Rape BnaCDPK10 gene for regulating pod length and application of rape BnaCDPK10 gene

Abstract

The invention discloses a rape BnaCDPK10 gene for regulating and controlling the pod length and application of the rape BnaCDPK10 gene, and relates to the technical field of plant molecular biology. The nucleotide sequence of the BnaCDPK10 gene is as shown in SEQ ID NO.3, and the amino acid sequence of the protein coded by the BnaCDPK10 gene is as shown in SEQ ID NO.4. A transgenic overexpression vector of the BnaCDPK10 gene is constructed, a transgenic arabidopsis thaliana plant over-expressing the BnaCDPK10 gene is obtained, it is found that the length of siliques can be remarkably increased after the gene is over-expressed, and it can be known that the gene has the function of positively regulating and controlling the length of the siliques. When the gene is transferred into crops and horticultural plants, the gene has good application potential in the aspect of improving the yield of the crops.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, and particularly relates to a rapeseed gene for regulating silique length BnaCDPK10 and its application. Background Art

[0002] The growth and development of plants are mainly regulated by genetic and environmental change information. Plant cell signal transduction refers to the process by which cells sense and transduce various environmental stimuli, regulate gene expression, and cause corresponding physiological and biochemical reactions. A large number of studies have shown that Ca²⁺ acts as a second messenger in many plant stimulus responses. Almost all different extracellular stimulus signals, such as light, touch, gravity, plant hormones, pathogens, etc., can cause a transient and significant increase in the intracellular free calcium ion concentration [Ca²⁺], or a change in the gradient and regional distribution within the cell. The calcium concentration change signal can respond to different extracellular stimuli, ultimately leading to specific physiological effects on specific stimuli (J. Kudla, O. Batistič, K. Hashimoto. Calcium signals: the lead currency of plant information processing. Plant Cell, 2010, 22:541−563). When plants are subjected to different stimuli, it first causes a change in the calcium ion concentration in the cells. This change in calcium ion concentration is called a calcium signal. The decoding of the calcium ion signal depends on calcium sensors. In plants, there are mainly four different types of calcium sensors, namely calmodulin (CaM), CaM-like proteins (CML), calcineurin-like proteins (CBL), and CDPKs (calcium-dependent protein kinases) (DeFalco, T. A., Bender, K. W. and Snedden, W. A. Breaking the code: Ca²⁺ sensors in plant signalling. Biochem J, 2010, 425:27−40). Signal transduction is carried out by directly binding or phosphorylating and activating target proteins through different calcium sensors. Different from other calcium sensors, CDPKs not only have an EF hand domain that binds to calcium ions but also a kinase domain.When the intracellular calcium ion concentration is low, the auto-inhibitory linker domain of the calcium-dependent protein kinase binds to the kinase domain to inhibit its own activity. When the calcium ion concentration increases, calcium ions bind to the EF motif and change the conformation, activating the kinase (V. Chandran, E.J. Stollar, K. Lindorff-Larsen, J.F. Harper, W.J. Chazin, C.M. Dobson, B.F. Luisi, J. Christodoulou. Structure of the regulatory apparatus of a calcium-dependent protein kinase (CDPK): a novel mode of calmodulin-target recognition. J Mol Biol, 2006, 357:400−410).

[0003] Rapeseed ( Brassica napus L.) is one of the most important oil crops in the world and also the largest oil crop in China (Hanzhong Wang. Rapeseed industry development strategy guided by new demands. Chinese Journal of Oil Crop Sciences, 2018, 40(5):613−617). Increasing yield is one of the main breeding goals of rapeseed, and the silique length of rapeseed is a key factor affecting yield. As a major factor in transmitting signals, calcium-dependent protein kinases are involved in regulating multiple aspects such as plant stress resistance and plant growth. However, there are few reports on calcium-dependent protein kinases in rapeseed, and its regulatory mechanism during rapeseed silique development is still unclear. Therefore, exploring how CDPKs respond to calcium ion changes to regulate rapeseed silique development and studying its downstream target proteins and pathways not only have important significance for revealing the molecular mechanism of rapeseed silique development but also provide new ideas for rapeseed high-yield breeding. Summary of the Invention

[0004] The object of the present invention is a rapeseed BnaCDPK10 gene that responds to changes in intracellular calcium ion concentration and regulates rapeseed silique length. The research of the present invention shows that BnaCDPK10 as a factor that can respond to calcium ion concentration fluctuations and positively regulates rapeseed silique length, its regulatory mechanism is deeply studied in rapeseed to provide theoretical help for breeding work and may provide new gene resources for crop high-yield breeding.

[0005] To achieve the above object, the present invention provides a rapeseed BnaCDPK10 gene that regulates silique length. The BnaCDPK10 nucleotide sequence of this gene is shown in SEQ ID NO.3.

[0006] The present invention also provides the above rapeseedBnaCDPK10 The protein encoded by the gene, and the amino acid sequence of the protein is shown in SEQ ID NO. 4.

[0007] The Brassica napus provided by the present invention BnaCDPK10 The gene or the protein encoded thereby can be used to regulate the silique length of plants and can also be used to increase crop yield.

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

[0009] The present invention also provides an overexpression vector for regulating silique length, and the overexpression vector contains the Brassica napus BnaCDPK10 gene with the nucleotide sequence shown in SEQ ID NO. 3.

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

[0011] The present invention also provides a recombinant expression bacterium for regulating silique length, and the recombinant expression bacterium contains the above-mentioned Brassica napus BnaCDPK10 gene or overexpression vector.

[0012] Preferably, the above recombinant expression bacterium is selected from Agrobacterium tumefaciens, and more preferably from Agrobacterium tumefaciens GV3101.

[0013] The present invention also provides a method for increasing the silique length of plants, comprising: constructing an expression vector containing the Brassica napus BnaCDPK10 gene with the nucleotide sequence shown in SEQID NO. 3, introducing the expression vector into the plant, and the obtained plant overexpressing the Brassica napus BnaCDPK10 gene is a transgenic plant with increased silique length.

[0014] The Brassica napus BnaCDPK10 gene for regulating silique length and its application of the present invention have the following advantages: The present invention provides a BnaCDPK10 gene obtained from Brassica napus for controlling the silique length of plants, constructs a plant transgenic expression vector containing the gene, and transfers the gene into plant cells and expresses it through transgenic technology, which can effectively affect the silique length of plants. Therefore, transferring the gene into crops and horticultural plants has good application potential in increasing crop yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the expression level of the Brassica napus BnaCDPK10 gene in the siliques of ZS11 induced by 10 mmol / L Ca²⁺ for 24 hours.

[0016] Figure 2 This is the Brassica napus BnaCDPK10Expression levels of genes in siliques at different development stages and other tissues of ZS11

[0017] Figure 3 For the rapeseed of the present invention BnaCDPK10 RNA electrophoresis results of overexpression plants of the gene of the present invention

[0018] Figure 4 Electrophoresis results of amplifying the target fragment of the present invention

[0019] Figure 5 Electrophoresis results of detecting positive clones of the transformation and ligation of the overexpression vector of the present invention

[0020] Figure 6 Electrophoresis results of restriction enzyme digestion of the expression vector pBin35SRed3 plasmid of the present invention

[0021] Figure 7 For the present invention BnaCDPK10 Electrophoresis results of identifying transgenic positive plants

[0022] Figure 8 For the rapeseed of the present invention BnaCDPK10 Relative expression levels of the gene of the present invention in overexpression plants and WT

[0023] Figure 9 Results of morphological comparison of siliques between OE-BnaCDPK10 and WT of the present invention

[0024] Figure 10 Quantitative results of silique length of OE-BnaCDPK10 and WT of the present invention

[0025] Figure 11 Quantitative results of the number of seeds per silique of OE-BnaCDPK10 and WT of the present invention Specific implementation manners

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] The plant materials, vectors, and strains used in the following experimental examples are specifically as follows: The large-seed Brassica napus material DL704 was created by our research team through distant hybridization in the early stage. Zhongshuang 11 (ZS11) is a Brassica napus variety selected 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, BC 4 F 2Populations. DL704, ZS11, and BC 4 F 2 All the populations were planted in the experimental base of the Rapeseed Engineering and Technology Research Center in Beibei District, Chongqing. Arabidopsis thaliana was the Columbia ecotype wild type and was planted in a light incubator. The settings of the incubator were as follows: 8 h of dark conditions, 16 h of light conditions, 60% humidity under light conditions / 40% under dark conditions, 16 °C under light conditions / 22 °C under dark conditions, and light intensity of 0 Lx under dark conditions / 10000 Lx under light conditions.

[0028] The vector selected was the plant expression vector pBin35SRed3, and the strains used included the DH5α Escherichia coli competent strain (Qingke Biotechnology) and the GV3101 Agrobacterium tumefaciens competent strain (Qingke Biotechnology).

[0029] The culture media and reagents used in the following experimental examples are as follows: LB medium: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. For solid medium, 15 g / L agar powder also needs to be added, adjust the pH to 6.8, sterilize at 121 °C for 20 min.

[0030] CTAB Buffer: 20 g / L CTAB, 81.88 g / L NaCl, 40 mL EDTA (0.5 M, pH 8.0), and 100 mL Tris-HCl (1 M, pH 8.0).

[0031] 50x TAE Buffer (1 L): 242 g Tris, 37.2 g Na 2 EDTA·2H 2 O and 57.1 mL glacial acetic acid.

[0032] Antibiotics: kanamycin (kan) 100 mg / mL, rifampicin (Rif) 25 mg / mL.

[0033] Experimental Example 1 Screening of Rapeseed Genes Involved in Regulating Silique Development BnaCDPK10 of To find the calcium-dependent protein kinase genes involved in rapeseed silique development, the present invention carried out candidate gene screening and analysis, and the specific experimental process is as follows: 1. Construction of Extreme Bulk Pools and Transcriptome Sequencing During the flowering period of rapeseed, use woolen yarn of different colors to mark the flowering dates on the main inflorescence. Take the ovaries at 0 days, and the ovules at 14 days and 28 days after flowering. Immediately put them into liquid nitrogen after collection, and then store them at -80 °C. After the rapeseed seeds are matured and harvested, take 50 siliques on the main inflorescence of each individual plant, naturally dry them and then thresh them to examine the thousand-seed weight (Thousand seed weigth, TSW, g). According to the phenotypic examination results, select 5 individual plants with extremely large thousand-seed weight and 6 individual plants with extremely small thousand-seed weight from the BC 4 F 2 population to construct a pool with extremely large thousand-seed weight and a pool with extremely small thousand-seed weight, and send them to Biomarker Technologies Corporation for transcriptome sequencing to analyze differentially expressed genes.

[0034] 2. Screening of candidate genes Screen from the differentially expressed genes and found 6 genes with expression differences. BnaCDPKs Based on these 6 genes, analyze whether there is BnaCDPK (rape calcium-dependent protein kinase) gene affected by calcium ions and affects the development of rapeseed siliques. Treat the siliques at 7 days after pollination of ZS11 with 10 mmol / L Ca²⁺, and detect BnaCDPKs the change in gene expression level. It is found that within 1 - 3 hours of Ca²⁺ induction, BnaCDPK10 has a greater change in expression level, and there is a very significant change after 12 hours of induction, as shown in Figure 1 Therefore, it will have an obvious response to the fluctuation of calcium ion concentration.

[0035] Further analyze the transcriptional expression levels of this gene in different stages of rapeseed silique development and other parts, and find that BnaCDPK10 is also highly expressed in seeds and silique pericarp at different developmental stages, as shown in Figure 2 Therefore, BnaCDPK10 is used as a calcium-dependent protein kinase gene involved in rapeseed silique development for functional identification.

[0036] Experimental Example 2 Extraction of total RNA and synthesis of the first strand of cDNA Select the SteadyPure Plant RNA Extraction Kit of Aikerui Biotechnology Company. The specific steps are as follows: (1) Put 100 mg of rapeseed tissue frozen at ultra-low temperature (-80 °C) into a 2 mL centrifuge tube (RNase free), and use a multi-sample rapid grinder (Shanghai Jingxin Company) to break the sample into powder; (2) Transfer the powdered sample to a 1.5 mL centrifuge tube (RNase free) containing 500 μL of Buffer RLS lysis buffer (Buffer RLS is added with 50×DTT Solution before use to a final concentration of 1×DTT Solution, that is, 20 μL of 50×DTT Solution is added to every 1 mL of Buffer RLS). Use a pipette to pipette up and down repeatedly until there is no obvious precipitate. (3) Let it stand at room temperature for 2 min. (4) Centrifuge at 12,000 rpm at 4 °C for 5 min. (5) Carefully aspirate the supernatant into a new 1.5 mL centrifuge tube (RNase free). (6) Add an equal volume of absolute ethanol to the above filtrate, pipette and mix well to disperse the precipitate. (7) Transfer the above mixture to a Plant RNA Mini Column and centrifuge at 12,000 rpm at room temperature for 2 min, discard the filtrate. (8) Add 600 μL of Buffer RWA to the Plant RNA Mini Column and centrifuge at 12,000 rpm at room temperature for 1 min, discard the filtrate. (9) Add 750 μL of Buffer RWB (an appropriate amount of absolute ethanol has been added to Buffer RWB in advance, and the volume ratio of Buffer RWB to absolute ethanol is 3:7) to the Plant RNA Mini Column and centrifuge at 12,000 rpm at room temperature for 1 min, discard the filtrate. (10) DNase I digestion: Prepare and mix the DNase I reaction solution. The DNase I reaction solution contains: 4 μL of DNaseI (RNase free), 5 μL of 10× DNase I Buffer, and 41 μL of RNase free water. Add 50 μL of the DNaseI reaction solution to the center of the membrane of the Plant RNA Mini Column and let it stand at room temperature for 15 min. Add 350 μL of Buffer RWB to the center of the membrane of the Plant RNA Mini Column and centrifuge at 12,000 rpm at room temperature for 1 min, discard the filtrate.

[0037] (11) Add 750 μL of Buffer RWB to the Plant RNA Mini Column and centrifuge at 12,000 rpm at room temperature for 1 min, discard the filtrate. (12) Place the adsorption column of the Plant RNA Mini Column on a new 2.0 mL Collection Tube and centrifuge at 12,000 rpm for 2 min at room temperature; (13) Place the adsorption column of the Plant RNA Mini Column on a new RNase Free Tube. Add 100 μL of RNase Free Water to the center of the adsorption column membrane, let it stand at room temperature for 2 min, and then centrifuge at 12,000 rpm for 2 min at room temperature to elute the RNA, which can be used for subsequent experiments. If the subsequent experiments are not to be carried out immediately, the dissolved RNA can be stored at -80 °C;

[0038] (14) Measure the RNA concentration with a UV spectrophotometer and detect the RNA quality by 1% agarose gel electrophoresis. The results are as Figure 3 shown. Lanes 1-10 among them are technical replicate loadings. It can be seen that the band fragments are clear and can be used for the operations of subsequent experiments; (15) Select the reverse transcription kit of BIO-RAD company to reverse transcribe the extracted RNA to obtain cDNA. After the reaction is completed, dilute the cDNA to 50 ng / μL and store it at -20 °C for subsequent experiments.

[0039] Among them, the RNA reverse transcription reaction system: 4 μL of 5x iScript Reaction Mix, 1 μL of iScript Reverse Transcriptase, 1 μL of RNA template (1 μg), add Nuclease-free water to 20 μL. The RNA reverse transcription PCR reaction program: 25 °C, 5 min; 46 °C, 20 min; 95 °C, 1 min; 4 °C, ∞.

[0040] Experimental Example 3 Rapeseed BnaCDPK10 Gene Cloning Rapeseed BnaCDPK10 The primer sequences used for gene cloning are as follows: BnaCDPK10-F (SEQ ID NO.1): 5’-atttggagaggacacgaattcATGGGTAACTGTAACGTCTGC-3’, BnaCDPK10-R (SEQ ID NO.2): 5’-aagggctgcggccgcctcgagTCAAACAGGGACAGATTGTCC-3’.

[0041] Using the cDNA of ZS11 obtained in the early stage of the laboratory as a template, a PCR instrument was used to amplify the target fragment.

[0042] PCR reaction system (25 μL): 9.5 μL ddH 2 O, 12.5 μL 2×Taq Master Mix, 1 μL BnaCDPK10-F, 1 μL BnaCDPK10-R, and 1 μL cDNA.

[0043] PCR reaction program: pre-denaturation at 94 °C for 5 min; [denaturation at 94 °C for 25 s; annealing at 57 °C for 25 s; extension at 72 °C for 55 s], cycle 35 times; then extension at 72 °C for 5 min; stop at 16 °C for 5 min.

[0044] After PCR amplification of the target gene, agarose gel electrophoresis was carried out, and the target fragment was recovered from the gel. The amplification results are as Figure 4 shown. Lanes 1-3 in it are for technical replicate loading. It can be seen that there is an obvious band at 1650 bp, indicating that the BnaCDPK10 coding region sequence has been successfully cloned.

[0045] For the recovery of the target fragment, the Novoprotein FastPure® Gel DNA Extraction Mini Kit was selected, and the operation steps are as follows: (1) After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under ultraviolet light, weigh the gel, and 100 mg of gel is equivalent to 100 μL in volume, which is regarded as one gel volume; (2) Add an equal volume of Buffer GDP. Place it in a 55 °C oven and dissolve for 7-10 min until the gel block is completely dissolved;

[0046] (3) Place the FastPure DNA Mini Columns-G adsorption column in a 2 mL Collection Tubes collection tube, transfer the sol solution to the adsorption column, and centrifuge at 12,000 rpm for 60 s; (4) Discard the filtrate, and place the adsorption column in the collection tube. Add 300 µL of Buffer GDP to the adsorption column. Let it stand for 1 min. Centrifuge at 12,000 rpm for 60 s;

[0047] (5) Discard the filtrate, and place the adsorption column back in the collection tube. Add 700 µL of Buffer GW (anhydrous ethanol has been added) to the adsorption column, and centrifuge at 12,000 rpm for 60 s;

[0048] (6) Repeat step (5); (7) Discard the filtrate, place the adsorption column back into the collection tube. Centrifuge at 12,000 rp for 2 min;

[0049] (8) Place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 30 µL ddH 2 O to the center of the adsorption column, let it stand for 2 min. Centrifuge at 12,000 rpm for 1 min. Discard the adsorption column and store the DNA at -20 °C.

[0050] Sequence the recovered fragment to obtain the BnaCDPK10 gene nucleotide sequence and the encoded amino acid sequence as follows: BnaCDPK10 Gene (SEQ ID NO.3):

[0051] The encoded amino acid sequence is (SEQ ID NO.4): MGNCNVCVRPPNPEESKPTPKPKKTNQNRKLNPFTSDFIRSPVRTRAPKDAVIPTSHQTKITDKYILGRELGRGEFGITYLCTDRESREALACKSISKRKLRTAVDVEDVRREVSIMSTLPDHPNVVKLRATYEDGENVHLVMELCEGGELFDRIVARGHYTERAAAGVARTIAEVVMMCHVNGVVHRDLKPENFLFANKKENSALKAIDFGLSVFFKPGEKFKEIVGSPYYMAPEVLKRDYGPEVDVWSAGVIIYILLCGVPPFWAETEQGVALAILRGVIDFKRDPWPQISESAKSLVRQMLNPDPTKRLTAQQVLAHPWVQNAKKAPNVPLGDIVRSRLKQFSMMNRFKKKVLRVIAEHLSIQEVEVIKDMFSLMDEDNDGRITYLELKAGLQKVGSQLGEPEIKMLMEVADVDGNGFLDYGEFVAVIIHLQKIENDELFKLAFMFFDKDGSTYIELDELREALTDELGEPDVSVLNDIMREVDSDKDGRINYDEFVTMMKAGTDWRKASRQYSRERFKSLSINLMKDGSLHLHDALTGQSVPV*.

[0052] Experimental Example 4 Construction of Overexpression Vector 1. Plasmid Extraction Extract the plasmid of the expression vector (pBin35SRed3). Select the plasmid extraction kit from Aikrui Biotechnology Company. The operation steps are as follows: (1) Preparation of bacterial cells. Take 5 mL of overnight culture broth with a total OD of about 2 - 8. Centrifuge at 12,000 rpm for 2 min at room temperature and discard the supernatant;

[0053] (2) Add 250 μL of Buffer RS (containing RNase A) to the centrifuge tube and fully suspend the bacterial cell pellet until there are no remaining cell clumps in the suspension; (3) Add 250 μL of Buffer LS to the above suspension and gently invert the tube up and down 8 times. The solution becomes transparent and is relatively viscous at this time; (4) Add 350 μL of pre-cooled Buffer BS. At this time, white flocs appear in the solution. Gently invert the mixture up and down 8 times;

[0054] (5) Let it stand for 2 min, centrifuge at 12,000 rpm for 10 min at room temperature, and take the supernatant; (6) Transfer the above solution to a Plasmid DNA Mini Column. After standing at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the filtrate; (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; (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; (9) Repeat step (8) once; (10) Place the Mini Column on a new 2 mL Collection tube and centrifuge at 12,000 rpm for 2 min at room temperature; (11) Place the Mini Column on a new 1.5 mL centrifuge tube. Add 50 μL of sterilized water (pre-warmed) to the center of the Mini Column membrane and let it stand at room temperature for 1 min; (12) Centrifuge at 12,000 rpm for 1 min at room temperature to elute the DNA. Store at -20 °C.

[0055] 2. Double digestion of the vector Double digestion system of the plasmid containing the target gene (50 μL): 5 μL of 10×Fast Digest Green Buffer, 2.5 μL EcoRI , 2.5 μL of X hoI , 10 μL of the plasmid containing the target gene and 30 μL of ddH 2 O.

[0056] Incubate at 37 °C in an incubator for 2 h, perform gel extraction on the vector backbone, and detect by electrophoresis as Figure 5 shown.

[0057] 3. Ligate the vector Use the Pro-Ligation Cloning Kit homologous recombination kit from abm company. The homologous recombination enzyme ligation system is as shown in Table 1 below. Control the temperature with PCR at 50 °C for 1 h.

[0058] Table 1 Homologous recombination enzyme ligation system 4. Transformation and Recovery Use TreliefTM 5α Chemically Competent Cell from Tsingke Biological for transformation. The specific experimental steps are as follows: (1) Take out 100 μL of competent cells from the ultra-low temperature freezer and thaw them on ice. Add the above ligation product, mix gently, and place on ice for 30 min.

[0059] (2) Heat shock in a 42 °C water bath for 45 s, quickly transfer to an ice bath, and let stand for 2 min.

[0060] (3) Add 600 μL of antibiotic-free LB liquid culture medium to the centrifuge tube and recover at 37 °C / 200 rpm for 60 min.

[0061] (4) Centrifuge at 4000 rpm for 5 min, discard 400 μL of the supernatant, pipette and mix the remaining liquid, and take 100 μL of the recovered liquid and evenly spread it on a solid medium containing Kan (kanamycin, 100 mg / mL) antibiotic. Incubate overnight in an inverted position in a 37 °C incubator.

[0062] 5. Bacterial Inspection Pick normal-sized E. coli colonies and place them in a sterilized 1.5 mL EP tube. Add 700 μL of LB liquid culture medium in advance and culture at 37 °C / 200 rpm for 8 h, then perform bacterial inspection by PCR.

[0063] The PCR reaction system (25 μL) contains: 9.5 μL ddH 2 O, 12.5 μL of 2×Taq Master Mix, 1 μL of primer QW586F, 1 μL of primer BnaCDPK10-R, and 1 μL of bacterial solution.

[0064] Among them, the sequence of primer QW586F is (SEQ ID NO.5): 5’-CGCACAATCCCACTATCCTT-3’.

[0065] PCR reaction program: 94 °C, 6 min; 94 °C, 30 s; 58 °C, 30 s; 72 °C, 1 kb / min; step to 2 for 30 cycles; 72 °C, 10 min; 16 °C, ∞.

[0066] The results of bacterial inspection are as Figure 6As shown, lanes 1-9 are all samples of technical replicate reactions. It can be seen that except for the relatively weak band in lane 1, obvious bands are observed at 1650 bp in the rest, indicating that they are all positive clone bacteria containing the target band. The sequencing of the bacterial liquid with the correct target band size was completed by Tsingke Biological Company.

[0067] 6. Transformation of Agrobacterium tumefaciens with the recombinant plasmid For plasmid extraction, refer to the aforementioned "Plasmid Extraction". In this experiment, the Agrobacterium tumefaciens competent cells of GV3101 from Tsingke Biological Company were used, and the operation steps are as follows: Take 50 μL of Agrobacterium competent cells from the ultra-low temperature freezer. After thawing on ice, gently pipette and mix with the recombinant plasmid. After standing on ice for 5 min, place it in liquid nitrogen for 5 min, then in a 37 °C water bath for 5 min, and finally in an ice bath at 4 °C for 5 min. Then add 600 μL of antibiotic-free LB liquid culture medium to the centrifuge tube, and incubate with shaking at 28 °C for 3 h. Then centrifuge at 4000 rpm for 5 min to collect the bacteria, and retain about 200 μL. Resuspend the bacterial pellet by pipetting the supernatant, take 100 μL and spread it on an LB solid plate containing the corresponding antibiotics, and place it upside down in a 28 °C incubator for 2-3 days.

[0068] After incubation, pick a single Agrobacterium colony into a liquid medium containing the corresponding antibiotics (containing 50 μg / mL Kan and 20 μg / mL Rif), and incubate with shaking at 28 °C until the bacterial liquid becomes turbid for bacterial liquid PCR amplification. The bacterial liquid PCR system and program are the same as those in step 7. Add an equal volume of 50% glycerol to the bacterial liquid with the correct target band size, mix well, write the name of the bacterial liquid and the date, and store it in an ultra-low temperature freezer at -80 °C for standby.

[0069] Experimental Example 5 Agrobacterium-mediated transformation of Arabidopsis thaliana 1. Cultivation of Arabidopsis thaliana Sow Arabidopsis thaliana seeds in small flower pots filled with moist humus soil, cover with plastic wrap to keep the soil moist and prevent rapid water loss, and then place them in a light incubator. The incubator conditions are set as follows: in the first stage, 16 h of light, temperature 22 °C, humidity 40%, light intensity 10000 Lx; in the second stage, 8 h of darkness, temperature 16 °C, humidity 60%. Remove the plastic wrap about one week after the seeds germinate, and water once every 4-5 days until Arabidopsis thaliana flowers and bears fruit.

[0070] 2. Transformation of Arabidopsis thaliana by the floral dip method (1) Take 200 μL of the Agrobacterium bacterial liquid containing the recombinant plasmid and incubate it with shaking at 28 °C in 150 mL of LB liquid medium containing the corresponding antibiotics for 2-3 days; (2) Centrifuge at 5000 rmp for 20 min at room temperature, discard the supernatant, resuspend the Agrobacterium precipitate with 5% sucrose solution, adjust the OD value to 0.8, then add 0.2% surfactant, and pipette to mix well; (3) Cut off the siliques and flowers of Arabidopsis thaliana, immerse the inflorescence of Arabidopsis thaliana in the resuspended Agrobacterium liquid for 30 s, cover with plastic wrap, and perform dark treatment for 24 h; (4) Remove the plastic wrap and transfer to a light incubator for normal cultivation; (5) Repeat steps (1)-(4) for transformation again at an interval of one week, and perform a total of 3 times; (6) When the siliques of Arabidopsis thaliana begin to turn yellow and split, collect the shed seeds, dry the seeds in an incubator at 37 °C, and store the dried seeds at 4 °C.

[0071] 3. Detection of positive seedlings Harvest the T 0 -generation seeds, select the seeds showing red light under the green light irradiation of a fluorescence excitation lamp and sow them separately. Sow the T 0 -generation red seeds directly into flower pots and cultivate them in a light incubator. Extract DNA from their leaves at the seedling stage for PCR identification. The primers for PCR identification are QW586F and BnaCDPK10-R. The PCR reaction system and identification procedure are the same as those used for Escherichia coli detection. The identification results are as Figure 7 shown. Among them, lanes 1-16 are the amplification identification results of different seedling plants. It can be seen that the positive plants all have obvious bands at about 1700 bp. After the siliques of the positive plants mature, harvest the seeds, and continuously propagate and screen.

[0072] Take the leaves of WT (wild Arabidopsis thaliana) and OE-BnaCDPK10 positive plants, extract RNA, and perform qRT-PCR to analyze the expression of the target gene in each strain.

[0073] BnaCDPK10 The qRT-PCR primer sequences of the gene are as follows: qBnaCDPK10-F (SEQ ID NO.6): 5’-AGAAGGTTGGCTCACAACTTGG-3’, qBnaCDPK10-R (SEQ ID NO.7): 5’-ACACTGACATCTGGCTCGCCT-3’.

[0074] Select the Arabidopsis AtActin7 gene (AT5G09810) as the internal reference gene, and the primer sequences are as follows: AtActin-7-F (SEQ ID NO.8): 5’-GCCCCTGAGGAGCACCCAGTT-3’, AtActin-7-R (SEQ ID NO.9): 5’-CCGGTTGTACGACCACTGGCA-3’.

[0075] The qRT-PCR system (20 μL) contains: 7 μL ddH 2 O, 10 μL SYBR Green Master Mix, 1 μL qBnaCDPK10-F, 1 μL qBnaCDPK10-R, and 1 μL cDNA.

[0076] The qRT-PCR program is: 95 °C, 5 min; 95 °C, 5 s; 60 °C, 30 s; step to 2 for 45 cycles. Each sample is repeated 3 times.

[0077] The qRT-PCR results are as Figure 8 shown. It can be seen that the expression level of the rapeseed BnaCDPK10 gene is zero in WT, while the expression levels of the BnaCDPK10 gene in OE-BnaCDPK10 plants are all higher than that in WT, indicating that the rapeseed BnaCDPK10 gene is successfully overexpressed in Arabidopsis OE-BnaCDPK10 plants, and these OE-BnaCDPK10 plants can be used for phenotypic investigation.

[0078] Experimental Example 6 Observation of Phenotypes of Transgenic Arabidopsis WT and OE-BnaCDPK10 positive plants are cultured in a light incubator, the development process of siliques is observed, and after the siliques are mature, they are harvested and the silique lengths are counted. The results of the morphological comparison and quantitative analysis of silique lengths between OE-BnaCDPK10 and WT are as Figure 9 , 10 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 the average silique length of OE-BnaCDPK10 is 12.30 cm, and the silique length of OE-BnaCDPK10 is increased by 11.29% compared with WT.

[0079] Meanwhile, the number of seeds per silique is investigated, and the statistical results are as Figure 11 shown. The number of seeds per silique of WT is 38.88, and the number of seeds per silique of OE-BnaCDPK10 is 44.91. The number of seeds per silique of OE-BnaCDPK10 is increased by 15.52% compared with WT. It can be seen that after overexpressing the BnaCDPK10 gene, not only the silique length of the plants is increased, but also the number of seeds per silique is increased, indicating that rapeseedBnaCDPK10 Genes have good application potential in improving crop yields.

[0080] In summary, the present invention discloses a rapeseed BnaCDPK10 gene, BnaCDPK10 The nucleotide sequence of the gene is shown in SEQ ID NO.3. The present invention constructs BnaCDPK10 a transgenic overexpression vector of the gene and obtains transgenic Arabidopsis thaliana plants overexpressing BnaCDPK10 the gene, and finds that overexpression of the gene can significantly increase the length of siliques. It can be seen that the gene has the function of positively regulating the length of siliques. By transferring the gene into crops and horticultural plants, it has good application potential in improving crop yields.

[0081] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be construed as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A rapeseed for regulating silique length BnaCDPK10 A gene characterized by Should BnaCDPK10 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. Rapeseed as claimed in claim 1 BnaCDPK10 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

4.

3. The rapeseed as claimed in claim 1 BnaCDPK10 Use of the gene or protein described in the claims in regulating the length of siliques of plants.

4. The rapeseed as claimed in claim 1 BnaCDPK10 Use of the gene or the protein described in the claim in increasing crop yield.

5. The use according to claim 3 or 4, characterized in that: The use comprises increasing silique length.

6. An overexpression vector for regulating silique length, characterized in that: The overexpression vector comprises the rapeseed according to claim 1 BnaCDPK10 Gene.

7. The overexpression vector according to claim 6, characterized in that The overexpression vector is selected from vector pBin35SRed3.

8. A recombinant expression bacterium for regulating silique length, characterized in that: The recombinant expression bacteria comprises the rapeseed described in claim 1 BnaCDPK10 The gene or the overexpression vector according to claim 5.

9. The recombinant expression bacteria according to claim 8, characterized in that: The recombinant expression bacteria are selected from Agrobacterium tumefaciens.

10. A method for increasing the length of siliques of a plant, characterized in that: The method comprises: constructing a rapeseed containing a nucleotide sequence as shown in SEQ ID NO.3 BnaCDPK10 The expression vector of the gene is introduced into the plant, and the resulting over-expressed rapeseed BnaCDPK10 The plants with the gene are transgenic plants with increased silique length.

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