Application of gene cclbd36 in regulating pepper leaf size

Abstract

The application discloses application of a gene CcLBD36 in regulating size of pepper leaves, and a nucleotide sequence of the gene CcLBD36 is shown as SEQ ID No. 2. The gene can regulate cell proliferation and morphological formation of the pepper leaves, realizes accurate regulation of leaf length, width and leaf area, and solves the bottleneck problem of the existing pepper leaf trait improvement technology.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics technology, specifically, it relates to the application of gene CcLBD36 in regulating the size of chili pepper leaves. Background Technology

[0002] As the core organ for photosynthesis and transpiration in plants, the size and characteristics of leaves directly determine photosynthetic efficiency and nutrient distribution patterns, thus significantly impacting crop yield. Horticultural crops accumulate 95% of their dry matter through leaf photosynthetic products, and leaf length, width, and area, as key agronomic traits, are not only important components of crop plant architecture but also closely related to optimized planting density, enhanced stress resistance, and final yield. As a widely cultivated horticultural crop globally, pepper leaf length and width are core elements in shaping plant architecture, directly affecting field ventilation and light penetration, planting density, and yield potential per unit area. Therefore, in-depth exploration of key genes regulating pepper leaf size and analysis of their molecular mechanisms have significant theoretical and applied value for improving pepper plant architecture and targeted breeding of high-quality, high-yield varieties.

[0003] Plant leaf development involves coordinated regulation at three polarity levels: basal-apical, mid-lateral, and proximal-distal. Multiple genes work synergistically through complex networks to influence cell proliferation, differentiation, and morphogenesis, ultimately determining leaf morphology and size. Leaf length and width are typical quantitative traits, their genetic regulation being finely controlled by multiple hormone signaling pathways, including auxin, gibberellin, and methyl jasmonate. Previous studies have shown that R2R3-MYB transcription factors CsRAXs can regulate cucumber leaf size and affect fruit setting through the auxin pathway, while PtoGRF9 regulates leaf development by activating PtoHB21 during cell proliferation and attenuating PtoLD expression during the transition phase. However, the conservation and specificity of these regulatory mechanisms in pepper remain unclear. The genus *Capsicum* comprises 43 species and tens of thousands of cultivated varieties. Long-term artificial selection has led to significant differences in leaf size and morphology among different germplasm resources. However, the physiological, cellular, and molecular regulatory basis behind these phenotypic differences remains unclear, greatly limiting the targeted improvement of pepper leaf traits.

[0004] The LBD (lateral organboundaries domain) transcription factor family (also known as the ASL (asymmetric leaves 2-like) gene family) is a class of transcriptional regulatory factors unique to plants. Their N-terminus contains a conserved LOB domain, and they can be divided into Class I and Class II based on sequence characteristics. Class I members contain a zinc finger-like structure (CX2CX6CX3C), a GAS region, and a leucine-like zipper motif, participating in DNA binding, protein dimerization, and biological function regulation; Class II members retain only the conserved zinc finger motif. This family of genes plays a multifunctional regulatory role in plant growth and development. Multiple members have been identified in species such as Arabidopsis thaliana, maize, rice, and tomato. In Arabidopsis, AtLBD28, AtLBD36 (AtASL1), and AtLBD12 are involved in leaf development and polarity formation; AtLBD16 and AtLBD18 mediate auxin signaling and lateral root formation; in tomato, SILBD1 and SILBD2 influence plant architecture through a transcriptional regulatory network; and in maize, ZmMS1 / ZmLBD30 controls pollen exine development. These findings suggest that the LBD family of genes exhibits high conservation and functional diversity in plant organ development. Although several transcription factors in the LBD family have been confirmed to be involved in organ morphogenesis, the LBD gene regulating leaf size in pepper has not yet been cloned and identified. The lack of functional genes and supporting technologies that can be directly applied to molecular breeding limits the precise improvement of pepper leaf traits.

[0005] In summary, identifying the key genes in chili peppers that regulate leaf size and establishing efficient genetic engineering improvement technologies are urgent needs for solving the aforementioned problems. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a gene CcLBD36 that regulates the leaf size of chili peppers and Arabidopsis thaliana, along with its encoded protein and applications.

[0007] Specifically, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a chili pepper CcLBD36 protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0009] Secondly, the present invention provides a chili pepper CcLBD36 gene, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0010] Thirdly, the present invention provides an expression cassette, recombinant vector, recombinant microorganism or recombinant cell containing the CcLBD36 gene.

[0011] Fourthly, the present invention provides the use of the CcLBD36 protein or the CcLBD36 gene or an expression cassette containing the CcLBD36 gene, a recombinant vector, a recombinant microorganism or a recombinant cell in any of the following:

[0012] (1) Regulating plant leaf size;

[0013] (2) Improve plant plant shape;

[0014] (3) Cultivating plant varieties.

[0015] In one or more embodiments, the regulation of plant leaf size includes regulating the length, width, and leaf area of ​​plant leaves; the improvement of plant plant shape is to optimize plant density adaptability and ventilation and light transmission efficiency by regulating plant leaf size; and the cultivation of plant varieties is to cultivate plant varieties with improved leaf size.

[0016] In this invention, regulating leaf size includes both increasing and decreasing the size of chili pepper leaves. Specifically, silencing or knocking out the CcLBD36 gene in chili peppers results in smaller leaves; overexpressing the CcLBD36 gene in chili peppers results in larger leaves.

[0017] It is well known to those skilled in the art that the size of a plant's leaves can alter or influence its plant form, thereby affecting density suitability and ventilation and light transmission efficiency. In this invention, if the chili pepper leaves are too large, the planting density needs to be reduced, potentially impacting the final yield; conversely, if the leaves are too small, while planting density can be increased, the small leaves can impair chlorophyll absorption, thus affecting the final yield. Therefore, the appropriate chili pepper leaf size is crucial for the final yield. This invention utilizes the CcLBD36 gene to regulate leaf size, thereby improving the plant form of chili peppers and providing a new approach to increasing chili pepper yield.

[0018] The CcLBD36 gene described in this invention is also crucial for breeding plant varieties. By regulating the size of pepper leaves through the CcLBD36 gene, pepper parents with optimal leaf size can be obtained, and after years of breeding, pepper varieties with optimal yield can be obtained.

[0019] Fifthly, the present invention provides a method for cultivating transgenic plants with larger leaves, the method comprising the step of increasing the content and / or activity of the CcLBD36 protein in a recipient plant to obtain a transgenic plant; wherein the leaf length and / or width and / or leaf area of ​​the transgenic plant are greater than those of the recipient plant.

[0020] In one or more embodiments, the increase in the content and / or activity of CcLBD36 protein in the recipient plant is achieved by overexpressing the CcLBD36 gene in the recipient plant;

[0021] Preferably, the method for overexpressing the CcLBD36 gene in the recipient plant is as follows: construct an overexpression vector of the CcLBD36 gene and transfer it into the recipient plant via Agrobacterium-mediated transformation.

[0022] In a sixth aspect, the present invention provides a method for cultivating transgenic plants with smaller leaves, the method comprising the step of reducing the content and / or activity of the CcLBD36 protein in a recipient plant to obtain a transgenic plant; wherein the leaf length and / or width and / or leaf area of ​​the transgenic plant is lower than that of the recipient plant.

[0023] In one or more embodiments, the reduction of the content and / or activity of CcLBD36 protein in the recipient plant is achieved by knocking out or silencing the CcLBD36 gene in the recipient plant.

[0024] Preferably, the method for knocking out or silencing the CcLBD36 gene in the recipient plant is as follows: construct a knockout vector or silencing vector for the CcLBD36 gene and transfer it into the recipient plant via Agrobacterium-mediated transformation.

[0025] In one or more embodiments, the plant used in the above-described application or method of the present invention is a chili pepper or Arabidopsis thaliana. Preferably, the chili pepper is the Hainan Yellow Lantern variety.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The gene described in this invention achieves precise control over leaf length, width, and area by regulating cell proliferation and morphogenesis in chili leaves, thus solving the bottleneck problem of existing chili leaf trait improvement technologies. Attached Figure Description

[0028] Figure 1 It is the cDNA sequence and amino acid sequence of the CcLBD36 gene.

[0029] Figure 2 This is a graph showing the phenotypic expression changes of the CcLBD36 gene in peppers at different developmental stages in Example 2; where a is a comparison of plants with different leaf ages, b is a comparison of expression levels at different leaf ages, and c is a comparison of phenotypic changes at different leaf ages. (Units in Figure c: leaf length, leaf width, and leaf circumference are in cm, leaf area is in cm²) 2 ).

[0030] Figure 3 This is a verification of the gel electrophoresis results after the construction of the silencing vector in Example 3.

[0031] Figure 4These are images showing the growth status of chili pepper plants after different treatments in Example 3; where a is a positive control albino plant image; b is a phenotypic image of the silent plant: where WT represents wild type, PTRV2 represents empty plant, PTRV2-LBD36-1 represents silent plant line 1, PTRV2-LBD36-2 represents silent plant line 2, and PTRV2-LBD36 represents silent plant line 3.

[0032] Figure 5 This is a comparison of the long phenotypes of chili leaves after different treatments in Example 3.

[0033] Figure 6 This is a comparison diagram of the width phenotype of chili leaves after different treatments in Example 3.

[0034] Figure 7 This is a comparison diagram of the phenotypic circumference of chili leaves after different treatments in Example 3.

[0035] Figure 8 This is a comparison chart of the phenotypic distribution of chili leaf area after different treatments in Example 3.

[0036] Figure 9 This is a comparison diagram of the phenotypic distribution of chili pepper plants after different treatments in Example 3.

[0037] Figure 10 This is a comparison diagram of chili plant height phenotypes after different treatments in Example 3.

[0038] Figure 11 This is a comparison chart of chlorophyll a content in peppers after different treatments in Example 3.

[0039] Figure 12 This is a comparison chart of chlorophyll b content in peppers after different treatments in Example 3.

[0040] Figure 13 This is a comparison chart of the total chlorophyll content of peppers after different treatments in Example 3.

[0041] Figure 14 This is a comparison chart of the expression levels of the CcLBD36 gene in chili peppers after different treatments in Example 4.

[0042] Figure 15These are anatomical diagrams of chili pepper leaves after different treatments in Example 4; where a (WT) is the anatomical diagram of wild-type chili pepper leaves, b (PTRV2) is the anatomical diagram of leaves of the empty control chili pepper, c (PTRV2-LBD36-1) is the anatomical diagram of leaves of silent plant line 1, d (PTRV2-LBD36-2) is the anatomical diagram of leaves of silent plant line 2, and e (PTRV2-LBD36-3) is the anatomical diagram of leaves of silent plant line 3 (the scale bar in the figure is not clear due to software export issues; the attached figure is a 50μm scale bar, used to characterize the actual size of the leaf slices; the scale bars of a, b, c, d, and e are all 50μm).

[0043] Figure 16 These are comparative diagrams of cell phenotype analysis of chili leaves after different treatments in Example 4; where a is the average area of ​​cells in the upper epidermal tissue of wild type, b is the average area of ​​cells in the lower epidermal tissue, c is the average area of ​​cells in the palisade tissue, d is the number of cells in the upper epidermal tissue, e is the number of cells in the lower epidermal tissue, and f is the number of cells in the palisade tissue.

[0044] Figure 17 This is a comparison diagram of the thickness of chili leaves after different treatments in Example 4.

[0045] Figure 18 This is a gel electrophoresis image of the heterologous overexpression recombinant plasmid bacterial culture in Example 5.

[0046] Figure 19 This is a phenotypic diagram of Arabidopsis thaliana treated with CcLBD36 overexpression in Example 5, where, from left to right, they are WT, OE1, OE2 and OE3.

[0047] Figure 20 This is a graph showing the relative expression levels of Arabidopsis thaliana under CcLBD36 overexpression treatment in Example 5. Detailed Implementation

[0048] In the following examples, silent plants of the CcLBD36 gene in pepper were constructed. The results showed that the leaf length, width and surface area of ​​the silent plants were significantly smaller, indicating that CcLBD36 affects leaf growth by regulating cell enlargement rather than proliferation.

[0049] Because constructing a genetic transformation system for chili peppers is quite difficult, the following examples also involve conducting a "heterologous overexpression experiment" on chili peppers in Arabidopsis thaliana. The results showed that the leaf length, width, and surface area of ​​the overexpressing plants were significantly increased.

[0050] The data in the following examples show that the CcLBD36 gene can be used to regulate the leaf size of peppers, including leaf length, width and surface area: silencing or knocking out the CcLBD36 gene can reduce the leaf size of peppers; overexpression of the CcLBD36 gene can increase the leaf size of peppers.

[0051] Example

[0052] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0053] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0054] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0055] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0056] Example 1: Cloning and Sequence Identification of Gene CcLBD36

[0057] Total RNA was extracted from leaves of Hainan yellow lantern pepper using the Novizan RNA Extraction Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: RC411), and cDNA was obtained by reverse transcription of RNA using the Novizan Reverse Transcription Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: R333). Full-length PCR primers were designed based on the CcLBD36 sequence (primers corresponding to CcLBD36 in Table 1), and PCR amplification was performed (reaction system is shown in Table 2, reaction procedure is shown in Table 3).

[0058] The amplified fragment was inserted into the Novizan pCE3 Blunt Vector and sequenced.

[0059] The results showed that the complete CcLBD36 ORF could be cloned from Hainan yellow lantern pepper. This gene is 900 bp in length and encodes 299 amino acids. Figure 1 ).

[0060] Table 1 PCR Primers

[0061]

[0062] Table 2 PCR reaction system

[0063]

[0064] Table 3 PCR reaction procedure

[0065]

[0066] Example 2: Analysis of CcLBD36 expression patterns at different developmental stages of pepper

[0067] The expression characteristics of CcLBD36 in leaves at different developmental stages were analyzed using q-PCR. Leaves of the "BD-HDL" variety of Hainan yellow lantern pepper, which exhibited significant phenotypic changes at different developmental stages, were selected as materials. The average leaf length, width, perimeter, and area were measured. Based on the phenotypic results, RNA was extracted from pepper leaves at 12, 19, 26, and 33 days of age and reverse transcribed to obtain cDNA. qPCR primers were designed based on the LBD36 sequence (primers corresponding to CcqpcrLBD36 in Table 1), and internal control primers (primers corresponding to CcAtin in Table 1) were also designed. Real-time quantitative PCR analysis was performed using the SYBR Green I fluorescent dye method. Each sample had at least three biological replicates and at least three technical replicates. Data analysis was performed using Excel 2016 according to the 2^-ΔΔCT method, and finally, graphs were plotted using GraphPad Prism 9.

[0068] The results are as follows Figure 2 As shown, with increasing leaf age, pepper leaves gradually become longer, wider, and larger, with significant differences; with increasing leaf age, the expression level of CcLBD36 gradually increases, reaching its highest value on day 26, and then gradually decreases. Figure 2 c).

[0069] Example 3: Construction of Silent Expression Vectors and Genetic Transformation Methods

[0070] 3.1 Construction of the silencing expression vector PTRV2-CcLBD36

[0071] After genome-wide association analysis, LBD36, a candidate gene regulating pepper leaf length, was identified. Using cDNA from the Hainan Yellow Lantern “BD-HDL” variety as a template, the interference fragment was amplified by PCR (as shown in SEQ ID No. 3). The amplified fragment was then ligated into the silencing expression vector PTRV2 (purchased from Hunan Fenghui Biotechnology Co., Ltd., catalog number: BR709) via homologous recombination. The vector was then transformed into E. coli by heat shock to obtain a positive plasmid.

[0072] Specifically, based on the CcLBD36 sequence, interference fragment PCR primers (PTRV2-LBD36 and PTRV2-PDS in Table 4) were designed using the BamHI restriction site on the PTRV2 vector, and PCR amplification was performed (the reaction procedure was the same as in Table 3). Simultaneously, the PTRV2 vector was digested at the BamHI restriction site according to the system in Table 5. Following the reaction system in Table 6, the amplified fragment was inserted into the PTRV2 vector, transformed into *E. coli* using the heat shock method, and sequenced after shaking. For those with successful sequencing alignment, plasmids were extracted and transformed into *Agrobacterium*.

[0073] Table 4. Primers for genes related to pepper silencing experiment

[0074]

[0075] Table 5. Linearization reaction of the carrier

[0076]

[0077] Table 6 Recombination Reaction System

[0078]

[0079] 3.2 Genetic transformation

[0080] When the chili peppers reached the stage of having two true leaves, plants of similar growth were divided into four experimental groups, with ten plants in each group. One group contained the PTRV2 empty vector; one group was injected with PTRV2-CcLBD36; one group was injected with PTRV2-PDS (PDS served as a positive control for the albino gene; if the plant turned albino, it indicated that the vector could silence the gene); and one group served as a blank control. Except for the blank control group, all other groups required mixing with PTRV1 (purchased from Hunan Fenghui Biotechnology Co., Ltd., product number: BR100) before injection. The chili peppers were thoroughly watered the day before injection, using the following method:

[0081] ①On the first day, take one tube each of PTRV2 empty vector Agrobacterium, PTRV2-CcLBD36 Agrobacterium, and PTRV1 Agrobacterium from the ultra-low temperature freezer, and add them to a 50 mL centrifuge tube containing 10 mL of yeast extract peptone broth medium (containing 25 μg / mL kanamycin + rifampin + gentamicin). Incubate at 28℃ and 200 rpm for 16-20 hours until the OD600 value is between 1.0 and 1.2.

[0082] ② On the second day, take 1 mL of the cultured bacterial solution and add it to 25 mL of Agrobacterium tumefaciens induction medium (IM) at a ratio of 1:25. Incubate at 28℃ and 200 rpm for 16-18 hours until the OD600 value is around 0.8-1.0.

[0083] ③ On the third day, centrifuge the cultured bacterial solution at 3500 rpm for 10 minutes at 4°C, discard the culture medium, then resuspend the bacterial cells with the same volume of methanesulfonic acid (MES), centrifuge again at 4°C for 10 minutes, and resuspend the bacterial cells with 1 / 2 volume of methanesulfonic acid (MES). The final OD600 value is about 0.8-1.0.

[0084] ④ Mix PTRV1 and PTRV2 in a 1:1 ratio (unloaded or linked), add 200 mM acetylsuccinone (AS) to a final concentration of 200 μM, and place in the dark at around 24°C for 4 hours.

[0085] ⑤ Use a 2mL syringe to draw an appropriate amount of the inoculum (the specific formula is shown in Table 7) and inject it into the chili pepper leaves;

[0086] ⑥ Treat in the dark at 16℃ for 24-48 hours, then place in a greenhouse for continued regular cultivation;

[0087] ⑦ After 40 days, leaves at 40 days of age were selected for chlorophyll content determination and qPCR detection of related genes (internal reference gene primers were the primers corresponding to CcAtin in Table 4, and expression level detection primers were the primers corresponding to vigsCcqpcrLBD36 in Table 4). Paraffin sections were then prepared for cytological analysis to observe the differences between the results and the control. Leaf cells were observed at the same magnification, photographed simultaneously, and then representative cell images were selected within the field of view. A region was framed using the same size field of view, and the average area and number of cells within that field of view were counted.

[0088] Results analysis:

[0089] like Figure 3 As shown in the gel electrophoresis image, the silencing vector was successfully constructed.

[0090] like Figure 4 As shown, the leaves of the positive control (PTRV2-PDS) exhibited an albino phenotype. Figure 4 a) confirms the effectiveness of the gene silencing system constructed in this embodiment; the leaf size of the three silenced plants (PTRV2-LBD36-1, PTRV2-LBD36-2 and PTRV2-LBD36-3) is significantly different from that of the control plants. Figure 4 b).

[0091] Combination Figures 4-13 Phenotypic analysis showed that, compared with the wild type (WT), the leaves of the CcLBD36 silent line were significantly shorter ( Figure 4 b and Figure 5 ), narrowing ( Figure 4 b and Figure 6 The leaf perimeter and leaf area were significantly reduced. Figure 4 b and Figure 7 , 8 The plant width decreased significantly ( Figure 4 b and Figure 9 Plant height showed no significant change. Figure 10 Compared with the wild type (WT), the chlorophyll content of the CcLBD36 silent lines was significantly reduced. The chlorophyll a content of the silent plants PTRV2-LBD36-1, PTRV2-LBD36-2, and PTRV2-LBD36-3 was reduced by 64.67%, 63.15%, and 60.86%, respectively, compared with the wild type (WT). Figure 11 The chlorophyll b content decreased by 25.72%, 18.79%, and 13.60%, respectively. Figure 12 The total chlorophyll content decreased by 37.46%, 32.11%, and 27.82%, respectively. Figure 13 ).

[0092] Table 7. Formulation of dye solution for the silence test

[0093]

[0094] Example 4: Expression analysis and cytological analysis of silent plants

[0095] Based on the phenotypic results, RNA was extracted from pepper leaves of the same age and reverse transcribed to obtain cDNA. qPCR primers were designed based on the LBD36 sequence (Table 1). Real-time quantitative PCR analysis was performed using the SYBR Green I fluorescent dye method. Each sample had at least three biological replicates and at least three technical replicates. Data analysis was performed using Excel 2016 according to the 2^-ΔΔCT method, and graphs were plotted using GraphPad Prism 9. Further qRT-PCR was used to detect the leaves of the gene-silenced lines, and the results are as follows: Figure 14As shown, the expression level of CcLBD36 in the silent plants PTRV2-LBD36-1, PTRV2-LBD36-2 and PTRV2-LBD36-3 decreased significantly by 62.3%, 73.1% and 90.4%, respectively.

[0096] To further determine the cytological reasons for the difference in leaf size between the silent lines and the control group, paraffin sections were examined on leaves of wild-type and silent individual plants PTRV2-LBD36-1, PTRV2-LBD36-2, and PTRV2-LBD36-3 at 40 days of leaf age. The results are as follows: Figure 15 As shown, silencing CcLBD36 significantly inhibited the growth of cells in the palisade tissue, upper epidermis, and lower epidermis.

[0097] Figure 16 and Figure 17 The cell phenotype analysis of pepper leaves in each group showed that, compared with the wild type, silencing of the CcLBD36 gene significantly reduced the epidermal cell area of ​​the leaves. Specifically, the average cell area per unit area of ​​epidermal tissue in the three silenced plants PTRV2-LBD36-1, PTRV2-LBD36-2, and PTRV2-LBD36-3 decreased by 17.4%, 16.7%, and 13.6%, respectively. Figure 16 a); CcLBD36 gene silencing resulted in a significant reduction in the lower epidermal cell area of ​​leaves. Compared with the wild type, the average cell area per unit area of ​​the lower epidermal tissue in the three silenced plants PTRV2-LBD36-1, PTRV2-LBD36-2, and PTRV2-LBD36-3 decreased by 15.8%, 32.2%, and 19.0%, respectively. Figure 16 b); Compared with the wild type, the mean cell area of ​​palisade tissue per unit area in the three silent individuals PTRV2-LBD36-1, PTRV2-LBD36-2, and PTRV2-LBD36-3 decreased by 26.9%, 32.4%, and 34.2%, respectively. Figure 16 c); Compared with the wild type, the leaf thickness per unit area of ​​the three silent individuals PTRV2-LBD36-1, PTRV2-LBD36-2, and PTRV2-LBD36-3 was significantly reduced, decreasing by 2.5%, 5.2%, and 6.2%, respectively. Figure 17 Furthermore, compared to the wild type, there was no significant difference in the average cell number per unit area in the upper epidermis, lower epidermis, and palisade tissue. Figure 16 (d, e, f) indicates that CcLBD36 affects leaf growth by regulating cell swelling rather than proliferation.

[0098] Example 5: Construction of Heterologous Overexpression Vectors and Genetic Transformation Methods

[0099] Based on the previous cloning of CcLBD36 in "BD-HDL" pepper, a plant heterologous expression vector pBI121-CcLBD36 was constructed and transformed into Arabidopsis thaliana.

[0100] 5.1 Plant materials

[0101] Colombian-type Arabidopsis thaliana (wild type) was purchased from Danling County Xinghuo Biotechnology Business Department, product number: col-0 (300ul). After vernalization at 4℃ for 2 days, the Arabidopsis seeds were evenly sown on a substrate (nutrient soil: vermiculite: perlite = 2:1:1). Once the cotyledons were fully expanded, the Arabidopsis seeds were transplanted into 10x10cm seedling trays. The cultivation conditions were 16 hours of light / 8 hours of darkness, temperature 24℃ / 18℃, and humidity 85%. Transformation was performed approximately 4 weeks after the Arabidopsis thaliana lateral branches developed numerous unopened flower buds.

[0102] 5.2 Experimental Methods

[0103] Using plasmids extracted from *E. coli* after the gene-linked topological cloning vector was cultured, primers with homologous arms of pBI121 were designed using CEdesign from the Novizan website. PCR was performed, and the plasmids were purified, ligated into a linearized pBI121 vector, and transformed into *E. coli* using the heat shock method. Sequencing was performed after culture. For plasmids with successfully aligned sequencing results, plasmids were extracted from the culture, transformed into *Agrobacterium*, and then used to infect *Arabidopsis thaliana*. The specific steps are as follows:

[0104] (1) Construction of the carrier

[0105] Based on the CcLBD36 sequence, primers containing the homologous arm of pBI121 (yybdLBD36smaI in Table 8) were designed using the SmaI restriction site on the pBI121 vector, and PCR amplification was performed (reaction procedure shown in Table 12). Simultaneously, the pBI121 vector was digested with enzymes at the SmaI restriction site according to the system in Table 9. Following the reaction system in Table 10, the amplified fragment was inserted into the pBI121 vector, transformed into *E. coli* using the heat shock method, and sequenced after shaking. For those with successful sequencing alignment, plasmids were extracted and transformed into *Agrobacterium*.

[0106] Table 8. Primers related to heterologous overexpression

[0107]

[0108] Table 9. Linearization reaction of the carrier

[0109]

[0110] Table 10 Recombination Reaction System

[0111]

[0112] (2) PCR verification of bacterial culture

[0113] The bacterial culture was amplified according to the PCR reaction system in Table 11 and the PCR reaction procedure in Table 12. The transformation of the recombinant plasmid was detected by gel electrophoresis. Then, the bacterial culture with clear and bright electrophoretic bands was selected for sequencing.

[0114] Table 11 PCR reaction system

[0115]

[0116] Table 12 PCR reaction procedure

[0117]

[0118] (3) Extraction of recombinant plasmids

[0119] Take 200 μl of the successfully sequenced bacterial culture and add it to 10 mL of LB broth (50 mL centrifuge tube) containing kanamycin resistance. Incubate at 37°C and 200 rpm for 16 hours. Extract plasmids using a kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: DC201-01).

[0120] (4) Freeze-thaw transformation of Agrobacterium and screening of positive clones

[0121] ① Remove the competent Agrobacterium cells placed at -80℃. When the bacterial solution is partially thawed, add 1 μL of recombinant plasmid to every 100 μL of Agrobacterium and gently tap to mix.

[0122] ② Let stand on ice for 5 minutes, then in liquid nitrogen for 5 minutes, then in a 37°C water bath for 5 minutes, and finally in an ice-water bath for 5 minutes;

[0123] ③ Add 700 μl of yeast extract peptone broth medium (Yep liquid medium, antibiotic-free), incubate at 28°C and 200 rpm for 3 hours;

[0124] ④ Take 50 μl of bacterial culture and spread it evenly on Yep plate (containing 25 μg / mL kanamycin + rifampin + gentamicin), and incubate at 28℃ for 40 hours;

[0125] ⑤ Pick a single colony, add 50 ddH2O, take 4 μl as a template, and perform bacterial PCR verification. If the verification is successful, add the remaining bacterial culture to 1 mL of yeast extract peptone broth medium (containing 25 μg / mL kanamycin + rifampin + gentamicin), incubate overnight, then add sterile glycerol (final glycerol concentration is 20%), and store at -80℃.

[0126] (5) Infection of Arabidopsis thaliana

[0127] Arabidopsis thaliana was infected with the infiltration solution prepared according to Table 13.

[0128] Table 13 Infection solution formulation

[0129]

[0130] ① On the morning of the day before or the day of infection, cut off the flowers and pods of Arabidopsis thaliana that have already turned white, water thoroughly, and then thaw the pBI121-LBD36 Agrobacterium tumefaciens in an ultra-low temperature freezer. Take 200 μL and add it to a 50 mL sterile centrifuge tube containing 25 mL of LYep liquid medium (shake 4 tubes for each gene). Incubate at 28 °C with shaking for about 16 hours. At this time, the OD600 value is about 1.0.

[0131] ② After centrifugation at 4000 rpm, discard the supernatant, resuspend in the invading staining solution, and adjust the OD600 value to around 0.8;

[0132] ③ Add surfactant Silwet-77 (final concentration 0.02%);

[0133] ④ Pour the infection solution into a 200mL petri dish, immerse the Arabidopsis thaliana below the surface of the solution for 30 seconds, and at the same time use a pipette to spot the flower buds that are not immersed.

[0134] ⑤ After 24 hours of dark treatment, it was transferred to a greenhouse for further cultivation, with light at 23℃ for 16 hours and darkness at 20℃ for 8 hours;

[0135] ⑥ Infect again after one week, for a total of three inoculations. Finally, collect the mature seeds and dry them in silica gel for storage.

[0136] (6) Disinfection of transformed Arabidopsis seeds

[0137] ① Place Arabidopsis seeds in a 5mL centrifuge tube, add an appropriate amount of sterile water to wash, remove excess water, and leave 1mL of solution;

[0138] ② Add 2.4 mL of anhydrous ethanol (final ethanol concentration of about 70%) to the centrifuge tube, shake and wash for 1 min, and then remove the liquid;

[0139] ③ Add sterile water, wash once, and leave 1 mL of solution;

[0140] ④ Add 1.6 mL of 8% sodium hypochlorite solution (the final concentration of sodium hypochlorite is approximately 5%), shake and wash for 2 minutes, then remove the liquid;

[0141] ⑤ Wash five times with sterile water, 4 minutes each time.

[0142] (7) Screening of Arabidopsis thaliana plates

[0143] ①Preparation of MS medium resistance plates (T0 generation seeds: 40 μg / mL kanamycin; T1 generation seeds: 50 μg / mL kanamycin);

[0144] ② Spread the disinfected seeds evenly on the flat plate;

[0145] ③ Vernalize at 4℃ for three days, then place in an incubator and incubate at 23℃ for 16 hours and 20℃ for 8 hours;

[0146] ④ After about 20 days of cultivation, the positive seedlings that are growing normally can be selected;

[0147] ⑤ Transplant the positive seedlings into the substrate and grow them to maturity, then collect the seeds;

[0148] ⑥ T1 and T2 generation seedlings were selected sequentially, and DNA verification and quantitative fluorescence analysis were performed.

[0149] 5.3 Experimental Results

[0150] like Figure 18 The gel electrophoresis results shown indicate that the overexpression vector was successfully constructed.

[0151] like Figure 19 The phenotypic analysis shown indicates that, compared with the wild type (WT), the Arabidopsis lines overexpressing CcLBD36 (randomly selected overexpression lines, named OE1, OE2 and OE3, respectively) had significantly longer and wider leaves, significantly larger leaf perimeter and leaf area, and significantly larger plant width.

[0152] like Figure 20 The CcLBD36 expression analysis shown indicates that, compared with the wild type (WT), the expression level of CcLBD36 in the CcLBD36 overexpression lines (OE1, OE2 and OE3) was significantly increased.

[0153] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The use of the chili pepper CcLBD36 protein or the chili pepper CcLBD36 gene, or an expression cassette, recombinant vector, recombinant microorganism, or recombinant cell containing the chili pepper CcLBD36 gene, in any of the following: (1) Regulating plant leaf size; (2) Improve plant plant shape; (3) Cultivating plant varieties; The amino acid sequence of the chili pepper CcLBD36 protein is shown in SEQ ID No. 1; the nucleotide sequence of the chili pepper CcLBD36 gene is shown in SEQ ID No.

2. The plants mentioned in (1)-(3) are pepper or Arabidopsis thaliana; The method of regulating plant leaf size in (1) is as follows: by silencing the CcLBD36 gene, the length of pepper leaves is shortened, the width is narrowed, and the leaf area is reduced; by overexpressing the CcLBD36 gene, the length, width, and leaf area of ​​Arabidopsis leaves are increased. The method of improving plant architecture in (2) is as follows: by silencing the CcLBD36 gene, the plant width of pepper is reduced; by overexpressing the CcLBD36 gene, the plant width of Arabidopsis is increased. The method of cultivating plant varieties in (3) is as follows: by silencing the CcLBD36 gene, a chili pepper variety with smaller leaves and smaller plant width is cultivated; by overexpressing the CcLBD36 gene, an Arabidopsis variety with larger leaves and larger plant width is cultivated.

2. A method for cultivating transgenic plants with larger leaves, characterized in that, The method includes the step of increasing the content of CcLBD36 protein in the recipient plant to obtain a transgenic plant; the leaf length and / or width and / or leaf area of ​​the transgenic plant is higher than that of the recipient plant; the plant is Arabidopsis thaliana; the amino acid sequence of the CcLBD36 protein is shown in SEQ ID No.

1.

3. The method as described in claim 2, characterized in that, The increase in the content of CcLBD36 protein in the recipient plant is achieved by overexpressing the CcLBD36 gene in the recipient plant; the nucleotide sequence of the CcLBD36 gene is shown in SEQ ID No. 2; The method for overexpressing the CcLBD36 gene in recipient plants is as follows: construct an overexpression vector for the CcLBD36 gene and transfer it into recipient plants via Agrobacterium-mediated transformation.

4. A method for cultivating transgenic plants with smaller leaves, characterized in that, The method includes the step of reducing the content of CcLBD36 protein in the recipient plant to obtain a transgenic plant; the leaf length and / or width and / or leaf area of ​​the transgenic plant is lower than that of the recipient plant; the plant is a pepper; the amino acid sequence of the CcLBD36 protein is shown in SEQ ID No.

1.

5. The method as described in claim 4, characterized in that, The reduction of CcLBD36 protein content in the recipient plant is achieved by knocking out or silencing the CcLBD36 gene in the recipient plant; the nucleotide sequence of the CcLBD36 gene is shown in SEQ ID No. 2; The method for knocking out or silencing the CcLBD36 gene in recipient plants is as follows: construct a knockout vector or silencing vector for the CcLBD36 gene, and transfer it into the recipient plant via Agrobacterium-mediated transformation.

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