A Chinese cabbage leaf size-related gene BrNAC2a and its application

Abstract

The present invention discloses a gene BrNAC2a related to the leaf size of Chinese cabbage and its application. The DNA sequence of the gene BrNAC2a related to the leaf size of Chinese cabbage is shown as SEQ ID No.1. Compared with the Columbia-type wild-type Arabidopsis thaliana, there is no obvious change in the leaf size of the T-DNA insertion mutant Arabidopsis thaliana material of the orthologous gene of this gene; through Agrobacterium-mediated infection, BrNAC2a with an enhanced promoter was transformed into Columbia-type Arabidopsis thaliana, and an Arabidopsis thaliana line overexpressing BrNAC2a was obtained. The results showed that overexpression of BrNAC2a would cause the leaves of Arabidopsis thaliana to become smaller. This indicates that heterologous expression of BrNAC2a in Chinese cabbage can inhibit the expansion of Arabidopsis thaliana leaves, and this gene can be applied to the breeding of Chinese cabbage vegetables and other horticultural plants, with good application prospects.

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 young 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 goes through two processes: first, a large number of cell proliferations in the leaf primordia cells, dividing new cells with almost constant size throughout the leaf primordia; in the second stage, the leaf primordia cells transform from cell proliferation to cell expansion and thus develop into mature leaves. These two stages are collectively called 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 petunias. 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. Its highly variable transcriptional activation region can bind a variety of different proteins, indicating the diversity of its functions. Currently, 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.It 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 of 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 of Chinese cabbage plays an important role in regulating leaf size and has great application potential in achieving high yield of Chinese cabbage. Summary of the Invention

[0005] The object of the present invention is to provide a gene BrNAC2a related to Chinese cabbage leaf size 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 Chinese cabbage leaf size, which is a gene cloned from 'Youqing Sijiu', and the BrNAC2a has:

[0007] (1) The nucleotide sequence shown in SEQ ID No.1; or

[0008] (2) The nucleotide sequence shown in SEQ ID No.1 with one or more nucleotides substituted, deleted and / or added; or

[0009] (3) A nucleotide sequence that hybridizes with the DNA sequence defined in (1) under stringent conditions.

[0010] The present invention provides a biological material containing the above-mentioned gene BrNAC2a related to Chinese cabbage leaf size, and the biological material is an expression vector, expression cassette, host cell or engineering bacterium.

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

[0012] Furthermore, the application is specifically:

[0013] By overexpressing BrNAC2a of Chinese cabbage, the leaves of plants are significantly reduced during the vegetative growth stage.

[0014] The present invention provides the application of the above-mentioned gene BrNAC2a related to Chinese cabbage leaf size or its corresponding biological material in the preparation of transgenic plants.

[0015] The present invention provides the application of the above-mentioned gene BrNAC2a related to Chinese cabbage leaf size or its corresponding biological material in the preparation of transgenic Arabidopsis thaliana.

[0016] The nucleotide sequence of the gene BrNAC2a related to the leaf size of Chinese cabbage provided by the present invention is shown in SEQ ID No.1. In the T-DNA insertion mutant material of the Arabidopsis gene AtNAC2 , the change in the expression of AtNAC2 does not affect the leaf size of the plant; by infecting with Agrobacterium, Chinese cabbage BrNAC2a with an enhanced promoter was transformed into Columbia-type Arabidopsis thaliana, and an Arabidopsis thaliana line overexpressing BrNAC2a was obtained. The phenotypic analysis experiment showed that the overexpression of Chinese cabbage BrNAC2a could inhibit the expansion of Arabidopsis thaliana leaves, while the AtNAC2 mutation of Arabidopsis thaliana could not affect the leaf size of the plant. This indicates that Chinese cabbage BrNAC2a is closely related to the leaf size of plants, and applying this gene to the breeding of Chinese cabbage or other cruciferous vegetables has good application prospects.

[0017] The beneficial effect of the present invention is that the present invention provides the function and expression analysis of the gene BrNAC2a related to the leaf size of Chinese cabbage, which is beneficial to the regulation of the leaf size of Chinese cabbage. The overexpression of BrNAC2a will cause the leaves of Arabidopsis thaliana to become smaller, which indicates that the 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. Description of the Drawings

[0018] Figure 1 is a schematic diagram of the BrNAC2a overexpression vector;

[0019] Figure 2 is AtNAC2 the electrophoresis diagram for detecting the homozygous mutant of the gene T-DNA insertion;

[0020] 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;

[0021] 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;

[0022] 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 is 1 cm);

[0023] Figure 6 For Figure 5 The sorting result diagram of the leaf sizes of three plant lines of SALK_137131 plants, Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ) and the control Arabidopsis thaliana plants (scale bar is 1 cm);

[0024] Figure 7 For the statistical chart of the maximum leaf length of three plant lines of T-DNA insertion mutant SALK_137131 plants, Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ) and the control Arabidopsis thaliana plants;

[0025] Figure 8 For the statistical chart of the maximum leaf width of three plant lines of T-DNA insertion mutant SALK_137131 plants, Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ) and the control Arabidopsis thaliana plants. Specific embodiments

[0026] The present invention will be described below through specific embodiments. The technical means not described in detail in the embodiments belong to the conventional techniques well-known to those skilled in the art. The embodiments are only used to illustrate the present invention, but do not limit the scope of the present invention. Any other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0027] The present invention provides a gene BrNAC2a related to the leaf size of Chinese cabbage. This gene is a gene cloned from the 'Youqing Sijiu' flowering Chinese cabbage, and its gene has the sequence shown in SEQ ID No.1.

[0028] The embodiments of the present invention also provide the application of the above-mentioned gene BrNAC2a related to the leaf size of Chinese cabbage in regulating the leaf size of cruciferous plants, which will be specifically described below.

[0029] Example 1: Construction of BrNAC2a Overexpression Vector

[0030] 1. Double digestion of pFGC1008 vector:

[0031] Sal Ⅰ and kpn Ⅰ were double digested, and the large fragment band was separated by electrophoresis, cut from the gel and recovered to obtain a linearized vector.

[0032] 2. Extract RNA by Trizol method:

[0033] Samples were taken from the leaves of "Youqing 49", and its RNA was extracted by 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; and 1.5 ml centrifuge tubes (RNAFree), RNAFree pipettes and tips, liquid nitrogen, centrifuge tube plates, placed in a high-pressure steam sterilizer at 121 °C 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 on ice; take the sample 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 on ice for 5 min; centrifuge at 4 °C with a rotation speed of 12000 rpm for 10 min, transfer 600 μl of the supernatant into a new 1.5 ml centrifuge tube, add 1 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 rotation 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 and 12000 rpm for 10 min, pour out the supernatant; centrifuge without adding any reagent for another 20 s to completely remove the supernatant, suck out 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.

[0034] 3. Prepare cDNA:

[0035] 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 ddH2O, and 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 ddH2O, at 37°C for 20 min, and react at 85°C for 5 s. Finally, cDNA is obtained and stored at -20°C for standby.

[0036] 4. Design specific primers and use high-fidelity KOD enzyme to amplify the CDS sequence of BrNAC2a. Among them, the sequence information of the designed specific primers is shown in Table 1. The amplified PCR products are separated by 0.8% agarose gel electrophoresis. If the length is consistent, cut the gel and recover the target fragment for standby.

[0037] Table 1: Primers used for constructing heterologous expression vectors

[0038]

[0039] 5. Homologous recombination ligation:

[0040] Ligate the above linearized vector and the PCR-amplified and purified fragment by homologous recombination: Use the single-fragment homologous recombination kit C112 from Novizan. The system is: 4 μl of 5×CEⅡBuffer, 2 μl of ExaseⅡ, 200 ng of the double-digested and recovered linearized vector, 20 ng of the CDS fragment of BrNAC2a, and make up to 20 μl with ddH2O; React at 37°C for 30 min.

[0041] 6. Transform Escherichia coli, verify the obtained bacterial liquid by PCR, extract the target plasmid and verify it by sequencing. Transform the plasmid with successful sequence alignment into Agrobacterium competent cells by electroporation. The transformed bacterial liquid has bands by PCR, and then culture it with an enlarged volume; Extract the plasmid and verify it by sequencing. The map of the BrNAC2a overexpression vector obtained is as Figure 1 shown. Preserve the strain of the bacterial liquid with successful sequencing verification and store the mother liquid at 4°C for standby.

[0042] Example 2: Transformation of Arabidopsis thaliana by the floral dip method and screening of positive transformants

[0043] 1. Transform Arabidopsis thaliana by the floral dip method:

[0044] The above-mentioned bacterial solution verified by sequencing was used as the mother solution, and Arabidopsis thaliana was transformed by the floral dip method as follows: 500 μl of the Agrobacterium tumefaciens solution 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 with shaking at 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 the bacterial solution was resuspended with 200 ml of 5% sucrose; Silwet-77 was added to a final concentration of 200 μl / L, and shaken at 28 °C and 200 rpm for 2 min; the wild-type WT Arabidopsis thaliana was removed of open flowers and siliques, the flower buds were immersed in the bacterial solution for 60 s, the excess bacterial solution was blotted dry, and cultured in the dark at 25 °C with humidity for 24 h, and then cultured normally, and the floral dip was repeated once after one week.

[0045] 2. Preparation of kanamycin sowing medium:

[0046] 2.22 g of MS powder and 10 g of sucrose were added with 2 M NaOH to adjust the pH to 5.8 to obtain MS medium. After adding 4 g of agar powder, it was autoclaved at 121 °C for 20 min, and then placed in a laminar flow hood and cooled to 50 - 60 °C. Kanamycin was added to a final concentration of 75 mg / L, and solid plates were poured.

[0047] 3. Screening of positive transformants:

[0048] The T1 generation of Arabidopsis thaliana seeds obtained by floral dip were sown on the kanamycin sowing medium as follows: disinfected with 10 vol% NaClO for 2 min; washed with 75 vol% ethanol for 2 min; rinsed with sterile water 5 times, 1 min each time; sown on the kanamycin sowing medium, sealed, and cultured in an incubator at 22 °C (16 h light, 8 h dark). After about two weeks, the positive plants with strong growth and dark green leaf color were removed and verified by PCR detection. Among them, the primers used for PCR detection are shown in Table 2.

[0049] Table 2: Primers used for PCR detection of transgenic Arabidopsis thaliana

[0050]

[0051] Example 3: Screening of T-DNA insertion mutant materials

[0052] 1. DNA extraction by rapid DNA extraction method: Add 200 μl of DNA extraction buffer and magnetic beads to a centrifuge tube containing Arabidopsis thaliana leaves, and use a crusher to crush for 120 s to break the leaf tissue, where 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 a new 1.5 ml centrifuge tube, 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 empty for 1 min), suck out the liquid, air-dry the precipitate for 5 min, add 25 - 50 μl of ddH2O, and store at -20 °C for later use.

[0053] 2. PCR detection by three - primer method: The three primers are shown in Table 3; 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, 2 μl of Primer R; The program is set for pre - denaturation at 98 °C for 2 min 30 s, and then enter 35 cycles of amplification (denaturation at 98 °C for 10 s, annealing at 55 °C for 10 s, extension at 72 °C for 10 s), and finally fully extend at 72 °C for 2 min to ensure complete amplification of the fragment; The PCR products amplified by LP + RP primers and the PCR products amplified by RP+BP primers are separated by 0.8 wt% agarose gel electrophoresis, and 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 individual containing only a single small fragment is the mutant material SALK_137131, and seeds are harvested for subsequent experiments.

[0054] Table 3: Primers used for screening T - DNA insertion mutant materials

[0055]

[0056] Example 4: Real - time fluorescence quantitative PCR

[0057] 1. Analyze the change in BrNAC2a expression level by real - time fluorescence quantitative PCR: Take Arabidopsis thaliana pFGC1008:For the BrNAC2a overexpressing plant leaves and the control group of wild-type Arabidopsis thaliana, at least 10 plants of each were sampled together, marked, and quickly placed in liquid nitrogen for fixation. After all the sampling was completed, total RNA was extracted and reverse transcribed into cDNA, and then qRT-PCR analysis was carried out. The primers used for qPCR analysis were designed by Primer Premier 6 and are shown in Table 4. 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: 30 s, 40 cycles (95°C: 5 s, 55°C: 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.

[0058] Finally, the qRT-PCR identification results of three lines of BrNAC2a overexpressing plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ) and the control group of Arabidopsis plants are shown in Figure 3 . It can be seen from this that BrNAC2a is up-regulated in the overexpressing plants compared with the control group of Arabidopsis plants.

[0059] Table 4: Primers used for qRT-PCR analysis of Arabidopsis plants

[0060]

[0061] Example 5: Identification of leaf size of transgenic Arabidopsis plants

[0062] 1. Observation of phenotypes of transgenic Arabidopsis:

[0063] The homozygous T-DNA insertion mutant plant SALK_137131 and the BrNAC2a overexpressing plants were sown simultaneously with the wild-type WT Arabidopsis thaliana in a light incubator and cultured for about 14 d, and the leaf development was observed; 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 leaves were measured. According to the calculation results, the significance of the difference in leaf size among the plants in each group was analyzed.

[0064] The observation results of the phenotypes of the T-DNA insertion mutant SALK_137131 plants, Chinese cabbage BrNAC2a overexpressing plants, and the control group of wild-type WT Arabidopsis plants after sowing for 14 d are shown in Figure 4As shown, it can be seen that the leaf size of the Chinese cabbage BrNAC2a overexpression plants is significantly smaller than that of the T-DNA insertion mutant SALK_137131 plants and the control group plants. After overexpressing this gene in wild-type WT Arabidopsis thaliana, the leaves of the overexpression lines are smaller than those of the wild type, while there is no significant difference in the leaf size between the T-DNA insertion Arabidopsis mutant plants and the wild type.

[0065] 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 overexpression plants, and the control wild-type WT Arabidopsis plants were obtained, as Figure 5 shown; Figure 5 The leaves of the SALK_137131 plants, Chinese cabbage BrNAC2a overexpression plants, and the control wild-type WT Arabidopsis plants in Figure 6 were sorted from smallest to largest, and the sorting results of the leaf sizes of the three lines of SALK_137131 plants, Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ) and the control wild-type WT Arabidopsis plants were obtained; the maximum leaf lengths of the T-DNA insertion mutant SALK_137131 plants, three lines of Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a -2 and pFGC1008: BrNAC2a -3 ) and the control wild-type WT Arabidopsis plants were statistically analyzed, and the results are as Figure 7 shown. Different letters therein represent significant differences. It can be seen that the maximum leaf length of the Chinese cabbage BrNAC2a overexpression plants is significantly smaller than that of the control group, while there is no significant difference in the maximum leaf length between the T-DNA insertion mutant SALK_137131 plants and the control; the maximum leaf widths of the T-DNA insertion mutant SALK_137131 plants, three lines of Chinese cabbage BrNAC2a overexpression plants ( pFGC1008: BrNAC2a -1 , pFGC1008: BrNAC2a - 2 and pFGC1008: BrNAC2a -3 ) and the control wild-type WT Arabidopsis plants were statistically analyzed, and the results are as Figure 8As shown, where different letters represent significant differences. It can be seen that the maximum leaf width of the Chinese cabbage BrNAC2a overexpression plants is significantly smaller than that of the control group, while there is no significant difference in the maximum leaf width of the T-DNA insertion mutant SALK_137131 plants compared with the control. Comparative analysis of the maximum leaf length and maximum leaf width of each group of plants is as Figure 5 - Figure 8 shown. It can be seen 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. There is no significant difference in the maximum leaf length and maximum leaf width of the SALK_137131 Arabidopsis line compared with the wild-type WT Arabidopsis. The results show that Chinese cabbage BrNAC2a plays a negative regulatory role in the development of plant leaves, while AtNAC2 Arabidopsis does not have this function.

[0066] The above are the preferred specific embodiments of the present invention. These embodiments are for better explaining the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. However, all details are not described in detail in these embodiments, 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. Application of overexpression of Chinese cabbage leaf size-related gene BrNAC2a in negatively regulating Arabidopsis leaf size, characterized in that, The BrNAC2a has a nucleotide sequence shown in SEQ ID No.1.

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