Application of GhUGT73C3 gene or protein coded by GhUGT73C3 gene in improving oil yield and seed size of plant seeds

Abstract

The invention discloses an application of a GhUGT73C3 gene or a protein coded by the GhUGT73C3 gene in improving the oil yield and the seed size of plant seeds, and belongs to the technical field of gene engineering. According to the invention, the GhUGT73C3 gene is subjected to heterologous expression in arabidopsis thaliana, the oil content and fatty acid components of transgenic line seeds are determined by adopting GC (Gas Chromatography), and the application of the GhUGT73C3 gene in the aspects of improving the cottonseed oil yield and the seed size is defined by determining the grain length, the grain width and the thousand grain weight of the transgenic line seeds. Meanwhile, the application of the protein UGT expressed by the GhUGT73C3 gene in improving the cottonseed oil yield and the seed size is also clear, and the protein UGT has wide application space and market prospect in the agricultural field.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of a GhUGT73C3 gene or a protein encoded by it in improving the oil yield and seed size of plant seeds. Background Art

[0002] The global supply of vegetable oil is far lower than the demand. The demand for vegetable oil in 2023 / 24 will increase by about 5.6 million tons, while the supply will increase by about 2.6 million tons, with a gap of 3 million tons, which cannot meet people's needs for healthy living. Cotton is one of the important fiber and oil crops in the world, and plays an important role in ensuring clothing and food. Cottonseed is the main by-product of cotton, accounting for about 60% of the total cotton production. The oil content of cottonseed after shelling accounts for 27.19-39.89%, which is the fourth most important source of edible oil in the world.

[0003] With the development of my country's economy and the improvement of people's living standards, people are paying more and more attention to nutrition and health. Compared with other vegetable oils, cottonseed oil contains a large amount of fatty acids necessary for the human body and has a higher nutritional value. For example, the content of unsaturated fatty acids such as oleic acid and linoleic acid is close to 80%, which makes cottonseed oil have a strong antioxidant capacity. Among them, oleic acid can lower cholesterol in the blood, prevent cholesterol from depositing on the blood vessel wall, and is beneficial to cardiovascular and cerebrovascular diseases; linoleic acid can be used to treat hyperlipidemia and arteriosclerosis. In addition, cottonseed oil contains a high level of vitamin E, which has the effect of improving the body's ability to resist cell aging and reducing the incidence of cardiovascular and cerebrovascular diseases.

[0004] Cottonseed oil also has broad application prospects as a bioenergy fuel. 99% of the fatty acid carbon chain lengths in cottonseed oil are concentrated between C16 and C18, which is extremely similar to the composition of diesel. The efficiency of conversion into biodiesel is as high as 95%. In addition, cottonseed oil is rich in oxygen and does not contain sulfur. The converted biodiesel burns more fully, is environmentally friendly and does not pollute the environment. In recent years, my country's cotton planting area has shown an overall downward trend from 50.0894 million mu in 2019 to 42.5745 million mu in 2024. Therefore, using genetic engineering to increase the oil content of cottonseed is the only way to maintain the total output of cottonseed oil and even improve the utilization efficiency of cotton by-products.

[0005] UGT (UDP-glucosyltransferase) genes belong to the uridine diphosphate (UDP)-glycosyltransferase gene family. They use UDP glucose as a sugar donor to catalyze the glycosylation of substrate molecules and are UDP-dependent glycosyltransferases. UGTs usually catalyze the glycosylation modification of small molecule compounds, such as flavonoids, alkaloids and plant hormones, through the formation of glycosidic bonds (Li et al. 2018). The UGT gene family has many members, which are mainly involved in plant secondary metabolic pathways, plant detoxification reactions, and plant responses to biotic and abiotic stresses. In rice, Dong et al. found that the UGT83A1 gene is involved in the regulation of cell proliferation and expansion during spikelet development, indirectly affecting auxin levels and the expression of auxin-related genes, and regulating grain size development (Dong et al. 2020); Wu et al. found a major QTL, qEDR5.1, encoding a UDP glycosyltransferase gene, which regulates the difference in endosperm development between upland rice and rice (Wu et al. 2022). Cao et al. found that the UGT71C4 gene reshapes the morphological development of cotton seeds by affecting the transformation of metabolic flow in the phenylpropane metabolic pathway (Cao et al. 2024). In cotton, there are relatively few studies on the function of UGT family genes, and there are no studies on the regulation of cottonseed oil content and seed size by UGT genes. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the GhUGT73C3 gene or a protein encoded by the gene in improving the oil yield and seed size of plant seeds, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is the use of the GhUGT73C3 gene or the protein encoded by it in improving the oil yield of plant seeds.

[0009] The second technical solution of the present invention is a method for increasing the oil yield of plant seeds, which overexpresses the GhUGT73C3 gene to increase the oil content of plant seeds.

[0010] The third technical solution of the present invention is the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the GhUGT73C3 gene in improving the oil yield of plant seeds.

[0011] The fourth technical solution of the present invention is the use of the GhUGT73C3 gene or the protein encoded by it in regulating the size of plant seeds.

[0012] The fifth technical solution of the present invention is a method for regulating the size of plant seeds, which overexpresses the GhUGT73C3 gene to increase the grain length, grain width and thousand-grain weight of plant seeds.

[0013] The sixth technical solution of the present invention is the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the GhUGT73C3 gene in regulating plant seed size.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention heterologously expresses the GhUGT73C3 gene in Arabidopsis thaliana, uses gas chromatography (GC) to determine the oil content and fatty acid composition of transgenic strain seeds, and determines the grain length, grain width and 1000-grain weight of the transgenic strain seeds, thereby clarifying the application of the GhUGT73C3 gene in improving the oil yield and seed size of cotton seeds. At the same time, it also clarifies the application of the protein UGT expressed by the GhUGT73C3 gene in improving the oil yield and seed size of cotton seeds, which will have broad application space and market prospects in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the cloning of the GhUGT73C3 gene.

[0018] Figure 2 Identification of bacterial solution of GhUGT73C3 connected to pEarleyGate 101 vector.

[0019] Figure 3 Schematic diagram of the subcellular localization of the protein encoded by the GhUGT73C3 gene.

[0020] Figure 4 Screening of GhUGT73C3 transgenic Arabidopsis positive seedlings for Basta.

[0021] Figure 5 PCR identification of GhUGT73C3 transgenic Arabidopsis positive seedlings.

[0022] Figure 6 The results of qRT-PCR of GhUGT73C3 transgenic Arabidopsis positive seedlings.

[0023] Figure 7 Western Blot identification of GhUGT73C3 transgenic Arabidopsis positive seedlings.

[0024] Figure 8 Seed oil content (a) and fatty acid content (b) of GhUGT73C3-transfected Arabidopsis thaliana.

[0025] Fig. 9 Comparison of seed size (a) and grain length (b), grain width (c), and 1000-grain weight (d) of transgenic Arabidopsis thaliana with the GhUGT73C3 gene. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0032] The embodiment of the present invention provides the use of the GhUGT73C3 gene or the protein encoded by it in improving the oil yield of plant seeds.

[0033] In some specific embodiments, the nucleotide sequence of the GhUGT73C3 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.2.

[0034] In some specific embodiments, overexpression of the GhUGT73C3 gene increases the oil content of plant seeds.

[0035] The embodiment of the present invention also provides a method for increasing the oil yield of plant seeds, which overexpresses the GhUGT73C3 gene to increase the oil content of plant seeds.

[0036] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the GhUGT73C3 gene in improving the oil yield of plant seeds.

[0037] The embodiments of the present invention also provide the use of the GhUGT73C3 gene or the protein encoded by it in regulating the seed size of plants.

[0038] In some specific embodiments, the nucleotide sequence of the GhUGT73C3 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.2.

[0039] In some specific embodiments, overexpression of the GhUGT73C3 gene increases the grain length, grain width and thousand-grain weight of plant seeds.

[0040] The embodiment of the present invention also provides a method for regulating the size of plant seeds, overexpressing the GhUGT73C3 gene to increase the grain length, grain width and thousand-grain weight of the plant seeds.

[0041] The embodiments of the present invention also provide the use of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the GhUGT73C3 gene in regulating the size of plant seeds.

[0042] The present invention heterologously expresses the GhUGT73C3 gene in Arabidopsis thaliana, and uses gas chromatography (GC) to determine the oil content and fatty acid component content of transgenic seeds, thereby clarifying the application of the GhUGT73C3 gene and its expression protein UGT in improving the oil yield of plant seeds. At the same time, the grain length, grain width and thousand-grain weight of the transgenic seeds are determined, thereby clarifying the application of the GhUGT73C3 gene and its expression protein UGT in increasing the size of plant seeds.

[0043] Example 1

[0044] Cloning of GhUGT73C3 Gene in Cotton

[0045] 1.1 Required enzymes, kits and drugs

[0046] (1) Enzyme and kit: 2× Max Master Mix (P515) high-fidelity enzyme, 2×Taq Plus Master Mix II (Dye Plus), Invitrogen TM Gateway TM BP Clonase TM II Enzyme mix, Invitrogen TM Gateway TM LR Clonase TM II Enzyme mix, FastPure plant total RNA extraction kit (for samples rich in polysaccharides and polyphenols) were purchased from Nanjing Novozyme Biotechnology Co., Ltd., HiScript III RT SuperMix reverse transcription kit, Green Realtime PCR qRT-PCR fluorescence quantitative enzyme was purchased from TOYOBO Biotechnology Co., Ltd. Plasmid MiniPrep Kit was purchased from Beijing Quanshijin Company. The QuickGel Extraction Kit was purchased from Beijing Quanshijin Company, the competent Escherichia coli DH5α was purchased from Kangwei Century, the competent Agrobacterium GV3101 was purchased from Weidi Biotechnology, and the primers used in the experiment were purchased from Zhengzhou Shangya Bioengineering Company.

[0047] (2) Other drugs: Agarose was purchased from Quanshijin Biotechnology Co., Ltd.; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. were domestic analytical grade; kanamycin and ampicillin, etc. were purchased from Bao Biotechnology Dalian Co., Ltd.

[0048] (3) Solution preparation: All reagents mentioned but not listed in this article were prepared according to the methods in the third edition of the Molecular Cloning Experiment Guide. Biochemical reagents were of analytical grade or above.

[0049] (4) LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L;

[0050] LB solid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, agar powder 15 g / L, fixed volume to 1 L;

[0051] LB selection medium: Before spreading LB plates, add antibiotics of corresponding concentration when the medium is sterilized by high pressure and cooled to 55°C, shake well and spread on the plates.

[0052] (5) Main instruments: PCR amplification instrument (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (ABI7500), vacuum freeze dryer ( Alpha I-5; Martin Christ), gas chromatograph (Nexis GC-2030, Shimadzu Corporation, Kyoto, Japan), and automatic sample grinder (JXFSTRBP-64, Shanghai Jingxin).

[0053] 1.2 Cloning of GhUGT73C3 gene

[0054] The reference sequence of GhUGT73C3 comes from the TM-1 (Cotton Research Institute, Chinese Academy of Agricultural Sciences) genome, and its gene number is Gh_D11G379500. Primers were designed using Oligo 6 software, and the complete CDS sequence of 1473 bp was amplified from the upland cotton material Zhimian No. 3 using PCR (Polymerase Chain Reaction) technology, encoding 490 amino acid residues. The gene CDS sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0055] >GhUGT73C3 gene CDS sequence is (SEQ ID NO.1):

[0056]

[0057] >The amino acid sequence of GhUGT73C3 protein is (SEQ ID NO.2):

[0058] MGEELHFLLIPLMSPGHLLPMVDMAHLLATHGVTVSIITTPLNALRFTSVVDSAVASGLRIQVHHLPFPAKEFGLPENCENMDQLPSRDLIMNFLMAAANELQQRFEELFNKLKPKPSCMVSGK NLPWTVKTATKFNVPRIVFDGMGCFSFVCTHKLELSKVHEMVSEFESFKIPGLLHEIELKKAQLPENLNPVSNDLINIRDIRKAELVCDGIVVNTFEELENEYVKEFKSIKGNGKVWCIGPVS AINKLSSDKAERGQKQCRFETLQPWLDSKEPGSVIYACLGSISGLTKWQLIELGLGLESSGKPFIWVIRENPKSNEIEKWILDEKFEDRVKDRGIIIHGWSPQLWVLSHPAIGAFLTHCGWNS TMEAVSAGVPVITCPLFAEQFINEKLVVDVLGIGVSAGVESAVTWGLEDKFGLLMKRERVKNAINEVMEKSEAGEERRRKAKQIGETANKAIEKGGSSYQEMEMLIQFVLQRTKEVAQTSS*.

[0059] 1.3 Specific process of gene cloning

[0060] (1) Experimental materials Zhongzhimian No. 3 was planted in the experimental field of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and managed as a normal field. The parts taken included roots, stems, leaves, flowers, buds, hypocotyls, ovules at different developmental stages, and fibers. The materials were quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use. Plant DNA was extracted using the modified CTAB method, and plant total RNA was extracted using a kit from Novozymes.

[0061] (2) Reverse transcribe 1 μg RNA into cDNA, and dilute the reverse transcribed cDNA solution 10 times as a PCR reaction template.

[0062] The extracted total RNA of cotton ovules at different stages was used as a template and reverse transcribed into cDNA using the reverse transcription kit of Novozyme. The specific steps are as follows:

[0063] ① Genomic DNA removal system: RNA template 1μg, 4×gDNA wiper Mix 4μL, RNase-free ddH 2 Add 3% HO to 16 μL.

[0064] After preparing the above system, mix well with a pipette, and then place in a PCR instrument at 42°C for 2 minutes.

[0065] ②Reverse transcription reaction system: 5×HiScriptⅢqRT SuperMix 4μL, step ① reaction solution 16μL.

[0066] After preparing the above system, mix it with a pipette, then place it in a PCR instrument and react at 37℃ for 15min and 85℃ for 5sec. After the reaction, dilute the cDNA 10 times, divide it into aliquots and store it in a -80℃ ultra-low temperature refrigerator.

[0067] (3) Perform PCR reaction to amplify the target gene

[0068] The full-length primers of GhUGT73C3 gene were designed using Oligo 6 software, and the mixed cDNA obtained by reverse transcription was used as a template for PCR amplification of cotton GhUGT73C3 gene. Max Master Mix (P515) high-fidelity enzyme was used for amplification.

[0069] The reaction system was: 2×Phanta Max Master Mix 25 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, cDNA template 1.6 μL and ddH 2 O 19.4 μL.

[0070] The PCR reaction program was: 95°C for 3 min; 95°C for 15 sec, 56°C for 15 sec, 72°C for 60 sec, repeated for 35 cycles; 72°C for 5 min; and stored at 12°C.

[0071] (4) After the PCR reaction is completed, PCR detection is performed using 1.2% agarose gel electrophoresis. If the band size is consistent with the expected design, it is considered valid. Figure 1 As shown. Figure 1 It can be seen that the band size is between 1000-2000 bp, which is in line with expectations and can be used for subsequent experiments.

[0072] (5) Using the kit from Beijing Quanshijin Co., Ltd. Quick Gel Extraction Kit was used to recover the target size band from gel.

[0073] Example 2

[0074] Construction of GhUGT73C3 gene expression vector

[0075] The Gateway system was used to construct the GhUGT73C3 gene expression vector. The cloned GhUGT73C3 gene fragment was connected to the expression vector pEarleyGate 101 using BP and LR reactions. The specific steps were as follows:

[0076] 2.1PDONR / Zeo vector ligation

[0077] BP reaction primers were designed according to the pDONR / Zeo map. The primer sequences are as follows:

[0078] GhUGT73C3-BP-F (SEQ ID NO.3):

[0079] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGGAGAAGAGCTTCATT TCCTTT-3';

[0080] GhUGT73C3-BP-R(SEQ ID NO.4):

[0081] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAAGAACTAGTTTGAGCTACCT CTTTA-3'.

[0082] The recovered DNA fragments were connected to the pDONR / Zeo vector by BP reaction. The reaction system is shown in Table 1.

[0083] Table 1 BP reaction system

[0084]

[0085] Prepare the above reaction system in a 200 μL centrifuge tube and place it at 25°C for 1 hour. Then transform the ligation product into DH5α, grow it overnight in LB solid medium containing bleomycin, pick a single clone, use M13-F and M13-R primers to identify the bacterial solution, and select the positive bacterial solution for plasmid extraction.

[0086] M13F (SEQ ID NO.5): 5'-GTTGTAAAACGACGGCCAG-3';

[0087] M13R (SEQ ID NO. 6): 5'-CAGGAAACAGCTATGAC-3'.

[0088] 2.2 pEarleyGate 101 expression vector ligation

[0089] The above plasmid was connected to the expression vector pEarleyGate 101 through LR reaction. The reaction system is shown in Table 2.

[0090] Table 2LR reaction system

[0091]

[0092] Prepare the above reaction system in a 200 μL centrifuge tube and place it at 25°C for 1 hour. Then, transform the ligation product into E. coli competent DH5α, pick a single clone, use 35S-F and EYFP-R primers to identify the bacterial solution, select the correct positive bacterial solution for plasmid extraction, transform Agrobacterium competent GV3101, pick a single clone, and use 35S-F and EYFP-R primers to identify the bacterial solution ( Figure 2 ), use 50% glycerol to preserve bacteria for later use.

[0093] 35S-F (SEQ ID NO.7): 5'-cccactatccttcgcaag-3';

[0094] EYFP-R (SEQ ID NO. 8): 5'-gaacttgtggccgtttac-3'.

[0095] 2.3 Subcellular localization of UGT protein expressed by GhUGT73C3 gene

[0096] After culturing Nicotiana benthamiana in a greenhouse at 16 h light, 8 h dark, and 25 °C for 4-5 weeks, Agrobacterium tumefaciens containing pEarleyGate101-GhUGT73C3 vector and empty vector control was shaken and then treated with 20 mM MES, 150 nM ASA, 10 mM MgCl 2 Resuspend in 1% ethanol solution, OD 600 The value was adjusted to about 1.0, and after being placed in the dark for 3 hours, Agrobacterium was injected into tobacco leaves. After culturing for about 36 hours, the GFP signal of the leaves was collected using Olympus FV1200 (Olympus, Japan). It can be observed that the GhUGT73C3-GFP fluorescence signal appears in the cell nucleus and cell membrane, indicating that the GhUGT73C3 protein is localized in the cell nucleus and cell membrane ( Figure 3 ).

[0097] Example 3

[0098] Obtaining Arabidopsis thaliana overexpressing GhUGT73C3 gene

[0099] 3.1 Obtaining GhUGT73C3-transgenic Arabidopsis

[0100] a. Wild-type Arabidopsis thaliana (Col-0) was planted in an Arabidopsis greenhouse at 22°C and 16 h of photoperiod and watered appropriately one day before infection.

[0101] b. In a clean bench, take 10 μL of the Agrobacterium culture medium carrying the pEarlyGate101-GhUGT73C3 target gene vector and streak it on LB solid culture medium containing kanamycin and rifampicin, then place the plate upside down in a 28°C constant temperature incubator for overnight culture.

[0102] c. In a clean bench, pick up the colony with a sterile pipette tip and place it in 100 mL LB liquid medium (containing kanamycin and rifampicin) at 28°C and 180 rpm in a shaker for overnight culture. 600 Stop the culture when the concentration is between 1.0 and 1.6.

[0103] d. Transfer 100 mL of bacterial solution to a 50 mL sterile centrifuge tube, centrifuge at 5000 rpm for 10 min, discard the supernatant, and resuspend the cells with resuspension solution (Table 3) to an OD of 600 0.8, and place in the dark for 2 hours.

[0104] Table 3 Resuspension formula

[0105]

[0106] e. Pour the resuspended liquid into the culture dish, immerse all the Arabidopsis inflorescences in each pot into the bacterial solution for 1 min, shake the Arabidopsis back and forth to improve the transformation efficiency, then cover with plastic wrap, keep in the dark for 24 hours, and continue daily cultivation in the Arabidopsis greenhouse to harvest T0 generation seeds.

[0107] f. After the T0 generation seeds were mixed and harvested, they were sown into culture trays (nutrient soil: vermiculite volume = 1:1), watered appropriately, covered with plastic wrap and placed in an Arabidopsis greenhouse. After the seedlings emerged, 10% Basta herbicide was sprayed every morning and evening. Negative seedlings withered and died, while positive seedlings were tender green and grew normally until Figure 4 The positive seedlings were transplanted into new nutrient pots, and the rosette leaves were taken for DNA extraction after bolting. Then PCR test was performed to confirm the positive seedlings. Figure 5 .

[0108] 3.2 RNA extraction from transgenic Arabidopsis and qRT-PCR verification of gene expression

[0109] (1) The total RNA of the control strain and transgenic Arabidopsis thaliana was extracted using a plant total RNA rapid extraction kit, reverse transcription was performed using a reverse transcription kit from Novazonics, and qRT-PCR verification was performed using a fluorescence quantitative kit from Novazonics.

[0110] (2) The qRT-PCR reaction system is shown in Table 4.

[0111] Table 4 qRT-PCR reaction system

[0112]

[0113]

[0114] The qRT-PCR reaction steps are as follows: ① Pre-denaturation: qRT-PCR starts at 95℃ and lasts for 30sec; ② Cyclic reaction: denaturation at 95℃ (10sec), primer annealing at 60℃ (30sec), 40 cycles; ③ Melting curve: 95℃ (15sec), 60℃ (60sec), 95℃ (15sec). The Actin gene of Arabidopsis thaliana was selected as the internal reference gene, and each template was repeated 3 times, and 2 -ΔΔCT Methods The relative gene expression was calculated.

[0115] Fluorescence quantitative primer sequence:

[0116] qRT-AtActin_F (SEQ ID NO.9): 5'-TAACTCTCCCGCTATGTATGTC-3';

[0117] qRT-AtActin_R (SEQ ID NO.10): 5'-GAGAAACCCTCGTAGATTGG-3';

[0118] qRT-GhUGT73C3_F (SEQ ID NO.11): 5'-TTAAAATTCCTGGTTTACTCCATGA-3';

[0119] qRT-GhUGT73C3_R (SEQ ID NO. 12): 5'-ACTGGGCCGATACACCATACTTT AC-3'.

[0120] The results are as follows Figure 6 As shown: the expression level of GhUGT73C3 gene in the three transgenic positive strains was significantly higher than that in the control strain (p<0.01).

[0121] Western Blot experiments were then performed on the three strains. Compared with the control, the GhUGT73C3 protein in the three overexpression strains was extremely highly expressed ( Figure 7 ).

[0122] 3.3 Determination of oil content and size of transgenic Arabidopsis seeds

[0123] The oil and fatty acid content of seeds of the three overexpression lines were determined. Figure 8 Compared with Arabidopsis WT, the oil content of seeds of the three transgenic lines increased by 4.91%, 6.89% and 10.56%, respectively; the content of oleic acid (C18:1) in fatty acid components increased by 17.16%, 22.32% and 14.67%, respectively; the content of linoleic acid (C18:2) increased by 1.71%, 3.54% and 5.35%, respectively, all reaching significant or extremely significant levels.

[0124] The seeds of the three overexpression lines were further compared in size and their length, width, and thousand-grain weight were measured. Fig. 9 Compared with the control, the seeds of the three transgenic lines were significantly larger, among which the grain length increased by 15.35%, 27.95%, and 22.44%, respectively, all reaching extremely significant levels; the grain width increased by 12.93%, 21.77%, and 19.05%, respectively, all reaching extremely significant levels; and the thousand-grain weight increased by 7.07%, 23.23%, and 34.35%, respectively, reaching significant or extremely significant levels.

[0125] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of the GhUGT73C3 gene or the protein encoded by it in improving the oil yield of plant seeds.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the GhUGT73C3 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: Overexpression of the GhUGT73C3 gene increases the oil content of plant seeds.

4. A method for increasing the oil yield of plant seeds, characterized in that: Overexpression of the GhUGT73C3 gene increases the oil content of plant seeds.

5. Application of a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the GhUGT73C3 gene in improving the oil yield of plant seeds.

6. Application of the GhUGT73C3 gene or the protein encoded by it in regulating plant seed size.

7. The use according to claim 6, characterized in that: The nucleotide sequence of the GhUGT73C3 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.

2.

8. The use according to claim 6, characterized in that: Overexpression of the GhUGT73C3 gene increases the grain length, grain width and thousand-grain weight of plant seeds.

9. A method for regulating plant seed size, characterized in that: Overexpression of the GhUGT73C3 gene increases the grain length, grain width and thousand-grain weight of plant seeds.

10. Use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the GhUGT73C3 gene in regulating plant seed size.

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