BjuDA1 R356K Genes and their encoded proteins and their applications

Abstract

The present invention relates to the field of plant molecular biology, and discloses a BjuDA1 gene of mustard (pickled mustard) containing a single mutation site, and a cloning method and application thereof. The present invention obtains the coding region of the BjuDA1 gene cDNA of the mustard by homologous cloning, and obtains the BjuDA1 gene of the mustard with a single mutation site by changing the 1067th base of the cDNA from G to A. R356K The coding region of the gene cDNA, the full length of the sequence is shown in Seq ID No.3, and the amino acid sequence of the encoded protein is shown in Seq ID No.4. R356K The gene overexpression vector was used to genetically transform wild-type Arabidopsis. The results showed that the seeds and leaves of the overexpression Arabidopsis positive strains were significantly enlarged. The hypocotyls of the tumorous mustard were infected by Agrobacterium-mediated method, and BjuDA1 R356K The gene overexpression vector is used to genetically transform the tumorous stem of the stem tumorous mustard, and the tumorous stem of the obtained positive plant is significantly enlarged. The single mutation site of the stem tumorous mustard BjuDA1 created by the present invention R356K The gene has the characteristic of increasing the size of the tubercle stems, and is of great value in increasing the yield of mustard tuber.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to application of a mustard tuber BjuDA1 gene containing a single mutation site in regulating the expansion of tumorous stems of mustard tuber. Background Art

[0002] Tumored mustard (Brassica juncea var.tumida Tsen et Lee) belongs to the Brassica genus of the Cruciferae family and is a variant of mustard. Tumored stems are its main product organ. The finished product made from tumored stems is "Zhacai", one of the three most famous pickled vegetables in the world. Due to its tender and crisp texture, delicious flavor and rich nutrition, it is widely planted in Sichuan, Chongqing, Jiangsu and Zhejiang (Liu Peiying, 1996). Zhacai, as a specialty vegetable in my country, has become a dominant specialty industry with the largest production and sales scale, the highest brand awareness and the strongest radiation driving ability in the rural economy in Chongqing, the main planting area. Continuously increasing the output of Zhacai is an important way to increase farmers' income.

[0003] The DA1 gene is a receptor for an E3 ubiquitin ligase discovered and cloned in Arabidopsis by Li Yunhai et al. (2008). As a ubiquitin receptor protein, AtDA1 can be bound by a variety of E3 ubiquitin ligases, or interact with downstream growth regulatory factors to regulate their activity, or degrade related substrates, ultimately affecting the size of seeds and organs, and negatively regulating the size of seeds and organs. In Arabidopsis, when the 358th amino acid of the DA1 gene mutates from arginine (R) to lysine (K), the mutant produces larger seeds and organs (Peng et al. 2015). Wang et al. (2017) overexpressed AtDA1 in Brassica napus. R358K It was found that overexpression of exogenous genes reduced the expression level of BnDA1 in Brassica napus, and organs such as seeds, leaves, flowers and siliques of transgenic plants were significantly enlarged.

[0004] At present, the DA1 gene has been reported in Arabidopsis, rapeseed, corn and rice, but not in mustard tuber. The BjuDA1 gene was cloned in mustard tuber using homologous cloning method, and the mutation site was introduced by in vitro artificial gene synthesis technology to create a point mutation BjuDA1 from the mustard tuber itself. R356K It is a method worth exploring to identify the gene and then cultivate new genetically modified mustard tuber-stemmed swollen germplasm through genetic engineering. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a stem tumor mustard BjuDA1 R356K Genes, their encoded proteins and their applications.

[0006] First, the present invention provides a BjuDA1 protein of Tumor Brassica juncea, which is:

[0007] 1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

[0008] 2) A protein derived from 1) with equivalent activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2.

[0009] The 356th position of the amino acid sequence of the protein is mutated from R (AGA, arginine) to A (AAA, lysine), and the amino acid sequence is shown in SEQ ID No.4.

[0010] The invention also provides a gene encoding the tumorous stem mustard BjuDA1.

[0011] Preferably, the sequence of the gene is shown as SEQ ID No.1 or SEQ ID No.3.

[0012] The invention also provides vectors, host cells and engineering bacteria containing the gene.

[0013] The invention also provides use of the gene in promoting the enlargement of tumorous stems of Brassica juncea.

[0014] In one embodiment of the present invention, the gene is transferred into the genome of the tumorous stem mustard and overexpressed in the transgenic tumorous stem mustard to promote the expansion of the tumorous stem of the tumorous stem mustard.

[0015] The invention also provides a method for promoting the swelling of tumorous stems of tumorous stem mustard, which comprises transferring a vector containing the gene into the genome of the tumorous stem mustard and overexpressing the gene in the transgenic tumorous stem mustard.

[0016] The present invention adopts homologous cloning method to clone BjuDA1 gene in mustard tuber, and obtains BjuDA1 of mustard tuber with single mutation site by changing the 1067th base of cDNA from G to A. R356K Gene, and then constructed BjuDA1 using genetic engineering methods R356K The overexpression vector of the gene was used to infect the inflorescence of wild-type Arabidopsis thaliana by Agrobacterium-mediated method, and finally the T3 generation homozygous transgenic positive strains were screened and identified. It was found that the seeds and leaf organs of the transgenic positive plants were significantly enlarged. Subsequently, the hypocotyls of mustard tuber were infected by Agrobacterium-mediated method, and BjuDA1 R356K The gene overexpression vector was used to genetically transform mustard tuber, and the tumorous stems of the obtained positive plants were significantly enlarged. R356K The gene has the characteristic of increasing the size of the tubercle stems and has great application prospects in increasing the yield of mustard tuber. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown is a cluster diagram of DA1 genes of different species in the Cruciferae family in Example 1, wherein UIM represents ubiquitin-interacting motif, and LIM represents zinc-binding domain (from LIN-11, Isl1 and MEC-3).

[0018] Figure 2 Shown is the single mutation site of mustard tuber BjuDA1 in Example 2 R356K Gene sequence alignment diagram, where BjuVA06G14890 is the reference genome v2.0 sequence of Brassica juncea, BjuDA1 is the sequence cloned from Brassica juncea in this study, and BjuDA1 R356K This is the point mutation sequence of BjuDA1.

[0019] Figure 3 The figure shows the electrophoresis diagram of the restriction enzyme digestion verification of the overexpression vector in Example 3, wherein the marker is the DNA molecular weight standard, pBin35SRed3-H is the vector band, and BjuDA1 R356K The target gene band.

[0020] Figure 4 The figure shows the screening and phenotypic identification of transgenic Arabidopsis positive plants in Example 5. A: Under the red filter, the seeds are illuminated by the LUYOR portable excitation light source, and the seeds that fluoresce are T0 generation seeds; B: T2 generation homozygous transgenic seeds; C: PCR identification of T2 generation transgenic positive plants, where Marker is a DNA molecular weight standard, 1-7 are 7 randomly selected T2 generation transgenic positive plants, CK is the control Arabidopsis Col-0, and P is the positive control overexpression vector pBin35SRed3-H-BjuDA1 R356K , N is the negative control (the template is water); D: expression analysis, where Col-0 is the control, Line 1-Line 3 are 3 randomly selected positive plants, * and ** indicate significant differences at the 0.01 and 0.05 levels, respectively; E: seeds of transgenic Arabidopsis Line 1 and Line 2, Col-0 is the control, Bar=500μm; F: seeds of transgenic Arabidopsis Line 1 and Line 2, the leftmost is the control Col-0, Bar=0.2cm; GI: 1000-grain weight, relative value of seed area and relative value of petal area of ​​transgenic Arabidopsis Line 1 and Line 2; J: cotyledons of transgenic Arabidopsis Line 1 and Line 2 sown for 9 days, the leftmost is CK, Bar=0.2cm; K: the whole of transgenic Arabidopsis Line 1 and Line 2 sown for 30 days, the leftmost is CK, Bar=1.8cm.

[0021] Figure 5The figure shows the screening and phenotypic identification of transgenic mustard tuber positive plants in Example 7. A: PCR identification of transgenic mustard tuber positive plants, where M is a DNA molecular weight standard, 1-7 are the 7 transgenic positive plants obtained, and P is the positive control overexpression vector pBin35SRed3-H-BjuDA1 R356K , N is negative control; B: 7 transgenic plants were amplified by point mutation primers, and the two bands indicated by arrows (Line 5 and Line 7) are positive lines with single mutation sites; C: Sequencing results of target fragments of Line 5 and Line 7, the arrows indicate the mutation sites (from G to A); D: Leaf size of transgenic positive plants, WT is wild type, Bar=1.2cm; E: Flower size of transgenic positive plants, the leftmost is WT, Bar=0.6cm; FG: Siliques and seeds of transgenic positive plants, Bar=0.6cm, the top is WT; H: Tuberous stems of transgenic positive plants, Bar=2cm; IL: Histograms of leaf area, petal area, silique width and 1000-grain weight of transgenic positive plants, * and ** indicate significant differences at the 0.05 and 0.01 levels, respectively. DETAILED DESCRIPTION

[0022] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0023] Example 1 Cloning of the cDNA sequence of the BjuDA1 gene of mustard tuber

[0024] 1) Extraction of total RNA from mustard tuber leaves at seedling stage:

[0025] The operation steps refer to the instructions of Tiangen RNA Extraction Kit (Cat. No.: DP432), collect 80-120 mg of fresh mustard tuber leaves at the 8-10 leaf stage, quickly put them into a mortar pre-cooled with liquid nitrogen, add an appropriate amount of liquid nitrogen and fully grind them into powder, immediately transfer them into a 1.5 ml enzyme-free centrifuge tube, add 450 μL of cell lysis solution RL (add β-mercaptoethanol with a final concentration of 1% in advance), vortex and shake to mix; transfer the above grinding solution to the filter column CS (CS is placed in the collection tube), centrifuge at 12000rpm for 5min, carefully pipette the supernatant in the collection tube into an RNase-free centrifuge tube; add 250 μL of anhydrous ethanol, mix gently, transfer the solution to the adsorption column CR3, centrifuge at 12000rpm for 50s, and discard the filtrate; add 350 μL of deproteinization solution RWl to CR3, centrifuge at 12000rpm for 45s, and discard the filtrate; add 80 μL of DNase in mid-air I working solution was placed in the center of CR3 adsorption membrane, allowed to stand at room temperature for 15 minutes, 350 μL RW1 was added, centrifuged at 12000 rpm for 45 seconds, and the filtrate was discarded; 500 μL rinsing solution RW (before the first use, 48 ml of anhydrous ethanol was added to 12 ml RW) was added dropwise to the adsorption column CR3, allowed to stand at room temperature for 2 minutes, centrifuged at 12000 rpm for 45 seconds, and the waste liquid was discarded. This step was repeated once; centrifuged at 12000 rpm for 2 minutes, the tube cover was opened, the waste liquid was poured out, CR3 was allowed to stand at room temperature for 4-6 minutes, and the residual rinsing solution was dried; CR3 was placed in a new RNase-free centrifuge tube, 30-100 μL RNase-free ddH2O was added dropwise in the center of the adsorption membrane in mid-air, allowed to stand at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 2 minutes to obtain RNA solution.

[0026] RNA concentration and integrity test: The concentration test was performed by taking 2 μL of the diluted RNA solution and placing it on an ultra-micro spectrophotometer (Nanodrop 2000) to measure the RNA concentration, ensuring that the peak value was correct and the OD value was between 1.8 and 2.1. The integrity test was performed by soaking the instrument and the electrophoresis tank with hydrogen peroxide for 20 minutes, and then preparing 1.0% agarose gel on an enzyme-free, clean operating table. The total RNA was immediately subjected to electrophoresis after extraction. When the electrophoresis result graph showed at least two clear bands, and the brightness of the upper 28S band was about twice that of the middle 18S band, it proved that the integrity of the extracted mustard tuber total RNA was good and could be used for subsequent experiments. Finally, it was stored at -80°C for future use.

[0027] 2) First-strand cDNA synthesis:

[0028] The RNA concentration was uniformly adjusted to 100 ng / μl, and a 14.5 μL reaction system {total RNA: 4 μL, Oligo(dT) 15Primer: 1μL (50μmol / L), pd(N)6: 0.5μL (50μmol / L), RNase Free ddH2O: 9μL} were placed in a 200μL centrifuge tube and placed on a PCR amplification instrument for denaturation reaction (program: 70℃10min, 4℃2min) to obtain product A. Then, 20μL reverse transcription reaction system {A: 14.5μL, 5×M-MLV buffer: 4μL, M-MLV: 0.25μL, dNTP (10mM): 1μL, Recombinant RNase Inhibitor, 0.25μL} was prepared in a 200μL centrifuge tube and placed on a PCR amplification instrument for reaction (program: 42℃60min, 70℃10min, 4℃2min) to obtain the first-strand cDNA, which was stored at -80℃ for future use.

[0029] 3) Cloning of the BjuDA1 gene from Zhacai:

[0030] According to the TAIR website (https: / / www.arabidopsis.org), the Arabidopsis DA1 gene (AT1G19270) was searched. According to its full-length CDS sequence, the Brassica database (http: / / brassicadb.cn) was used for Blast (parameter Nucleotide databases: Braju tum V 2.0cds), and the homologous gene BjuVA06G14890 of mustard tuber was obtained. The primers were designed using the Oligo 7 software: forward primer F1: 5'-ATGGGTTGGTTTAACAAGATCTTC-3' (Seq ID No.5), reverse primer R1: 5'-AACCGGGAATCTACCGGTCATCTGTAT-3' (Seq ID No.6). The first-strand cDNA after reverse transcription was used as a template, and the above forward and reverse primer combinations were used to amplify the BjuDA1 gene by PCR. The PCR amplification system (50 μL) was: 25 μL of 2×Fast Pfu Master Mix enzyme, 2 μL of forward and reverse primers, 2 μL of template cDNA, and 19 μL of ddH2O. The PCR amplification program was: 94°C for 90 s; 94°C for 20 s, 58°C for 20 s, 72°C for 49 s, 30 cycles; 72°C for 5 min.

[0031] After the PCR reaction is completed, the target band is cut out after detection by 1.5% (w / v) agarose gel electrophoresis, and the target band is recovered using a gel recovery / DNA purification kit (DC301). For specific steps, refer to the instructions of the kit. -Blunt Simple was connected and transformed into Escherichia coli DH-5α competent cells. Monoclonal plaques were picked for detection, and the correct plaques were sent to the company for sequencing. The coding region sequence of the BjuDA1 gene was verified. According to the sequence splicing results, the coding region sequence of the BjuDA1 gene cDNA of mustard tuber was obtained (Seq ID No.1), and the BjuDA1 gene plasmid was obtained at the same time.

[0032] The coding sequence of the BjuDA1 gene cDNA of mustard tuber was translated to obtain the amino acid sequence of BjuDA1 (Seq ID No. 2). Further, the amino acid sequence was compared with the genomes of Arabidopsis thaliana, Brassica rapa, Brassica oleracea, Brassica nigra, Brassica napus, Brassica juncea and Brassica rapa published in the Cruciferae family. The results showed that BjuDA1 was clustered with Brassica rapa BraA06g015110.3.5C.2, Brassica napus A06p15420.1_BnaDAR and Brassica juncea BjuVA06G14890, which were co-distributed on chromosome A06. They had the same domain structure and had the highest sequence similarity with Brassica juncea BjuVA06G14890 (98.2%), indicating that the BjuDA1 gene of mustard tuber was successfully cloned ( Figure 1 ).

[0033] Example 2: Mustard tuber containing a single mutation site DA1R356K Acquisition of genes

[0034] Point mutation primers were designed according to Oligo 7 software: forward primer F2: 5'-CTATCACATGCCAGAGACAAAAGGACTCTGCCTTTCAGAAG-3' (Seq ID No.7), reverse primer R2: 5'-CTTCTGAAAGGCAGAGTCCTTTTGTCTCTGGCATGTGATAG-3' (Seq ID No.8). BjuDA1 gene plasmid was used as template and PCR amplified with primers F2 and R2. The reaction system (25 μL) was: 2×Fast Pfu Master Mix enzyme 13 μL, primers F2 and R2 1 μL each, BjuDA1 gene plasmid 1 μL, ddH2O 9 μL. PCR amplification program was: 94℃90s; 94℃20s, 58℃20s, 72℃50s, 32 cycles; 72℃5min. After obtaining the PCR product, the product was immediately placed on ice, 1 μL of Dpn I enzyme was added, mixed, and incubated at 37°C for 2 h. The incubated PCR product was transformed into Escherichia coli, positive clones were screened, and plasmids were extracted and sent to the company for sequencing to obtain the mustard tuber BjuDA1 containing a single mutation site. R356K Gene sequence (Seq ID No. 3).

[0035] Example 3: Mustard tuber DA1R356K Construction of overexpression vector

[0036] The expression vector used was pBin35Sred3-H, and the restriction sites were XbaⅠ and Pac I. PCR amplification was used to amplify the target gene BjuDA1 R356K Primers were designed at both ends of the coding region, and the corresponding restriction sites and protective bases were added to the 5' end of the primers. The target gene plasmid was used as a template, and F3: 5'-GCTCTAGAATGGGTTGGTTTAACAAGATC-3' (Seq ID No.9, introducing XbaⅠ restriction site and protective base GC) and R3: 5'-GTTAATTAATTAAACCGGGAATCTACCGG-3' (Seq ID No.10, introducing Pac I restriction site and protective base G) were used as primers, and 2×Fast Pfu Master Mix enzyme was used for PCR amplification. The reaction system (25μL) was: 2×Fast Pfu Master Mix enzyme 13μL, primers F3 and R3 1μL each, BjuDA1 R356K 1 μL of gene plasmid, 9 μL of ddH2O. PCR amplification program: 94℃90s; 94℃20s, 58℃20s, 72℃50s, 32 cycles; 72℃5min.

[0037] After the PCR reaction, the PCR product was subjected to 1.5% (w / v) agarose gel electrophoresis, and the PCR product was recovered using a gel recovery / DNA purification kit (DC301). The product and the plasmid of the expression vector were subjected to double enzyme digestion reaction respectively. The enzyme digestion reaction system (50 μL) was: 21 μL of vector or gene gel recovery product, 5 μL of Fastcut Buffer, 1.5 μL of Xba I, 1.5 μL of Pac I, and 21 μL of ddH2O. After mixing, it was placed in a 37°C water bath for 2 hours. After the enzyme digestion was completed, DC301 was used to recover the target gene and the expression vector to obtain the enzyme digestion product. T4-DNA ligase was used to connect the target gene after enzyme digestion with the expression vector. The connection system (10 μL) was: 5 μL of Solution I (containing T4-DNA ligase), 4 μL of the target gene, and 1 μL of the expression vector. Use a small centrifuge to fully mix and react in a 16°C metal bath for 2 hours. After the connection was completed, it was transformed into competent cells of Escherichia coli DH-5α, and the culture plate was placed in an incubator at 37°C for overnight culture. The primers Seq ID No.9 and QW586R (5′-AAAAGACAAAAGTGGGGTAG-3′, Seq ID No.11) were used to detect the colonies, and the positive colonies were shaken and sent to the company for sequencing verification. After verification, the overexpression vector pBin35SRed3-H-BjuDA1 was obtained. R356K , double enzyme digestion verification was performed using Xba I and Pac I, and agarose gel electrophoresis was performed after enzyme digestion. The results showed that two bands appeared (the 11000 bp band was the vector pBin35SRed3-H band, and the 1600 bp band was the target gene BjuDA1 R356K Stripe)( Figure 3 ), proving that the overexpression vector pBin35SRed3-H-BjuDA1 R356K Build successful.

[0038] Agrobacterium transformation: Take 2 μL pBin35SRed3-H-BjuDA1 R356KThe recombinant plasmid was added to 100 μL of Agrobacterium competent cells GV3101 melted on ice in advance and mixed evenly; ice bath for 5 minutes, liquid nitrogen freezing for 5 minutes, quickly placed in a 37°C water bath for 5 minutes, and ice bath for 5 minutes; 800 μL of YEB liquid medium without antibiotics was added, and the culture was shaken at 28°C and 250 rpm for 2 hours; centrifuged at 6000 rpm for 1 minute to collect the bacterial solution, and about 100 μL of the supernatant was gently blown and resuspended, and evenly coated on YEB solid medium (containing 60 μg / mL Kan and 25 μg / mL Rif antibiotics), and inverted in a 28°C incubator for 48 hours, and single colonies were detected with primers Seq ID No. 9 and Seq ID No. 11, and positive colonies were shaken and preserved (700 ul bacterial solution + 300 ul of 60% glycerol) to obtain pBin35SRed3-H-BjuDA1. R356K The Agrobacterium culture medium containing the recombinant plasmid was used for the genetic transformation of Arabidopsis thaliana and Brassica juncea as described below.

[0039] Example 4 Overexpression vector pBin35SRed3-H-BjuDA1 R356K Genetic transformation of Arabidopsis thaliana

[0040] 1) Sowing: Wild-type Arabidopsis Col-0 was used as the material. Full seeds were selected and placed on moist filter paper and placed in a 4°C light incubator for 48 h. After germination, they were transplanted into nutrient soil (perlite: vermiculite: 6 mm peat = 1:4:5) and grown under the conditions of 14 h light / 10 h dark, 22°C, and 70% relative humidity.

[0041] 2) Activation and propagation: Use an inoculation loop to pick up a small amount of the culture medium containing pBin35SRed3-H-BjuDA1 R356K The Agrobacterium liquid of the recombinant plasmid was streaked on a YEB solid medium containing 60μg / mL Kan and 25μg / mL Rif, and cultured in a 28℃ constant temperature incubator for 2 days. After colony PCR detection, positive colonies were picked and shaken, and 4ml of the bacterial liquid was aspirated and activated in 50mL YEB liquid medium (containing 60μg / mL Kan and 25μg / mL), and propagated to OD 600 It is 0.8~1.2.

[0042] 3) Bacteria collection: centrifuge the above bacterial solution at 4000 rpm for 10 min, discard the supernatant, and collect the bacteria at the bottom of the centrifuge tube;

[0043] 4) Suspension: Resuspend the bacterial mass in 1 / 2MS liquid medium (containing 5% sucrose and 3% Silwet L-77 surfactant) to an OD of 600 =0.4~0.6, used to infect Arabidopsis inflorescence.

[0044] 5) Infection culture: Soak the Arabidopsis inflorescence (pods removed in advance) in the bacterial solution for about 30 seconds, place it on its side in a foam box, culture it in the dark for 24 hours away from light, and then culture it under normal light conditions. Repeat this twice with an interval of 7 days. After the seeds mature, screen the positive seeds.

[0045] Example 5 Screening and phenotypic identification of transgenic Arabidopsis positive plants

[0046] After Agrobacterium infection, the harvested T0 generation seeds were screened for red fluorescence. The specific operation was as follows: the seeds were illuminated with a LUYOR-3415RG dual-wavelength fluorescent protein observation lamp, and the seeds that fluoresced through a red filter were transgenic positive seeds ( Figure 4 A). Sow the fluorescent T0 generation positive seeds and harvest them individually, screen the harvested T1 generation seeds for red fluorescence according to the above method, select the transgenic lines that meet the Mendelian trait segregation ratio of 3:1, continue to sow T1 generation positive seeds individually, and select the lines that all fluoresce after harvesting the seeds, which are the T2 generation homozygous transgenic seeds ( Figure 4 B). Sowing T2 homozygous transgenic seeds, at the 4-6 leaf stage, extracting DNA from the leaves using the CTAB method, specifically: taking 0.5 g of leaves and placing them in a 1.5 mL centrifuge tube, grinding them with a grinding rod for several times, and then adding 200 μL of DNA extraction buffer (DNA buffer composition: 200 mmol / L Tris-Hcl, pH=8.0; 250 mmol / L NaCl; 25 mmol / L EDTA, PH=8.0; 0.5% SDS); after fully grinding, adding 200 μL of extraction buffer, letting stand at room temperature for 2 min; centrifuging at 12000 rpm for 5 min, taking the supernatant and placing it in a new centrifuge tube; adding 300 μL of isopropanol, mixing by inverting, and settling at -20°C for 5 min; centrifuging at 12000 rpm for 6 min, discarding the supernatant and retaining the precipitate; adding 300 μL of isopropanol, centrifuging at 12000 rpm for 2 min, discarding the supernatant, aspirating the residual liquid at the bottom of the centrifuge tube, and letting stand at room temperature for 5 min; adding 50 μL ddH2O, flick to dissolve and obtain DNA.

[0047] Arabidopsis Col-0 was used as the control (CK), water was used instead of template as the negative control (N), and pBin35SRed3-H-BjuDA1 R356K As the positive control (P), PCR identification was performed on 7 randomly selected T2 transgenic Arabidopsis positive plants using primers Seq ID No.9 and Seq ID No.11. The agarose gel electrophoresis results showed that the target bands were detected in all 7 transgenic lines and the positive control, while no bands were detected in CK and negative controls ( Figure 4C). Three positive plants were randomly selected for fluorescence quantitative qRT-PCR expression analysis. RNA extraction and first-chain cDNA synthesis were performed on the rosette leaves of the selected plants, as described in Example 1. The kit used for fluorescence quantitative PCR analysis was SYBR qPCR SuperMix Plus (E096-01A), the fluorescence quantitative PCR instrument was CFX96 Touch fluorescence quantitative PCR instrument, the primers were F4: 5′-TGCGAGGTCACTGCCATT-3′ (Seq ID No. 12) and R4: 5′-ACGTCTTGGCTCAGCGTC-3′ (Seq ID No. 13), and the Arabidopsis thaliana internal reference gene TUB2 was used as the control primers F5: 5′-ATCCGTGAAGAGTACCCAGAT-3′ (Seq ID No. 14) and R5: 5′-AAGAACCATGCACTCATCAGC-3′ (Seq ID No. 15). The expression analysis results showed that BjuDA1 R356K The expression level of the gene was significantly increased in transgenic Arabidopsis ( Figure 4 D). Further phenotypic observation and statistical analysis revealed that the cotyledons, seeds, petals and overall plant shape of the transgenic lines were significantly larger than those of the control ( Figure 4 EK). Description of mustard tuber BjuDA1 R356K Genetic regulation of organ size in Arabidopsis thaliana.

[0048] Example 6 Genetic transformation of mustard tuber with overexpression vector pBin35SRed3-H-BjuDA1R356K

[0049] 1) Pre-cultivation for sowing: Select 20 mustard tuber seeds with full grains, put them into a 200ml sterile tissue culture bottle, soak them in 75% alcohol for 45 seconds, then wash them with sterile deionized water for 60 seconds, soak them in 10% NaClO solution for 15 minutes, shake them continuously, continue to wash them with sterile deionized water for 60 seconds, repeat the washing 5 times, and complete the seed disinfection. Sow the disinfected seeds on MS solid culture medium, place them in the culture room for 7 days, cut the germinated seedling hypocotyls into small sections of about 1 cm, and culture them on the pre-culture medium (MS solid culture medium + 3mg / L 6-BA + 0.2mg / L NAA) for 2 days.

[0050] 2) Activation of dark culture: Use an inoculation loop to take a small amount of the culture medium containing pBin35SRed3-H-BjuDA1 R356KThe Agrobacterium liquid of the recombinant plasmid was streaked on a YEB solid medium containing 25 μg / mL Rif and 60 μg / mL Kan, and cultured in a 28°C constant temperature incubator for 2 days. After colony PCR detection, positive colonies were picked and shaken, and 4 ml of the liquid was aspirated and activated in 50 mL YEB liquid medium (containing 25 mg / L Rif and 60 mg / L Kan), and propagated to OD 600 is 0.5-0.6; transfer 50mL of bacterial solution to a 50mL sterile centrifuge tube, centrifuge at 4000rpm at 4℃ for 10min, discard the supernatant, resuspend the bacteria with 25mL pre-cooled MS liquid culture medium, and use the resuspended solution to infect the pre-cultured hypocotyls for 10min. During the infection process, shake gently continuously. Finally, dry the bacterial solution on the hypocotyls with sterile filter paper, spread it on the preculture medium (MS solid culture medium + 3mg / L 6-BA + 0.2mg / L NAA), and culture it in the dark for 2d.

[0051] 3) Differentiation and rooting culture: After dark culture, the co-cultured hypocotyls were transferred to MS screening medium (MS solid medium + 3mg / L 6-BA + 0.2mg / L NAA + 25mg / L Hyg + 400mg / L Cb), and cultured at 25°C, 16h of light, and 8h of darkness. The screening medium was replaced every 7 days until the hypocotyls differentiated into adventitious buds. After multiple subcultures, the surviving adventitious buds were cut off and inserted into rooting medium (MS solid medium + 0.2mg / L NAA + 25mg / L Hyg + 400mg / L Cb) to induce rooting. After the roots grew to 3-5cm, the tissue culture bottle was opened to harden the seedlings for 2-3 days, and then the culture medium on the roots was removed and the seedlings were transplanted into the matrix to obtain the single mutation site BjuDA1 of mustard tuber. R356K Gene overexpression transgenic lines.

[0052] Example 7 Screening and phenotypic identification of transgenic mustard tuber positive plants

[0053] The DNA extraction method in Example 5 was used to extract DNA from non-transgenic mustard tuber and transgenic mustard tuber positive plants as templates, Seq ID No.9 and Seq ID No.11 were used as primers, and 2×Taq PCR Master Mix enzyme was used for PCR amplification. The results of agarose gel electrophoresis showed that 3 positive strains were screened out from 7 transgenic materials, and the bands were consistent with the positive control ( Figure 5 A). Further, primers (F6: 5′-ACACTTTTCAATGCCAACCT-3′ (Seq ID No. 16) and Seq ID No. 11) were designed before and after the mutation site, PCR amplification was performed, and the target fragment was recovered for sequencing, and finally two transgenic mustard tuber positive strains with single mutation sites were obtained ( Figure 5 BC). The transgenic strains were planted together with wild-type mustard tubers in a test field with the same environmental conditions. Phenotypic observation and statistical analysis showed that the leaves, tuberculous stems, petals and 1000-grain weight of the transgenic strains were significantly larger than those of the wild-type mustard tubers, and they grew normally ( Figure 5 In summary, the point mutation of BjuDA1 in Zhacai can significantly increase the number of product organs, which has important practical significance for increasing the yield of Zhacai.

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of the tumorous stem mustard BjuDA1 protein in promoting the enlargement of tumorous stems of tumorous stem mustard, characterized in that: Overexpression of the protein in tumorous stem mustard promotes the enlargement of tumorous stems of the tumorous stem mustard, wherein the amino acid sequence of the tumorous stem mustard BjuDA1 protein is shown in SEQ ID No.

4.

2. The use according to claim 1, characterized in that The gene encoding the protein is transferred into the genome of the tumorous mustard plant and overexpressed in the transgenic tumorous mustard plant to promote the swelling of the tumorous stem of the tumorous mustard plant.

3. The use according to claim 2, characterized in that The gene nucleotide sequence is shown in SEQ ID No.

3.

4. The use according to claim 2, characterized in that The gene encoding the protein is transferred into the genome of Brassica juncea by Agrobacterium-mediated method.

5. A method for promoting the enlargement of tumorous stems of Brassica juncea, characterized in that: The gene encoding the BjuDA1 protein of the stem tumor mustard is transferred into the genome of the stem tumor mustard and overexpressed in the transgenic stem tumor mustard, wherein the amino acid sequence of the BjuDA1 protein of the stem tumor mustard is shown in SEQ ID No.

4.

6. The method according to claim 5, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.3.

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