A rapeseed gene BnNAC022 and its application

Abstract

The present invention relates to a rapeseed gene BnNAC022 and its application, belonging to the technical field of plant genetic engineering. This gene has a nucleotide sequence as shown in SEQ ID NO.1. In the present invention, by constructing a recombinant plasmid and introducing the rapeseed gene BnNAC022 as the target gene into the genome of cruciferous plants, it is found that overexpression of this gene can improve the insect resistance of cruciferous plants, indicating that the rapeseed gene BnNAC022 is involved in the regulation of insect resistance in cruciferous plants. The discovery of the gene in the present invention can provide gene resources for crop breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a rapeseed gene BnNAC022 and an application thereof. Background Art

[0002] At present, insect pests are one of the major problems affecting rapeseed production, causing huge economic losses to agricultural production. The methods for controlling insect pests are mainly divided into traditional methods such as chemical control, biological control, physical control and agricultural control. Among them, agricultural control is an important means of insect pest control because it takes the overall concept of agricultural ecosystem as its starting point. Its goal is to increase crop yields, improve farmland environment, select suitable crop varieties, create an environment suitable for crop growth but not conducive to the survival and growth of pests, so as to control the pest density within the range allowed by economic losses. In the long-term "competition and confrontation" with pests, plants have evolved a variety of means and mechanisms to resist insect pests. In actual production, the use of insect-resistant varieties and strengthening of plant defense systems are one of the most environmentally friendly and economically feasible insect control methods.

[0003] NAC transcription factors are a complex plant-specific family and the fourth largest transcription factor family in plants. They are widely present in many species. NAC transcription factors can participate in regulating many biological processes of plant growth and development, including response to external stress, floral organ formation, establishment of organ boundaries and plant morphology, secondary cell wall thickening, stem and root apical meristem formation, lateral root development, fiber development, senescence regulation and fruit development. In addition, more and more studies have shown that NAC transcription factors also play an important regulatory role in fruit ripening. Although NAC transcription factors were originally discovered due to their functions in plant development, their role in plant response to various stresses (including abiotic and biotic stresses) has also received increasing attention. However, there are currently few reports on the mechanism of NAC transcription factors responding to insect pest stress and participating in insect resistance in rapeseed. The molecular pathways and working mechanisms of NAC transcription factors in regulating rapeseed insect resistance remain to be further explored. Summary of the invention

[0004] The purpose of the present invention is to provide a rapeseed gene BnNAC022 and its application in order to solve the above problems.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0006] A rapeseed gene BnNAC022 has a nucleotide sequence as shown in SEQ ID NO.1.

[0007] Application of a rapeseed gene BnNAC022 in regulating insect resistance of cruciferous plants.

[0008] As a further optimization scheme of the present invention, the overexpression of the rapeseed gene BnNAC022 can improve the insect resistance of cruciferous plants.

[0009] As a further optimization scheme of the present invention, the cruciferous plants are Arabidopsis thaliana and rapeseed.

[0010] As a further optimization scheme of the present invention, the insect is aphid.

[0011] A recombinant plasmid is obtained by transferring the rapeseed gene BnNAC022 onto a vector.

[0012] As a further optimization scheme of the present invention, the vector is pCAMBIA 1301a.

[0013] A method for obtaining a transgenic cruciferous plant variety with high insect resistance is to introduce the rapeseed gene BnNAC022 as a target gene into the genome of cruciferous plants for overexpression, and cultivate to obtain a transgenic cruciferous plant variety with high insect resistance.

[0014] The beneficial effects of the present invention are as follows:

[0015] By constructing a recombinant plasmid and introducing the rapeseed gene BnNAC022 as a target gene into the genome of cruciferous plants, it is found that the overexpression of this gene can improve the insect resistance of cruciferous plants, indicating that the rapeseed gene BnNAC022 is involved in the regulation of insect resistance in cruciferous plants. The discovery of the gene in the present invention can provide gene resources for crop breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a cloning diagram of the rapeseed gene BnNAC022;

[0017] Figure 2 is a schematic diagram of the pCAMBIA 1301a vector structure;

[0018] Figure 3 is a phenotypic diagram of BnNAC022 transgenic plants and wild-type plants (CK);

[0019] Figure 4 is a phenotypic analysis diagram of BnNAC022 transgenic plants and wild-type plants (CK) against aphids. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The following further describes the present application in detail with reference to the drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] 1. Materials

[0022] Unless otherwise specified, the methods used in this example are conventional methods known to those skilled in the art. The reagents and other materials used are commercially available products unless otherwise specified.

[0023] 2. Methods

[0024] 2.1 Extraction of total RNA (Trizol method)

[0025] (1) Prepare RNase-free EP tubes (2.0 mL) and pre-cool them in a 4°C centrifuge.

[0026] (2) Pre-cool a mortar and pestle with liquid nitrogen. Put an appropriate amount of leaf tissue of Brassica napus variety Heyou 202 into the mortar, add liquid nitrogen, wait for the liquid nitrogen to evaporate, quickly and thoroughly grind, add liquid nitrogen again, continue to grind, repeat once, until the sample becomes a fine powder, the whiter the color, the better. Then use the thick end of a pre-cooled 1 mL RNase-free pipette tip to transfer 200 - 300 mg of the powder to a pre-cooled 2.0 mL RNase-free EP tube.

[0027] (3) Add 1 mL of pre-cooled Trizol solution, cover the lid, quickly shake on a vortex shaker for 30 - 60 s to mix well, place on ice, protect from light, and let stand for 10 min.

[0028] (4) Add 500 μl of chloroform, invert and mix well, place on ice, protect from light, and let stand for about 5 min.

[0029] (5) Take out the sample and place it in a pre-cooled centrifuge, centrifuge at 4°C, 13000 rpm for 6 min.

[0030] (6) After centrifugation, take about 600 μl of the supernatant (the amount of the supernatant can be determined according to the actual situation) and transfer it to a new 1.5 mL RNase-free EP tube. Be sure to avoid sucking out the precipitate during this process. Then add an equal volume of isopropanol and invert to mix well, without vigorous shaking.

[0031] (7) Centrifuge the mixed solution obtained in step (6) at 4°C, 13000 rpm for 10 min.

[0032] (8) Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water), mix well, and can shake on a shaker for a few seconds. Then centrifuge at 4°C, 13000 rpm for 5 min, and discard the supernatant.

[0033] (9) Repeat step (8), discard the supernatant, centrifuge again, aspirate the remaining 75% ethanol solution with an RNase-free pipette tip, and air-dry in a fume hood for 10 min;

[0034] (10) Add 50 μl of DEPC water to dissolve the RNA, gently pipette to mix well, then measure the purity and concentration of the extracted RNA with a nucleic acid detector, and the integrity of the RNA can be detected by agarose gel electrophoresis. Store the remaining RNA at -80 °C.

[0035] 2.2 Obtain cDNA by reverse transcription of RNA

[0036] Perform reverse transcription reaction using the reverse transcription kit (323) from Novoprotein Scientific Inc., and operate strictly according to the actual instruction manual. The specific steps are as follows:

[0037] (1) Genomic DNA removal

[0038] Prepare the reaction solution in an RNase-free centrifuge tube (the components are shown in Table 1), gently pipette to mix well, and incubate at 42 °C for 2 min:

[0039] Table 1 Dosage of each component of the reaction solution

[0040] Reagent Name Dosage RNase-free ddH2O 16 μl 4×gDNA wiper Mix 4 μl Template RNA (Total RNA) 1 pg–1 μg

[0041] (2) Prepare the reverse transcription reaction system and perform the reverse transcription reaction

[0042] Take 16 μl of the reaction solution obtained in step (1), add 4 μl of 5×HiScript III qRT Super Mix, and gently pipette to mix well for reverse transcription reaction. The specific temperature of the reverse transcription reaction is shown in Table 2:

[0043] Table 2 Temperature and time of reverse transcription reaction

[0044] Reaction Temperature Time (*)37℃ 45 min 85℃ 5 sec

[0045] Note: If the template has a complex secondary structure or a high GC region, the reaction temperature (*) can be increased to 50 °C, which helps to increase the yield.

[0046] The product after the reverse transcription reaction is cDNA. Store the product at -20 °C, but it needs to be used within half a year; if stored for a long time (more than half a year), it should be aliquoted and stored at -80 °C. In addition, cDNA should be avoided from being repeatedly frozen and thawed to avoid degradation.

[0047] 2.3 Construct recombinant plasmid

[0048] Design primers for the published rapeseed gene BnNAC022 CDS sequence in the database (as shown in SEQ ID NO.1, sequence source: gene ID GSBRNA2T00087539001) using homologous recombination method and perform PCR amplification reaction. The PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4. As Figure 1 shown, the PCR product (the amplified rapeseed gene BnNAC022 sequence) is detected by 0.1% agarose gel electrophoresis.

[0049] Insert the cloned rapeseed gene BnNAC022 CDS sequence into the multiple cloning site (BAMHI restriction site) of the vector pCAMBIA 1301a as Figure 2 shown (the pCAMBIA 1301a vector inserts the CaMv35S promoter sequence at the MCS of pCAMBIA 1301 through double digestion with EcoRI and SacI. This promoter is used to initiate the target sequence to achieve the purpose of overexpression) to obtain the recombinant plasmid (pCAMBIA1301a-BnNAC022), and then it can be transformed into Escherichia coli.

[0050] Table 3 PCR reaction system for amplifying the target gene

[0051] Reagent Name Dosage 2×Phanta Max Master Mix (DyePlus) 25 μl SEQ ID NO.2: Forward Primer F (10 μM) 2 μl SEQ ID NO.3: Reverse Primer R (10 μM) 2 μl cDNA 2 μl ddH2O 19 μl Total (Total Volume) 50 μl

[0052] Table 4 PCR reaction program for amplifying the target gene

[0053]

[0054] Regarding the extraction of Escherichia coli plasmid, the specific steps are as follows:

[0055] (1) Add the overnight turbid bacterial liquid into a 2 ml centrifuge tube, centrifuge at 12000 r / min for 1 min, and discard the supernatant; add the bacterial liquid again and centrifuge once to enrich the bacterial liquid, discard the culture medium, and invert it on the absorbent paper to suck out the residual liquid;

[0056] (2) Add 250 μl of Buffer P1 (RNase A has been added to Buffer P1) to the centrifuge tube with the bacterial pellet, mix well with a pipette or vortex oscillator to fully disperse the bacterial pellet;

[0057] (3) Add 250 μl of Buffer P2 to the bacteria treated in step (2), gently invert and mix 8 - 10 times to fully lyse the bacteria;

[0058] (4) Add 350 μl of Buffer P3 to the bacterial cells treated in step (3), and immediately invert gently up and down 8 - 10 times to thoroughly neutralize Buffer P2. At this time, a white flocculent precipitate should appear. Centrifuge at 12,000 r / min for 10 min.

[0059] (5) Place the Fast Pure DNA Mini Column adsorption column in a 2 ml collection tube. Carefully transfer the supernatant from step (4) to the adsorption column using a pipette, taking care not to aspirate the precipitate. Centrifuge at 12,000 r / min for 30 - 60 s, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0060] (6) Add 600 μl of Buffer PW2 (diluted with absolute ethanol) to the adsorption column. Centrifuge at 12,000 r / min for 30 - 60 s, discard the waste liquid, and put the adsorption column back into the collection tube.

[0061] (7) Repeat step (6).

[0062] (8) Place the adsorption column back into the collection tube and centrifuge at 12,000 r / min for 1 min to dry the adsorption column, aiming to completely remove the residual washing solution in the adsorption column.

[0063] (9) Place the adsorption column in a new sterilized 1.5 ml centrifuge tube. Add 30 - 100 μl of Elution Buffer to the center of the membrane of the adsorption column. Let it stand at room temperature for 2 min and centrifuge at 12,000 r / min for 1 min to elute the plasmid.

[0064] (10) Discard the adsorption column. The obtained Escherichia coli plasmid is stored at -20 °C for later use and for sequencing and comparison results.

[0065] 2.4 Transformation of the recombinant plasmid into Agrobacterium tumefaciens (GV3101)

[0066] (1) Take out the competent cells of Agrobacterium tumefaciens GV3101 from the -80 °C refrigerator, place them on ice to thaw, add 2 μl of the recombinant plasmid (pCAMBIA 1301a - BnNAC022), and add the mixture to a 1.5 ml EP tube to transform Agrobacterium tumefaciens.

[0067] (2) Incubate on ice for 5 min, in liquid nitrogen for 5 min, heat shock at 37 °C for 5 min, and then incubate on ice for 5 min.

[0068] (3) Add 700 μl of antibiotic - free LB solution (10 g peptone, 10 g sodium chloride, 5 g yeast extract / L) to the EP tube, and shake the bacteria at 28 °C, 220 rpm in the dark for 3 - 5 h.

[0069] (4) Take 50 μl and spread it on an LB plate containing kanamycin and rifampicin resistance, and incubate it in the dark at 28 °C for 48 h;

[0070] (5) Wait for the appearance of colonies, verify by colony PCR, shake the bacteria, and expand the culture to obtain the Agrobacterium liquid containing the recombinant plasmid, and store it for later use.

[0071] 2.5 Sterilization treatment and planting of Arabidopsis thaliana seeds

[0072] Arabidopsis thaliana belongs to the Brassicaceae family, Angiospermae, Dicotyledoneae. The advantages of Arabidopsis thaliana are its small plant size and high seed production. The genome of Arabidopsis thaliana is the smallest among known plant genomes. Arabidopsis thaliana is a self-pollinating plant with highly homozygous genes. In the process of variety selection in rapeseed breeding, the model plant Arabidopsis thaliana of the same family is usually used as the initial research object because the two have similar morphological and structural characteristics and similar molecular regulatory mechanisms in many developmental processes.

[0073] 2.5.1 Surface sterilization treatment of Arabidopsis thaliana seeds

[0074] (1) Take an appropriate amount of Arabidopsis thaliana seeds into a 2.0 mL centrifuge tube, add 12% Kao Wang solution in a laminar flow hood and treat for 10 min. During this period, the centrifuge tube needs to be constantly inverted and shaken to make the seeds fully contact with the disinfectant;

[0075] (2) Aspirate the 12% Kao Wang solution in the centrifuge tube, and then wash it 6 - 8 times with sterile water, shaking well for 1 - 2 min each time;

[0076] (3) After washing, add an appropriate amount of sterile water, and place the Arabidopsis thaliana seeds at 4 °C for vernalization for 3 days under dark conditions.

[0077] 2.5.2 Planting of wild-type Arabidopsis thaliana

[0078] (1) Evenly sow the vernalized seeds on a 1 / 2 MS solid medium (Ms 2.2 g, MES 0.5 g, sucrose 10 g; adjust the pH to 5.7 - 5.8 with NaOH), place it vertically in a growth chamber, at 25 °C, with a light intensity of 16 h-light / 8 h-dark;

[0079] (2) Mix vermiculite and sieved black soil after autoclaving in a volume ratio of 3:1, divide them into small square pots (7 cm × 7 cm × 10 cm), place the square pots in a tray, add tap water to the bottom of the tray to slowly soak and moisten the nutrient soil and vermiculite;

[0080] (3) When Arabidopsis thaliana grows to 6 - 8 days old and the root length is about 6 cm, open the culture dish, gently transplant Arabidopsis thaliana to the pre-prepared nutrient soil with forceps, being careful not to damage the root system. Press the roots of Arabidopsis thaliana with an appropriate amount of soil and cover it with plastic wrap to prevent water loss from the seedlings;

[0081] (4) One week later, when the seedlings grow stably, the plastic wrap can be removed to allow them to grow normally. Water and fertilize at appropriate times, and pay attention to preventing diseases and pests. When Arabidopsis thaliana blooms, infect it.

[0082] 2.6 Agrobacterium - mediated transformation of Arabidopsis thaliana

[0083] (1) After activating the Agrobacterium liquid containing the recombinant plasmid (obtained in step 2.4), take 500 μl and transfer it to 50 mL of liquid LB medium containing antibiotics (kanamycin, rifampicin) for enlarged culture. Incubate in the dark at 28 °C and 220 rpm for 36 - 48 h;

[0084] (2) Collect the bacterial liquid with a 50 mL centrifuge tube, centrifuge at 3000 rpm for 10 min at room temperature, and discard the supernatant;

[0085] (3) Add 15 mL of Arabidopsis transformation buffer (0.22 g of Ms, 0.05 g of MES, 5 g of sucrose, 30 μl of silwetl7), fully suspend the bacterial cells and mix well;

[0086] (4) Use a Pasteur pipette to suck a certain amount of the suspension and drop it onto the stigmas of the Arabidopsis thaliana about to bloom to be infected (obtained in step 2.5.2) in turn. After the infection, cover the infected plants with a black plastic bag and remove it 24 h later;

[0087] (5) One week later, repeat the infection step as above to improve the transformation efficiency;

[0088] (6) After two infections, the growth state of Arabidopsis thaliana may deteriorate. Water and fertilize at appropriate times and pay attention to preventing diseases and pests;

[0089] (7) When most of the siliques of the infected Arabidopsis thaliana turn yellow and mature, stop watering and successively harvest the T0 generation of transgenic seeds.

[0090] 2.7 Screening of transgenic positive lines of Arabidopsis thaliana

[0091] (1) Disinfect the T0 generation of transgenic Arabidopsis thaliana seeds, vernalize them, and then evenly sow them on a 1 / 2MS solid medium containing 25 mg / L hygromycin. Place them vertically in an artificial climate chamber for growth at 25 °C with a light cycle of 16 h - light / 8 h - dark;

[0092] (2) Grow in the greenhouse for 7 - 10 days (such asFigure 3 As shown in the figure, the Arabidopsis thaliana that can grow normally on the plate is the transgenic positive seedling, and it is transplanted into the nutrient soil;

[0093] (3) When the transgenic positive seedling Arabidopsis thaliana grows to almost flowering, take 1-2 leaves from each plant in turn, extract DNA and perform PCR verification.

[0094] 3 Experimental verification

[0095] 3.1.1 DNA extraction from transgenic plants

[0096] (1) Put the young and tender leaves of the above-mentioned transgenic Arabidopsis thaliana into a 2 mL centrifuge tube, add steel beads and liquid nitrogen, and quickly freeze;

[0097] (2) Vigorously grind the leaves until they become powdery;

[0098] (3) Add 500 μl of 2×CTAB extraction buffer (pre-warmed at 65 °C), shake for 30 s, mix well and incubate in a water bath at 65 °C for about 1 h, during which invert the tube up and down;

[0099] (4) Take out the centrifuge tube, add an equal volume of chloroform and isoamyl alcohol (chloroform:isoamyl alcohol is 24:1), shake for 30 s, and centrifuge at 12000 r / min at room temperature for 15 min;

[0100] (5) Take 300 μl of the supernatant and transfer it to a new 1.5 mL centrifuge tube, add 2 volumes of absolute ethanol, invert and mix well, let stand for 15 min, centrifuge at 12000 r / min at room temperature for 15 min, and discard the supernatant;

[0101] (6) Add 1 mL of 75% alcohol, invert the tube several times to wash the precipitate, centrifuge at 12000 r / min for 5 min, and repeat once;

[0102] (7) After removing the washing solution, centrifuge again at 12000 r / min for 10 s to remove all the supernatant, and finally dry it in a fume hood;

[0103] (8) Add 50 μl of sterile water to the centrifuge tube, pipette and blow repeatedly to dissolve it completely to obtain the DNA of the transgenic plant, and store it for later use.

[0104] 3.1.2 Amplification of the target fragment of transgenic plants

[0105] Using the DNA extracted in 3.1.1 as a template, perform PCR amplification. The PCR reaction system is shown in Table 5, and the PCR reaction program is shown in Table 6. After the PCR reaction is completed, perform electrophoresis detection. The band with the correct size is the transgenic positive seedling:

[0106] Table 5 PCR reaction system for amplifying the target fragment of transgenic plants

[0107] Template DNA 2 μl SEQ ID NO.4: F-Primer 1 μl SEQ ID NO.5: R-Primer 1 μl Taq Mix Enzyme 12.5 μl ddH2O 8.5 μl Total Volume 25 μl

[0108] Table 6 PCR reaction procedure for amplifying target fragments of transgenic plants

[0109]

[0110] 3.1.3 Indoor identification experiment on aphid resistance of transgenic plants

[0111] When the transgenic positive seedlings (BnNAC022) and wild type (CK) grow to the five-leaf stage, they are used for insect inoculation identification. There are 12 transgenic positive seedlings and 12 wild types respectively. 2 aphids are inoculated on each plant, and the investigation is carried out once every 5 days. Record the total aphid amount of each plant (the transgenic plants and wild type plants are placed in insect cages respectively). Specifically, as Figure 4 shown

[0112] Experimental conclusion: Compared with the wild type, the transgenic Arabidopsis thaliana overexpressing the rapeseed gene BnNAC022 shows a stronger aphid-resistant phenotype, which can prove that the overexpression of the rapeseed gene BnNAC022 has the effect of improving the insect resistance of cruciferous plants

[0113] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention

Claims

1. Application of a rapeseed gene BnNAC022 in regulating insect resistance of cruciferous plants, characterized in that The rapeseed gene BnNAC022 has a nucleotide sequence as shown in SEQ ID NO.

1. Overexpression of the rapeseed gene BnNAC022 can improve the aphid resistance of Arabidopsis thaliana.

2. A method for obtaining a transgenic cruciferous plant variety with high insect resistance, characterized in that, Introduce the rapeseed gene BnNAC022 as the target gene into the Arabidopsis thaliana genome for overexpression, and cultivate a transgenic Arabidopsis thaliana variety with high aphid resistance; the rapeseed gene BnNAC022 has the nucleotide sequence shown in SEQ ID NO.1.

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