A BnDAR2 gene for increasing plant grain weight, biological materials and applications

Abstract

The present invention provides a BnDAR2 gene, biological material and application for increasing plant grain weight, belonging to the technical field of molecular biology and genetic breeding. The present invention provides a BnDAR2 gene for increasing plant grain weight, and the nucleotide sequence of the BnDAR2 gene is shown in SEQ ID NO.3. The present invention has found that the BnDAR2 gene significantly increases the grain weight of Brassica napus, with the increase in grain weight reaching 41.2%. Therefore, the specific positive regulatory role of the BnDAR2 gene in increasing plant grain weight provides a theoretical basis and gene resources for Brassica napus breeding, which is of great significance for obtaining strains with high grain weight and high oil yield in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and genetic breeding, and in particular relates to a BnDAR2 gene for improving plant grain weight, a biological material and an application thereof. Background Art

[0002] Rapeseed is one of the important oil and energy crops, and increasing rapeseed yield has become an urgent problem to be solved in rapeseed breeding today. The increase in grain weight is one of the important reasons for the increase in rapeseed yield. Grain weight is a quantitative trait controlled by multiple genes and is regulated by the environment and genotype. As the main cultivated type of rapeseed, there is still a lot of room for growth in the thousand-grain weight of Brassica napus. Aggregating large-grain alleles from different germplasms can effectively increase the grain weight of existing varieties, thereby increasing yield. Its molecular regulation mechanism is also a hot topic of current research. Therefore, it is crucial to deeply analyze the mechanism of rapeseed grain weight regulation and obtain a gene related to improving rapeseed grain weight traits. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a BnDAR2 gene, biological material and application for increasing plant grain weight, wherein the BnDAR2 gene can significantly increase the grain weight of Brassica napus.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a BnDAR2 gene for increasing plant grain weight. The nucleotide sequence of the BnDAR2 gene is shown in SEQ ID NO.3.

[0006] The present invention also provides a protein encoded by the BnDAR2 gene.

[0007] Preferably, the amino acid sequence of the protein is shown in SEQ ID NO.4.

[0008] The present invention also provides a biological material containing the above-mentioned BnDAR2 gene, which is any one of the following:

[0009] (1) a recombinant expression vector comprising the above-mentioned BnDAR2 gene;

[0010] (2) a recombinant microorganism comprising the above-mentioned BnDAR2 gene;

[0011] (3) a recombinant microorganism comprising the recombinant expression vector described in (1);

[0012] (4) transgenic plant materials containing the above-mentioned BnDAR2 gene;

[0013] (5) transgenic plant material containing the recombinant expression vector described in (1);

[0014] (6) transgenic plant materials containing the recombinant microorganism described in (2) or (3);

[0015] The transgenic plant material includes transgenic plant cell lines, transgenic plant tissues or transgenic plant organs.

[0016] The present invention also provides an application of the BnDAR2 gene, protein or biological material in preparing plants with high grain weight.

[0017] The present invention also provides an application of the BnDAR2 gene, protein or biological material in increasing plant grain weight.

[0018] Preferably, the plant comprises Brassica napus.

[0019] The present invention also provides a method for preparing high-grain-weight plants, comprising the steps of increasing the expression level of the above-mentioned BnDAR2 gene in a recipient plant to obtain a transgenic plant.

[0020] Preferably, the method comprises the step of introducing the above-mentioned BnDAR2 gene or biological material into a recipient plant to obtain a transgenic plant.

[0021] In the above method, the plant preferably comprises Brassica napus.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a BnDAR2 gene, biological material and application for increasing plant grain weight. The present invention found that the BnDAR2 gene significantly increased the grain weight of Brassica napus, with the increase in grain weight reaching 41.2%. Therefore, the specific positive regulatory role of the BnDAR2 gene in increasing plant grain weight provides a theoretical basis and gene resources for Brassica napus breeding, which is of great significance for obtaining strains with high grain weight and high oil yield in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The electrophoresis diagram of the total RNA of Brassica napus prepared in Example 1;

[0025] Figure 2 The gDNA results extracted from wild-type plants, BnDAR2 gene overexpression plants and BnDAR2-RNAi plants, where M is DL 15000 DNA marker, 1 to 4 are BnDAR2-RNAi DNA, 5 to 9 are BnDAR2-OE DNA, and 10 is ZS11 DNA;

[0026] Figure 3The results of PCR amplification of Hyg gene in wild-type plants and plants with overexpression of BnDAR2 gene are shown, where M is DL 2000DNA marker, 1 is ZS11 HYG PCR, and 2 to 6 are BnDAR2-OE HYG PCR;

[0027] Figure 4 The results of PCR amplification of Hyg gene in BnDAR2-RNAi plants, where M is DL 2000DNA marker, and 1 to 4 are BnDAR2-RNAi HYG PCR;

[0028] Figure 5 The total RNA extracted from BnDAR2 gene overexpression plants and BnDAR2-RNAi plants, where M is DL2000DNA marker, 1 to 5 are BnDAR2-OE RNA, and 6 to 8 are BnDAR2-RNAi RNA;

[0029] Figure 6 The results of BnDAR2 gene expression in wild-type plants, BnDAR2 gene overexpression plants and BnDAR2-RNAi plants;

[0030] Figure 7 The results of 1000-grain weight of Brassica napus under different treatments;

[0031] Figure 8 The results of the effects of different treatments on the grain weight of Brassica napus;

[0032] Fig. 9 The results show the effects of different treatments on the circumference of Brassica napus seeds. DETAILED DESCRIPTION

[0033] The present invention provides a BnDAR2 gene for increasing plant grain weight. The nucleotide sequence of the BnDAR2 gene is shown in SEQ ID NO.3.

[0034] The BnDAR2 gene of the present invention is obtained by extracting total RNA from Brassica napus and amplifying it by reverse transcription to obtain cDNA, and then using the cDNA as a template to amplify the BnDAR2 gene by primers BnDAR2-F and BnDAR2-R. The nucleotide sequences of BnDAR2-F and BnDAR2-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0035] The present invention also provides a protein encoded by the BnDAR2 gene.

[0036] In the present invention, the amino acid sequence of the protein is preferably as shown in SEQ ID NO.4.

[0037] The present invention also provides a biological material containing the above-mentioned BnDAR2 gene, which is any one of the following:

[0038] (1) a recombinant expression vector comprising the above-mentioned BnDAR2 gene;

[0039] (2) a recombinant microorganism comprising the above-mentioned BnDAR2 gene;

[0040] (3) a recombinant microorganism comprising the recombinant expression vector described in (1);

[0041] (4) transgenic plant materials containing the above-mentioned BnDAR2 gene;

[0042] (5) transgenic plant material containing the recombinant expression vector described in (1);

[0043] (6) transgenic plant materials containing the recombinant microorganism described in (2) or (3);

[0044] The transgenic plant material includes transgenic plant cell lines, transgenic plant tissues or transgenic plant organs.

[0045] In the present invention, the backbone plasmid of the recombinant expression vector includes the pCAMBIA1300-35S vector. The preparation method of the recombinant expression vector includes inserting the above-mentioned BnDAR2 gene into the pCAMBIA1300-35S vector to obtain the recombinant expression vector. The insertion position is between the endonucleases SacⅠ and XbaⅠ of the pCAMBIA1300-35S vector. The recombinant microorganism is preferably a bacterium or a fungus, such as Escherichia coli or Agrobacterium, the Escherichia coli is Escherichia coli DH5α, and the Agrobacterium is GV3101. The preparation method of the recombinant microorganism includes transforming the above-mentioned BnDAR2 gene or the recombinant expression vector into Escherichia coli or Agrobacterium to obtain the recombinant microorganism. The preparation method of the transgenic plant material includes transforming the above-mentioned BnDAR2 gene, the recombinant expression vector or the recombinant microorganism into a transgenic plant material (such as a transgenic plant cell line, a transgenic plant fine tissue or a transgenic plant organ) to obtain the transgenic plant material. The transgenic plant organ includes a cotyledon petiole or a hypocotyl.

[0046] The present invention also provides an application of the BnDAR2 gene, protein or biological material in preparing plants with high grain weight.

[0047] The present invention also provides an application of the BnDAR2 gene, protein or biological material in increasing plant grain weight.

[0048] In the present invention, the plant preferably includes Brassica napus, such as Zhongshuang No. 11.

[0049] The present invention also provides a method for preparing a high-grain-weight plant, comprising the steps of increasing the expression level of the BnDAR2 gene in a recipient plant to obtain a transgenic plant. The plant preferably comprises Brassica napus, such as Zhongshuang No. 11.

[0050] As a preferred embodiment, the method for preparing high-grain-weight plants comprises the step of introducing the BnDAR2 gene or biological material into a recipient plant to obtain a transgenic plant. The plant preferably comprises Brassica napus, such as Zhongshuang No. 11.

[0051] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] In the following embodiments or application examples, the preparation method of the YEB medium (also called YEB liquid medium) is as follows (400 mL): 0.40 g of yeast powder, 0.20 g of MgSO4·7H2O, 2.00 g of sucrose and 2.00 g of trypsin are weighed respectively and dissolved in 400 mL of double distilled water, the pH is adjusted to 7.0-7.2, and sterilized in an autoclave at 121° C. for 20 min.

[0053] YEB solid medium is prepared by adding 6.00 g of agar to the above YEB medium.

[0054] Example 1

[0055] Construction of BnDAR2 overexpression vector

[0056] 1. Total RNA extraction and detection of Brassica napus (rapeseed variety: Zhongshuang 11, referred to as ZS11)

[0057] 1.1 Extract total RNA using Trizol method. The specific steps are as follows:

[0058] (1) Take about 1 g of rapeseed ZS11 leaves and grind them into powder in a pre-cooled mortar with liquid nitrogen;

[0059] (2) Quickly transfer the leaf powder into a 1.5 mL EP tube, add 1 mL of Trizol lysis buffer, mix immediately, and place on ice for 5 min;

[0060] (3) Place in a refrigerated centrifuge at 12000 rpm, 4°C, centrifuge for 10 min, take the supernatant and add 0.2 mL of chloroform to a spare RNase-free EP tube, mix for 2 min, and place on ice for 5 min;

[0061] (4) Centrifuge at 12000 rpm, 4°C for 10 min, and transfer the supernatant to a new RNase-free EP tube;

[0062] (5) Add isopropanol equal to the volume of the supernatant, mix well, and place in a -20°C refrigerator for 1 h to precipitate RNA;

[0063] (6) After centrifugation at 12,000 rpm, 4°C, for 10 min, discard the supernatant, add 1 mL of 75% ethanol solution for elution, and then place in a refrigerated centrifuge at 12,000 rpm, 4°C, and centrifuge for 5 min;

[0064] (7) Remove the supernatant, add 0.4 mL of anhydrous ethanol, and centrifuge at 12000 rpm, 4°C for 2 min;

[0065] (8) Discard the supernatant, dry for 5 min to evaporate the ethanol, add 20 μL of DEPC water to dissolve, obtain total RNA of Brassica napus, and store it in a -80°C refrigerator.

[0066] 1.2 Nucleic acid agarose gel electrophoresis detection and concentration purity determination

[0067] (1) Prepare 25 mL of 1 wt.% agarose gel: Accurately weigh 0.25 g of agarose powder and 25 mL of 1×TAE, pour them into a 100 mL small conical flask, heat in a microwave oven to fully boil and dissolve the gel, and after slightly cooling, pour it into a rubber plate with a comb of the desired pore size and wait for condensation.

[0068] (2) Place the prepared gel in an electrophoresis tank, pour in 1×TAE to submerge the surface of the gel, mix 2 μL of the sample to be tested (i.e., total RNA from Brassica napus) with 1 μL of Loading Buffer, select DL-15000 Marker, set the electrophoresis tank to 120V, and incubate for 30 min.

[0069] (3) After the electrophoresis is completed, the gel blocks are taken out and observed using a gel imaging system. The results are shown in Figure 1 The results showed that the total RNA bands of Brassica napus obtained were clear, indicating that the RNA integrity was high.

[0070] (4) The total RNA concentration of Brassica napus was determined by Nanodrop to be approximately 1000 ng / μL, 1.9 <OD260 / 280<2.1,OD260 / 230> 2. The total RNA purity of Brassica napus is relatively high.

[0071] 2. Reverse transcription of cDNA

[0072] (1) Genomic DNA removal: Construct a reaction system according to Table 1, place the system at 42°C, react for 2 min, and obtain a mixed solution;

[0073] Table 1 Reaction system for removing genomic DNA

[0074]

[0075] (2) The mixed solution in step (1) is reacted according to the system in Table 2 and the procedure in Table 3, and the obtained cDNA is placed in a -20°C refrigerator for later use;

[0076] Table 2 cDNA synthesis system

[0077]

[0078]

[0079] Table 3 cDNA synthesis program

[0080]

[0081] 3. BnDAR2 gene CDS sequence amplification and electrophoresis

[0082] Primer Premier 5.0 software was used to design specific primers for amplifying the BnDAR2 fragment, as shown in Table 4. The specific PCR amplification reaction system is shown in Table 5. The PCR reaction amplification program is shown in Table 6. After amplification, the PCR amplification product of the BnDAR2 gene was obtained and the size and quality of the target band were detected by agarose gel electrophoresis.

[0083] Table 4 PCR primer sequences

[0084]

[0085] Table 5 PCR reaction system

[0086]

[0087] Table 6 PCR reaction amplification program

[0088]

[0089]

[0090] 4. Recovery of BnDAR2 gene fragments

[0091] The target fragment was recovered using the FastPure Gel DNA Extraction Mini Kit from Nanjing Novozymes:

[0092] (1) Mix 2 μL of the BnDAR2 gene PCR reaction product obtained in the above step with 2 μL of loading buffer and load it for agarose gel electrophoresis detection. After the electrophoresis is completed, cut the agarose gel with the target fragment and weigh it in a 2 mL EP tube;

[0093] (2) Add an equal volume of buffer GDP solution to the agarose gel in the EP tube (1) and incubate in a 55°C water bath until the gel is completely dissolved;

[0094] (3) After dissolution, centrifuge briefly, transfer the solution to the adsorption column and place it for 2 min, centrifuge at room temperature at 12000 rpm for 60 s, and discard the filtrate in the collection tube;

[0095] (4) Add 300 μL of buffer GDP to the adsorption column, let it stand for 1 min, centrifuge at 12000 rpm for 60 s at room temperature, and discard the filtrate in the collection tube;

[0096] (5) Take 600 μL of Buffer GW rinse solution and add it to the adsorption column. Centrifuge at 12000 rpm for 60 s and remove the waste liquid in the collection tube.

[0097] (6) Repeat step 5 above;

[0098] (7) Centrifuge at 12000 rpm for 2 min to remove the rinse solution, and let the adsorption column dry at room temperature for 2 min;

[0099] (8) Add 20 μL of 65°C preheated ddH2O to the center of the adsorption column to dissolve the BnDAR2 fragment. Let stand for 2 min and then centrifuge at 12000 rpm for 2 min.

[0100] (9) After recovery, the recovered BnDAR2 gene fragment was stored in a -20°C refrigerator.

[0101] 5. Double restriction enzyme digestion of BnDAR2 gene fragment and pCAMBIA1300-35S vector

[0102] The BnDAR2 fragment and pCAMBIA1300-35S vector recovered in the above steps were double digested with SacⅠ and XbaⅠ according to the system in Table 7 (the digestion sites were GAGCTC and TCTAGA, respectively), and digested at 37°C for 8 hours; after digestion, the samples were loaded for electrophoresis detection and then excised and recovered on agarose gel to obtain the BnDAR2 fragment and pCAMBIA1300-35S fragment after digestion, respectively.

[0103] Table 7 SacⅠ, XbaⅠ double restriction enzyme digestion system

[0104]

[0105]

[0106] 6. Ligation of BnDAR2 fragment and pCAMBIA1300-35S vector fragment

[0107] The above-mentioned BnDAR2 fragment after enzyme digestion and the pCAMBIA1300-35S fragment were connected at 16°C overnight according to the system in Table 8 to obtain a recombinant cloning plasmid.

[0108] Table 8 BnDAR2 fragment and pCAMBIA1300-35S vector fragment connection system

[0109]

[0110] 7. E. coli transformation and identification

[0111] 7.1 E. coli transformation

[0112] (1) Take out 50 μL of E. coli DH5α competent cells from the -80°C ultra-low temperature freezer and immediately put them in ice to allow them to freeze and thaw on ice.

[0113] (2) Take 5 μL of the recombinant cloning plasmid ligation solution obtained in the above step and add it to the competent cells in step (1), flick the tube wall to mix, and let it stand for 25 minutes.

[0114] (3) Heat shock at 42°C for 45 seconds.

[0115] (4) After the heat shock was completed, the mixture was immediately placed in ice to cool for 3-4 min, and then 600 μL of LB medium without antibiotics was added and cultured at 37°C with a shaker at 200 rpm for 1 h.

[0116] (5) Aseptically spread 100 μL of the solution onto LB solid medium containing 50 mg / L Kana antibiotic.

[0117] (6) Incubate at 37°C for 14 h. Pick a single colony and shake it in LB liquid medium with 50 mg / L Kana. See Table 5 for the PCR identification reaction system and Table 6 for the PCR reaction procedure.

[0118] (7) Extract plasmids from bacterial cultures that are positive by PCR.

[0119] Note: LB medium (200 mL): Weigh 1.00 g of yeast powder, 2.00 g of tryptone, and 2.00 g of NaCl respectively and dissolve them in 20 mL of double distilled water. Sterilize in an autoclave at 121°C for 20 min (for solid culture medium, add 3.00 g of agar before sterilization).

[0120] 7.2 Plasmid extraction and identification: The plasmid was extracted using the FastPure Plasmid Mini Kit from Nanjing Novezan, and DNA sequencing was performed on the extracted recombinant clone plasmid. The correctly sequenced recombinant clone plasmid and bacterial solution were stored at -80°C. The sequencing results showed that the nucleotide sequence of the BnDAR2 gene was as shown in SEQ ID NO.3, specifically:

[0121]

[0122] The amino acid sequence of the BnDAR2 gene is shown in SEQ ID NO.4, specifically:

[0123] MDSSSYGVSHVSHISNPCIFGAGSSSSPEKKWNLMKWVSKLFKSGSNGGTGGARTNRHPPQFQEDENMVFPLPPSSSDDWSRASRDKEELDRALPVSLADDTNRPYGYGWSMDNNSDFPRPFRSGLNPS FIPPYEPSYQVRRPQRICGGCNSDIGLGNYLGCMGTFFHPDCFCCDSCRYPITEHEFSLSGTKPYHQICFKELTHPKCEVCHHFIPTNDAGLIEYRCHPFWNQKYCPSHEHDRTARCCSCERLESWEVR YYTLDDGRSLCLECMETAITDTGDCQPLYHAIRDYYEGMYMKLEQQIPMLLVQREALNDAIVGEKHGYHHMPETRGLCLSEEQTVTSVLKRPRLGARRLVGMRTQPQKLTRKCEVTAILVLYGLPRLLT GAILAHELMHGWLRLKGYRNLNPEVEEGICQVLSYMWLESEVLSDPSSRSMPSTSTATSSSSSSSSNKKGGKTNVEKKLGEFFKHQIAHDASPAYGGGFRAANAAVCKYGLRRTLDHIRFTGTFPL.

[0124] 8. Agrobacterium transformation and identification

[0125] (1) Add 1 μL of the extracted recombinant plasmid to 100 μL of GV3101 Agrobacterium competent medium that has been frozen and thawed for 5 minutes in a sterilized clean bench;

[0126] (2) After mixing, place in ice bath for 5 min and freeze in liquid nitrogen for 5 min;

[0127] (3) Take out and place in a 37°C constant temperature water bath for 5 min, and then in an ice bath for 5 min;

[0128] (4) After adding YEB medium, incubate at 28°C in a shaker at 200 rpm / min for 2 h;

[0129] (5) Centrifuge at 5000 rpm for 2 min at room temperature, remove part of the supernatant and resuspend the bacteria, spread on YEB medium containing 50 mg / L Rif (rifampicin) and 50 mg / L Kana (kanamycin) and culture for 24 h, pick a single colony and shake it on the YEB medium with Kana. The PCR identification reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 6. The positive bacterial solution is stored in a -80°C refrigerator and recorded as BnDAR2 overexpression positive Agrobacterium.

[0130] Comparative Example 1

[0131] Construction of BnDAR2-RNAi vector

[0132] (1) PrimerPremier 5.0 was used to design a 350 bp target sequence for the third exon of BnDAR2. The specific PCR primer sequence information is shown in Table 9. The specific sequence is as follows: Forward sequence: CACTTTACCATGCAATACGTGACTATTACGAAGGAATGTACATGAAACTTGAGCAACAAATCCCCATGCTTCTTGTTCAGCGAGAAGCTCTCAACGACGCTATCGTCGGAGAGAAACACGGATACCATCACATGCCTGAGACAAGGGGTTTATGTTTGTCTGAAGAACAGACAGTCACAAGTGTTCTTAAAAGACCGAGACTTGGCGCTCACCGTCTTGTTGGTATGAGAACTCAGCCTCAAAAGCTTACACGTAAATGTGAAGTCACTGCGATTCTCGTTCTTTACGGCCTCCCTAGACTATTAACTGGAGCAATTCTTGCCCACGAGCTGATGCATGGATGGCTAAGG (SEQ ID NO.9), reverse sequence: CCTTAGCCATCCATGCATCAGCTCGTGGGCAAGAATTGCTCCAGTTAATAGTCTAGGGAGGCCGTAAAGAACGAGAATCGCAGTGACTTCACATTTACGTGTAAGCTTTTGAGGCTGAGTTCTCATACCAACAAGACGGTGAGCGCCAAGTCTCGGTCTTTTAAGAACACTTGTGACTGTCTGTTCTTCAGACAAACATAAACCCCTTGTCTCAGGCATGTGATGGTATCCGTGTTTCTCTCCGACGATAGCGTCGTTGAGAGCTTCTCGCTGAACAAGAAGCATGGGGATTTGTTGCTCAAGTTTCATGTACATTCCTTCGTAATAGTCACGTATTGCATGGTAAAGTG (SEQ ID NO.10). PCR amplification was performed using the reverse transcribed cDNA as a template according to the system and reaction procedure in Table 10 to obtain a PCR amplification product.

[0133] Table 9 PCR primer sequence information

[0134]

[0135] Table 10 PCR reaction system and reaction procedure

[0136]

[0137]

[0138] (2) The PCR amplification product in step (1) is subjected to agarose gel electrophoresis detection and the target fragment is recovered to obtain the target fragment.

[0139] (3) Enzyme digestion and ligation: The target fragment forward sequence recovered in step (2) and pCAMBIA1300-35SI (purchased from Wuhan Transduction Biological Co., Ltd.) were double-digested with BamHI and SpeI (the restriction sites were GGATCC and ACTAGT, respectively), and the digestion products were ligated to obtain ligation products. The specific reaction system and reaction conditions are shown in Table 11. The target fragment reverse sequence recovered in step (2) and the pCAMBIA1300-35SI-forward sequence obtained in step (3) were ligated with Xgf Double enzyme digestion was performed with BglII (the enzyme cutting sites were TCTAGT and GGATCT respectively), and the enzyme cutting products were connected to obtain a connection product. The specific reaction system and reaction conditions are shown in Table 12.

[0140] Table 11 Enzyme ligation system and procedure

[0141]

[0142] Table 12 Enzyme ligation system and procedure

[0143]

[0144] (4) Take 5 μL of the ligation product in step (3) to transform competent E. coli cells. Spread on a resistant plate containing 50 mg / L R and 50 mg / L Kana, culture at 37°C for 12 h, perform colony PCR identification and DNA sequencing identification, PCR primers are shown in Table 13, reaction system and procedure are shown in Table 14; after the DNA sequencing results come out, take a tube of plasmid corresponding to the correct sequencing (the method of extracting plasmid is the same as that of 7.2 in Example 1), transfer the extracted plasmid into Agrobacterium, and store the Agrobacterium liquid identified as positive in a -80°C refrigerator, which is recorded as BnDAR2-RNAi positive Agrobacterium, wherein the nucleotide sequence of siRNA is as shown in SEQ ID NO.As shown in Figure 11, specifically: 5’-CACTTTACCATGCAATACGTGACTATTACGAAGGAATGTACATG AAACTTGAGCAACAAATCCCCATGCTTCTTGTTCAGCGAGAAGCTCTCAACGACGCTATCGTCGGAGAGAAACACGGATACCATCACATGCCTGAGACAAGGGGTTTATGTTTGTCTGAAGAACAGACAGTCACAAGTGTTCTTAAAAGACCGAGACTTGGCGCTCACCGTCTTGTTGGTATGAGAACTCAGCCTCAAAAGCTTACACGTAAATGTGAAGTCACTGCGATTCTCGTTCTTTACGGCCTCCCTAGACTATTAACTGGAGCAATTCTTGCCCACGAGCTGATGCATGGATGGCTAAGGACTAGTAGAGGGTAATTAAGCAAAACTTATCCAAAACTAAACATTTTACTATTATTTTGACCTTTTTATTCCACTTTTCTTAGACAATGATTTAACCTCGTAATCAATTGTTAGGATTTCTAGTCCTTAGCCATCCATGCATCAGCTCGTGGGCAAGAATTGCTCCAGTTAATAGTCTAGGGAGGCCGTAAAGAACGAGAATCGCAGTGACTTCACATTTACGTGTAAGCTTTTGAGGCTGAGTTCTCATACCAACAAGACGGTGAGCGCCAAGTCTCGGTCTTTTAAGAACACTTGTGACTGTCTGTTCTTCAGACAAACATAAACCCCTTGTCTCAGGCATGTGATGGTATCCGTGTTTCTCTCCGACGATAGCGTCGTTGAGAGCTTCTCGCTGAACAAGAAGCATGGGGATTTGTTGCTCAAGTTTCATGTACATTCCTTCGTAATAGTCACGTATTGCATGGTAAAGTG-3’.

[0145] Table 13 Positive Agrobacterium Identification PCR Primer Sequences

[0146]

[0147] Table 14 PCR Reaction System and Reaction Program

[0148]

[0149] Application Example 1

[0150] Obtaining BnDAR2 overexpression and RNAi plants

[0151] 1. Genetic transformation of rapeseed

[0152] (1) Rapeseed planting

[0153] Take the seeds of Brassica napus Zhongshuang 11 (ZS11) in a 1.5mL EP tube, soak them with sterile water, and place them in a 4℃ refrigerator for 1 day for vernalization. After vernalization, add sterile water to wash them 5 times, then add 1mL of 75% alcohol to wash for 30s, and then rinse them with sterile water 5 times after removing the alcohol; then disinfect them with 1mL of 2% sodium hypochlorite for 5min, and then rinse them with sterile water 5 times. Remove excess water from the tube, inoculate the treated seeds in MS0 medium (MS0: MS medium + 3wt.% sucrose + 0.7wt.% agar, pH 5.8-6.0), and place them in a culture room with 16h light, 8h dark and 24℃ for 5-7d until the rapeseed seedlings grow cotyledons.

[0154] (2) Preparation of agricultural stem infection solution

[0155] The BnDAR2 overexpression positive Agrobacterium prepared in Example 1 and the BnDAR2-RNAi positive Agrobacterium prepared in Comparative Example 1 were taken out from the -80°C refrigerator, and streaked on YEB solid culture medium (the YEB solid culture medium contained 50 mg / L Rif and 50 mg / L Kana) for activation, and single colonies were picked and inoculated in YEB liquid culture medium with the same resistance (the YEB liquid culture medium contained 50 mg / L Rif and 50 mg / L Kana) at 28°C and 180 rpm for 16 h. 5 mL of the bacterial solution was added to 50 mL of YEB liquid culture medium with the same resistance and shaken until the bacterial solution OD 600 =0.6~0.8. Then, the cells were enriched by centrifugation and resuspended in MS1 ​​(MS1: MS liquid medium + 3% sucrose + 1.0 mg / L 2,4-D + 200 μmol / LAS (acetosyringone) + 500 mg / LMES (fatty acid methyl ester sulfonate), pH 5.8~6.0) liquid medium to adjust the bacterial liquid OD 600 =0.1-0.2 and then allowed to stand for 1 hour for use, thereby obtaining BnDAR2 overexpression agrostem infection fluid and BnDAR2-RNAi agrostem infection fluid, respectively.

[0156] (3) Explant preparation and pre-culture

[0157] Take out the rapeseed seedlings grown for 5-7 days in step (1), cut the cotyledon petiole (excluding the growth point) of about 2 mm and the hypocotyl of about 0.5 cm in length with a scalpel in the clean bench, and place them in the rapeseed pre-culture medium MS2 (MS2: MS0 + 1 mg / L 2,4-D) for dark culture for 2 days for the next transformation experiment. Note that in this experiment, the cotyledon petiole is always inserted in the culture medium, and the hypocotyl is flattened in the culture medium.

[0158] (4) Agrobacterium infection and co-cultivation

[0159] The cotyledon petiole and hypocotyl pre-cultured for 2 to 3 days in step (3) are respectively immersed in the BnDAR2 overexpression agro-stalk infection solution and the BnDAR2-RNAi agro-stalk infection solution in step (2) for 8 minutes, and placed on a shaker at 28°C and 180 rpm for shaking and mixing to fully infect. After the infection is completed, the cotyledon petiole and hypocotyl are taken out and placed on filter paper for filtration and drying, and the sterilized filter paper is spread on the solid culture medium MS1 (0.7 wt.% agar is added to MS1), and the excess bacterial solution is removed with the filter paper, and then placed on the co-culture medium for dark culture for 2 to 3 days.

[0160] (5) Degerming and delayed screening

[0161] The cotyledon petioles and hypocotyls after co-cultivation in step (4) were placed in sterile water containing 500 mg / L Cef and washed three times for 5 minutes each time, then rinsed three times with sterile water and transferred to MS3 (MS3: MS0+5 mg / L AgNO3+0.1 mg / L NAA (α-naphthylacetic acid)+3 mg / L6-BA+250 mg / L Cef (cephalosporin), pH 5.8-6.0) medium for light culture for 7 days.

[0162] (6) Antibiotic screening and rescreening

[0163] The petioles and hypocotyls in the delayed screening medium of step (5) were transferred to the corresponding screening differentiation medium MS4 (MS4: MS0 + 3mg / L 6-BA + 0.1mg / L NAA + 5mg / L AgNO3 + 250mg / L Cef + 13mg / L Hyg (hygromycin), pH 5.8-6.0) according to the strain and vector used for infection, and placed in a light culture room with 16h light and 8h dark for primary screening (low antibiotic concentration, 13mg / mL), and subcultured every 2 weeks until the callus differentiates into buds. The explants with differentiated small buds were then transferred to the screening medium MS5 for rescreening (MS5: MS0 + 3mg / L 6-BA + 0.1mg / L NAA + 5mg / LAgNO3 + 250mg / L Cef + 15mg / L Hyg, pH 5.8-6.0).

[0164] (6) Rooting, transplanting and hardening of transformed seedlings

[0165] When the regenerated plants after Hyg rescreening in step (5) grow to 2-3 cm, they are cut off 0.5 cm from the bottom of the new stem and transferred to rooting medium MS6 (MS6: MS0+0.2 mg / L NAA+250 mg / L Cef+13 mg / L Hyg, pH 5.8-6.0), and the medium is renewed every two weeks until the roots are fully grown. Then, the seedlings are hardened and moved to the field for cultivation after they adapt to the external environment.

[0166] 2. Identification of BnDAR2 overexpression (BnDAR2-OE) and BnDAR2-RNAi plants

[0167] 2.1 The steps for identifying overexpression plants are as follows:

[0168] Extract gDNA from the BnDAR2 gene overexpressing plants obtained by the above steps (see Figure 2 ).

[0169] The gDNA was extracted using the CTAB method. The steps are as follows:

[0170] (1) Take about 2 g of rapeseed ZS11 leaves and grind them into powder in a pre-cooled mortar with liquid nitrogen;

[0171] (2) Transfer the leaf powder into a 1.5 mL EP tube and add 0.7 mL of 2% CTAB extraction buffer preheated at 65°C. Mix well and place in a 65°C water bath for 1 h. Shake and mix 2-3 times every 10 minutes during the water bath.

[0172] (3) Cool to room temperature, add 0.7 mL (volume ratio = 24:1) chloroform-isoamyl alcohol solution, mix well, and centrifuge at 12000 rpm for 10 min;

[0173] (4) Take the supernatant and put it into a new 1.5 mL EP tube. Add isopropanol of equal volume to the supernatant, mix well, and place in a -20 °C refrigerator for 1 h to precipitate gDNA.

[0174] (6) Centrifuge at 12000 rpm for 10 min, discard the supernatant, add 0.8 mL of 75% ethanol solution for elution, and repeat once;

[0175] (7) Centrifuge at 12000 rpm for 1 min, discard the supernatant, dry for 5 min to evaporate the ethanol, add 50 μL of ddH2O to dissolve, obtain Brassica napus gDNA, and store in a -20°C refrigerator.

[0176] Using gDNA as template, PCR detection was performed using primers HYG-F and HYG-R ( Figure 3 ); Extraction of total RNA ( Figure 5 ), reverse transcribed into cDNA, and qRT-PCR was used to identify the expression level of BnDAR2 gene to further screen positive seedlings. Figure 3 The results showed that 5 positive plants were able to successfully amplify the Hyg gene fragment, and Figure 6 The results showed that the expression levels of BnDAR2 gene in three strains were higher than that in the wild type, and they were recorded as: BnDAR2-OE-1, BnDAR2-OE-2, and BnDAR2-OE-3;

[0177] Total RNA extraction and reverse transcription system were the same as those in 1 and 2 of Example 1 except that the BnDAR2 gene overexpressing plants were used as experimental materials.

[0178] The PCR reaction system is the same as the reaction system in Table 5 in Example 1 except for the primers and cDNA template;

[0179] The PCR reaction program differs from the reaction program in Table 6 in Example 1 in that the annealing temperature is 55° C., and the others are the same.

[0180] The nucleotide sequence information of the primers is as follows:

[0181] HYG-F: 5'-GGCGAGTACTTCTACACAGC-3' (SEQ ID NO. 14);

[0182] HYG-R: 5'-CATGTGTATCACTGGCAAAC-3' (SEQ ID NO. 15);

[0183] BnDAR2-qrt-F: 5'-AACATCAACTGCCACCTCGT-3' (SEQ ID NO. 16);

[0184] BnDAR2-qrt-R: 5'-CGACGCAGACCGTACTTACA-3' (SEQ ID NO. 17).

[0185] 2.2 RNAi plant identification steps are as follows:

[0186] Extract gDNA from the BnDAR2 gene silenced plants obtained by the above steps (see Figure 2 ), the method is the same as above, using gDNA as template and primers HYG-F and HYG-R for PCR detection (see Figure 4 ); Extraction of total RNA ( Figure 5 ), reverse transcribed into cDNA, and qRT-PCR was used to identify the expression level of BnDAR2 gene to further screen positive seedlings. Figure 4The results showed that 3 positive plants were able to successfully amplify the Hyg gene (hygromycin gene) fragment, and Figure 6 The results showed that the expression level of BnDAR2 gene in three strains was lower than that in the wild type, which were recorded as: BnDAR2-RNAi-1, BnDAR2-RNAi-2 and BnDAR2-RNAi-3;

[0187] Total RNA extraction and reverse transcription system were the same as those in 1 and 2 of Example 1 except that BnDAR2 gene RNAi plants were used as experimental materials.

[0188] The PCR reaction system is the same as the reaction system in Table 5 in Example 1 except for the primers and cDNA template;

[0189] The PCR reaction program differs from the reaction program in Table 6 in Example 1 in that the annealing temperature is 55° C., and the others are the same.

[0190] 3. Sequencing

[0191] All samples with the correct band size mentioned above shall be sent for testing and comparison.

[0192] 4. Grain weight phenotype analysis of BnDAR2 overexpression and BnDAR2-RNAi plants

[0193] Overexpression plants BnDAR2-OE-1, BnDAR2-OE-2 and BnDAR2-OE-3, wild type ZS11 (control group, including ZS11-1, ZS11-2 and ZS11-3) and silenced plants BnDAR2-RNAi-1, BnDAR2-RNAi-2 and BnDAR2-RNAi-3 were planted in the field for sowing. When the materials treated reached the flowering period, they were bagged and self-pollinated. 35 days after flowering, the fully mature plants were harvested from the field, dried (2 to 3 days) and threshed.

[0194] The steps for measuring seed weight are as follows: 100 seeds were weighed using an analytical balance. Three groups of seeds from each treatment were weighed and the average value was taken. The results are shown in Tables 15 and Figure 7 (* indicates p<0.05, which means there is a significant difference between the experimental group and the control group; ** indicates p<0.01, which means there is an extremely significant difference between the experimental group and the control group; the scale between the experimental group and the control group is 1 cm). All experiments were performed with three biological replicates, and SPSS16.0 was used for significant difference analysis.

[0195] A scanner (Cano Scan, 5600F) and EXCEL software were used to take phenotypic photos and record data, and GraphPadPrism software was used to draw graphs. The results are shown in Tables 15 and Figures 7-8 .

[0196] Table 15 Statistics of 100-grain weight of seeds of BnDAR2 overexpression and RNAi plants (unit: g)

[0197] Plant name 1 group 2 groups 3 groups Mean ZS11-1 0.51 0.46 0.49 0.49 ZS11-2 0.50 0.50 0.52 0.51 ZS11-3 0.51 0.46 0.48 0.48 BnDAR2-OE-1 0.67 0.70 0.67 0.68 BnDAR2-OE-2 0.70 0.66 0.65 0.67 BnDAR2-OE-3 0.66 0.73 0.82 0.74 BnDAR2-RNAi-1 0.27 0.30 0.31 0.29 BnDAR2--RNAi-2 0.28 0.31 0.26 0.28 BnDAR2--RNAi-3 0.31 0.30 0.26 0.29

[0198] Table 15 and Figures 7-8 The results show that the 100-grain weight of BnDAR2 overexpressing seeds is significantly greater than that of the wild type, while the 100-grain weight of BnDAR2-RNAi plants is significantly less than that of the wild type. Statistical results show that the 100-grain weight of overexpressing plants increased by 41.2% compared with that of wild-type plants, while the 100-grain weight of RNAi plants decreased by 41.8% compared with that of wild-type plants.

[0199] The seed circumference was measured and counted using the image analysis software CaseVeiwer, and the data were recorded and averaged. The experimental results were the mean ± SD of 3 times. SPSS16.0 was used for significant difference analysis, and GraphPad Prism software was used for drawing.

[0200] Table 16 Statistics of seed circumference of BnDAR2 overexpression and RNAi plants (unit: mm)

[0201] Plant name 1 group 2 groups 3 groups Mean ZS11-1 0.565 0.575 0.575 0.572 ZS11-2 0.597 0.568 0.593 0.586 ZS11-3 0.597 0.603 0.60 0.6 BnDAR2-OE-1 0.691 0.688 0.666 0.682 BnDAR2-OE-2 0.691 0.659 0.685 0.678 BnDAR2-OE-3 0.722 0.707 0.788 0.739 BnDAR2-RNAi-1 0.487 0.502 0.496 0.495 BnDAR2-RNAi-2 0.408 0.424 0.446 0.426 BnDAR2-RNAi-3 0.468 0.465 0.465 0.466

[0202] The results are shown in Table 16 and Fig. 9 It was found that the average circumference of overexpression mature seeds was 7.00mm, the average circumference of wild-type mature seeds was 5.86mm, and the average circumference of RNAi mature seeds was 4.62mm. The circumference of overexpression mature seeds was significantly larger than that of wild-type, and the circumference of RNAi mature seeds was significantly smaller than that of wild-type.

[0203] In summary, the seed size and grain weight of BnDAR2 overexpressing plants were significantly larger than those of the wild type, and the increase in grain weight was as high as 41.2%. Therefore, the BnDAR2 gene has a profound effect on rapeseed grain weight.

[0204] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A BnDAR2 gene for increasing plant grain weight, characterized in that: The nucleotide sequence of the BnDAR2 gene is shown in SEQ ID NO.

3.

2. The protein encoded by the BnDAR2 gene according to claim 1.

3. The protein according to claim 2, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

4.

4. A biological material containing the BnDAR2 gene according to claim 1, characterized in that: Any of the following: (1) a recombinant expression vector comprising the BnDAR2 gene according to claim 1; (2) A recombinant microorganism comprising the BnDAR2 gene according to claim 1.

5. The biomaterial according to claim 4, characterized in that The biological material is a recombinant microorganism comprising the recombinant expression vector described in (1).

6. Use of the BnDAR2 gene according to claim 1, the protein according to claim 2, or the biological material according to claim 4 or 5 in the preparation of high-grain-weight Brassica napus.

7. Use of the BnDAR2 gene according to claim 1, the protein according to claim 2, or the biological material according to claim 4 or 5 in increasing the grain weight of Brassica napus.

8. A method for preparing high-grain-weight Brassica napus, characterized in that: The method comprises the steps of increasing the expression level of the BnDAR2 gene as claimed in claim 1 in a recipient Brassica napus to obtain transgenic Brassica napus.

9. The method according to claim 8, characterized in that The method comprises the steps of introducing the BnDAR2 gene of claim 1 or the biological material of claim 4 or 5 into a recipient Brassica napus to obtain a transgenic Brassica napus.

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