Gene BrNAC2a related to leaf size of Chinese cabbage and application of gene BrNAC2a

Abstract

The invention discloses a Chinese cabbage leaf size related gene BrNAC2a and application thereof. The DNA (deoxyribonucleic acid) sequence of the Chinese cabbage leaf size related gene BrNAC2a is shown as SEQ ID No. 1. Compared with Columbia wild type arabidopsis thaliana, the T-DNA of the orthologous gene of the gene inserted into a mutant arabidopsis thaliana material has the advantages that the leaf size is not obviously changed; the BrNAC2a with the enhanced promoter is transformed into Columbia type arabidopsis thaliana through agrobacterium infection, a BrNAC2a overexpression arabidopsis thaliana strain is obtained, and the result shows that the overexpression of the BrNAC2a can cause the reduction of the leaves of the arabidopsis thaliana. The heterologous expression of the Chinese cabbage BrNAC2a can inhibit the expansion of the leaves of arabidopsis thaliana, and the gene can be applied to breeding of Chinese cabbage vegetables and other horticultural plants and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, specifically relates to the technical field of the Chinese cabbage BrNAC2a gene and its application in regulating plant leaf size, and particularly relates to a Chinese cabbage leaf size-related gene BrNAC2a and its application. Background Art

[0002] Leaves are the main organs for plant photosynthesis and transpiration. Their size and shape affect plant development, the ability to resist biotic and abiotic stresses, and yield, and thus have great research value. Leaves originate from the shoot apical meristem (SAM). A group of cells on the periphery of the SAM differentiates to form leaf primordia, which then become juvenile leaves containing ventral and dorsal sides through apical growth and marginal growth, and finally form mature leaves after a series of developmental processes. The development of leaf primordia generally undergoes two processes: first, a large number of leaf primordium cells proliferate, dividing into new cells of almost constant size throughout the leaf primordium; in the second stage, the leaf primordium cells change from cell proliferation to cell expansion and thus develop into mature leaves. These two stages are collectively referred to as intercalary growth. Research shows that the normal development of leaves is the result of the combined action of different genes and hormones. These genes can have an important impact on the hormone pathways including biosynthesis, transport, and signal transduction. At the same time, hormones can also feedback-regulate the transcription and translation of the above genes.

[0003] NAC Transcription factors ( NAM , ATAF1 / 2 , CUC1 / 2 ) are one of the largest transcription factor families in plants and are a kind of plant-specific proteins encoded by large gene families. The first NAC transcription factor ( petunia no apical meristem , NAM ) was discovered in Petunia hybrida. Subsequently, two other members of NAC were discovered in Arabidopsis thaliana, cup- shaped cotyledons ( CUC ) and arabidopsis transcription activation factor 1 / 2 ( ATAF1 / 2 ). Research shows that typical NAC proteins contain a conserved NAC domain and a highly variable transcriptional activation region, and its highly variable transcriptional activation region can bind a variety of different proteins, indicating the diversity of its functions. At present, people have conducted relatively in-depth research on the NAC transcription factor gene family in various plants and found that it plays an important role in plant growth, lateral root formation, leaf senescence, fruit ripening and softening, and responses to abiotic stresses such as drought and low temperature.

[0004] Chinese cabbage ( Brassica rapa L.(Brassica rapa L.) is a biennial herbaceous plant of the genus Brassica in the family Brassicaceae. It has a long cultivation history, and now Chinese cabbage has become one of the vegetables widely cultivated globally. The yield of Chinese cabbage is extremely abundant, laying a solid foundation for the sustainable development of the Chinese cabbage industry. In recent years, high-quality and high-yield as important agronomic traits are important breeding goals for Chinese cabbage, and as the product organ of Chinese cabbage, the molecular mechanism of leaf formation has always been a research hotspot in molecular breeding. BrNAC2a in Chinese cabbage plays an important role in regulating leaf size and has great application potential in achieving high yields of Chinese cabbage. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide a gene BrNAC2a related to the leaf size of Chinese cabbage and its application in view of the deficiencies of the prior art.

[0006] The object of the present invention is achieved by the following technical solutions: The present invention provides a gene BrNAC2a related to the leaf size of Chinese cabbage, which is a gene cloned from 'Youqing 49', and the BrNAC2a has: (1) The nucleotide sequence shown in SEQ ID No.1; or (2) The nucleotide sequence shown in SEQ ID No.1 with one or more nucleotides substituted, deleted, and / or added; or (3) A nucleotide sequence that hybridizes with the DNA sequence defined in (1) under stringent conditions.

[0007] The present invention provides a biological material containing the above-mentioned gene BrNAC2a related to the leaf size of Chinese cabbage, and the biological material is an expression vector, expression cassette, host cell or engineered bacterium.

[0008] The present invention provides the application of the above-mentioned gene BrNAC2a related to the leaf size of Chinese cabbage or its corresponding biological material in regulating the leaf size of plants.

[0009] Furthermore, the application is specifically: By overexpressing BrNAC2a of Chinese cabbage, the leaves of plants are significantly reduced during the vegetative growth stage.

[0010] The present invention provides the application of the above-mentioned gene BrNAC2a related to the leaf size of Chinese cabbage or its corresponding biological material in the preparation of transgenic plants.

[0011] The present invention provides the application of the above-mentioned gene BrNAC2a related to the leaf size of Chinese cabbage or its corresponding biological material in the preparation of transgenic Arabidopsis thaliana.

[0012] The nucleotide sequence of the gene BrNAC2a related to the leaf size of Chinese cabbage provided by the present invention is as shown in SEQ ID No.1. In the T-DNA insertion mutant material of the Arabidopsis thaliana gene AtNAC2 ​AtNAC2 The change in expression does not affect the size of plant leaves; through Agrobacterium infection, Chinese cabbage BrNAC2a with an enhanced promoter was transformed into Columbia-type Arabidopsis thaliana to obtain BrNAC2a overexpressing Arabidopsis thaliana lines. Phenotypic analysis experiments showed that overexpression of Chinese cabbage BrNAC2a could inhibit the expansion of Arabidopsis thaliana leaves, while AtNAC2 the mutation of Arabidopsis thaliana could not affect the size of plant leaves. This indicates that Chinese cabbage BrNAC2a is closely related to the size of plant leaves, and applying this gene to the breeding of Chinese cabbage or other cruciferous vegetables has good application prospects.

[0013] The beneficial effects of the present invention are as follows: the present invention provides the function and expression analysis of the gene BrNAC2a related to the size of Chinese cabbage leaves, which is beneficial to the regulation of the size of Chinese cabbage leaves. Overexpression of BrNAC2a will lead to smaller leaves of Arabidopsis thaliana, indicating that heterologous expression of Chinese cabbage BrNAC2a can inhibit the expansion of Arabidopsis thaliana leaves. This gene can be applied to the breeding of Chinese cabbage vegetables and other horticultural plants, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a schematic diagram of the BrNAC2a overexpression vector; Figure 2 is AtNAC2 the electrophoresis diagram for detecting the homozygous mutant of the gene T-DNA insertion; Figure 3 is the qRT-PCR identification result diagram of three lines of BrNAC2a overexpressing plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ) and the control group of Arabidopsis thaliana plants; Figure 4 is the phenotypic observation result diagram of the T-DNA insertion mutant SALK_137131 plants, Chinese cabbage BrNAC2a overexpressing plants and the control group of Arabidopsis thaliana plants 14 days after sowing; Figure 5 is the observation result diagram of the leaf size of the T-DNA insertion mutant SALK_137131 plants, Chinese cabbage BrNAC2a overexpressing plants and the control group of Arabidopsis thaliana plants 7 days after transplantation (the scale bar is 1 cm); Figure 6 is Figure 5 the SALK_137131 plants in pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a-2 and pFGC1008: BrNAC2a -3 ), and the sorting result diagram of the leaf sizes of the Arabidopsis thaliana plants in the control group (the scale bar is 1 cm); Figure 7 For the T-DNA insertion mutant SALK_137131 plant, Chinese cabbage BrNAC2a Three overexpression plant lines ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ), and the statistical chart of the maximum leaf length of the Arabidopsis thaliana plants in the control group; Figure 8 For the T-DNA insertion mutant SALK_137131 plant, three overexpression plant lines of Chinese cabbage BrNAC2a ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ), and the statistical chart of the maximum leaf width of the Arabidopsis thaliana plants in the control group. Specific implementation manners

[0015] The present invention will be described below through specific examples. The technical means not described in detail in the examples belong to the conventional techniques well-known to those skilled in the art. The examples are only used to illustrate the present invention, but do not limit the scope of the present invention. Any other examples obtained by those skilled in the art based on the examples of the present invention without creative efforts fall within the protection scope of the present invention.

[0016] The present invention provides a Chinese cabbage leaf size-related gene BrNAC2a, which is a gene cloned from 'Youqing Sijiu' flowering Chinese cabbage, and its gene has the sequence shown in SEQ ID No.1.

[0017] The embodiment of the present invention also provides the application of the above-mentioned Chinese cabbage leaf size-related gene BrNAC2a in regulating the leaf size of cruciferous plants, which will be specifically described below.

[0018] Example 1: Construction of the BrNAC2a overexpression vector

[0019] 1. Double digestion of the pFGC1008 vector: Sal Ⅰ and kpn Ⅰ are used for double digestion, and the large fragment bands are separated by electrophoresis, and the gel is cut and recovered to obtain a linearized vector.

[0020] 2. Extract RNA using the Trizol method: Take samples from the leaves of "Youqing 49", and extract its RNA using the Trizol method: Calculate the number of samples, prepare the corresponding mortar, pestle and small iron spoon, wrap them with tin foil and bake at 180 °C for 4 - 5 h; as well as 1.5 ml centrifuge tubes (RNAFree), RNAFree pipettes and pipette tips, liquid nitrogen, centrifuge tube plates, place them at 121 °C and sterilize them by high-pressure steam for 40 min. Take the corresponding number of centrifuge tubes and number them; add 1 ml of Trizol reagent to each centrifuge tube in the fume hood and place them on ice; take the samples from liquid nitrogen into a pre-cooled mortar, add liquid nitrogen and grind 3 - 5 times until it becomes powdery, transfer the powder into the centrifuge tube containing Trizol reagent, and shake and vortex thoroughly; add 200 μl of chloroform in the fume hood, shake vigorously for 15 s, and then place it on ice for 5 min; centrifuge at 4 °C with a speed of 12000 rpm for 10 min, transfer 600 μl of the supernatant into a new 1.5 ml centrifuge tube, add an equal volume of isopropanol and invert to mix evenly, and let it stand at -20 °C for 30 min; centrifuge again at 4 °C with a speed of 12000 rpm for 10 min to remove the supernatant; add 1 ml of pre-cooled 75 vol% ethanol dissolved in diethyl pyrocarbonate (DEPC) water, centrifuge at 4 °C with a speed of 12000 rpm for 10 min, pour out the supernatant; centrifuge without adding any reagent for another 20 s to completely remove the supernatant, aspirate the liquid, and dry it in the fume hood. Then add 50 μl of DEPC water to dissolve the RNA precipitate and measure the concentration for standby.

[0021] 3. Prepare cDNA: Use the Takara reverse transcription kit to remove genomic DNA to obtain cDNA. The specific steps can refer to the Takara reverse transcription kit instruction manual: The reaction system contains 2.0 μl of 5×gDNA Eraser Buffer, 1.0 μl of gDNA Eraser, 1.0 μg / μl of Total RNA, 6.0 μl of RNAFree ddH 2 O, incubate in a metal bath at 42 °C for 2 min; Reverse transcription: The reaction system contains 10 μl of the genomic DNA-removed product obtained above, 4.0 μl of 5xPrimerscript Buffer, 1.0 μl of Primerscript RT Enzyme Mix, 1.0 μl of RT Primer Mix, 4.0 μl of RNase Free ddH 2 O, incubate at 37 °C for 20 min, react at 85 °C for 5 s, and finally obtain cDNA, which is stored at -20 °C for standby.

[0022] 4. Design specific primers and amplify the CDS sequence of BrNAC2a using high-fidelity KOD enzyme. The sequence information of the designed specific primers is shown in Table 1. The amplified PCR products were separated by 0.8% agarose gel electrophoresis. After the length was confirmed to be consistent, the gel was cut and recovered, and the target fragment was recovered for standby.

[0023] Table 1: Primers used for constructing heterologous expression vectors

[0024] 5. Homologous recombination ligation: Perform homologous recombination ligation on the above linearized vector and the purified PCR amplified fragment: Use the single-fragment homologous recombination kit C112 from Novoprotein. The system is as follows: 4 μl of 5×CEⅡ Buffer, 2 μl of ExaseⅡ, 200 ng of the linearized vector recovered by double digestion, 20 ng of the CDS fragment of BrNAC2a, and ddH 2 O was added to make up to 20 μl; reacted at 37°C for 30 min.

[0025] 6. Transform Escherichia coli, verify the resulting bacterial liquid by PCR, extract the target plasmid and verify it by sequencing. The plasmid with successful sequence alignment was transformed into Agrobacterium competent cells by electroporation. The transformed bacterial liquid showed bands by PCR, and the volume was expanded for culture; the plasmid was extracted and verified by sequencing. The map of the BrNAC2a overexpression vector obtained is as Figure 1 shown. The bacterial liquid with successful sequencing verification was stored as a strain and the mother liquor was stored at 4°C for standby.

[0026] Example 2: Arabidopsis thaliana transformation by floral dip method and screening of positive transformants

[0027] 1. Transform Arabidopsis thaliana by floral dip method: Use the above-mentioned bacterial liquid verified by sequencing as the mother liquor and transform Arabidopsis thaliana by the floral dip method. The steps are as follows: 500 μl of the Agrobacterium bacterial liquid containing the target vector was added to 200 ml of liquid LB medium containing kanamycin and rifampicin (50 mg / L), and cultured at 28°C and 200 rpm for about 30 h until the OD 600 reached 1.2; then centrifuged at 8000 rpm for 10 min to obtain the Agrobacterium precipitate, and resuspended the bacterial liquid with 200 ml of 5% sucrose; added Silwet-77 to a final concentration of 200 μl / L, and oscillated at 28°C and 200 rpm for 2 min; Remove the open flowers and siliques from wild-type WT Arabidopsis thaliana, immerse the flower buds in the bacterial liquid for 60 s, blot dry the excess bacterial liquid, and culture in the dark at 25°C for 24 h, and then culture normally. Repeat the floral dip once after one week.

[0028] 2. Prepare kanamycin sowing medium: Add 2.22 g of MS powder and 10 g of sucrose, adjust the pH to 5.8 with 2 M NaOH to obtain MS medium. After adding 4 g of agar powder, autoclave at 121 °C for 20 min, then place it in a laminar flow hood and cool to 50 - 60 °C. Add kanamycin to a final concentration of 75 mg / L and pour solid plates.

[0029] 3. Screening of positive transformants: Sow the Arabidopsis thaliana seeds T 1 obtained by floral dip on the kanamycin sowing medium. The steps are as follows: Disinfect with 10 vol% NaClO for 2 min; wash the seeds with 75 vol% ethanol for 2 min; rinse with sterile water 5 times, 1 min each time; sow on the kanamycin sowing medium, seal the mouth, and place it in an incubator at 22 °C for cultivation (16 h light, 8 h dark). After about two weeks, transfer out the positive plants with strong growth and dark green leaves, and perform PCR detection and verification. Among them, the primers used for PCR detection are shown in Table 2.

[0030] Table 2: Primers used for PCR detection of transgenic Arabidopsis thaliana

[0031] Example 3: Screening of T-DNA insertion mutant materials

[0032] 1. Extract DNA by rapid DNA extraction method: Add 200 μl of DNA extraction buffer and magnetic beads to a centrifuge tube containing Arabidopsis thaliana leaves, use a crusher to crush for 120 s to break the leaf tissue, and the frequency of the crusher is 60 Hz; after grinding, transfer all the tissue fluid into a 1.5 ml centrifuge tube and centrifuge at 13000 rpm for 8 min; prepare new 1.5 ml centrifuge tubes, add 100 μl of isopropanol to each tube, take 100 μl of the supernatant and transfer it into a centrifuge tube containing an equal volume of isopropanol, gently shake about 50 times, and let it stand at room temperature (about 25 °C) for 5 min; centrifuge at 13000 rpm for 6 min to remove the supernatant; then wash the precipitate twice with 1 ml of 70 vol% ethanol (shake up and down 20 times, centrifuge at 13000 rpm for 3 min, discard the supernatant, repeat once; centrifuge again for 1 min), suck out the liquid, air-dry the precipitate for 5 min, add 25 - 50 μl of ddH 2 O and store at -20 °C for later use.

[0033] 2. PCR detection by the three-primer method: The three primers are shown in Table 3 below. The 1.1x T3 Super Mix PCR reaction system is as follows: 44 μl of 1.1x T3 super Mix, 2 μl of Template, 2 μl of Primer F, and 2 μl of Primer R. The program is set for pre-denaturation at 98°C for 2 min 30 s, followed by 35 cycles of amplification (denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 10 s). Finally, a sufficient extension at 72°C for 2 min is performed to ensure complete amplification of the fragment. The PCR products amplified by the LP + RP primers and the PCR products amplified by the RP+BP primers are separated by 0.8 wt% agarose gel electrophoresis. The results are as Figure 2 shown, where the M lane is the DNA marker, and the lane where the rectangular box is located is the homozygous mutant plant. As Figure 2 shown, the homozygous individuals containing only a single small fragment are the mutant material SALK_137131, which is harvested for subsequent experiments.

[0034] Table 3: Primers used for screening T-DNA insertion mutant materials

[0035] Example 4: Real-time fluorescence quantitative PCR

[0036] 1. Analysis of the change in BrNAC2a expression level by real-time fluorescence quantitative PCR: At least 10 leaves of Arabidopsis thaliana pFGC1008: BrNAC2a overexpressing plants and the wild-type Arabidopsis thaliana in the control group were mixed and sampled. After making marks, they were quickly placed in liquid nitrogen for fixation. After all sampling was completed, total RNA was extracted and reverse transcribed into cDNA, and then qRT-PCR analysis was performed. The primers used for qPCR analysis were designed by Primer Premier 6 and are shown in Table 4 below. The reaction system was 15 μL: 7.5 μL of SYBRGreen Master Mix, 0.3 μL of each forward and reverse primer, 1 μL of template, and 5.9 μL of double-distilled water. The qRT-PCR reaction procedure was: 95°C for 30 s, followed by 40 cycles (95°C for 5 s, 55°C for 45 s). The specificity of the reaction was determined by the melting curve, and the internal reference gene was Atactin7 , and the relative expression level of the gene was calculated by the 2 -ΔΔCt method. Three biological replicates were set during sampling.

[0037] Finally, three lines of BrNAC2a overexpressing plants were obtained ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a-3 ), and the qRT-PCR identification results of Arabidopsis thaliana plants in the control group are as Figure 3 shown. It can be seen from this that the expression of BrNAC2a is up-regulated in the overexpressing plants compared with that in the Arabidopsis thaliana plants in the control group.

[0038] Table 4: Primers used for qRT-PCR analysis of Arabidopsis thaliana plants

[0039] Example 5: Identification of leaf size of transgenic Arabidopsis thaliana plants

[0040] 1. Observation of phenotypes of transgenic Arabidopsis thaliana: The homozygous T-DNA insertion mutant plant SALK_137131 and the overexpressing BrNAC2a plants were sown simultaneously with the wild-type WT Arabidopsis thaliana, and cultured in a light incubator for about 14 d to observe the leaf development; three plants with consistent growth vigor in each group were selected for transplantation, and the leaf development was observed one week later, and the length and width of the largest leaf were measured. According to the calculation results, the significance of the difference in leaf size among the plants in each group was analyzed.

[0041] The observation results of the phenotypes of the T-DNA insertion mutant SALK_137131 plants, Chinese cabbage BrNAC2a overexpressing plants and the control wild-type WT Arabidopsis thaliana plants after sowing for 14 d are as Figure 4 shown. It can be seen from this that the leaf size of the Chinese cabbage BrNAC2a overexpressing plants is significantly smaller than that of the T-DNA insertion mutant SALK_137131 plants and the control plants. After overexpressing this gene in wild-type WT Arabidopsis thaliana, the leaves of the overexpressing lines are smaller than those of the wild-type, while there is no significant difference in the leaf size between the T-DNA inserted Arabidopsis mutant plants and the wild-type.

[0042] One week after transplantation, the leaf sizes of the plants in each group were observed, and the observation results of the leaf sizes of the T-DNA insertion mutant SALK_137131 plants, Chinese cabbage BrNAC2a overexpressing plants and the control wild-type WT Arabidopsis thaliana plants were obtained, as Figure 5 shown; the leaves of the SALK_137131 plants, Chinese cabbage BrNAC2a overexpressing plants and the control wild-type WT Arabidopsis thaliana plants in Figure 5 were sorted from small to large, and the three lines of the SALK_137131 plants, Chinese cabbage BrNAC2a overexpressing plants ( Figure 6 BrNAC2a pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3), and the results of ranking the leaf sizes of the wild-type WT Arabidopsis thaliana plants in the control group; the maximum leaf lengths of the T-DNA insertion mutant SALK_137131 plants and three lines of Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ), and the wild-type WT Arabidopsis thaliana plants in the control group are as shown in Figure 7 . Different letters therein represent significant differences. It can be seen from this that the maximum leaf length of the Chinese cabbage BrNAC2a overexpression plants is significantly smaller than that of the control group, while the maximum leaf length of the T-DNA insertion mutant SALK_137131 plants has no significant difference compared with the control; the maximum leaf lengths of the T-DNA insertion mutant SALK_137131 plants and three lines of Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a - 2 and pFGC1008: BrNAC2a -3 ), and the wild-type WT Arabidopsis thaliana plants in the control group are as shown in Figure 8 . Different letters therein represent significant differences. It can be seen from this that the maximum leaf width of the Chinese cabbage BrNAC2a overexpression plants is significantly smaller than that of the control group, while the maximum leaf width of the T-DNA insertion mutant SALK_137131 plants has no significant difference compared with the control. The maximum leaf lengths and maximum leaf widths of the plants in each group are compared and analyzed, and the results are as shown in Figure 5 - Figure 8 . It can be seen from this that both the maximum leaf length and maximum leaf width of the Chinese cabbage BrNAC2a overexpression lines are significantly lower than those of the wild-type WT in the control group, and there is no significant difference in the maximum leaf length and maximum leaf width of the SALK_137131 Arabidopsis thaliana line compared with the wild-type WT Arabidopsis thaliana. The results show that Chinese cabbage BrNAC2a plays a negative regulatory role in the development of plant leaves, while AtNAC2 of Arabidopsis thaliana does not have this function.

[0043] The above is the preferred specific embodiment of the present invention. These examples are for better explaining the principle and practical application of the present invention, so that those skilled in the relevant technical field can well understand and utilize the present invention. However, these embodiments do not describe all details in detail, and some improvements or modifications can be made on this basis, which are obvious to any person skilled in the art. Therefore, these modifications and improvements made on the basis of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A cabbage leaf size-related gene BrNAC2a, characterized in that: The BrNAC2a has a nucleotide sequence as shown in SEQ ID No.

1.

2. A biological material containing the cabbage leaf size-related gene BrNAC2a according to claim 1, characterized in that: The biological material is an expression vector, an expression cassette, a host cell or an engineered bacterium.

3. Use of the cabbage leaf size-related gene BrNAC2a according to claim 1 or the biological material according to claim 2 in regulating plant leaf size.

4. Use of the cabbage leaf size-related gene BrNAC2a according to claim 1 or the biological material according to claim 2 in preparing transgenic plants.

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