medium, and the correctly identified Agrobacterium strain was expanded and propagated in 10 mL sterile centrifuge tubes and stored at -80℃ for later use.

[0028] Example 3: PyYABBY Stable Transformation of Arabidopsis thaliana and Leaf Phenotypic Observation

[0029] 1. Stable transformation of Arabidopsis thaliana. Agrobacterium containing the PyYABBY overexpression vector was used to infect Col-0 Arabidopsis thaliana using the flower-spreading method (Clough & Bent, 1998).

[0030] The specific method is as follows:

[0031] (1) Using a solution containing 50 mg / L K + and 100mg / LR + Agrobacterium was activated by streaking solid LB medium and incubated at 28°C for 36-48 hours.

[0032] (2) Use a sterile toothpick or pipette tip to pick up the monoclonal antibodies from the thread and place them in a 100mL Erlenmeyer flask. Add 30mL of solution containing 50mg / LK. + and 100mg / LR + The liquid LB medium was incubated at 28°C and 200 rpm for 12 hours in a shaker.

[0033] (3) Collect bacterial cells by centrifuging at 5000 rpm for 5 minutes in a 50 mL centrifuge tube;

[0034] (4) The bacterial cells were resuspended in an equal volume of conversion medium [1 / 2 MS; 5% sucrose (W / V); 10 μg / L 6-BA; pH adjusted to 5.7 with KOH; 0.025% surfactant (V / V)].

[0035] (5) For the first conversion, the horns of Arabidopsis should be removed;

[0036] (6) Soak Arabidopsis inflorescences in a transformation medium containing bacteria, use a vacuum filter pump to evacuate to 380 mmHg, and soak for 5 minutes; infect once a week, for a total of 3-4 times;

[0037] (7) Place it in a 22°C incubation room in the dark for 24 hours, and then place it in a 22°C long-day (16 hours of light / 8 hours of darkness) environment for incubation.

[0038] Two-week-old positive T1 generation Arabidopsis plants resistant to hygromycin were harvested. RNA was extracted from rosette leaves and the expression level of PyYABBY was detected by quantitative real-time PCR using primers SEQ ID NO.5 and SEQ ID NO.6. Three lines (OE-3, OE-6, and OE-8) showed high expression levels, and their homozygotes were used for subsequent experiments. Seeds harvested after replanting were sterilized and sown together with wild-type seeds on germination medium. Wild-type seeds were sown on MS medium (3% sucrose (w / v) and 0.75% agar (w / v)). Transgenic Arabidopsis seeds required germination medium supplemented with resistance: 50 mg / ml hygromycin and 250 mg / ml termethin. After germination, seedlings were transplanted into nutrient soil and cultured at 22°C under long-day conditions (16 hours light / 8 hours dark).

[0039] 2. Phenotypic observation of wild-type, yabby mutant (YABBY gene deletion), and complemented Arabidopsis thaliana (PyYABBY infection).

[0040] (1) Materials preparation: wild-type Arabidopsis thaliana (Col-0), the target gene yabby mutant (YABBY gene deletion), and the reinfection-infected PyYABBY line. Wild-type Arabidopsis thaliana and yabby mutant (AT2G45190, purchase number N2101886) were purchased from Arashare (https: / / www.arashare.cn / index / ).

[0041] (2) Instruments and equipment: Canon camera, ImageJ software, vernier calipers (for calibration).

[0042] Auxiliary tools: white background, sterile forceps, filter paper.

[0043] (3) The specific method is as follows:

[0044] ① Plant culture and sampling

[0045] (1) After disinfecting Arabidopsis seeds with 75% ethanol for 2 min, rinse them with sterile water 5 times for 1 min each time. Then disinfect them with 10% sodium hypochlorite for 5 min, rinse them with sterile water 5 times for 1 min each time, and then sow them in 1 / 2 MS solid medium. Vernalize them at 4℃ for 48 hours and then transfer them to a light incubator (22±1℃, 16h light / 8h darkness).

[0046] (2) When the plant has grown to 14-21 days (when the rosette leaves are fully unfolded), select the 3rd-5th true leaves with consistent growth and gently pick them with sterile tweezers to avoid mechanical damage.

[0047] ②Standardized image acquisition

[0048] (1) Lay the leaf flat on the white filter paper, making sure it is fully spread out without overlap.

[0049] (2) Take photos using a camera;

[0050] (3) Open ImageJ software, import the blade image, and convert it to an 8-bit grayscale image (“Image→Type→8-bit”). Set a uniform threshold (“Image→Adjust→Threshold”) to separate the blade from the background and manually correct edge errors. Select “Analyze→Measure” to obtain the blade area (unit: mm). 2 ), to record data.

[0051] ③ Data validation and statistics

[0052] At least 10 leaves were analyzed in each group. The mean ± standard deviation was calculated, and a t-test was used to analyze the significance of differences between groups (P < 0.05). The results are as follows: Figure 4 As shown.

[0053] (4) The leaf area of ​​the reinfected PyYABBY strain of Arabidopsis thaliana was significantly larger than that of the mutant and wild-type Arabidopsis thaliana.

[0054] Ten days after transplanting into the soil, the leaf area of ​​the replanted Arabidopsis thaliana strain was 0.5119 ± 0.0636 cm². 2 The length was significantly larger than that of the wild type (0.3287±0.0470cm). 2 (P<0.001) and yabby mutant (0.2462±0.0217cm). 2 (P<0.001) Figure 4 (a&c)). Quantitative fluorescence analysis showed that the gene expression level of YABBY in the reinfected PyYABBY Arabidopsis strain was significantly higher than that in the mutant and wild-type Arabidopsis (P<0.001). Figure 4 (b)). The above results indicate that the PyYABBY gene in pear plays a positive regulatory role in leaf size development and can promote leaf enlargement.

[0055] Main References

[0056] Bao,L.,Chen,K.,Zhang,D.,Cao,Y.,Yamamoto,T.,andTeng,Y.(2007).Geneticdiversity and similarity of pear(Pyrus L.)cultivars native toEastAsiarevealedby SSR(simple sequencerepeat)markers.Genetic Resources andCrop Evolution 54,959-971.

[0057] Baxevanis AD,Landsman D(1995).The HMG-1 box protein family:classification and functional relationships.Nucleic Acids Research,23(9):1604–1613

[0058] Bowman JL(2000).The YABBY gene family and abaxial cell fate.CurrentOpinion in Plant Biology,3(1):17–22

[0059] Clough SJ,Bent AF.(1998).Floral dip:a simplified methodforAgrobacterium-mediated transformation of Arabidopsis thaliana.PlantJournal for Cell&Molecular Biology 16(6):735.

[0060] Hussain Q,Asim M,Zhang R,et al(2021).Transcription fac-tors interactwith ABA through gene expression and sig-naling pathways to mitigate droughtand salinity stress.Biomolecules,11(8):1159

[0061] Kanaya E,Nakajima N,Okada K(2002).Non-sequence-spe-cific DNA bindingby the FILAMENTOUS FLOWER protein from Arabidopsis thaliana is reduced byEDTA.Journal of Biological Chemistry,277(14):11957–11964

[0062] Li H,SunMh,Qi Mf,et al(2018).Alteration of SlYABBY2b gene expressionimpairs tomato ovary locule number and endogenous gibberellin content.Journalof Zhejiang University-SCIENCE B(Biomedicine&Biotechnology),19(6):445–457

[0063] Li JM,Zhang MY,Li XL,et al(2022).Pear genetics:recent advances,newprospects,and a roadmap for the future.Horticulture Research,9.

[0064] Potter D,Eriksson T,Evans R C,et al(2007).Phylogeny andclassification of Rosaceae.Plant Systematics and Evolution,2007,266(1-2):5-43.

[0065] Pu,F.and Wang,Y.(1963).Pomology of China.Pears,vol 3.Shanghai:Shanghai Science and Technology Press.

[0066] Sawa S,Watanabe K,Goto K,et al(1999).FILAMENTOUS FLOWER,a meristemand organ identity gene of Arabidopsis,encodes a protein with a zinc fingerand HMG-related domains.Genes Development,13(9):1079–1088.

[0067] Stahle MI,Kuehlich J,Staron L,et al(2009).YABBYs and thetranscriptional corepressors LEUNIG and LEUNIG_HOMOLOG maintain leaf polarityand meristem activity inArabidopsis.The Plant Cell,21(10):3105–3118

[0068] Tanaka W,Toriba T,Hirano H-Y(2017).Three TOB1-related YABBY genes arerequired to maintain proper function of the spikelet and branch meristems inrice.New Phytologist,215:825–839

[0069] Yamaguchi T,Nagasawa N,Kawasaki S,et al(2004).The YABBY gene DROOPINGLEAF regulates carpel specification and midrib development in Oryzasativa.The Plant Cell,16(2):500–509

[0070] Yu,D.(1979)Taxonomy of the fruit tree in China.Beijing:AgriculturePress.

[0071] Villanueva JM,Broadhvest J,Hauser BA,et al(1999).INNER NO OUTERregulates abaxial-adaxial patterning in Arabidopsis ovules.Genes Development,13(23):3160–3169

[0072] Wu,J.,Wang,Y.,Xu,J.,et al.(2018).Diversification and independentdomestication of Asian and European pears.Genome Biology 19,77.

[0073] Zhang S,Wang L,Sun epidermises of leaves and arrests the development of shoot apical meristen in Arabidopsi. Plant Science, 170(2):364–371

[0074] Chen Yigao, Hao Mingzhuo, Chu Dongsheng, et al. (2013). Comparison of leaf growth traits and physiological indicators of different privet varieties. Forestry Science and Technology Development, 27(4):48-51. Guo Rui, Cao Yufen, Zhang Ying, Yang Xiang, Nie Hanyu (2022). Comparative analysis of flowering and leaf growth characteristics of different pear resources. Journal of Fruit Resources, 3(2):05-12. Teng Yuanwen (2017). Research progress on phylogeny of Pyrus species and origin of Oriental pear varieties. Journal of Fruit Science, 34(3):370-378.

[0075] SEQ ID No. 1:

[0076] ATGTCATCCTCTTCAACCTTGTCCCTCGACCAGCTCCCTCCTTCCGAGCAGCTCTGTTATGTCCATTGCAACATTTGCGACACTGTCCTTGCGGTGAGTGTTCCTTGCACAAGTTTGTTCAAGACTGTGACGGTGCGATGTGGGCACTGCACCAATCTGCTGCCAGTGAACATGCGTGGGCAGCTTCTGCCTTCTTCAAATCAGTTTCATCATCTTGGTCACTCTTTTTTTTCTTCCCCTAACAATTCCCTTAATCTTCTGGAAGAGATTCCAAATGTTCCGACCCCAAATTTTCTGGTTAACCAAACAAATGTGAATGACTTTGCTGCAACACCAAGAGGTGGAGCTGATGAACATTTTCCAACGCCCGCAGTCATTAACAGACCCCCGGAGAAGAGACAGAGAGTCCCCTCTGCATACAACCGTTTCATCAAGGACGAGATCCAACGCATCAAGTCTGTGAATCCTGATATATCACATAGAGAAGCCTTCAGTGCAGCTGCCAAGAATTGGGCTCACTTTCCACACATTCACTTTGGTCTTATGCCTGACCAGACTGTGAAGAAGACAACCATTCGCCAGCAGGAAGGAGAGGATGTTCTGATGAAAGATGGGTTTTTCTCTTCAGCTAATAATGTGCGTGTCTCCCCTTACTAA

[0077] SEQ ID No.2:

[0078] MSSSSTLSLDQLPPSEQLCYVHCNICDTVLAVSVPCTSLFKTVTVRCGHCTNLLPVNMRGQLLPSSNQFHHLGHSFFSSPNNSLNLLEEIPNVPTPNFLVNQTNVNDFAATPRGGADEHFPTPAVINRPPEKRQRVPSAYNRFIKDEIQRIKSVNPDISHREAFSAAAKNWAHFPHIHFGLMPDQTVKKTTIRQQEGEDVLMKDGFFSSANNVRVSPY*

[0079] SEQ ID No.3:

[0080] gcccttgctcaccatggatccATGTCATCCTCTTCAACCTTGTCC

[0081] SEQ ID No.4:

[0082] gagaacacgggggactctagaGTAAGGGGAGACACGCACATTAT

[0083] SEQ ID No.5:

[0084] TGAACATTTTCCAACGCCCG

[0085] SEQ ID No.6:

[0086] AGTGAGCCCAATTCTTGGCA

Claims

1. A PyYABBY gene isolated from 'Dangshan Crisp Pear' that promotes pear leaf enlargement, the nucleotide sequence of which is shown in SEQ ID No. 1, and the amino acid sequence encoded by which is shown in the sequence listing SEQ ID No.

2.

2. A recombinant expression vector containing the PyYABBY gene as described in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that, The recombinant expression vector uses pCAMBIA1301 as the starting vector, and the insertion site of the PyYABBY gene is between Xba I and BamHI.

4. Genetically engineered bacteria containing the PyYABBY gene as described in claim 1.

5. The primer pair for cloning the PyYABBY gene cDNA sequence as described in claim 1, characterized in that, The sequence of the upstream primer PyYABBY-F1 is shown in SEQ ID No. 3, and the sequence of the downstream primer PyYABBY-R1 is shown in SEQ ID No.

4.

6. The application of the PyYABBY gene as described in claim 1 in promoting leaf enlargement.

7. The application of the recombinant expression vector according to claim 2 or 3 in promoting leaf enlargement.

8. The application of the genetically engineered bacteria according to claim 4 in promoting leaf enlargement.

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