BnaCDPK10 gene for regulating length of silique in brassica napus and application thereof

Abstract

The application discloses rape with regulated silique length BnaCDPK10 Gene and application thereof, and relates to the technical field of plant molecular biology. BnaCDPK10 The nucleotide sequence of the gene is shown as SEQ ID NO. 3, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4. BnaCDPK10 The application constructs a transgenic overexpression vector of the gene, and obtains a transgenic Arabidopsis thaliana plant overexpressing the gene. BnaCDPK10 It is found that the length of the silique can be obviously increased after the gene is overexpressed, and the gene has the function of positively regulating the length of the silique. The gene is transferred into crops and horticultural plants, and has good application potential in increasing crop yield.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, specifically to a method for regulating silique length in rapeseed. BnaCDPK10 Genes and their applications. Background Technology

[0002] Plant growth and development are primarily regulated by genetic and environmental changes. Plant cell signal transduction refers to the process by which cells sense and transduce various environmental stimuli, regulate gene expression, and elicit corresponding physiological and biochemical responses. Numerous studies have shown that Ca²⁺ acts as a second messenger in numerous plant stimulus responses. Almost all different extracellular stimulus signals, such as light, touch, gravity, plant hormones, and pathogens, can cause a transient and significant increase in intracellular free calcium ion concentration [Ca²⁺], or changes in its gradient and regional distribution within the cell. Calcium concentration changes can respond to different extracellular stimuli, ultimately leading to specific physiological effects on specific stimuli (J. Kudla, O. Batistič, K. Hashimoto. Calciumsignals: the lead currency of plant information processing. Plant Cell, 2010, 22:541−563). When plants are stimulated by different factors, the concentration of calcium ions in the cells changes first. This change in calcium ion concentration is called calcium signaling. Decoding calcium ion signals depends on calcium sensors. In plants, there are four main types of calcium sensors: calmodulin (CaM), CaM-like protein (CML), calcineurin-like protein (CBL), and CDPKs (calcium-dependent protein kinases) (DeFalco, TA, Bender, KW and Snedden, WABreaking the code: Ca²⁺ sensors in plant signalling. Biochem J, 2010, 425:27−40). These sensors directly bind to or phosphorylate target proteins to activate them, thereby transducing signals. Unlike other calcium sensors, CDPKs not only have an EF-hand domain that binds to calcium ions but also a kinase domain.When the concentration of calcium ions in the body is low, the self-inhibition linker domain of calcium-dependent protein kinase binds to the kinase domain to inhibit its own activity. When the concentration of calcium ions increases, calcium ions bind to the EF motif, changing the conformation and activating the kinase (V. Chandran, EJStollar, K. Lindorff-Larsen, JF Harper, WJ Chazin, CM Dobson, BFLuisi, J. Christodoulou. Structure of the regulatory apparatus of acalcium-dependent protein kinase (CDPK): a novel mode of calmodulin-target recognition. J Mol Biol, 2006, 357:400−410).

[0003] rape( Brassica napus Rapeseed (L.) is one of the most important oilseed crops in the world and the largest oilseed crop in my country (Wang Hanzhong. Development Strategy of Rapeseed Industry Guided by New Demands. Chinese Journal of Oil Crops, 2018, 40(5):613−617). Increasing yield is one of the main breeding goals of rapeseed, and the length of rapeseed siliques is a key factor affecting yield. Calcium-dependent protein kinases (CDPKs) are the main signal transduction factors, participating in the regulation of plant stress resistance and plant growth, but there are few reports on CDPKs in rapeseed, and their regulatory mechanism in rapeseed silique development is still unclear. Therefore, exploring the regulation of rapeseed silique development by CDPKs in response to changes in calcium ions, and studying their downstream target proteins and pathways, is not only of great significance for revealing the molecular mechanism of rapeseed silique development, but also provides new ideas for high-yield breeding of rapeseed. Summary of the Invention

[0004] The purpose of this invention is to regulate the length of rapeseed siliques in response to changes in intracellular calcium ion concentration. BnaCDPK10 Genes and their applications, this invention demonstrates BnaCDPK10 As a factor that can respond to fluctuations in calcium ion concentration and positively regulate the length of rapeseed pods, this study delves into its regulatory mechanism in rapeseed to provide theoretical support for breeding work and may offer new genetic resources for high-yield crop breeding.

[0005] To achieve the above objectives, the present invention provides a rapeseed method for regulating silique length. BnaCDPK10 Genes, the BnaCDPK10 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0006] The present invention also provides the above-mentioned rapeseed BnaCDPK10 The protein encoded by the gene, the amino acid sequence of which is shown in SEQ ID NO.4.

[0007] The rapeseed provided by this invention BnaCDPK10 Genes or the proteins they encode can be used to regulate the length of siliques in plants and can also be used to increase crop yield.

[0008] Preferably, the application includes increasing the length of the silique.

[0009] The present invention also provides an overexpression vector for regulating silique length, the overexpression vector comprising rapeseed with the nucleotide sequence shown in SEQ ID NO.3. BnaCDPK10 Gene.

[0010] Preferably, the overexpression vector is selected from the vector pBin35SRed3.

[0011] The present invention also provides a recombinant expression bacterium for regulating silique length, the recombinant expression bacterium comprising the above-mentioned rapeseed. BnaCDPK10 Gene or overexpression vector.

[0012] Preferably, the recombinant expression bacteria are selected from Agrobacterium rhizogenes, and more preferably from Agrobacterium GV3101.

[0013] This invention also provides a method for increasing the length of siliques in rapeseed plants, comprising: constructing a rapeseed plant containing a nucleotide sequence as shown in SEQ ID NO.3. BnaCDPK10 Gene expression vectors are used to introduce gene expression vectors into plants, resulting in overexpressed rapeseed. BnaCDPK10 The plant with the gene is a transgenic plant with increased silique length.

[0014] The rapeseed of the present invention for regulating silique length BnaCDPK10 Genes and their applications have the following advantages:

[0015] This invention provides a method for controlling the length of siliques in rapeseed. BnaCDPK10 The gene was developed, and a transgenic expression vector containing this gene was constructed. This gene was then transferred into plant cells and expressed, effectively influencing the length of the siliques. Therefore, transferring this gene into crops and horticultural plants has great potential for increasing crop yields. Attached Figure Description

[0016] Figure 1 The rapeseed of this invention BnaCDPK10 Gene expression levels in ZS11 siliques after 24 hours of induction with 10 mmol / L Ca²⁺.

[0017] Figure 2 The rapeseed of this invention BnaCDPK10Results of gene expression levels at different developmental stages and in other tissues of ZS11 siliques.

[0018] Figure 3 The rapeseed of this invention BnaCDPK10 Results of RNA electrophoresis in gene-overexpressing plants.

[0019] Figure 4 The electrophoresis results are for the amplified target fragment of this invention.

[0020] Figure 5 The electrophoresis results of positive clones of the transformed and ligated overexpression vector in this invention are shown.

[0021] Figure 6 The results of enzyme digestion and electrophoresis of the expression vector pBin35SRed3 plasmid of this invention are shown.

[0022] Figure 7 For the present invention BnaCDPK10 Electrophoresis results for identifying transgenic positive plants.

[0023] Figure 8 The rapeseed of this invention BnaCDPK10 Relative expression levels of genes in overexpressed plants and WT.

[0024] Figure 9 This is a comparison of the morphological results of OE-BnaCDPK10 and WT siliques in this invention.

[0025] Figure 10 This is the quantitative result of the OE-BnaCDPK10 and WT silique length of the present invention.

[0026] Figure 11 This is the quantitative result of the number of fruits per corner of OE-BnaCDPK10 and WT according to the present invention. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0028] The plant materials, vectors, and strains used in the following experimental examples are detailed below:

[0029] DL704, a large-grained Brassica napus-type rapeseed, was created by our research team through distant hybridization in the previous stage. Zhongshuang 11 (ZS11) is a Brassica napus-type rapeseed variety bred by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences. Using ZS11 as the recurrent parent and DL704 as the recipient parent, a BC4F2 population was constructed. DL704, ZS11, and BC4F2 populations were all planted at the experimental base of the Rapeseed Engineering Technology Research Center in Beibei District, Chongqing. Arabidopsis thaliana, Columbia ecotype wild type, was grown in a light-incubator. The incubator settings were: 8 hours in darkness, 16 hours in light, humidity 60% under light / 40% under darkness, temperature 16 ℃ under light / 22 ℃ under darkness, and light intensity 0 Lx under darkness / 10000 Lx under light.

[0030] The selected vector was the plant expression vector pBin35SRed3, and the strains used included DH5α Escherichia coli competent strain (Qingke Biotechnology) and GV3101 Agrobacterium rhizogenes competent strain (Qingke Biotechnology).

[0031] The culture media and reagents used in the following experimental examples are as follows:

[0032] LB medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride. For solid medium, add 15 g / L agar powder, adjust pH to 6.8, sterilize at 121 ℃ for 20 min.

[0033] CTAB Buffer: 20g / L CTAB, 81.88g / L NaCl, 40mL EDTA (0.5M, pH 8.0) and 100mL Tris-HCl (1M, pH 8.0).

[0034] 50 x TAE Buffer (1L): 242g Tris, 37.2g Na2EDTA·2H2O and 57.1 mL glacial acetic acid.

[0035] Antibiotics: Kanamycin (KAN) 100 mg / mL, Rifampin (RIF) 25 mg / mL.

[0036] Example 1: Rapeseed genes involved in regulating silique development BnaCDPK10 Filtering

[0037] To identify the calcium-dependent protein kinase gene involved in rapeseed silique development, this invention conducted candidate gene screening and analysis. The specific experimental procedure is as follows:

[0038] 1. Construction of extreme mixing pools and transcriptome sequencing

[0039] During the rapeseed flowering period, different colored yarns were used to mark the flowering date on the main inflorescence. Ovules were collected at day 0, 14 days, and 28 days after flowering. Immediately after collection, the ovaries were placed in liquid nitrogen and then stored at -80°C. After rapeseed seed harvest, 50 siliques from the main inflorescence of each plant were collected, naturally dried, and threshed to determine the thousand-seed weight (TSW, g). Based on the phenotypic results, 5 plants with extremely high and 6 plants with extremely low thousand-seed weights were selected from the BC4F2 population to construct mixed pools with extremely high and extremely low thousand-seed weights, respectively. These pools were sent to Biomarker for transcriptome sequencing to analyze differentially expressed genes.

[0040] 2. Candidate gene screening

[0041] Screening of differentially expressed genes revealed six genes with differential expression. BnaCDPKs Based on these 6 genes, we can analyze whether there is... BnaCDPK The (rapeseed calcium-dependent protein kinase) gene is affected by calcium ions, thus influencing silique development in rapeseed. Seven days after pollination, ZS11 siliques were treated with 10 mmol / L Ca²⁺, and the effects were measured. BnaCDPKs Changes in gene expression levels were observed within 1–3 hours after Ca²⁺ induction. BnaCDPK10 Expression levels changed more significantly, and there were very significant changes after 12 hours of induction. See [link to relevant documentation]. Figure 1 As shown, it will therefore respond significantly to fluctuations in calcium ion concentration.

[0042] Further analysis of the transcriptional expression levels of this gene at different stages of rapeseed silique development and in other parts revealed... BnaCDPK10 It is also expressed at high levels in seeds and siliques at different developmental stages, see [reference]. Figure 2 As shown, therefore BnaCDPK10 Functional identification was performed on a calcium-dependent protein kinase gene involved in the development of rapeseed siliques.

[0043] Experimental Example 2: Extraction of Total RNA and Synthesis of First Strand cDNA

[0044] The SteadyPure plant RNA extraction kit from Aikerui Biotechnology Co., Ltd. was used. Specific steps included:

[0045] (1) Place 100 mg of rapeseed tissue frozen at ultra-low temperature (-80 ℃) into a 2 mL centrifuge tube (RNase free), and use a multi-sample rapid grinder (Shanghai Jingxin Company) to grind the sample into powder;

[0046] (2) Transfer the powdered sample to a 1.5 mL centrifuge tube containing 500 μL of Buffer RLS lysis buffer (RNase free) (add 50×DTT Solution to Buffer RLS before use, until the final concentration is 1×DTT Solution, that is, add 20 μL of 50×DTT Solution to each 1 mL of Buffer RLS), and repeatedly pipette until there is no obvious precipitation;

[0047] (3) Let stand at room temperature for 2 min;

[0048] (4) Centrifuge at 12,000 rpm and 4 ℃ for 5 min;

[0049] (5) Carefully aspirate the supernatant into a new 1.5 mL centrifuge tube (RNase free);

[0050] (6) Add half the volume of anhydrous ethanol to the above filtrate, mix well by pipetting, and disperse the precipitate; (7) Transfer the above mixture to a Plant RNA Mini Column, centrifuge at 12,000 rpm at room temperature for 2 min, and discard the filtrate;

[0051] (8) Add 600 μL of Buffer RWA to the Plant RNA Mini Column, centrifuge at 12,000 rpm at room temperature for 1 min, and discard the filtrate;

[0052] (9) Add 750 μL of Buffer RWB (an appropriate amount of anhydrous ethanol has been added to the RWB in advance, and the volume ratio of Buffer RWB to anhydrous ethanol is 3:7), centrifuge at 12,000 rpm at room temperature for 1 min, and discard the filtrate.

[0053] (10) DNase I digestion: Prepare and mix the DNase I reaction solution, which contains: 4 μL DNase I (RNase free), 5 μL 10× DNase I Buffer and 41 μL RNase free water. Add 50 μL of DNase I reaction solution to the center of the Plant RNA Mini Column membrane and incubate at room temperature for 15 min. Add 350 μL of Buffer RWB to the center of the Plant RNA Mini Column membrane, centrifuge at 12,000 rpm at room temperature for 1 min, and discard the filtrate;

[0054] (11) Add 750 μL of Buffer RWB to the Plant RNA Mini Column, centrifuge at 12,000 rpm at room temperature for 1 min, and discard the filtrate;

[0055] (12) Place the adsorption column of the Plant RNA Mini Column onto a new 2.0 mL Collection Tube and centrifuge at 12,000 rpm at room temperature for 2 min;

[0056] (13) Place the Plant RNA Mini Column onto a new RNase-free Tube, add 100 μL of RNase-free Water to the center of the adsorption column membrane, let stand at room temperature for 2 min, then centrifuge at 12,000 rpm at room temperature for 2 min to elute the RNA, which can be used for subsequent experiments. If subsequent experiments are not to be performed immediately, the dissolved RNA can be stored at -80℃;

[0057] (14) The RNA concentration was determined using a UV spectrophotometer, and the RNA quality was detected by 1% agarose gel electrophoresis. The results are as follows: Figure 3 As shown, lanes 1-10 are for repeated sample loading, and the band fragments are clear and can be used for subsequent experimental operations.

[0058] (15) The extracted RNA was reverse transcribed using the BIO-RAD reverse transcription kit to obtain cDNA. After the reaction was completed, the cDNA was diluted to 50 ng / μL and stored at -20 ℃ for subsequent experiments.

[0059] The RNA reverse transcription reaction system consisted of 4 μL 5x iScript Reaction Mix, 1 μL iScriptReverse Transcriptase, and 1 μL RNA template (1 μg), with nuclease-free water added to a final volume of 20 μL. The RNA reverse transcription PCR reaction program was as follows: 25 ℃, 5 min; 46 ℃, 20 min; 95 ℃, 1 min; 4 ℃, ∞.

[0060] Experiment Example 3: Rapeseed BnaCDPK10 Cloning of genes

[0061] rape BnaCDPK10 The primer sequences used for gene cloning are as follows:

[0062] BnaCDPK10-F (SEQ ID NO.1):

[0063] 5'-atttggagaggacacgaattcATGGGTAACTGTAACGTCTGC-3',

[0064] BnaCDPK10-R (SEQ ID NO.2):

[0065] 5'-aagggctgcggccgcctcgagTCAAACAGGGACAGATTGTCC-3'.

[0066] Using the cDNA of ZS11 obtained in the laboratory in the early stage as a template, the target fragment was amplified by PCR.

[0067] PCR reaction system (25 μL): 9.5 μL ddH2O, 12.5 μL 2×Taq Master Mix, 1 μL BnaCDPK10-F, 1 μL BnaCDPK10-R and 1 μL cDNA.

[0068] PCR reaction procedure: pre-denaturation 94 ℃ 5 min; [denaturation 94 ℃ 25 s; annealing 57 ℃ 25 s; extension 72 ℃ 55 s], 35 cycles; further extension 72 ℃ 5 min; stop 16 ℃ 5 min.

[0069] After PCR amplification of the target gene, agarose gel electrophoresis was performed, and the target fragment was recovered from the gel. The amplification results are as follows: Figure 4 As shown, lanes 1-3 represent repeated sample loading. A clear band is visible at 1650bp, indicating successful cloning. BnaCDPK10 The coding region sequence.

[0070] The target fragment was recovered using the Novizan FastPure® Gel DNA Extraction Mini Kit, and the procedure is as follows:

[0071] (1) After DNA electrophoresis, the gel containing the target DNA fragment is quickly cut off under UV light, and the gel weight is weighed. 100 mg of gel is equivalent to 100 μL volume, which is taken as a gel volume.

[0072] (2) Add an equal volume of Buffer GDP. Place in a 55 ℃ oven to dissolve for 7–10 min, until the gel block is completely dissolved;

[0073] (3) Place the FastPure DNA Mini Columns-G adsorption column into a Collection Tube 2 mL collection tube, transfer the sol solution into the adsorption column, and centrifuge at 12,000 rpm for 60 s;

[0074] (4) Discard the filtrate and place the adsorption column in the collection tube. Add 300 µL of Buffer GDP to the adsorption column. Let stand for 1 min. Centrifuge at 12,000 rpm for 60 s;

[0075] (5) Discard the filtrate and reset the adsorption column in the collection tube. Add 700 µL of Buffer GW (with anhydrous ethanol added) to the adsorption column and centrifuge at 12,000 rpm for 60 s;

[0076] (6) Repeat step (5);

[0077] (7) Discard the filtrate and place the adsorption column back into the collection tube. Centrifuge at 12,000 rp for 2 min;

[0078] (8) Place the adsorption column in a 1.5 mL sterile centrifuge tube, add 30 µL ddH2O to the center of the adsorption column, and let stand for 2 min. Centrifuge at 12,000 rpm for 1 min. Discard the adsorption column and store the DNA at -20 ℃.

[0079] The recovered fragment was sequenced to obtain the... BnaCDPK10 The gene's nucleotide sequence and the encoded amino acid sequence are as follows:

[0080] BnaCDPK10 Gene (SEQ ID NO.3):

[0081]

[0082] The encoded amino acid sequence is (SEQ ID NO.4):

[0083] *

[0084] Experiment Example 4: Construction of Overexpression Vectors

[0085] 1. Plasmid extraction

[0086] The plasmid of the expression vector (pBin35SRed3) was extracted using a plasmid extraction kit from Aikerui Biotechnology Co., Ltd. The procedure is as follows:

[0087] (1) Cell preparation. Take 5 mL of overnight cultured bacterial solution with a total OD of approximately 2-8, centrifuge at 12,000 rpm at room temperature for 2 min, and discard the supernatant;

[0088] (2) Add 250 μL of Buffer RS ​​(containing RNase A) to the centrifuge tube to fully suspend the bacterial precipitate until there are no bacterial clumps remaining in the suspension;

[0089] (3) Add 250 μL of Buffer LS to the above suspension, gently invert and mix 8 times until the solution becomes transparent. At this time, the solution is relatively viscous.

[0090] (4) Add 350 μL of pre-cooled Buffer BS. At this point, white clumps will appear in the solution. Gently invert the container 8 times to mix.

[0091] (5) Let stand for 2 min, centrifuge at 12,000 rpm for 10 min at room temperature, and take the supernatant;

[0092] (6) Transfer the above solution to a Plasmid DNA Mini Column, let it stand at room temperature for 1 min, then centrifuge at 12,000 rpm for 1 min at room temperature and discard the filtrate;

[0093] (7) Add 500 μL of Buffer WA to the Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate;

[0094] (8) Add 750 μL of Buffer WB to the Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate;

[0095] (9) Repeat step (8) once;

[0096] (10) Place the Mini Column on a new 2 mL Collection tube and centrifuge at 12,000 rpm for 2 min at room temperature;

[0097] (11) Place the Mini Column on a new 1.5 mL centrifuge tube, add 50 μL of sterile water (preheated) to the center of the Mini Column membrane, and let it stand at room temperature for 1 min;

[0098] (12) Elute DNA by centrifuging at 12,000 rpm for 1 min at room temperature. Store at -20 ℃.

[0099] 2. Vector double enzyme digestion

[0100] Double digestion system containing the target gene plasmid (50 μL): 5 μL 10×Fast Digest Green Buffer, 2.5 μL EcoRI 2.5 μL X hoI 10 μL containing the target gene plasmid and 30 μL ddH2O.

[0101] Enzyme digestion was performed at 37 ℃ for 2 h, followed by gel recovery of the vector backbone and electrophoretic detection. Figure 5 As shown.

[0102] 3. Connecting carrier

[0103] The Pro-Ligation Cloning Kit from abm was used for homologous recombination. The homologous recombinase ligation system is shown in Table 1 below. PCR was performed at a controlled temperature of 50 °C for 1 h.

[0104] Table 1 Homologous recombinase ligation system

[0105]

[0106] 4. Transformation and recovery

[0107] The transformation was performed using Trelief™ 5α Chemically Competent Cell supercompetent cells from Qingke Biotechnology. The specific experimental steps are as follows:

[0108] (1) Take 100 μL of competent cells out of the ultra-low temperature freezer and thaw them on ice. Add the above ligation product, mix gently, and place on ice for 30 min.

[0109] (2) Heat shock in a 42 ℃ water bath for 45 s, then quickly transfer to an ice bath and let stand for 2 min.

[0110] (3) Add 600 μL of antibiotic-free LB liquid culture medium to the centrifuge tube and revive at 37 ℃ / 200 rpm for 60 min.

[0111] (4) Centrifuge at 4000 rpm for 5 min, discard 400 μL of supernatant, mix the remaining liquid by pipetting, take 100 μL of the recovery solution and spread it evenly on a solid culture medium containing Kan (kanamycin, 100 mg / mL) antibiotic, and incubate in an inverted incubator at 37 ℃ overnight.

[0112] 5. Microbial testing

[0113] Normal-sized E. coli plaques were picked and placed in sterilized 1.5 mL EP tubes. 700 μL of LB liquid medium was added in advance, and the tubes were incubated at 37 ℃ and 200 rpm for 8 h. The bacterial count was then performed by PCR.

[0114] The PCR reaction system (25 μL) contained: 9.5 μL ddH2O, 12.5 μL 2×Taq Master Mix, 1 μL primer QW586F, 1 μL primer BnaCDPK10-R and 1 μL bacterial culture.

[0115] The sequence of primer QW586F is (SEQ ID NO.5):

[0116] 5'-CGCACAATCCCACTATCCTT-3'.

[0117] PCR reaction program: 94 ℃, 6 min; 94 ℃, 30 s; 58 ℃, 30 s; 72 ℃, 1 kb / min; step to 2 for 30 cycles; 72 ℃, 10 min; 16 ℃, ∞.

[0118] Bacterial test results as follows Figure 6 As shown, lanes 1-9 are samples from repeated reactions. It can be seen that, except for a weaker band in lane 1, all the others have a clear band at 1650 bp, indicating that they are all positive clones containing the target band. The sequencing of the bacterial cultures with the correct target band size was performed by Qingke Biotechnology Co., Ltd.

[0119] 6. Recombinant plasmid transformation of Agrobacterium

[0120] Plasmid extraction was performed as described in the previous section on "Plasmid Extraction." This experiment used GV3101 Agrobacterium competent cells from Qingke Biotechnology Co., Ltd., and the procedure is as follows:

[0121] Take 50 μL of competent Agrobacterium cells from an ultra-low temperature freezer, thaw them on ice, gently mix them with the recombinant plasmid by pipetting, let them stand on ice for 5 min, then place them in liquid nitrogen for 5 min, followed by a 37 ℃ water bath for 5 min, and finally a 4 ℃ ice bath for 5 min. Add 600 μL of antibiotic-free LB liquid culture medium to a centrifuge tube, and incubate at 28 ℃ with shaking for 3 h. Centrifuge at 4000 rpm for 5 min to collect the bacterial cells, reserving about 200 μL. Resuspend the bacterial cells in supernatant by pipetting, take 100 μL, spread it on an LB agar plate containing the corresponding antibiotic, and incubate upside down at 28 ℃ for 2–3 days.

[0122] After cultivation, single-spot samples of *Agrostis spp.* were transferred to liquid culture medium containing the corresponding antibiotics (50 μg / mL Kan and 20 μg / mL Rif), and cultured at 28 °C with shaking until the culture became turbid. Then, bacterial PCR amplification was performed. The bacterial PCR system and procedure were the same as in step 7. A sample of the bacterial culture showing the correct target band size was added to an equal volume of 50% glycerol, mixed well, and labeled with the bacterial culture name and date. The culture was then stored at -80 °C for later use.

[0123] Experimental Example 5: Agrobacterium-mediated transformation of Arabidopsis thaliana

[0124] 1. Cultivation of Arabidopsis thaliana

[0125] Sow Arabidopsis seeds in small pots filled with moist humus, cover with plastic wrap to keep the soil moist and prevent excessive water loss, then place in a light incubator with the following conditions: Stage 1: 16 h light, 22°C, 40% humidity, 10000 Lx light intensity; Stage 2: 8 h darkness, 16°C, 60% humidity. Remove the plastic wrap about a week after germination, and water every 4-5 days until the Arabidopsis flowers and fruits.

[0126] 2. Transformation of Arabidopsis thaliana by flower soaking method

[0127] (1) Take 200 μL of Agrobacterium tumefaciens containing recombinant plasmid and culture it in 150 mL of LB liquid medium containing the corresponding antibiotic at 28 °C for 2–3 days with shaking.

[0128] (2) Centrifuge at 5000 rpm for 20 min at room temperature, discard the supernatant, resuspend the Agrobacterium precipitate with 5% sucrose solution to adjust the OD value to 0.8, add 0.2% surfactant, and mix by pipetting.

[0129] (3) Cut off the Arabidopsis pods and flowers, immerse the Arabidopsis inflorescence in a suspension of Agrobacterium bacteria for 30 seconds, cover with plastic wrap, and treat in the dark for 24 hours;

[0130] (4) Remove the plastic wrap and transfer it to a light incubator for normal incubation;

[0131] (5) Repeat steps (1) to (4) every week for a total of 3 times;

[0132] (6) When the Arabidopsis thaliana pods begin to turn yellow and crack, collect the fallen seeds and dry them in a 37 ℃ incubator. Store the dried seeds at 4 ℃.

[0133] 3. Positive vaccine detection

[0134] T0 generation seeds were harvested, and seeds that showed red light under green fluorescent excitation light were selected and sown separately. These T0 generation red seeds were directly sown in flowerpots and cultured in a light incubator. At the seedling stage, DNA was extracted from leaves for PCR identification. The primers used for PCR identification were QW586F and BnaCDPK10-R. The PCR reaction system and identification procedure were the same as those used for E. coli detection. The identification results are as follows: Figure 7 As shown in the figure, lanes 1-16 represent the amplification and identification results of different seedlings. It can be seen that positive plants all have obvious bands at around 1700 bp. After the siliques of the positive plants mature, seeds are harvested for continuous propagation and selection.

[0135] Leaves of WT (wild Arabidopsis thaliana) and OE-BnaCDPK10 positive plants were collected, RNA was extracted, and qRT-PCR was performed to analyze the expression of the target gene in each line.

[0136] BnaCDPK10 The qRT-PCR primer sequences for the gene are as follows:

[0137] qBnaCDPK10-F (SEQ ID NO.6):

[0138] 5'-AGAAGGTTGGCTCACAACTTGG-3',

[0139] qBnaCDPK10-R (SEQ ID NO.7):

[0140] 5'-ACACTGACATCTGGCTCGCCT-3'.

[0141] Select Arabidopsis thaliana AtActin7 The gene (AT5G09810) is an internal reference gene, and the primer sequences are as follows:

[0142] AtActin-7-F (SEQ ID NO.8):

[0143] 5'-GCCCCTGAGGAGCACCCAGTT-3',

[0144] AtActin-7-R (SEQ ID NO.9):

[0145] 5'-CCGGTTTGTACGACCACTGGCA-3'.

[0146] The qRT-PCR system (20 μL) contains: 7 μL ddH2O, 10 μL SYBR Green Master Mix, 1 μL qBnaCDPK10-F, 1 μL qBnaCDPK10-R and 1 μL cDNA.

[0147] The qRT-PCR program was as follows: 95 ℃, 5 min; 95 ℃, 5 s; 60 ℃, 30 s; step to 2 for 45 cycles. Each sample was repeated 3 times.

[0148] qRT-PCR results are as follows Figure 8 As shown, it can be seen that rapeseed BnaCDPK10 The gene expression level was zero in WT, while BnaCDPK10 The expression levels of the gene in OE-BnaCDPK10 plants were higher than those in WT plants, indicating that rapeseed... BnaCDPK10 The gene was successfully overexpressed in Arabidopsis thaliana OE-BnaCDPK10 plants, which can be used for phenotypic evaluation.

[0149] Experiment Example 6: Phenotypic Observation of Transgenic Arabidopsis

[0150] WT and OE-BnaCDPK10 positive plants were cultured in a light incubator, and the development process of siliques was observed. Siliques were harvested after maturity, and their length was measured. The morphological comparison and quantitative results of silique length between OE-BnaCDPK10 and WT plants are shown below. Figure 9 , 10 As shown, the silique length of OE-BnaCDPK10 is significantly longer than that of WT. The average silique length of WT is 11.05 cm, while that of OE-BnaCDPK10 is 12.30 cm. The silique length of OE-BnaCDPK10 is 11.29% longer than that of WT.

[0151] At the same time, the number of fruits per corner was examined, and the statistical results are as follows: Figure 11 As shown, the number of fruits per pod in WT was 38.88, while the number of fruits per pod in OE-BnaCDPK10 was 44.91, representing a 15.52% increase in the number of fruits per pod compared to WT. This indicates overexpression. BnaCDPK10 After gene modification, not only did the length of the siliques in the plant increase, but the number of seeds per silique also increased, indicating that rapeseed... BnaCDPK10 Genes have great potential for increasing crop yields.

[0152] In summary, this invention discloses a rapeseed... BnaCDPK10 Gene, BnaCDPK10 The nucleotide sequence of the gene is shown in SEQ ID NO.3. This invention constructs... BnaCDPK10 Gene transgenic overexpression expression vector, and obtaining overexpression BnaCDPK10 Transgenic Arabidopsis plants after gene transfer showed that overexpression of this gene significantly increased silique length, indicating that the gene has a positive regulatory function on silique length. Transferring this gene into crops and horticultural plants has great potential for improving crop yields.

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

Claims

1. Rapeseed BnaCDPK10 The application of genes or their encoded proteins in increasing the length of Arabidopsis siliques, among which, The rapeseed BnaCDPK10 The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

4.

2. Rapeseed BnaCDPK10 The application of genes or their encoded proteins in increasing Arabidopsis yield, among which, The rapeseed BnaCDPK10 The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

4.

3. A method for increasing the length of siliques in plants, characterized in that, This method includes: constructing a rapeseed plant containing a nucleotide sequence as shown in SEQ ID NO.

3. BnaCDPK10 Gene expression vectors are used to introduce these expression vectors into plants, resulting in overexpressed rapeseed. BnaCDPK10 The plant with the gene is a transgenic plant with increased silique length; the plant in question is Arabidopsis thaliana.

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