Genes related to leaf wrinkling in *Brassica oleracea* and their applications

Abstract

This invention belongs to the field of plant genetic engineering technology, and proposes a gene related to leaf wrinkling in *Brucea javanica*, which provides... BcNGA1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1, and whose conserved domain nucleotide sequence is shown in SEQ ID NO.2, is described. This invention also proposes a vector containing the aforementioned *Brucea javanica* leaf wrinkling-related gene and its application. This invention relates to... BcNGA1 Gene functional identification and analysis revealed its function in regulating plant leaf morphology. Overexpression and silencing recombinant vectors were prepared and applied to the breeding of new plant leaf morphology varieties, successfully screening and cultivating new varieties. BcNGA1 Overexpression in Arabidopsis thaliana and silent Chinese cabbage plants resulted in changes in the leaf morphology of both plants. This invention not only deepens the theoretical understanding of the mechanism of plant leaf morphogenesis, but also provides a theoretical basis for the early identification of leaf wrinkling in Chinese cabbage in production practice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a gene related to plant leaf wrinkling and its application. Background Technology

[0002] Black cabbage ( Brassica campestris L. ssp. Chinensis var. rosularis *Brassica oleracea* (also known as Chinese cabbage) is a variety of this subspecies, widely cultivated in the Yangtze-Huaihe River basin and an important leafy vegetable in my country during autumn and winter. Its distinctive feature is the presence of wrinkled, bubbly leaves, which differentiates it from other non-heading Chinese cabbage subspecies. This wrinkled, bubbly trait is closely related to the quality and stress tolerance of *Brassica oleracea*. Currently, the molecular mechanism of *Brassica oleracea* leaf wrinkling development is not fully understood. Identifying key genes involved in *Brassica oleracea* leaf wrinkling development and elucidating its molecular mechanism are crucial for further application of biotechnology to improve this trait. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the first objective of this invention is to provide a gene related to leaf wrinkling in *Brassica oleracea*, which will help to rapidly create *Brassica oleracea* breeding materials with different wrinkled and bubbly types using biotechnology, and also provide new insights into the mechanisms of morphological changes in plant leaves.

[0004] The second objective of this invention is to propose a vector containing the aforementioned gene related to leaf wrinkling in *Brassica oleracea*.

[0005] A third objective of this invention is to propose the application of the gene in the breeding of diverse new varieties of cruciferous plants with stable bubbly traits.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A gene related to leaf wrinkling in *Brassica oleracea*, for BcNGA1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1; and whose conserved domain nucleotide sequence is shown in SEQ ID NO.2.

[0007] The conserved domain nucleotide sequence is from black spinach BcNGA1 The nucleic acid sequence of the gene was compared with the NGA1 gene sequence in species such as Arabidopsis thaliana, Brassica napus, and Brassica rapa, and the regions with high homology were selected as its conserved structural domains.

[0008] A vector containing the aforementioned *Brucea javanica* leaf wrinkling-related gene, wherein the vector is a vector containing the aforementioned gene. BcNGA1 overexpression recombinant vectors of the gene and containing the above BcNGA1 Gene silencing recombination vector.

[0009] Wherein, the overexpression recombinant vector is a vector that expresses recombinant DNA. BcNGA1Gene insertion into the pCambia1305-35S-nFLAG-cMYC overexpression vector Bam HI and Xba The silencing recombinant vector is obtained by I restriction site; BcNGA1 Insertion of conserved gene domains into PCVA silencing vectors Xba I and Kpn Obtained from the I restriction site.

[0010] Furthermore, the method for constructing the overexpression recombinant vector is as follows: (1) Using monoclonal fragments, in order to Bam HI and Xba I represents the double enzyme digestion site. Recombinant primers BcNGA1-1305-F and BcNGA1-1305-R were designed using double enzyme digestion linearization. Using *Brucea javanica* cDNA as a template, PCR amplification was performed to obtain PCR amplification products. The obtained PCR amplification products were then purified. (2) Extract the plasmid from the overexpression vector pCambia1305-35S-nFLAG-cMYC, and use the extracted plasmid... Bam HI and Xba The product was double-digested with enzyme I and purified using a product purification kit. (3) The enzyme-digested pCambia1305-35S-nFLAG-cMYC vector plasmid and the target gene were combined. BcNGA1 Fragment linking forms an overexpression recombinant vector; The method for constructing the silent recombination vector is as follows: (4) Use monoclonal fragments, in order to Xba I and Kpn I represents the double enzyme digestion site. Recombinant homologous arm primers BcNGA1-PCVA-F and BcNGA1-PCVA-R were designed using double enzyme digestion linearization. Using *Brucea javanica* cDNA as a template, PCR amplification was performed to obtain PCR amplification products. The obtained PCR amplification products were then purified. (5) Extract the plasmid from the silencing vector PCVA, and use the extracted plasmid... Xba I and Kpn The product was double-digested with enzyme I and purified using a product purification kit. (6) Digest the PCVA vector plasmid and the target gene. BcNGA1 Conservative structural domain segments are connected to form a silent recombination vector.

[0011] Preferably, in step (1), the nucleotide sequences of primers BcNGA1-1305-F and BcNGA1-1305-R are as shown in SEQ ID NO.3 and SEQ ID NO.4; in step (4), the nucleotide sequences of primers BcNGA1-PCVA-F and BcNGA1-PCVA-R are as shown in SEQ ID NO.6 and SEQ ID NO.7.

[0012] More preferably, the PCR amplification system in step (1) is: 12.5 μL of 2×Phanta Max Master Mix (Dye Plus), 9.5 μL of ddH2O, 1 μL of cDNA, and 1 μL each of BcNGA1-1305-F and BcNGA1-1305-R; the PCR amplification system in step (4) is: 12.5 μL of 2×Phanta Max Master Mix (Dye Plus), 9.5 μL of ddH2O, 1 μL of cDNA, and 1 μL each of BcNGA1-PCVA-F and BcNGA1-PCVA-R; 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 30 s, for 35 cycles, and 72℃ final extension for 10 min.

[0013] Further, the double digestion system described in step (2) is: 5 μL of 10×Quiekcut Buffer. Bam 2.5 μL of HI rapid digestion enzyme Xba 2.5 μL of rapid digestion enzyme, 15 μL of plasmid, and 25 μL of ddH2O; the double digestion system described in step (5) is: 5 μL of 10×Quiekcut Buffer. Xba 2.5 μL of rapid digestion enzyme I, Kpn I rapid digestion enzyme 2.5 μL, plasmid 15 μL, ddH2O 25 μL.

[0014] The present invention BcNGA1 Application of genes in breeding to alter plant leaf morphology.

[0015] The leaf morphology of the plant changes with the gene expression level, and the plant is Arabidopsis thaliana or Chinese cabbage.

[0016] A preferred embodiment of the present invention is to apply the above-mentioned... BcNGA1 The method for genetically breeding Arabidopsis thaliana leaves to achieve morphological completion includes the following steps: 1) Transform the pCambia1305-35S-BcNGA1-nFLAG-cMYC overexpression vector plasmid into Agrobacterium GV3101; 2) Using two pairs of primers, BcNGA1-1305-F and BcNGA1-1305-R, and BcNGA1-1305-F and 1305-R, the Agrobacterium tumefaciens culture obtained in step 1) was used as a template for PCR amplification and verification; the sequence of primer 1305-R is shown in SEQ ID NO.5; 3) After reviving the Agrobacterium bacterial suspension, prepare a resuspension with buffer solution, and use the inflorescence infection method to infect Arabidopsis inflorescences to obtain infected T0 generation Arabidopsis seeds. 4) T0 generation Arabidopsis seeds were sown on 1 / 2 MS agar plates containing hygromycin B to screen for overexpressing Arabidopsis plants. The selected transgenic plants were sown on 1 / 2 MS agar plates containing hygromycin B to screen for T2 generation transgenic plants. In step 4), T0 generation Arabidopsis seeds can be sown on 1 / 2 MS agar plates containing 30 μg / mL hygromycin B to screen for overexpressing Arabidopsis plants. The selected transgenic plants can then be sown on 1 / 2 MS agar plates containing 50 μg / mL hygromycin B for further screening.

[0017] Applications BcNGA1 The method for genetically breeding Chinese cabbage leaves to achieve plant morphology includes the following steps: The BcNGA1-PCVA silencing vector plasmid was transformed into Agrobacterium GV3101 and cultured to obtain Agrobacterium bacterial culture. Using two pairs of primers, BcNGA1-PCVAF and BcNGA1-PCVA-R, and BcNGA1-PCVA-F and PCVA-R, the Agrobacterium tumefaciens culture was used as a template for PCR amplification and verification; the sequence of primer PCVA-R is shown in SEQ ID NO.8. After resuscitating the Agrobacterium tumefaciens bacterial suspension, a resuspension was prepared using buffer solution. The suspension was then injected into the cotyledons of Chinese cabbage. Positive silent plants were obtained after identifying the expression level.

[0018] This invention provides a gene related to leaf wrinkling in *Brucea javanica* and its application. Compared with existing technologies, it has the following advantages: 1. This invention identifies the function of the BcNGA1 gene and finds that it has the function of regulating plant leaf morphology. Overexpression recombinant vector and silence recombinant vector were prepared and applied to the cultivation of new plant leaf morphology varieties. BcNGA1 overexpressing Arabidopsis plants and silenced Chinese cabbage plants were successfully screened and cultivated. The leaf morphology of both plants changed. 2. This invention not only deepens the theoretical understanding of the mechanism of plant leaf morphogenesis, but also provides a theoretical basis for the early identification of leaf wrinkling in *Brassica oleracea* in production practice. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is an electrophoresis diagram of the gene clone used to construct the BcNGA1 overexpression recombinant vector in Example 1 of this invention. Figure 2 This is an electrophoresis diagram of the gene clone used to construct the BcNGA1 gene silencing recombinant vector in Example 2 of this invention. Figure 3 This is a diagram showing the culture status of wild-type Arabidopsis thaliana and transgenic plants in a petri dish in Example 3 of the present invention; Figure 4 This is a PCR electrophoresis image of wild-type Arabidopsis thaliana and T2 generation transgenic plants in Example 3 of this invention; Figure 5 This is an electrophoresis diagram showing the expression level of gene BcNGA1 in wild-type Arabidopsis and T2 generation transgenic Arabidopsis in Example 3 of the present invention. Figure 6 This is a PCR electrophoresis image of a silent plant and a control plant in Example 4 of this invention. Figure 7 Electrophoresis diagrams of gene BcNGA1 expression levels in silent and control plants in Example 4 of this invention; Figure 8 This is a comparison of the growth status of wild-type Arabidopsis thaliana and T2 generation transgenic Arabidopsis thaliana plants in Example 5 of this invention. The scale bar in the lower right corner of each photo is 1 cm. Figure 9 This is a comparison chart of the growth status of silent plants and control plants in Example 6 of the present invention; the scale bar in the chart is 1 cm. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] This application provides a gene related to leaf wrinkling in *Brucea javanica* and its application, which solves the problem that the molecular mechanism of leaf wrinkling and blistering development in *Brucea javanica* is still unclear, identifies key genes for leaf wrinkling and blistering development, and elucidates the molecular mechanism of wrinkling and blistering development, thus addressing the technical problem of further applying biotechnology to improve the wrinkling and blistering trait.

[0023] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0024] Example 1: Construction of overexpression recombinant vector: (1) In order to obtain BcNGA1 The gene sequence was analyzed using CE Design V1.04 software, employing a single-clone fragment approach. Bam HI and Xba I represents the double enzyme digestion site. Recombinant primers BcNGA1-1305-F and BcNGA1-1305-R were designed using double enzyme digestion linearization. Using cDNA from the WS-1 germplasm of *Brucea javanica* as a template, PCR amplification was performed to obtain the PCR amplification product.

[0025] The PCR amplification system consisted of: 12.5 μL of 2×Phanta Max Master Mix (Dye Plus), 9.5 μL of ddH2O, 1 μL of cDNA, and 1 μL each of BcNGA1-1305-F and BcNGA1-1305-R. 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 30 s, 35 cycles, and 72℃ final extension for 5 min.

[0026] The nucleotide sequence of primer BcNGA1-1305-F is shown in SEQ ID NO.3; The nucleotide sequence of primer BcNGA1-1305-R is shown in SEQ ID NO.4.

[0027] Table 1. Nucleotide sequences of primers used for BcNGA1 gene cloning and vector plasmid identification. The obtained PCR amplification products were purified (purification instrument purchased from Beijing TransGen Biotech Co., Ltd.), and the concentration of the recovered products was determined using a nucleic acid quantification instrument after purification.

[0028] (2) Using a plasmid extraction kit (purchased from Beijing TransGen Biotech Co., Ltd.), the plasmid of the overexpression vector pCambia1305-35S-nFLAG-cMYC was extracted according to the instructions. The extracted plasmid was then subjected to... BamHI and Xba Double digestion with enzyme I.

[0029] The double digestion system consisted of 5 μL of 10×Quiekcut Buffer. Bam 2.5 μL of HI rapid digestion enzyme Xba I rapid digestion enzyme 2.5 μL, plasmid 15 μL, ddH2O 25 μL.

[0030] The product was then purified using a gel extraction kit (purchased from Beijing TransGen Biotech Co., Ltd.), and the concentration of the recovered product was determined using a nucleic acid quantification instrument.

[0031] (3) Using a gene recombination kit (purchased from Beijing TransGen Biotech Co., Ltd.), the purified pCambia1305-35S-nFLAG-cMYC vector plasmid and the target gene were recombined. BcNGA1 Fragments are linked together to form a recombinant vector.

[0032] like Figure 1 As shown, the amplification product bands are clear and single, with no obvious non-specific amplification or primer dimers. The band positions are approximately between 750 bp and 1000 bp, which is consistent with the expected size of the target gene fragment, indicating that the PCR amplification was successful.

[0033] Example 2: Construction of the silent recombination vector: (4) Analysis BcNGA1 The gene sequence was obtained and its conserved domain nucleotides were selected (as shown in SEQ ID NO.2). Homologous arm primers for the PCVA vector were designed using the primer design software CE Design. The designed primer sequences were then sent to General Biotech (Anhui) Co., Ltd. for primer synthesis. The specific homologous arm primer sequences are shown in Table 2. Table 2 Primer sequences for silencing vector construction (5) Using a plasmid extraction kit (purchased from Beijing TransGen Biotech Co., Ltd.), extract the plasmid of the silencing vector PCVA according to the instructions. Then, use the extracted plasmid... Xba I and Kpn Double digestion with enzyme I.

[0034] The double digestion system consisted of 5 μL of 10×Quiekcut Buffer. Xba 2.5 μL of rapid digestion enzyme I, Kpn I rapid digestion enzyme 2.5 μL, plasmid 15 μL, ddH2O 25 μL.

[0035] The product was then purified using a gel extraction kit (purchased from Beijing TransGen Biotech Co., Ltd.), and the concentration of the recovered product was determined using a nucleic acid quantification instrument.

[0036] (6) The purified PCVA vector plasmid and the target gene BcNGA1 fragment were ligated using a gene recombination kit (purchased from Beijing TransGen Biotech Co., Ltd.) to form a recombinant vector.

[0037] like Figure 2 As shown, the amplification product bands are clear and single, with no obvious non-specific amplification or primer dimers. The band positions are approximately 200 bp, which is consistent with the expected size of the target gene fragment, indicating that the PCR amplification was successful.

[0038] Example 3: Cultivation and Validation of Arabidopsis thaliana Overexpression Plants 1) Using a plasmid extraction kit (purchased from Beijing TransGen Biotech Co., Ltd.), the plasmid of the overexpression vector pCambia1305-35S-nFLAG-cMYC (obtained in Example 1) was extracted according to the instructions and transformed into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) according to the instructions.

[0039] 2) Using primer pairs BcNGA1-1305-F and BcNGA1-1305-R, and BcNGA1-1305-F and 1305-R, the Agrobacterium tumefaciens culture obtained in the above steps was used as a template for PCR amplification and verification. The sequence of primer 1305-R is SEQ ID NO.5: AATTCCCGATCTAGTAACATA.

[0040] 3) After reviving the Agrobacterium tumefaciens culture, prepare a resuspension with OD560=0.8-1 using buffer solution. Infect Col-0 Arabidopsis thaliana inflorescences using the inflorescence infection method to obtain infected T0 generation Arabidopsis thaliana seeds. The buffer solution was prepared as follows: 200 mL 1 / 2 MS, 10 g sucrose, 50 μL Silwet-77, with the pH adjusted to 5.8.

[0041] 4) T0 generation Arabidopsis thaliana were sown on 1 / 2 MS agar plates containing 30 μg / mL hygromycin B (Invitrogen, Carlsbad, California) to screen for overexpressing Arabidopsis thaliana plants. The screened transgenic plants were sown on 1 / 2 MS agar plates containing 50 μg / mL hygromycin B (Invitrogen, Carlsbad, California) until T2 generation transgenic plants were screened.

[0042] like Figure 3As shown, the upper half is wild-type Arabidopsis thaliana, and the lower half is T2 generation transgenic plants. It can be seen that only T2 generation transgenic plants can grow normally on 1 / 2 MS agar plates containing hygromycin B.

[0043] PCR amplification was performed on T1 generation transgenic plants and wild-type Arabidopsis using specific primers (BcNGA1-1305-F and BcNGA1-1305-R). A specific band was detected in Arabidopsis plants overexpressing the BcNGA1 gene, but not in wild-type plants. This indicates that pCambia1305-35S-BcNGA1-nFLAG-cMYC has been successfully introduced into the Arabidopsis genome and successfully expressed. Figure 4 As shown, lanes OE-1, OE-2, OE-3, OE-4, and OE-5 represent BcNGA1 gene overexpression lines 1, 2, 3, 4, and 5, respectively, all being the first generation of overexpression lines. Lane Col-0 is a wild-type Arabidopsis thaliana, belonging to the "standard model material." Figure 4 The samples were used as negative controls. Clear, single target bands were amplified in OE-1 through OE-5, indicating successful integration of the exogenous gene into the Arabidopsis genome. Col-0: No band was observed, ruling out a false positive.

[0044] Using fluorescent primers (YG-BcNGA1-F and YG-BcNGA1-R, sequences shown in Table 3), qPCR was performed on T2 generation transgenic plants and wild-type Arabidopsis thaliana. The results showed that the expression level of BcNGA1 in all T2 generation overexpression lines was significantly higher than that in wild-type (WT), with an increase of 40-80 times (e.g., ...). Figure 5 (As shown in the image). These results all indicate that the genetic transformation of transgenic Arabidopsis plants was successful, and subsequent experiments can be conducted using transgenic plants.

[0045] Example 4: Cultivation and Verification of Silent Bok Choy Plants This embodiment provides a method for cultivating bok choy leaf morphology into a plant, including the following operations: The BcNGA1-pCVA silencing vector plasmid was transformed into Agrobacterium GV3101, and Agrobacterium culture was obtained. Using two pairs of primers, BcNGA1-pCVA-F and BcNGA1-PCVA-R, and BcNGA1-PCVA-F and PCVA-R, the Agrobacterium culture was used as a template for PCR amplification and verification.

[0046] Sow SYM (Chinese bok choy germplasm variety "March Slow") about one week in advance. When the plants have grown to the point where the two cotyledons are fully developed, they can be injected with Agrobacterium tumefaciens inoculation containing the virus silencing vectors pCVA, pCVB, and BcNGA1-pCVA. For bacterial resuscitation, gently shake for about 24 hours: add 4 mL LB + 4 µL Kana + 8 µL Rif + 40 µL bacterial solution to a 10 mL centrifuge tube. Then shake vigorously for about 16 hours: shake the system to 100 mL in a 250 mL Erlenmeyer flask, add 100 mL LB + 100 µL Kana + 200 µL Rif + 1000 µL bacterial solution.

[0047] When the bacterial culture becomes turbid, centrifuge at 6000 rpm for 5 min, precipitate the bacterial cells and discard the supernatant. Add 5 mL of buffer (5 mg / mL glucose, 10 mM MES, 2 mM Na3PO4·12H2O) to the precipitated bacterial cells, pipette to mix well, resuspend the bacterial cells, centrifuge again and discard the supernatant; resuspend the bacterial plaques with buffer, adjust the OD600 of the bacterial culture to about 1.2, and add 100 μM AS (acetylsyleugenol). After storing the resuspended bacterial culture in the dark for 3 h, inject the mixed PCVA and PCVB, and BcNGA1-PCVA and PCVB into the leaves of Chinese cabbage using a syringe. After injection, treat in a dark, wind-protected environment for 24 h, and then continue to culture under light until the phenotype appears.

[0048] PCR amplification was performed on silent plants and control plants using specific primers (BcNGA1-PCVA-F and BcNGA1-PCVA-R). Specific bands were detected in silent plants but not in control plants. This indicates that PCVA has been successfully introduced into the pak choi plants. Figure 6 As shown in the figure, SYM-PDS: positive control vector for the VIGS (viral silencing vector) experiment (PDS is used to verify whether the VIGS system is working properly); SYM-NGA1: VIGS vector of the target gene NGA1, which is the focus of this screening; SYM-PCVA: negative control. The numbers 145-192 represent the silencing of strains 145 to 192.

[0049] qPCR was performed on silent and control plants using fluorescent primers (YG-NGA1-PCVA-F and YG-NGA1-PCVA-R, sequences shown in Table 3). The results showed that the expression level of BcNGA1 in all mutant lines was significantly lower than that in the control line PCVA, with line 183 showing the lowest expression level (approximately 30% of the control). Figure 7 As shown, Figure 7The leftmost line in the middle is the control line PCVA. From left to right, the silent plants are: the 157th silent *Phyllanthus edulis* (March Slow), the 158th silent *Phyllanthus edulis* (March Slow), the 161st silent *Phyllanthus edulis* (March Slow), the 168th silent *Phyllanthus edulis* (March Slow), the 172nd silent *Phyllanthus edulis* (March Slow), and the 183rd silent *Phyllanthus edulis* (March Slow). These results all indicate that the genetic transformation of the silent plants was successful. Table 3 Example 5: Comparison of leaf growth status between transgenic Arabidopsis thaliana plants and wild-type common Arabidopsis thaliana plants WT (wild-type Arabidopsis thaliana) and OE (overexpressing Arabidopsis thaliana) seedlings were planted on 1 / 2 MS plates under the conditions of 22℃ / 18℃, 16 h / 8 h (day and night), 60% relative humidity, and 12000 LUX light intensity, and then transplanted into nutrient pots for three weeks.

[0050] This experiment used wild-type Arabidopsis thaliana and T2 generation transgenic plants (obtained using the method in Example 3), and the experimental data were repeated three times. After three weeks of normal culture, the growth status of the plants was as follows: Figure 8 As shown, the leaves of the transgenic plant are long and narrow, while the leaves of the wild-type plant grow normally. This indicates that the leaf morphology of the transgenic plant has changed.

[0051] Example 6: Comparison of leaf growth status between silent bok choy plants and control group bok choy plants Both the control group of Chinese cabbage plants and the plants after the silencing treatment were cultured at 22℃ / 18℃ for 16 h / 8 h (day and night), with a relative humidity of 60% and a light intensity of 12000 LUX until the phenotype appeared.

[0052] like Figure 9 As shown in Figure A, the control strain PCVA has leaves that are spread out and have smooth edges, while the mutant strain has leaves that are... Figure 9 All of them (B to G) showed varying degrees of morphological distortion, including leaf shrinkage, edge wrinkling, and asymmetrical curling.

[0053] In summary, compared with the prior art, the present invention has the following beneficial effects: 1. This invention identifies the function of the BcNGA1 gene and finds that it has the function of regulating plant heat resistance. A recombinant vector was prepared and applied to the cultivation of new plant leaf morphology. BcNGA1 overexpressing plants were successfully screened and cultivated, which can change leaf morphology. 2. This invention not only deepens the theoretical understanding of the mechanism of plant leaf morphogenesis, but also provides a theoretical basis for the early identification of leaf wrinkling in *Brassica oleracea* in production practice.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene related to leaf wrinkling in *Brucea javanica*, characterized in that, for BcNGA1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1; and whose conserved domain nucleotide sequence is shown in SEQ ID NO.

2.

2. A vector containing the leaf wrinkling-related gene of *Brucea javanica* as described in claim 1, characterized in that, The carrier contains the aforementioned BcNGA1 overexpression recombinant vectors of the gene and / or containing the above BcNGA1 Gene silencing recombination vector.

3. The carrier as described in claim 2, characterized in that, The overexpression recombinant vector is used to... BcNGA1 Gene insertion into the pCambia1305-35S-nFLAG-cMYC overexpression vector Bam HI and Xba The silencing recombinant vector is obtained by I restriction site; BcNGA1 Insertion of conserved gene domains into PCVA silencing vectors Xba I and Kpn Obtained from the I restriction site.

4. The carrier as described in claim 3, characterized in that, The method for constructing the overexpression recombinant vector is as follows: (1) Using monoclonal fragments, in order to Bam HI and Xba I represents the double enzyme digestion site. Recombinant primers BcNGA1-1305-F and BcNGA1-1305-R were designed using double enzyme digestion linearization. Using *Brucea javanica* cDNA as a template, PCR amplification was performed to obtain PCR amplification products. The obtained PCR amplification products were then purified. (2) Extract the plasmid from the overexpression vector pCambia1305-35S-nFLAG-cMYC, and use the extracted plasmid... Bam HI and Xba The product was double-digested with enzyme I and purified using a product purification kit. (3) The enzyme-digested pCambia1305-35S-nFLAG-cMYC vector plasmid and the target gene were combined. BcNGA1 Fragment linking forms an overexpression recombinant vector; The method for constructing the silent recombination vector is as follows: (4) Use monoclonal fragments, in order to Xba I and Kpn I represents the double enzyme digestion site. Recombinant homologous arm primers BcNGA1-PCVA-F and BcNGA1-PCVA-R were designed using double enzyme digestion linearization. Using *Brucea javanica* cDNA as a template, PCR amplification was performed to obtain PCR amplification products. The obtained PCR amplification products were then purified. (5) Extract the plasmid from the silencing vector PCVA, and use the extracted plasmid... Xba I and Kpn The product was double-digested with enzyme I and purified using a product purification kit. (6) Digest the PCVA vector plasmid and the target gene. BcNGA1 The conservative structural domain segments are connected to form a silent recombination carrier.

5. The carrier as described in claim 4, characterized in that, In step (1), the nucleotide sequences of primers BcNGA1-1305-F and BcNGA1-1305-R are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; in step (4), the nucleotide sequences of primers BcNGA1-PCVA-F and BcNGA1-PCVA-R are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

6. The carrier as described in claim 4, characterized in that, The PCR amplification system in step (1) is: 12.5 μL of 2×PhantaMax Master Mix (Dye Plus), 9.5 μL of ddH2O, 1 μL of cDNA, and 1 μL each of BcNGA1-1305-F and BcNGA1-1305-R; the PCR amplification system in step (4) is: 12.5 μL of 2×PhantaMax Master Mix (Dye Plus), 9.5 μL of ddH2O, 1 μL of cDNA, and 1 μL each of BcNGA1-PCVA-F and BcNGA1-PCVA-R. 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 30 s, 35 cycles, and 72℃ final extension for 10 min.

7. The carrier as described in claim 4, characterized in that, The double digestion system described in step (2) is: 5 μL of 10×Quiekcut Buffer. Bam 2.5 μL of HI rapid digestion enzyme Xba 2.5 μL of rapid digestion enzyme, 15 μL of plasmid, and 25 μL of ddH2O; the double digestion system described in step (5) is: 5 μL of 10×Quiekcut Buffer. Xba 2.5 μL of rapid digestion enzyme I, Kpn I rapid digestion enzyme 2.5 μL, plasmid 15 μL, ddH2O 25 μL.

8. The claim 1 BcNGA1 Application of genes in breeding to alter plant leaf morphology.

9. The application as described in claim 8, characterized in that, The leaf morphology of the plant changes with the gene expression level; the plant in question is Arabidopsis thaliana or Chinese cabbage.

10. The application as described in claim 9, characterized in that, Applications BcNGA1 The method for genetically breeding Arabidopsis thaliana leaves to achieve morphological completion includes the following steps: 1) Transform the pCambia1305-35S-BcNGA1-nFLAG-cMYC overexpression vector plasmid into Agrobacterium GV3101; 2) Using two pairs of primers, BcNGA1-1305-F and BcNGA1-1305-R, and BcNGA1-1305-F and 1305-R, the Agrobacterium tumefaciens culture obtained in step 1) was used as a template for PCR amplification and verification; the sequence of primer 1305-R is shown in SEQ ID NO.5; 3) After reviving the Agrobacterium bacterial suspension, prepare a resuspension with buffer solution, and use the inflorescence infection method to infect Arabidopsis inflorescences to obtain infected T0 generation Arabidopsis seeds. 4) T0 generation Arabidopsis seeds were sown on 1 / 2 MS agar plates containing hygromycin B to screen for overexpressing Arabidopsis plants. The selected transgenic plants were sown on 1 / 2 MS agar plates containing hygromycin B to screen for T2 generation transgenic plants. Applications BcNGA1 The method for genetically breeding Chinese cabbage leaves to achieve plant morphology includes the following steps: The BcNGA1-pCVA silencing vector plasmid was transformed into Agrobacterium GV3101 and cultured to obtain Agrobacterium bacterial culture. Using two pairs of primers, BcNGA1-pCVA-F and BcNGA1-PCVA-R, and BcNGA1-PCVA-F and PCVA-R, the Agrobacterium tumefaciens culture was used as a template for PCR amplification and verification; the sequence of primer PCVA-R is shown in SEQ ID NO.8; After resuscitating the Agrobacterium tumefaciens bacterial suspension, a resuspension was prepared using buffer solution. The suspension was then injected into the cotyledons of Chinese cabbage. Positive silent plants were obtained after identifying the expression level.

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