Use of AaTPS gene in regulating plant seed size

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to application of AaTPS gene in regulating plant seed size. The present application provides application of AaTPS gene in regulating plant seed size. The present application clones AaTPS gene from Artemisia vulgaris var. nanshanica, successfully constructs a 35S constitutive promoter driven overexpression vector and transforms Arabidopsis. Compared with wild type control plants, the expression of three transgenic Arabidopsis T3 generation lines is significantly increased. Compared with wild type control plants, the 1000-grain weight of seeds of the three transgenic Arabidopsis pure lines is increased by 40.58%, 46.12% and 53.74% respectively, reaching a significant level; meanwhile, the length of seeds of the transgenic lines is significantly increased, reaching a significant level, and the width of seeds of the OE7 line is also significantly increased. The present application can provide a new gene resource for improving plant seed size, and has important application value in plant breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the AaTPS gene in regulating plant seed size. Background Technology

[0002] Artemisia argyi is a perennial herb belonging to the Asteraceae family. Its dried leaves have significant therapeutic effects on menstrual-related symptoms, eczema, diarrhea, and tuberculosis. To date, the main chemical components isolated from Artemisia argyi include terpenes, flavonoids, and phenylpropanoids. Modern science has studied several characteristic components, discovering that it possesses various health benefits such as antioxidant, antitumor, anti-inflammatory, anticoagulant, antibacterial, neuroprotective, and immunomodulatory properties. The development and utilization of Artemisia argyi integrates pharmaceuticals, health products, industrial raw materials, and cultural and creative products, possessing a very wide range of uses and development prospects, earning it the reputation of "diamond among herbs."

[0003] Terpene synthases (TPSs) are crucial regulatory enzymes in the terpene biosynthesis pathway, particularly in regulating terpene compound diversity. The terpene synthesis pathway is a vital part of plant aroma formation, and TPSs are key enzymes involved in terpene synthesis, playing a significant role in plant aroma formation. TPSs regulate the downstream synthesis of a vast number and structurally diverse range of terpene compounds using geraniyl pyrophosphate (GPP), neroliyl pyrophosphate (NPP), farnesyl pyrophosphate (FPP), and geraniyl pyrophosphate (GGPP) as direct precursors. They are rate-limiting enzymes in the terpene metabolic pathway and play a vital role in the formation of plant terpene aroma compounds. TPSs include monoterpene synthases, sesquiterpene synthases, and diterpene synthases, and are the most extensively studied enzymes in terpene biosynthesis. However, to date, no research reports have documented the involvement of the AaTPS gene in regulating seed weight and size in Artemisia argyi.

[0004] As one of the "Four Famous Artemisia species," Artemisia argyi is not only a distinctive and authentic Chinese medicinal herb of Henan Province, but also occupies an important position in the field of traditional Chinese medicine. Artemisia argyi has a long history of application in traditional medicine and has played a vital role in safeguarding public health. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the AaTPS gene in regulating plant seed size.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of the AaTPS gene in regulating plant seed size, wherein the amino acid sequence encoded by the AaTPS gene is shown in SEQ ID NO: 2.

[0008] Preferably, the nucleotide sequence encoded by the AaTPS gene is shown in SEQ ID NO: 1.

[0009] Preferably, the regulation of plant seed size involves increasing the thousand-seed weight of the plant seeds, as well as increasing the length and width of the plant seeds.

[0010] Preferably, the plant seeds are Arabidopsis thaliana seeds or Artemisia argyi seeds.

[0011] Preferably, the AaTPS gene is an overexpressed AaTPS gene.

[0012] The present invention also provides the application of the AaTPS gene in plant breeding, wherein the goal of the breeding is to increase the size of plant seeds.

[0013] The present invention also provides the application of the expression vector containing the AaTPS gene in regulating plant seed size.

[0014] The present invention also provides the application of engineered bacteria containing the AaTPS gene in regulating plant seed size.

[0015] The present invention also provides a method for regulating plant seed size by influencing the expression of the AaTPS gene.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] To further explore the medicinal value of Artemisia argyi and promote its industrialization, we successfully resolved the reference genome of Artemisia argyi. Based on the genomic data, this study cloned the Artemisia argyi EVM0144301 (AaTPS) gene from Artemisia argyi in Tangyin County, successfully constructed a 35S constitutive promoter-driven overexpression vector, and transformed it into Arabidopsis thaliana. Compared with wild-type control plants, the expression levels of the three transgenic Arabidopsis thaliana T3 generation lines were significantly increased. Compared with the wild-type control, the thousand-seed weight of the three transgenic Arabidopsis thaliana pure lines increased by 40.58%, 46.12%, and 53.74%, respectively, reaching a significant level; simultaneously, the seed length of the transgenic lines increased significantly, reaching a significant level, with the seed width of the OE7 line also significantly increased. This invention can provide new gene resources for improving plant seed size and has important application value in plant breeding. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 This is a clone of the AaTPS gene.

[0020] Figure 2 Screening for T1 generation Arabidopsis thaliana resistant plants.

[0021] Figure 3 Electrophoresis image of PCR identification of T1 generation Arabidopsis thaliana resistant plants.

[0022] Figure 4 The relative expression levels of the AaTPS gene in the siliques of three transgenic Arabidopsis thaliana T3 generation plants.

[0023] Figure 5 The weight of 1000 seeds of AaTPS-transgenic Arabidopsis thaliana.

[0024] Figure 6 The length and width of AaTPS transgenic Arabidopsis seeds.

[0025] Figure 7 Seed size diagram of three T3 generation pure lines of AaTPS transgenic Arabidopsis thaliana. Detailed Implementation

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1: Cloning of the AaTPS gene in Artemisia argyi

[0028] 1.1 Materials required for the experiment

[0029] Enzymes and kits: Plant total RNA extraction kit (DP432) was purchased from Tiangen Biotech (Beijing) Co., Ltd.; RNA reverse transcription kit TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311-02), real-time fluorescence enzyme TransStart Top Green qPCR SuperMix (AQ131-04), cloning vector pEASY-T5 Zero Cloning Kit (CT501-01) and plasmid extraction kit EasyPure HiPure PlasmidMiniPrep Kit (EM111-01) were purchased from TransGen Biotech Co., Ltd.; high-fidelity PCR amplification enzyme KOD-Plus-Neo (KOD-401) was purchased from TOYOBO Biotechnology Co., Ltd.; DNA Fragment Purification Kit ver4.0 gel recovery kit and DNA Purification Kit PCR product purification kits were purchased from TaKaRa Biotechnology Co., Ltd.; infusion ligase was purchased from Novizan Biotechnology Co., Ltd.; competent E. coli DH5α cells were purchased from Shanghai Sangon Biotech Co., Ltd.; restriction endonucleases XbaI and PacI were both from NEB.

[0030] Other medicines: Agarose was purchased from Beijing TransGen Biotech Co., Ltd.; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. were domestically produced analytical grade; ampicillin (IA0340), kanamycin (YZ-130556), rifampin (IR0110), etc. were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0031] Solution preparation: All reagents mentioned in this article but not listed were prepared according to the methods in the third edition of Molecular Cloning Laboratory Manual. Biochemical reagents were of analytical grade or higher.

[0032] LB medium: 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride (NaCl), finally adjusted to 1 L with ddH2O; LB solid medium: 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), 15 g / L agar powder, and 10 g / L tryptone, finally adjusted to 1 L with ddH2O.

[0033] 2.1 Main instruments: PCR amplification instrument (BIO-RAD), clean bench, electrophoresis equipment (BIO-RAD), fluorescence PCR instrument (ABI7500), gel imaging system (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), Zeiss AxioImagerA1 microscope.

[0034] 2.2 Cloning of AaTPS

[0035] The full-length CDS sequence of the EVM0144301(AaTPS) gene was retrieved from the genome of Artemisia argyi from Tangyin in this experiment. Primer sequences were designed using Primer 5.0 software, and the complete open reading frame (ORF) of 1497 bp, encoding 498 amino acid residues, was amplified from the leaf tissue of Artemisia argyi from Tangyin using PCR (Polymerase Chain Reaction) technology.

[0036] The gene CDS sequence is as follows:

[0037] AaTPS

[0038]

[0039] The amino acid sequence is as follows:

[0040] AaTPS

[0041] MIQKSGNSLRVLELVDELQRLGISYLFEEEEISNLLETIYYNYYKFPENWNKINLNLKALGFRLLRQHGYHVPQEIFLKFKDKNQNLNSYLLNDVVEMLNLYEASYHSFEDENILHDARDITTKY LKESLEKIDGSIFSLVTHALEQPLHWRVQRVEAKWFIELYEKKNSMSPTLVELAKLDFDLVQAIHLEDLKHASRWWRNTSWDTKLTFARDLIVENFLWTIGFSYLPNFSRGRRTITKVAVMITTV DDVYDVFGTLGELEQFTDVINRWDIKAIEQLPDYMKICFFGLYNSINDITHETLASKGFLILPYLKKAWADLCKAYLVEARWYHRGHIPTLNEYLDNACVSISGPVALMHVHFLTSVSSIEEIH QCIQRTENIVRYVSLIFRLTDDLGTSLGEMERGDTLKSIQLYMHETGATEQEARSYIKSLIDKTWKKLNKERAIVNSESSREFIDYATNLARMAQFMYGEGDKDFGLDVIKSHVLSLLFTPIQGI

[0042] As shown in SEQ ID NO: 1 and 2.

[0043] 2.3 The process of cloning the AaTPS gene

[0044] (1) The experimental material was Artemisia argyi from Tangyin. The tissues collected were a mixture of leaf, stem and root tissues of Artemisia argyi from Tangyin. The collected materials were quickly frozen in liquid nitrogen and stored at -80℃ for later use. Total RNA extraction from plants was performed using a kit from Tiangen Pharmaceuticals.

[0045] (2) 500 ng of RNA was reverse transcribed into cDNA, and the cDNA solution of the reverse transcription product was diluted 2 times as a template for PCR reaction. Using total RNA extracted from the leaves of Artemisia argyi in Tangyin as a template, it was reverse transcribed into cDNA using the TransGold reverse transcription kit. The cDNA synthesis system is as follows.

[0046] Table 1 cDNA synthesis system

[0047]

[0048] Note: Incubate at 42°C for 30 min in a PCR instrument, then inactivate at 85°C for 5 s. Store at 4°C for later use.

[0049] (3) Perform PCR reaction to amplify the target gene.

[0050] Primer sequences:

[0051] AaTPS-F: 5′-ATGATACAAAAGTCAGGAAATTC-3′

[0052] AaTPS-R: 5′-TTATATCCCTTGGATTGGAGGTAAAC-3′

[0053] As shown in SEQ ID NO: 3 and 4.

[0054] The AaTPS gene of *Artemisia argyi* from Tangyin was amplified by PCR using synthetic primers AaTPS-F and AaTPS-R as a template with cDNA obtained from reverse transcription. The target gene amplification system is as follows.

[0055] Table 2 PCR reaction system

[0056]

[0057] The conditions required for the PCR reaction are set as follows:

[0058] Table 3 PCR reaction conditions

[0059]

[0060] Note: Perform 28 cycles of amplification from Step 2 to Step 4.

[0061] (4) After the reaction, the sample was stored at 4℃ and detected by 0.8% agarose gel electrophoresis to obtain a PCR amplification product of size 1497 bp. Figure 1 ).

[0062] (5) The reaction product of the target fragment was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit ver4.0.

[0063] (6) The product recovered from the above gel was ligated into the cloning vector pEASY-T5 and transformed into competent Escherichia coli DH5α cells.

[0064] (7) Incubate overnight at 37°C, pick single clones and culture them overnight at 37°C in LB medium containing 500 μl kan.

[0065] (8) Bacterial culture PCR verification: send bacterial culture containing the target gene fragment size band for sequencing, and name the clone with correct sequencing as pEASY-AaTPS.

[0066] 3. Construction of pCAMBIA2300-AaTPS plant seed-specific expression vector

[0067] 3.1 Plasmid extraction and enzyme digestion

[0068] The plasmid vector selected was pCAMBIA2300. The plasmid was extracted using the TransGen plasmid extraction kit. The plasmid concentration was measured to be 150 ng / μl. Agarose gel electrophoresis showed no protein contamination, meeting the experimental requirements. The restriction enzymes XbaI and PacI were used for double digestion.

[0069] 3.2 Construction of the pCAMBIA2300-AaTPS expression vector

[0070] (1) Amplification of the target gene ORF sequence: Based on the multiple cloning site on the final vector pCAMBIA2300, XbaI and PacI were designed as insertion sites and primers were synthesized.

[0071] The primer sequences are as follows:

[0072] AaTPS-XbaI-F: 5′-AGAACACGGGGGAC TCTAGA ATGATACAAAAGTCAGGAAA TTC-3' (The underlined portion contains the recognition sequence of the restriction endonuclease XbaI)

[0073] As shown in SEQ ID NO: 5.

[0074] AaTPS-PacI-R: 5'-TCTTTCTCCTTTACCCATG TTAATTAA TATCCCTTGGATTGGAGT AAAC-3' (The underlined portion contains the recognition sequence for the restriction endonuclease PacI)

[0075] As shown in SEQ ID NO: 6.

[0076] The AaMLP target gene sequence containing XbaI and PacI restriction sites was amplified using KOD-Plus-Neo high-fidelity enzyme. The reaction system is as follows.

[0077] Table 4 PCR reaction system

[0078]

[0079] The PCR reaction conditions were set as follows: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 68℃ extension for 45 s, 30 cycles; 68℃ extension for 5 min.

[0080] (2) Construction of fusion expression vector:

[0081] 1) The plant expression vector pCAMBIA2300 was digested with restriction endonucleases XbaI and PacI, and the vector backbone was recovered.

[0082] 2) Recover and purify the AaTPS amplification product containing XbaI and PacI restriction sites.

[0083] 3) Connect the vector skeleton obtained in step 1) and the recovered product obtained in step 2) to form the plant overexpression vector pCAMBIA2300-AaTPS.

[0084] 4) The ligation product from step 3) was heat-shocked and transformed into E. coli DH5α. The mixture was then incubated overnight at 37°C with the inverted incubator. Positive clones were selected for sequencing. The clones with the correct sequencing results were named pCAMBIA2300-AaTPS / DH5α.

[0085] 3.3 Transformation of the vector plasmid GV3101

[0086] a. Take 100 μl of GV3101 competent cells thawed on ice, and under aseptic conditions, add 3 μl (about 450 μg) of the target plasmid to the competent cells, mix gently, and let stand in an ice water bath for 5 minutes.

[0087] b. Place the centrifuge tubes in liquid nitrogen for 5 minutes to freeze quickly;

[0088] c. Then quickly place the centrifuge tube in a 37°C water bath for 5 minutes, and then place the centrifuge tube back in an ice-water bath for 5 minutes.

[0089] d. Spread 100 μL of bacterial culture onto LB selection medium containing kanamycin and rifampin, incubate at 28°C for about 3 days, select positive clones, incubate on LB liquid medium at 28°C for 48 hours, preserve glycerol bacteria with a final glycerol concentration of about 50%, and store at -80°C for later use.

[0090] 4. Genetic transformation, identification, and gene expression analysis of the pCAMBIA2300-AaTPS overexpression vector in Arabidopsis thaliana.

[0091] 1) Using Arabidopsis thaliana Columbia as the recipient, genetic transformation was performed using the flower dip method. Transgenic seeds of the T0 generation were harvested, and positive transformants were screened on MS medium containing kanamycin (50 mg / L). Figure 2T1 generation resistant plants were obtained, and seeds were collected after flowering and maturity. Forty days after transplanting the T1 generation resistant plants, DNA was extracted from leaves of the Arabidopsis plants, and the resistance markers NPTII (KanF: 5'-CACTGAAGCGGGAAGGGACT-3' and KanR: 5'-CGATACCGTAAAGCACGAGGAA-3', as shown in SEQ ID NO: 7 and 8) were amplified by PCR. Positive Arabidopsis plants were further screened. Results are shown in […]. Figure 3 The results show that the target band was obtained around 795bp in the agarose gel electrophoresis image, which preliminarily proves that the overexpression vector of this gene was successfully transformed.

[0092] Seeds from individual plants of generation T1 were harvested, and approximately 100 seeds were distributed on MS+Kan (50 mg / L) medium. After the cotyledons of the seedlings were fully expanded (approximately 10 days), the segregation ratio of yellow to green seedlings was calculated and a chi-square test was performed. Plants that showed a segregation ratio of green to yellow seedlings of 3:1 were preliminarily predicted to be single-copy insertion lines, and seeds were harvested separately for each line for subsequent analysis.

[0093] 5. Weigh the seeds, and measure their length and width.

[0094] Three T3 generation pure lines with high expression levels of the AaTPS gene in transgenic Arabidopsis siliques were selected and grown on MS + Kan (50 mg / L) medium. Figure 4 These three lines were named AaTPS OE1, OE5, and OE7, respectively, with 20 plants transplanted from each line. After maturity, seeds from the T3 generation were harvested for subsequent 1000-seed weight and seed size measurements. For seed weight analysis, under identical conditions, plants grew simultaneously, and 1000 mature seeds of each genotype were dried at 24°C for 7 days and weighed. Data are expressed as the mean ± SD of at least four independent experiments. Results are as follows... Figure 5 As shown, the thousand-seed weights of wild-type Arabidopsis thaliana and T3 generation homozygous transgenic Arabidopsis thaliana lines OE1, OE5, and OE7 were 18.05±1.10, 25.38±0.41, 26.38±3.54, and 27.75±2.19 mg, respectively. The thousand-seed weights of the three lines increased by 40.58%, 46.12%, and 53.74% compared with the control, respectively. This result indicates that AaTPS overexpression can significantly increase the thousand-seed weight.

[0095] The length and width of the seeds were photographed using a Zeiss Axio Imager A1 microscope and then measured using ImageJ software. Data were measured with at least eight independent seeds, and are expressed as mean ± SD. The aspect ratio of the seeds was calculated. Results are shown below. Figure 6As shown, compared with the wild-type control, the length of mature dry seeds of AaTPS OE was significantly increased, and the seed width of the OE7 line was also significantly increased. The seeds of AaTPS OE were larger than the wild-type seeds (WT). Figure 7 The results indicate that AaTPS overexpression can significantly increase seed length.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of AaTPS gene overexpression in increasing Arabidopsis seed size, characterized in that, The amino acid sequence encoded by the AaTPS gene is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the AaTPS gene is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The increase in Arabidopsis seed size refers to an increase in the thousand-seed weight of Arabidopsis seeds, and an increase in the length and width of Arabidopsis seeds.

4. The application of an expression vector containing the AaTPS gene as described in claim 1 in increasing the seed size of Arabidopsis thaliana, characterized in that, Overexpression of the AaTPS gene increases the seed size of Arabidopsis thaliana.

5. The application of engineered bacteria containing the AaTPS gene as described in claim 1 in increasing the seed size of Arabidopsis thaliana, characterized in that, Overexpression of the AaTPS gene increases the seed size of Arabidopsis thaliana.

6. A method for increasing the size of Arabidopsis thaliana seeds, characterized in that, Overexpression of the AaTPS gene as described in claim 1 increases the seed size of Arabidopsis thaliana.

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