CN116535476A - Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene - Google Patents

Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene Download PDF

Info

Publication number
CN116535476A
CN116535476A CN202310409132.9A CN202310409132A CN116535476A CN 116535476 A CN116535476 A CN 116535476A CN 202310409132 A CN202310409132 A CN 202310409132A CN 116535476 A CN116535476 A CN 116535476A
Authority
CN
China
Prior art keywords
gene
bjuda1
mustard
stem
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310409132.9A
Other languages
Chinese (zh)
Inventor
魏大勇
赵飒
吴杰
管婧汝
汤青林
王志敏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southwest University
Original Assignee
Southwest University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Southwest University filed Critical Southwest University
Priority to CN202310409132.9A priority Critical patent/CN116535476A/en
Publication of CN116535476A publication Critical patent/CN116535476A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention relates to the field of plant molecular biology, and discloses a stem tumor mustard (hot pickled mustard tuber) BjuDA1 gene containing a single mutation site, a cloning method and application thereof. The invention obtains the coding region of the BjuDA1 gene cDNA of the stem tumor mustard by a homologous cloning method, and obtains the BjuDA1 of the stem tumor mustard with single mutation site by changing the 1067 th base of the cDNA from G to A R356K Gene cThe full length of the coding region of the DNA is shown as SEQ ID No.3, and the amino acid sequence of the coding protein is shown as SEQ ID No. 4. BjuDA1 was isolated by Agrobacterium-mediated methods R356K The gene over-expression vector is used for genetically transforming the wild arabidopsis, and the result shows that the seed and leaf organs of the over-expression arabidopsis positive strain are obviously increased; infection of the hypocotyl of the Phoma mustard by Agrobacterium-mediated method, bjuDA1 R356K The gene over-expression vector is used for genetically transforming the stem nodule, and the tumor stem of the obtained positive plant is obviously increased. The invention creates single mutation site of the stem tumor mustard BjuDA1 R356K The gene has the characteristic of increasing the tumor-shaped stems, and has great utilization value for increasing the yield of the hot pickled mustard tuber.

Description

Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to an application of a tuber mustard BjuDA1 gene containing a single mutation site in regulation and control of tuber mustard tumor-shaped stem expansion.
Background
The stem mustard (Brassica junsea var. Tumida Tsen et Lee) belongs to Brassica crops of cruciferae, is one of the varieties of Brassica juncea, the neoplastic stem is a main product organ thereof, and the processed finished product taking the neoplastic stem as a raw material is the preserved szechuan pickle which is one of three major pickled vegetables in the world, and is widely planted in the Chongyu and Jiangzhe areas (Liu Peiying, 1996) due to the tender and crisp texture, delicious flavor and rich nutrition. The hot pickled mustard tuber is taken as a special vegetable in China, and is the dominant special industry with the largest production and marketing standard, the highest brand awareness and the highest radiation driving capability in rural economy in Chongqing city in the main planting area. The continuous improvement of the yield of the hot pickled mustard tuber is an important way for increasing the income of peasants.
DA1 gene is Li Yunhai (2008) and is a receptor of E3 ubiquitin ligase found and cloned in Arabidopsis thaliana, atDA1 is taken as ubiquitin receptor protein, can be combined by a plurality of E3 ubiquitin ligases or interacts with downstream growth regulating factors to regulate the activity of the E3 ubiquitin ligases or degrade related substrates, finally affects the size of seeds and organs, and has negative regulation effect on the size of the seeds and the organs. In Arabidopsis thaliana, when the 358 th amino acid of DA1 gene is mutated from arginine (R) to lysine (K), the mutant is producedLarger seeds and organs (Peng et al 2015). Wang (2017) et al by overexpression of AtDA1 in brassica napus R358K The over-expression of the exogenous gene is found to reduce the expression level of the brassica napus BnDA1, and the seed, leaf, flower, pod and other organs of the transgenic plant are obviously increased.
At present, DA1 genes are reported in Arabidopsis thaliana, rape, corn and rice, but are not reported in preserved szechuan pickle. Cloning BjuDA1 gene in hot pickled mustard tuber by homologous cloning method, introducing mutation site by in vitro artificial gene synthesis technology, and creating point mutation BjuDA1 from hot pickled mustard tuber species R356K The gene is a method worthy of exploration by cultivating new germplasm of the tuber mustard with enlarged transgenic tumor-shaped stems through genetic engineering.
Disclosure of Invention
To solve the above problems, the present invention provides a Chlamydia mustard BjuDA1 R356K Genes and proteins encoded thereby and uses thereof.
First, the present invention provides a stem tumor mustard BjuDA1 protein which is:
1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)
2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
Wherein, the 356 th site of the protein amino acid sequence is mutated from R (AGA, arginine) to A (AAA, lysine), and the amino acid sequence is shown in SEQ ID No. 4.
The invention also provides a gene encoding the stem tumor mustard BjuDA 1.
Preferably, the sequence of the gene is shown as SEQ ID No.1 or SEQ ID No. 3.
The invention also provides a vector, a host cell and engineering bacteria containing the gene.
The invention also provides application of the gene in promoting the expansion of the tumorous stem mustard.
In one embodiment of the invention, the gene is transferred into the genome of the stem tumor mustard and is overexpressed in the transgenic stem tumor mustard to promote expansion of the stem tumor mustard's neoplastic stem.
The invention also provides a method for promoting the expansion of the tumorous stem mustard, which is to transfer a vector containing the gene into the genome of the tumorous stem mustard and to overexpress the tumorous stem mustard in the transgenic tumorous stem mustard.
The invention adopts a homologous cloning method to clone BjuDA1 gene in preserved szechuan pickle, and obtains the preserved szechuan pickle BjuDA1 with single mutation site by changing 1067 th base of the cDNA from G to A R356K Gene, bjuDA1 is constructed by using a genetic engineering method R356K The over-expression vector of the gene is used for infecting inflorescence of wild arabidopsis thaliana by adopting an agrobacterium-mediated method, and finally, T is obtained by screening and identification 3 The generation of homozygous transgenic positive plants, and the seed and leaf organs of the transgenic positive plants are found to be obviously increased; subsequently, the hypocotyl of the preserved szechuan pickle is continuously infected by an agrobacterium-mediated method, and BjuDA1 is added R356K The gene over-expression vector is used for genetically transforming the hot pickled mustard tuber, and the tumor-shaped stem of the obtained positive plant is obviously increased. The invention creates the hot pickled mustard tuber BjuDA1 with single mutation site R356K The gene has the characteristic of increasing the tumor-shaped stems, and has great application prospect for increasing the yield of the hot pickled mustard tuber.
Drawings
FIG. 1 is a cluster diagram showing DA1 genes of different species of crucifer in example 1, wherein UIM represents ubiquitin-interacting motif and LIM represents zinc-binding domain (from LIN-11, isl1 and MEC-3).
FIG. 2 shows the single mutation site of BjuDA1 from hot pickled mustard tuber of example 2 R356K Gene sequence alignment diagram, wherein BjuVA06G14890 is mustard reference genome v2.0 sequence, bjuDA1 is cloned sequence in hot pickled mustard tuber in this study, bjuDA1 R356K Is BjuDA1 point mutation sequence.
FIG. 3 is a schematic diagram showing the enzyme digestion verification electrophoresis of the overexpression vector of example 3, wherein Marker is the DNA molecular weight standard, pBin35SRed3-H is the vector strip, and BjuDA1 R356K Is the target gene band.
FIG. 4 shows the screening and phenotypic identification of transgenic Arabidopsis positive plants in example 5. A: under the red filter sheet, the seed is irradiated by a LUYOR portable excitation light source, and the fluorescent light is T 0 Seed generation; b: t (T) 2 Substitute pureSynthesizing transgenic seeds; c: t (T) 2 PCR identification of transgenic positive plants, wherein Marker is DNA molecular weight standard, 1-7 is randomly selected 7T plants 2 Transgenic positive plants, CK is control Arabidopsis Col-0, P is positive control super-expression vector pBin35SRed3-H-BjuDA1 R356K N is a negative control (template is water); d: expression analysis, wherein Col-0 is control, line 1-Line 3 is randomly selected 3 positive plants, and x represent significant levels of difference at 0.01 and 0.05 levels, respectively; e: seeds of transgenic arabidopsis Line 1 and Line 2, col-0 as control, bar=500 μm; f: seeds of transgenic arabidopsis Line 1 and Line 2, the leftmost is control Col-0, bar=0.2 cm; G-I: thousand kernel weight, seed area relative value and petal area relative value of transgenic arabidopsis Line 1 and Line 2; j: sowing the cotyledons of the 9-day transgenic arabidopsis thaliana Line 1 and Line 2, wherein CK is the leftmost part, and bar=0.2 cm; k: the whole of Line 1 and Line 2 of transgenic Arabidopsis was sown for 30 days, with CK at the far left and Bar=1.8 cm.
FIG. 5 shows the screening and phenotypic identification of positive plants of transgenic tuber mustard in example 7. A: PCR identification of transgenic preserved szechuan pickle positive plants, wherein M is DNA molecular weight standard, 1-7 are 7 obtained transgenic positive plants, and P is positive control overexpression vector pBin35SRed3-H-BjuDA1 R356K N is a negative control; b: amplifying 7 transgenic plants by using a point mutation primer, wherein two bands (Line 5 and Line 7) shown by arrows are positive lines with single mutation sites; c: line 5 and Line 7 target fragment sequencing results, arrow indicates mutation site (G to A); d: leaf size of transgenic positive plants, WT wild type, bar=1.2 cm; e: flower size of transgenic positive plants, WT, bar=0.6 cm on the far left; F-G: the fruits and seeds of transgenic positive plants, bar=0.6 cm, with WT uppermost; h: tumor stem bar=2cm of transgenic positive plants; I-L: leaf area, petal area, pod width and thousand kernel weight histograms of transgenic positive plants represent the difference significance at 0.05 and 0.01 levels, respectively.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
EXAMPLE 1 cloning of cDNA sequence of BjuDA1 Gene of Hot pickled mustard tube
1) Extracting total RNA of leaf blades of the hot pickled mustard tuber in a seedling stage:
referring to a specification of a root RNA extraction kit (product number: DP 432), collecting 80-120 mg of fresh tuber mustard leaves in 8-10 leaf stage, rapidly placing the fresh tuber mustard leaves into a mortar after liquid nitrogen precooling, adding a proper amount of liquid nitrogen, fully grinding the fresh tuber mustard into powder, immediately transferring the powder into a 1.5ml enzyme-free centrifuge tube, dropwise adding 450 mu L of cell lysate RL (beta-mercaptoethanol with the final concentration of 1% is added in advance), and uniformly mixing the powder by vortex vibration; transferring the above grinding fluid into a filter column CS (CS is placed in a collecting tube), centrifuging at 12000rpm for 5min, and carefully sucking the supernatant in the collecting tube into an RNase-free centrifuge tube; adding 250 mu L of absolute ethyl alcohol, gently mixing, transferring the solution into an adsorption column CR3, centrifuging at 12000rpm for 50s, and discarding the filtrate; 350. Mu.L deproteinized liquid RWl was added dropwise to CR3, centrifuged at 12000rpm for 45s, and the filtrate was discarded; suspending and dripping 80 μL DNase I working solution in the center of CR3 adsorption film, standing at room temperature for 15min, adding 350 μLRWl, centrifuging at 12000rpm for 45s, and discarding filtrate; dropwise adding 500 μl of rinse solution RW (before first use, adding 48ml of absolute ethanol into 12ml RW) into an adsorption column CR3, standing at room temperature for 2min, centrifuging at 12000rpm for 45s, discarding the waste liquid, and repeating the steps once; centrifuging at 12000rpm for 2min, opening a tube cover, pouring out waste liquid, standing CR3 at room temperature for 4-6 min, and airing residual rinse liquid; placing CR3 on a new RNase-free centrifuge tube, suspending and dripping 30-100 mu L of RNase-free ddH 2 O is placed in the center of the adsorption film, kept stand for 2-5 min at room temperature, and centrifuged at 12000rpm for 2min to obtain RNA solution.
RNA concentration and integrity detection: concentration detection by sucking 2. Mu.L diluted RNA solution, and placing on an ultra-micro spectrophotometer (Nanodrop 2000) to measure RNA concentration, ensuring that the peak value is correct and the OD value is between 1.8 and 2.1. Integrity test by immersing the device and electrophoresis tank in hydrogen peroxide for 20min, preparing 1.0% agarose gel on an enzyme-free clean operation table, immediately performing electrophoresis test on total RNA after extraction, displaying at least two clear bands when an electrophoresis result diagram shows that the brightness of the upper 28S band is about twice that of the middle 18S band, proving that the integrity of the total RNA of the preserved szechuan pickle is better, and being applicable to subsequent tests, and finally preserving at low temperature of-80 ℃ for standby.
2) First strand cDNA Synthesis:
the RNA concentration was uniformly adjusted to 100 ng/. Mu.l, and 14.5. Mu.L of the reaction system { total RNA:4 mu L Oligo (dT) 15 Primer：1μL(50μmol/L)，pd(N) 6 ：0.5μL(50μmol/L)，RNase Free ddH 2 O: 9. Mu.L } in a 200. Mu.L centrifuge tube, and put on a PCR amplification apparatus for denaturation reaction (procedure: 10min at 70 ℃ C., 2min at 4 ℃ C.) to obtain a product A. Next, 20. Mu.L of a reverse transcription reaction system { A: 14.5. Mu.L, 5 XM-MLV buffer: 4. Mu.L, M-MLV: 0.25. Mu.L, dNTP (10 mM): 1 μl, recombinant RNase Inhibitor,0.25 μl } is placed in a 200 μl centrifuge tube, and placed on a PCR amplification instrument for reaction (procedure: 42 ℃ C. 60min,70 ℃ C. 10min,4 ℃ C.2 min), to obtain first strand cDNA, and stored at-80 ℃ C. For later use.
3) Cloning of the Hot pickled mustard tuber BjuDA1 Gene:
the Arabidopsis DA1 gene (AT 1G 19270) was searched according to the TAIR website (https:// www.arabidopsis.org), blast (parameter Nucleotide databases: braju tum V2.0 CDS) was performed on the brassica database (http:// brasicadb. Cn) according to its CDS full-length sequence, and the homologous gene BjuVA06G14890 of the preserved szechuan pickle was obtained, and primers were designed using Oligo 7 software: forward primer F1:5'-ATGGGTTGGTTTAACAAGATCTTC-3' (Seq ID No. 5), reverse primer R1:5'-AACCGGGAATCTACCGGTCATCTGTAT-3' (Seq ID No. 6). The BjuDA1 gene was amplified by PCR using the reverse transcribed first strand cDNA as a template and the above-described forward and reverse primer combinations. The PCR amplification system (50. Mu.L) was: 25. Mu.L of 2X Fast Pfu Master Mix enzyme, 2. Mu.L of each of forward and reverse primers, 2. Mu.L of template cDNA, ddH 2 O19. Mu.L. The PCR amplification procedure was: 94 ℃ for 90s;94℃for 20s,58℃for 20s,72℃for 49s,30 cycles; and at 72℃for 5min.
After completion of the PCR reaction, the target band was excised after detection by 1.5% (w/v) agarose gel electrophoresis, and the target band was recovered using a gel recovery/DNA purification kit (DC 301), and specific steps were referred to the kit instructions. Recovering the products from the gel and cloning vectorspost-Blunt Simple connectionTransforming into E.coli DH-5 alpha competent cells, picking up monoclonal bacterial plaques, detecting, sequencing the correct bacterial plaques by a company, verifying the coding region sequence of the BjuDA1 gene, and obtaining the coding region sequence (Seq ID No. 1) of the cDNA of the BjuDA1 gene of the preserved szechuan pickle according to the sequence splicing result, and obtaining the BjuDA1 gene plasmid.
The coding region sequence of the cDNA of the BjuDA1 gene of the hot pickled mustard tuber is translated to obtain a BjuDA1 amino acid sequence (Seq ID No. 2), and further, the amino acid sequence is subjected to homologous sequence alignment analysis with the published genomes of Arabidopsis thaliana, chinese cabbage, black mustard, cabbage type rape, mustard and Erussian mustard, and the BjuDA1 is obtained by carrying out the homologous sequence alignment analysis, so that the BjuDA1 gene is gathered together with the Chinese cabbage Bra06g015110.5C.2 distributed on the A06 chromosome, the brassica napus A06p15420.1_BnaDAR and the mustard BjuVA06G14890, has the same structural domain and has the highest sequence similarity with the mustard BjuVA06G14890 (98.2%), and the cloning of the BjuDA1 gene of the hot pickled mustard tuber is proved to be successful (figure 1).
Example 2 Hot pickled mustard tuber Bju containing Single mutation site DA1R356K Gene acquisition
The point mutation primers were designed according to Oligo 7 software: forward primer F2:5'-CTATCACATGCCAGAGACAAAAGGACTCTGCCTTTCAGAAG-3' (Seq ID No. 7), reverse primer R2:5'-CTTCTGAAAGGCAGAGTCCT TTTGTCTCTGGCATGTGATAG-3' (Seq ID No. 8). The BjuDA1 gene plasmid is used as a template, and is subjected to PCR amplification by using primers F2 and R2, and a reaction system (25 mu L) is as follows: 13. Mu.L of 2X Fast Pfu Master Mix enzyme, 1. Mu.L of each of primers F2 and R2, 1. Mu.L of BjuDA1 gene plasmid, ddH 2 O9. Mu.L. The PCR amplification procedure was: 94 ℃ for 90s;94℃for 20s,58℃for 20s,72℃for 50s,32 cycles; and at 72℃for 5min. After obtaining PCR products, immediately placing the products on ice, adding 1 mu L of Dpn I enzyme, uniformly mixing, incubating for 2 hours at 37 ℃, converting the incubated PCR products into escherichia coli, screening positive clones, extracting plasmids, and sending to a company for sequencing to obtain the hot pickled mustard tuber BjuDA1 containing single mutation sites R356K Gene sequence (Seq ID No. 3).
Example 3 Hot pickled mustard tuber Bju DA1R356K Construction of the overexpression vector
The expression vector used is pBin35Sred3-H, and the enzyme cutting sites are XbaI and Pac I. CollectingPCR amplification method is used to amplify the target gene BjuDA1 R356K Primers are designed at two ends of the coding region of the (E) and corresponding enzyme cutting sites and protecting bases are added at the 5' end of the primers. PCR was performed using the target gene plasmid as a template, F3:5'-GCTCTAGAATGGGTTGGTTTAACAAGATC-3' (Seq ID No.9, xbaI cleavage site and protecting base GC introduced) and R3:5'-GTTAATTAATTAAACCGGGAATCTACCGG-3' (Seq ID No.10, pac I cleavage site and protecting base G introduced) as primers, and 2X Fast Pfu Master Mix enzyme. The reaction system (25. Mu.L) was: 13. Mu.L of 2X Fast Pfu Master Mix enzyme, 1. Mu.L each of primers F3 and R3, bjuDA1 R356K Gene plasmid 1. Mu.L, ddH 2 O9. Mu.L. The PCR amplification procedure was: 94 ℃ for 90s;94℃for 20s,58℃for 20s,72℃for 50s,32 cycles; and at 72℃for 5min.
After the PCR reaction was completed, the PCR product was subjected to 1.5% (w/v) agarose gel electrophoresis, and recovered using a gel recovery/DNA purification kit (DC 301), and the plasmids of the product and the expression vector were subjected to a double digestion reaction, respectively, in which the digestion reaction system (50. Mu.L) was: vector or Gene gel recovery product 21. Mu.L, fastcut Buffer 5. Mu.L, xba I1.5. Mu.L, pac I1.5. Mu.L, ddH 2 O21. Mu.L. Mixing, and placing in a water bath kettle at 37 ℃ for 2h. After the enzyme digestion is completed, the target gene and the expression vector are recovered by using DC301 to obtain enzyme digestion products, and T is used 4 The DNA ligase ligated with the target gene after cleavage to the expression vector was a ligation system (10. Mu.L) of: solution I (containing T) 4 DNA ligase) 5. Mu.L, gene of interest 4. Mu.L, expression vector 1. Mu.L. And (3) fully and uniformly mixing by using a small centrifugal machine, and reacting for 2 hours in a metal bath at the temperature of 16 ℃. After the connection is completed, the mixture is transformed into competent cells of escherichia coli DH-5 alpha, a culture plate is placed in a 37 ℃ incubator for inversion and overnight culture, a colony is detected by using a primer Seq ID No.9 and QW586R (5'-AAAAGACAAAAGTGGGGTAG-3', seq ID No. 11), positive colonies are subjected to shaking, the positive colonies are subjected to company sequencing verification, and after the verification, the positive colonies are subjected to shaking and plasmid extraction, and an overexpression vector pBin35SRed3-H-BjuDA1 is obtained R356K Double enzyme digestion verification is carried out by using Xba I and Pac I, agarose gel electrophoresis detection is carried out after enzyme digestion, and 2 bands (a vector pBin35SRed3-H band is arranged at 11000bp and a target gene BjuDA1 is arranged at 1600 bp) appear R356K Band) (FIG. 3), demonstrating the overexpression vectorSomatic pBin35SRed3-H-BjuDA1 R356K The construction was successful.
Agrobacterium transformation: mu.L of pBin35SRed3-H-BjuDA1 was taken R356K The recombinant plasmid is added into 100 mu L of agrobacteria competent cells GV3101 which are melted on ice in advance, and the mixture is uniformly mixed; ice bath for 5min, freezing with liquid nitrogen for 5min, and rapidly placing in water bath at 37deg.C for 5min, ice bath for 5min; adding 800 mu L of YEB liquid culture medium without antibiotics, and oscillating at 28 ℃ and 250rpm for 2 hours; centrifuging at 6000rpm for 1min to collect bacterial liquid, collecting about 100 μl of supernatant, gently blowing to resuspension, uniformly coating on YEB solid culture medium (containing 60 μg/mL Kan and 25 μg/mL Rif antibiotics), culturing at 28deg.C in incubator upside down for 48 hr, detecting single colony with primers Seq ID No.9 and Seq ID No.11, shake-preserving positive colony (700 ul bacterial liquid+300 ul 60% glycerol) to obtain strain containing pBin35SRed3-H-BjuDA1 R356K The agrobacterium solution of the recombinant plasmid is used for genetic transformation of the following arabidopsis thaliana and the preserved szechuan pickle.
EXAMPLE 4 overexpression vector pBin35SRed3-H-BjuDA1 R356K Genetically transformed Arabidopsis thaliana
1) Sowing: the material is prepared from wild Arabidopsis Col-0, selecting plump seeds, placing on wet filter paper, placing in a 4 ℃ illumination incubator for 48 hours, transplanting to nutrient soil (perlite: vermiculite: 6mm turf=1:4:5) after germination, and growing under the conditions of 14 hours illumination/10 hours darkness, temperature 22 ℃ and relative humidity 70%.
2) Activating and expanding propagation: a small amount of pBin35SRed3-H-BjuDA1 was dipped in the inoculating loop R356K The recombinant plasmid Agrobacterium solution was streaked on a YEB solid medium containing 60. Mu.g/mL Kan and 25. Mu.g/mL Rif, and incubated in a constant temperature incubator at 28℃for 2d. After colony PCR detection, positive colony is picked up for shaking, 4mL of bacterial liquid is absorbed and activated in 50mL of YEB liquid culture medium (containing 60 mug/mL Kan and 25 mug/mL) and propagated to OD 600 0.8 to 1.2.
3) And (3) thallus collection: centrifuging the bacterial liquid at 4000rpm for 10min, discarding supernatant, and collecting thalli at the bottom of the centrifuge tube;
4) Suspending: the pellet was resuspended in 1/2MS liquid medium (containing 5% sucrose and 3% Silwet L-77 surfactant) to OD 600 =0.4 to 0.6 for infection of arabidopsis thalianaInflorescences.
5) Infection culture: soaking an arabidopsis inflorescence (pod removed in advance) in a bacterial liquid for about 30s, laterally placing the arabidopsis inflorescence in a foam box, culturing the arabidopsis inflorescence in dark for 24h, culturing the arabidopsis inflorescence under normal illumination conditions, and repeating the steps for 2 times at intervals of 7 d; after the seeds are mature, positive seeds are screened.
Example 5 screening and phenotypic characterization of transgenic Arabidopsis positive plants
T harvested after Agrobacterium infection 0 The generation seeds are subjected to red fluorescence screening, and the specific operation is as follows: seeds were irradiated with LUYOR-3415RG dual-wavelength fluorescent protein observation lamp and the fluorescent seeds were positive transgenic seeds through red filters (FIG. 4A). T to fluoresce 0 Sowing the seeds of the generation positive and harvesting the seeds singly, and carrying out T 1 The seeds harvested in the generation are subjected to red fluorescence screening according to the method, and the separation ratio meeting the Mendelian characters is 3:1, continuing to sow T by single plant 1 The positive seeds are generated, and all the lines which fluoresce are selected after the seeds are harvested, namely T 2 Homozygous transgenic seed (fig. 4B). Sowing T 2 The generation of homozygous transgenic seeds, 4-6 leaf stage, adopts CTAB method to extract DNA of leaf, and the specific operation is: placing 0.5g of the leaf into a 1.5mL centrifuge tube, grinding the leaf by a grinding rod, and adding 200 mu L of DNA extraction buffer (DNA buffer component: 200mmol/L Tris-HCl, pH=8.0; 250mmol/L NaCl;25mmol/L EDTA, pH=8.0; 0.5% SDS); adding 200 mu L of extraction buffer solution after fully grinding, and standing at room temperature for 2min; centrifuging at 12000rpm for 5min, and collecting supernatant, and placing into a new centrifuge tube; adding 300 μl of isopropanol, mixing, and settling at-20deg.C for 5min; centrifuging at 12000rpm for 6min, and discarding supernatant to obtain precipitate; adding 300 μl of isopropanol, centrifuging at 12000rpm for 2min, discarding supernatant, sucking residual liquid at bottom of centrifuge tube, and standing at room temperature for 5min; add 50. Mu.L ddH 2 O, dissolving by flicking to obtain DNA.
Arabidopsis Col-0 was used as Control (CK), water was used as negative control (N), pBin35SRed3-H-BjuDA1 was used as a template R356K For positive control (P), 7 strains T were randomly selected by primers Seq ID No.9 and Seq ID No.11 2 PCR identification is carried out on the transgenic arabidopsis positive plants, and agarose gel electrophoresis knots are formedThe results showed that the 7 transgenic lines detected the band of interest with the positive control, whereas CK and the negative control did not (fig. 4C). 3 positive plants were randomly selected and analyzed by fluorescent quantitative qRT-PCR expression, and the rosette leaves of the selected plants were subjected to RNA extraction and cDNA first strand synthesis as described in example 1. The kit used for fluorescent quantitative PCR analysis isSYBR qPCR SuperMix Plus (E096-01A), the fluorescent quantitative PCR instrument is a CFX96 Touch fluorescent quantitative PCR instrument, the primers are F4:5'-TGCGAGGTCACTGCCATT-3' (Seq ID No. 12) and R4:5'-ACGTCTTGGCTCAGCGTC-3' (Seq ID No. 13), the Arabidopsis internal reference gene TUB2 is used as a reference primer F5:5'-ATCCGTGAAGAGTACCCAGAT-3' (Seq ID No. 14) and R5:5'-AAGAACCATGCACTCATCAGC-3' (Seq ID No. 15), and the expression analysis result shows that BjuDA1 R356K The expression level of the gene in transgenic arabidopsis was significantly increased (fig. 4D). Further phenotypic observations and statistical analysis found that the cotyledons, seeds, petals and overall plant types of the transgenic lines were significantly greater than the controls (FIGS. 4E-K). Description of Hot pickled mustard tuber BjuDA1 R356K The gene regulates the organ size of arabidopsis thaliana.
EXAMPLE 6 overexpression vector pBin35SRed3-H-BjuDA1R356K Gene transformed Hot pickled mustard tuber
1) Sowing and preculture: selecting 20 full preserved szechuan pickle seeds, placing the preserved szechuan pickle seeds into a 200ml sterile tissue culture bottle, soaking and sterilizing the preserved szechuan pickle seeds for 45s by using 75% alcohol, cleaning the preserved szechuan pickle seeds for 60s by using sterile deionized water, soaking and sterilizing the preserved szechuan pickle seeds for 15min by using 10% NaClO solution, continuously shaking the preserved szechuan pickle seeds, continuously cleaning the preserved szechuan pickle seeds by using the sterile deionized water for 60s, and repeatedly cleaning the preserved szechuan pickle seeds for 5 times to finish seed sterilization. The sterilized seeds were sown on MS solid medium, placed in a culture room for 7d, the germinated seedling hypocotyl was cut into small pieces of about 1cm, and cultured on preculture medium (MS solid medium+3 mg/L6-BA+0.2 mg/L NAA) for 2d.
2) Activating dark culture: a small amount of pBin35SRed3-H-BjuDA1 was dipped in the inoculating loop R356K The recombinant plasmid Agrobacterium solution was streaked on a YEB solid medium containing 25. Mu.g/mL Rif and 60. Mu.g/mL Kan, and incubated in a constant temperature incubator at 28℃for 2d. After colony PCR detection, positive is picked upShaking colony, sucking 4mL bacterial liquid, activating in 50mL YEB liquid culture medium (containing 25mg/L Rif and 60mg/L Kan), and propagating to OD 600 0.5-0.6; transferring 50mL of bacterial liquid into a 50mL sterile centrifuge tube, centrifuging at 4000rpm at 4 ℃ for 10min, discarding the supernatant, resuspending the bacterial cells with 25mL of precooled MS liquid culture medium, infecting the pre-cultured hypocotyl with the resuspension for 10min, continuously gently shaking during the infection process, finally sucking the bacterial liquid on the hypocotyl with sterile filter paper, spreading on the pre-culture medium (MS solid culture medium+3 mg/L6-BA+0.2 mg/L NAA), and carrying out dark culture for 2d.
3) Differentiation rooting culture: after the dark culture, the co-cultured hypocotyls were transferred to MS screening medium (MS solid medium+3 mg/L6-BA+0.2 mg/L NAA+25mg/L Hyg+400mg/L Cb) and cultured at 25℃for 16h under light and 8h in the dark, and the screening medium was changed every 7d until the hypocotyls differentiated adventitious buds. Cutting off the surviving adventitious bud after subculturing for multiple times, inserting into rooting culture medium (MS solid culture medium+0.2 mg/L NAA+25mg/L Hyg+400mg/L Cb) to induce rooting, opening tissue culture bottle to acclimate for 2-3d after root system grows to 3-5cm, removing culture medium on root system, and transplanting seedling into matrix to obtain single mutation site preserved szechuan pickle BjuDA1 R356K Transgenic lines of gene overexpression.
Example 7 screening and phenotypic identification of transgenic Hot pickled mustard tuber Positive plants
The DNA extraction method in example 5 was used to extract DNA from non-transgenic and transgenic positive plants of hot pickled mustard tuber as templates, and PCR amplification was performed using 2X Taq PCR Master Mix enzyme using Seq ID No.9 and Seq ID No.11 as primers, and the agarose gel electrophoresis results showed that 3 positive lines were selected from 7 transgenic materials, and the bands were consistent with the positive control (FIG. 5A). Further, primers (F6: 5'-ACACTTTTCAATGCCAACCT-3' (Seq ID No. 16) and Seq ID No. 11) are designed before and after the mutation site, PCR amplification is carried out, target fragments are recovered and sequenced, and finally two transgenic preserved szechuan pickle positive strains with single mutation sites (fig. 5B-C) are obtained.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The stem tumor mustard BjuDA1 protein is:
1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)
2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
2. The BjuDA1 protein of the stem tumor mustard as claimed in claim 1, wherein the 356 th 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.
3. A gene encoding the phoma mustard BjuDA1 of claim 1 or 2.
4. The gene of claim 2, wherein the sequence is as set forth in SEQ ID No.1 or 3.
5. A vector comprising the gene of claim 3 or 4.
6. A host cell comprising the vector of claim 5.
7. An engineered bacterium comprising the gene of claim 3 or 4.
8. Use of the gene of claim 3 or 4 for promoting the enlargement of a tumorous stem mustard.
9. The use according to claim 7, wherein the gene is transferred into the genome of the tumorous stem mustard and is overexpressed in the transgenic tumorous stem mustard to promote the enlargement of the tumorous stem mustard.
10. A method of promoting the expansion of a tumorous stem mustard, characterized in that a vector comprising the gene of claim 3 or 4 is transferred into the genome of said tumorous stem mustard and overexpressed in the transgenic tumorous stem mustard.
CN202310409132.9A 2023-04-17 2023-04-17 Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene Pending CN116535476A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310409132.9A CN116535476A (en) 2023-04-17 2023-04-17 Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310409132.9A CN116535476A (en) 2023-04-17 2023-04-17 Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene

Publications (1)

Publication Number Publication Date
CN116535476A true CN116535476A (en) 2023-08-04

Family

ID=87453370

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310409132.9A Pending CN116535476A (en) 2023-04-17 2023-04-17 Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene

Country Status (1)

Country Link
CN (1) CN116535476A (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103865897A (en) * 2014-04-15 2014-06-18 西南大学 Site-directed mutagenesis system of mustard genome and application of site-directed mutagenesis system
CN105039392A (en) * 2007-10-11 2015-11-11 植物生物科学有限公司 Methods of controlling plant seed and organ size
CN105073994A (en) * 2013-04-05 2015-11-18 拜尔作物科学公司 Brassica plants comprising mutant DA1 alleles
CN105612171A (en) * 2013-08-14 2016-05-25 中国科学院遗传与发育生物学研究所 Methods of modulating seed and organ size in plants
US20160194651A1 (en) * 2013-08-14 2016-07-07 Institute Of Genetics And Developmental Biology Methods of modulating seed and organ size in plants
CN111378683A (en) * 2013-11-11 2020-07-07 植物生物科学有限公司 Method for modulating seed and organ size in plants
CN113924882A (en) * 2021-09-18 2022-01-14 西南大学 Application of melatonin in regulation of mustard bud bolting

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105039392A (en) * 2007-10-11 2015-11-11 植物生物科学有限公司 Methods of controlling plant seed and organ size
CN105073994A (en) * 2013-04-05 2015-11-18 拜尔作物科学公司 Brassica plants comprising mutant DA1 alleles
US20160040180A1 (en) * 2013-04-05 2016-02-11 Bayer Cropscience Nv Brassica plants comprising mutant da1 alleles
CN105612171A (en) * 2013-08-14 2016-05-25 中国科学院遗传与发育生物学研究所 Methods of modulating seed and organ size in plants
US20160194651A1 (en) * 2013-08-14 2016-07-07 Institute Of Genetics And Developmental Biology Methods of modulating seed and organ size in plants
CN111378683A (en) * 2013-11-11 2020-07-07 植物生物科学有限公司 Method for modulating seed and organ size in plants
CN103865897A (en) * 2014-04-15 2014-06-18 西南大学 Site-directed mutagenesis system of mustard genome and application of site-directed mutagenesis system
CN113924882A (en) * 2021-09-18 2022-01-14 西南大学 Application of melatonin in regulation of mustard bud bolting

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
HUXIAN YANG 等: ""Genome-Wide Analysis of DA1-Like Genes in Gossypium and Functional Characterization of GhDA1-1A Controlling Seed Size"", 《FRONT. PLANT SCI》, vol. 12, 20 May 2021 (2021-05-20), pages 10 *
JIE-LI WANG 等: ""Down-regulation of BnDA1, whose gene locus is associated with the seeds weight, improves the seeds weight and organ size in Brassica napus"", 《PLANT BIOTECHNOL J》, vol. 15, no. 8, 20 February 2017 (2017-02-20), pages 1024 - 1033 *
NCBI: ""PREDICTED: Brassica napus protein DA1 (LOC106346972), transcript variant X2, mRNA"", 《GENBANK》, 23 June 2022 (2022-06-23), pages 013786452 *
NCBI: ""protein DA1 isoform X1 [Brassica rapa]"", 《GENBANK》, 7 December 2020 (2020-12-07), pages 009149402 *
YUANCHENG PENG 等: ""The Ubiquitin Receptors DA1, DAR1, and DAR2 Redundantly Regulate Endoreduplication by Modulating the Stability of TCP14/15 in Arabidopsis"", 《PLANT CELL》, vol. 27, no. 3, 10 March 2015 (2015-03-10), pages 649 *
YUNHAI LI 等: ""Control of final seed and organ size by the DA1 gene family in Arabidopsis thaliana"", 《GENES DEV》, vol. 22, no. 10, 15 May 2008 (2008-05-15), pages 1331 - 1336, XP002512707, DOI: 10.1101/GAD.463608 *
崔超 等: ""泛素/26S蛋白酶体途径调节作物种子大小的研究进展"", 《中国农业科技导报》, vol. 23, no. 9, 30 September 2021 (2021-09-30), pages 19 - 29 *
李利军: ""甘蓝型油菜附加系与芸薹属A基因组种间杂交的研究"", 《中国优秀硕士学位论文全文数据库 (农业科技辑)》, no. 3, 15 March 2012 (2012-03-15), pages 048 - 15 *
陈嘉盈: ""利用基因编辑创建甘蓝型油菜种子大小变异的新种质"", 《中国优秀硕士学位论文全文数据库 (基础科学辑)》, no. 2, 15 February 2023 (2023-02-15), pages 047 - 926 *

Similar Documents

Publication Publication Date Title
CN110317815B (en) Gene for regulating and controlling adventitious root generation of populus euphratica, detection primer, expression vector and application
CN109423492B (en) Application of SlTOE1 gene in regulation and control of flowering time and yield of tomatoes
CN116515888A (en) Application of GmMTAs protein in regulating and controlling soybean plant height
CN107630022B (en) Application of tomato SlMYB75 gene in enhancing rot resistance and prolonging shelf life of tomato fruits
CN111635904B (en) Gene CsWRKY10 for enhancing cucumber target spot disease resistance and application thereof
CN116656698B (en) Application of corn gene Zm00001d018037 in improving drought resistance of monocotyledonous crops
CN111996192A (en) Method for creating green-free green-shoulder tomato material and application thereof
CN116064572B (en) MdWOX11 gene and protein for promoting adventitious root development and application thereof
CN107557384B (en) Genetic transformation system for inducing plant dwarfing and construction and application thereof
CN116535476A (en) Stem tumor mustard BjuDA1 R356K Gene, protein coded by gene and application of gene
CN114106121A (en) FvGR3 protein and coding gene and application thereof
CN111471691A (en) Application of FveCHA1 gene and RNAi vector in promoting strawberry callus formation and isolated bud regeneration
CN117511892B (en) Application of FTO protein in promotion of tree breeding
CN117051014B (en) Cloning and application of cold-resistant gene MYB97 of swallow flower
CN109097388B (en) Application of GmCOP1a and/or GmCOP1b in plant height regulation
CN110922459B (en) Application of SlSNAT1 protein and related biological material thereof in regulation and control of plant seed aging resistance
CN117431256B (en) Wheat yellow mosaic disease-resistant gene TaRx-2D, protein encoded by same and application thereof
CN116769799B (en) Soybean mutant gene for improving yield of leguminous crops and application thereof
CN116004646B (en) Tobacco NtSWEET gene and application thereof
CN115806999B (en) Tobacco NtEIJ gene and application thereof
CN115011630B (en) Application of tomato SlGID1L2 gene in regulation and control of drought tolerance of tomatoes and cultivation of drought tolerance tomatoes
CN116444637A (en) Protein GmWRKY100 related to plant leaf senescence and yield regulation, and coding gene and application thereof
CN118308369A (en) Gene IbNF-YB1 related to root growth and development of sweet potato, and encoding protein and application thereof
CN115991756A (en) Method for improving tomato fruit yield and tomato fruit lycopene content under dim light condition
CN117126863A (en) Tobacco NtHSP70-8b gene and application thereof in regulating and controlling tobacco seed size

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination