LU500576B1 - Soybean Oleosin Gene GmOLEO1 and Its Coding Protein and Application - Google Patents

Abstract

The invention discloses a soybean oleosin gene GmOLEO1, its coding protein and an application thereof. The nucleotide sequence of the gene is shown as Seq ID NO.1. The invention clones a soybean oleosin gene GmOLEO1 from the soybean genome for the first time. The invention uses the plant expression vector to introduce the GmOLEO1 gene into plant cells, thus transgenic plants with increased oil content can be obtained. Compared with non-transgenic soybeans, the soybean overexpressing the GmOLEO1 gene of the invention has significantly increased seed oil content, indicating that the GmOLEO1 gene plays an important role in improving the oil content of plant seeds, especially soybean seeds. The results of the present invention can be applied to regulate the expression of soybean oleosin gene through biotechnology. The results of the invention have important significance for cultivating soybean varieties with high oil content and have good application prospects.

Description

1 LU5S00576

DESCRIPTION Soybean Oleosin Gene GmOLEOI and Its Coding Protein and Application

TECHNICAL FIELD The invention relates to the technical field of genetic engineering, particularly to a soybean oleosin gene GmMOLEO I, its coding protein and application thereof.

BACKGROUND Oil in plants plays an important role in the process of plant cell division, growth and development. Vegetable oil is not only one of the important foods for human beings, but also a renewable and environmentally friendly biomass energy. It is an important energy source for human production and life. The synthesis of oil in plant seeds can be divided into three stages. The first stage is to synthesize fatty acids with sucrose as the main carbon source in plastids, the second stage is to synthesize triacylglycerol in endoplasmic reticulum, and the third stage is to combine the produced triacylglycerol with oleosin genes to form oil bodies. Oil body is a subcellular organ for storing triacylglycerol in plant seeds. The liquid triacylglycerol is stored in its interior, and its exterior is wrapped by a semi-unit membrane consisting of a phospholipid monolayer and oleosin genes embedded in it. There are a lot of oil bodies and oleosin genes in oil crop seeds such as soybean, peanut and rape. Oleosin protein is a highly hydrophobic basic protein with small molecular weight. It is of utmost importance to maintain the stability of oil body structure. The size and oil content of oil bodies in plant seeds are directly related to the oleosin gene content. After inhibiting the expression of seed oil body protein in transgenic soybeans by RNAi, a new small oil body with a diameter of about 50 nm was produced. They gradually merged to produce an oil body

2 LU5S00576 with a larger volume than that of wild type soybean (Schmidt and Herman, 2008). In rapeseed and Arabidopsis thaliana, the oleosin gene level of high oil content seeds is about 20% higher than that of low oil content seeds (Parthibane et al., 2012). The analysis of deletion mutants of oleosin genes in Arabidopsis thaliana showed that the oil content in seeds decreased (Siloto et al., 2000).

Soybean is the most important crop for both grain and oil. Soybean oil accounts for 28% of the global vegetable oil output, ranking first in the world vegetable oil output. Therefore, improving the oil content of soybean seeds is of great significance to human health and solving the economic and energy problems in China. With the development of current technology, DGAT2A, SLC 1, AtDGATI and other genes have been developed at home and abroad to increase soybean oil fat content and oil quality. However, the research on the effect of oleosin gene on soybean oil content is still blank.

Based on the above analysis, it is of great significance to study the influence of oleosin gene on soybean oil content and explore the oleosin gene of soybean for cultivating soybean varieties with high oil content. The invention has a good application prospect.

SUMMARY

1. Technical problems to be solved by the invention The object of the present invention is to provide a soybean oleosin gene GMOLFOI. Another object of the present invention is to provide the protein encoded by the gene. Another object of the present invention is to provide a vector containing the above gene. Another object of the present invention is to provide the application of the above gene or vector in the cultivation of high-fat soybean varieties.

2. Technical scheme

3 LU5S00576 In order to achieve the above object, the technical scheme adopted by the invention is as follows. A soybean oleosin gene GmOLEQO 1, whose nucleotide sequence is shown in the sequence table Seq ID NO. 1, encodes a protein consisting of 147 amino acids. In addition, the invention provides a protein encoded by soybean oleosin gene GmOLEOI. Amino acid sequence of the protein is shown in the sequence table Seq ID NO.2, and the molecular weight of the protein is 15.76 kDa and the isoelectric point is

7.81. It should be understood that considering the degeneracy of codons and the preference of codons of different species, those skilled in the art can use codons suitable for expression of specific species as needed. Therefore, the oleosin gene GMOLEOI of the present invention also includes the nucleotide sequence encoding the above protein obtained by substituting, deleting and/or adding one or several nucleotides to the nucleotide sequence shown in Seq ID NO.1. In addition, it should be understood that those skilled in the art can substitute, delete and/or add one or several amino acids according to the amino acid sequence (Seq ID NO.2) disclosed in the present invention without affecting its activity, so as to obtain the mutant sequence of the protein. Therefore, the coding protein of the invention also comprises a protein which is obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in Seq ID NO.2, has the same activity and is derived from the protein shown in Seq ID No.2. Furthermore, the present invention provides a vector containing the soybean oleosin gene GmOLEOI. The vector is a recombinant expression vector pCAMBIA3300-GmOLEO 1

4 LU5S00576 formed by inserting the soybean oleosin gene GMOLEOI into the pPCAMBIA3300 plant overexpression vector.

Furthermore, the invention provides the application of the soybean oleosin gene GmOLEOI or the vector in cultivating high-oil soybean varieties.

Further, the present invention provides a method for transforming soybean by using oleosin gene GMOLEOI. The method is to use the recombinant expression vector pCAMBIA3300-GmOLEOI to transform the gene of Claim 1 into soybean cotyledon nodes, and then cultivate the transformed soybean cells into transgenic plants.

3. Beneficial effects (1) The invention clones a soybean oleosin gene GMOLEOI from the soybean genome for the first time. Transgenic plants with increased oil content can be obtained by introducing the GmOLEQOI gene of the present invention into plant cells using plant expression vectors. Compared with non-transgenic soybeans, the soybean overexpressing the GmOLEOI gene of the invention has significantly increased seed oil content, indicating that the GMOLEO1 gene plays an important role in improving the oil content of plant seeds, especially soybean seeds.

(2) Transgenic plants with significantly increased seed oil content can be obtained by introducing the GmMOLEOI gene into plant cells using any vector that can guide the expression of exogenous genes in plants.

(3) When using the gene of the present invention to construct a plant expression vector, any enhanced promoter or inducible promoter can be added before the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed. For example,

LU5S00576 adding selective marker genes (GUS genes, luciferase genes, etc.) or antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.) expressed in plants. Considering the safety of transgenic plants, it is also possible to screen transformed plants directly by phenotype without adding any selective marker genes.

(4) The plant expression vector carrying GmOLEOI of the present invention can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation, etc. It can also culture the transformed plant tissues into plants. The transformed host can be either monocotyledonous or dicotyledonous.

(5) The results of the present invention can be applied to regulate the expression of soybean oleosin gene through biotechnology. Therefore, new soybean varieties with significantly improved seed oil content will be cultivated. The results of the invention have important significance for cultivating soybean varieties with high oil content and have good application prospects.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 The vector map of soybean transformation expression vector pCAMBIA3300- GmOLEO1 Note: Bar gene was used as transgenic screening marker, and the pCAMBIA3300- GmOLEOI vector was followed by full-length gmoleol cDNA with 35S promoter. After after the vector was used for transgene, transgenic plants overexpressing GmOLEO] could be obtained.

Figure 2 The verification results of positive transgenic soybean plants.

6 LU5S00576 Note: Figure A shows the detection results of transgenic soybeans by the leaf painting of glufosinate, and half of the leaves marked in black indicate that no herbicide is applied. The leaves on the other side of the main vein were coated with 200 mg/L Basta herbicide. Figure B is the PCR validation electrophoresis diagram of transgenic soybean, M is 2000 bp Marker, C is blank control, + is plasmid positive control, and 1-10 represents samples 1-10 of independent transgenic lines. Figure C is the expression result of bar gene detected by LibertyLink® strip. WT is non-transgenic soybean, and 1-10 are 10 independent transgenic lines. Figure 3 A Southern blot analysis chart of T1 transgenic soybean Note: M, Marker; +, the DNA fragment of the target gene GMOLKO]; -, non-transgenic soybean, 1-4 are four independent transgenic lines overexpressed by GmOLEO]. Figure 4 An analysis chart of oil content in seeds of transgenic soybean lines with GmOLEOI gene. Note: WT is a non-transgenic soybean, and OE1-4 is four independent transgenic lines.

DESCRIPTION OF THE INVENTION Terms used in the present invention, unless otherwise stated, generally have meanings commonly understood by those of ordinary skill in the art. The following describes the invention in further detail with reference to specific preparation embodiments and application embodiments and data. It should be understood that these embodiments are only for illustrating the present invention, and do not limit the scope of the present invention in any way. In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The primers used are all marked when they first

7 LU5S00576 appear, and the same primers used afterwards are all marked with the same contents for the first time.

Unless otherwise specified, the methods used in the following embodiments are all conventional methods.

Embodiment 1: Cloning of soybean oleosin gene GMOLEOI and construction of plant expression vector (1) Primer design, RNA extraction and cDNA inversion: Total RNA of soybean Williams 82 leaves was extracted with plant total RNA extraction kit (DP432, Tiangen), and the integrity of RNA was detected by 1% agarose gel electrophoresis. The operation of cDNA synthesis was referred to TaKaRa Primer Script TMRT reagent kit with gDNA Eraser kit. The primer sequence for amplification of gene GmOLEOI 1s designed as follows: Seq ID NO.3: GmOLEOI-F 5°-CCTACCACATTAATTACTCACTCTTCACTCA-3’; Seq ID NO.4: GmOLEOI-R 5’-TCAACTTTAACGCTCATTCCTGCATTCAT-3.

(2) PCR amplification, the specific steps are as follows.

Step 1: Prepare PCR reaction solution (50 ul system) according to the following component sequence: 10xPCR Buffer (25 ul), ddH20 (9 ul), dNTP (10 ul), GmOLEQOI-F (1.5 ul), GmOLEOI-R (1.5 ul), cDNA (2 ul), KOD FX enzyme (1 pl).

Step 2: The reaction was carried out on BIO-RAD PTC-200 PCR instrument, and the reaction procedure was set as follows. Denaturation at 94°C for 2 min, then at 98°C for sec, 55°C for 30 sec and 68°C for 30 sec, a total of 33 cycles. Then extension at 68°C for 7 min. Preservation at 4°C.

8 LU5S00576 Step 3: After the PCR product is recovered, the PCR product is connected with PMD19-T vector (TaKaRa), transformed into Escherichia coli DH5a, blue-white screened, shaken and sequenced, and the sequence is shown in Seq ID NO. 1. (3) Construction of plant expression vector A homologous recombination linker primer was designed.

The T vector containing GmOLEOI gene obtained in the above (2) was used as a template to amplify the full- length GmOLEO] fragment with recombination linker, and the GmOLEOI gene was positively introduced into soybean expression vector pCAMBIA3300 by seamless cloning technology to construct a recombinant plant expression vector pCAMBIA3300- GmOLEOI (as shown in Figure 1). The vector pCAMBIA3300 carries a selectable marker gene bar in the T-DNA region.

The gene encodes the glufosinate acetyl-CoA transferase (PAT), and can catalyze the free amino acetylation of the glufosinate, thus inactivating the herbicide glufosinate.

The primer sequences used for seamless cloning are as follows: Seq ID NO.5: Upstream primer 5'-TTTGGAGAGAACACGTATGGCTGAGCTTCACTACCAAC-3'; Seq ID NO.6 : Downstream primer 5'-TCGGGGAAATTCGGGGTTAAGAAGCCTGCACCCCACTG- 3 Embodiment 2: Cultivation of transgenic soybean with GMOLEOI gene overexpression (1) Disinfection and germination of seeds The surface disinfection of soybean seeds was carried out by chlorine dry sterilization.

Mature, full, lesion-free, hard and clean seeds were selected, and then arranged in a

9 LU5S00576 90*15 mm petri dishes in a single layer. Petri dishes were opened and put in a dryer. A 500 ml glass beaker was placed in the dryer, and 75 ml of commercial bleaching water was measured with a 100 ml measuring cylinder and added into the beaker, and 3 ml of 12M HCI was measured with a 10ml measuring cylinder and slowly added along the cup wall. The dryer was covered to ensure that the vessel was sealed, and then left stand overnight for 10-16 hours. After sterilization, the petri dishes were covered and transferred to the sterile ultra-clean table, and then the covers of the petri dishes were opened, and blown with strong wind for 25-40 min to remove residual chlorine. The disinfected seeds were sown on the germination medium (GM) with the navel down. The petri dishes were stacked, wrapped with fresh-keeping film, and placed in the biological incubator at 24°C for 16-24 hours in the dark.

(2) Preparation of Agrobacterium The recombinant vector pPCAMBIA3300-GmOLEO1 plasmid DNA was extracted, and the recombinant vector was transferred into Agrobacterium strain LBA4404 by electroporation, and stored in 50% glycerol. Two days before transgene, 50 ul of Agrobacterium glycerol containing vector was absorbed into 5 ml of YEP liquid medium (10 g/L peptone, 5 g/L. yeast extract, 5 g/L sodium chloride, pH 7.0) added with antibiotics (1/1000), and cultured for 24-36 hours at 28°C and 250 rpm. Saturated bacterial liquid was absorbed into 250 ml YEP liquid medium added with antibiotics (1/2000) for expanded culture until OD650 nm = 0.8-1.0. The bacterial liquid was sub- packed into several 50 ml sterile centrifuge tubes and centrifuged (4000 rpm, 10 min, 25°C). The colonies were collected, gently blown with 25-50 ml liquid co-culture medium (LCCM), and resuspended for later use. The LCCM medium contained large

LU5S00576 number and trace 1/10 BS and vitamins (Gamborg et al, 1968), 3% sucrose, 3.9 g/L organic buffer 2(N- morpholine) ethanol sulfonic acid (MES), pH 5.4. It was sterilized at 120°C for 20 min, and added with Gibberellin (GA3) 0.25 mg/L, 6-benzyladenine (BAP)

1.67 mg/L, cysteine (Cys) 400 mg/L, dithiothreitol (DTT) 1542 mg/L. and acetosyringone (As) 200 umol/L in sterile environment. (3) Preparation and co-culture of explants The swollen soybean seeds were placed on sterile absorbent paper and then cut longitudinally along the hilum with a scalpel. Cotyledon and hypocotyl were evenly divided into two petals, and seed coats were removed for later use. The Agrobacterium suspension was poured into clean sterile petri dishes, and then about 50 explants were put into the petri dishes for infection for 20-30 mins at room temperature. During infection, the bacterial liquid was often stirred to make the explants fully contact with the fresh bacterial liquid. After infection, the explants were taken out, dried with sterile absorbent paper, and then placed on a co-culture medium (CM) with sterile filter paper. There were 7-10 explants in each dish, with the paraxial surface facing up and placed horizontally. The formula of CM medium was the same as LCCM, with the addition of 5 g/L agar (Difco Agar, Noble company). The petri dishes were stacked, sealed with plastic wrap, and then co-cultured in Percival incubator at 23°C in the dark for 3-5 days. (4) Screening and regeneration After 3-5 days of co-cultivation, the elongated hypocotyl was cut off and left about 0.5 cm. The left hypocotyl was inserted into bud induction (SI) medium with screening agent at an oblique angle of 30-45°. The SI medium contained large number and trace BS and vitamins, sucrose 30 g/L, MES 0.59 g/L, and agar 8g/L (Sigma, USA). After sterilization

11 LU5S00576 at 120°C for 20 min, the SI medium was added with BAP 1.67 mg/L, ticarcillin (Tic) 250mg/L and cephalosporin (Cef) 100mg/L under sterile conditions. Then the SI medium was sealed with 3M breathable tape and transferred to culture room (24°C, 18/6 light intensity 140 u moles/m?*/sec), cultured for 4 weeks, and changed with fresh SI medium every two weeks. The residual cotyledons were cut off and transferred to bud elongation (SE) medium after induction and screening of multiple shoots for 4 weeks. SE medium containd large number and trace MS and vitamins (Murashige and Skoog, 1962), sucrose 30 g/L, MES 0.59 g/L, agar (Sigma, USA) 8g/L, pH 5.8. After sterilization at 120°C for min, the SE medium was added with GA 30.5 mg/L, L-asparagine (L-ASP) 50 mg/L, glutamine (Glu) 50 mg/L, indoleacetic acid (IAA) 0.1 mg/L, zeatin (ZR) 1 mg/L, Tic 250 mg/L and Cef 100 mg/L under sterile conditions. The culture conditions of SE medium were the same as that of multiple shoot induction. It was cultured for 2-8 weeks, and the fresh SE medium was changed every 2 weeks. The buds with an elongation of 3-4 cm were cut off, dipped in indole butyric acid (IBA) for 30 s-1 min, and then inserted into rooting medium (RM). The rooting medium contained large number and trace MS and vitamins, sucrose 20 g/L, MES 0.59 g/L, agar (Sigma, USA) 8 g/L, IBA 0.1 mg/L, L-Asp 50 mg/L, Glu 50 mg/L, Tic 250 mg/L and Cef 100 mg/L. After 1-2 weeks, when the root length was about 2-3 cm, the rooted seedlings were taken out from the culture medium. The residual culture medium in the roots was cleaned, and then the rooted seedlings were transferred to the soil and moved to the greenhouse for cultivation. The culture conditions were 24°C, 18/6 light intensity of 140 u moles/m?/sec.

Embodiment 3: Validation of transgenic materials

12 LU5S00576 Since the vector used for the transgene contains the coding glufosinate acetyl-CoA transferase (PAT), it can catalyze the acetylation of the free amino group of glufosinate, thereby inactivating the herbicide glufosinate.

The herbicide Basta was used for identification.

The stock solution was sprayed on the transgenic seedlings after diluted 1000 times (the concentration was 200 mg/L), and the negative plants withered and died, while the positive plants showed obvious resistance and kept good growth.

DNA was extracted from the leaves of positive plants detected to be alive by herbicide (CTAB rapid extraction kit of plant genome DNA: Zhong Ding Company, item number DN14-100T), and the positive materials were further screened by PCR detection marker bar gene.

The primer sequence of bar gene is as follows.

Seq ID NO.7: upstream primer 5'-ATGAGCCCAGAACGACGC-3'; Seq ID NO.8: downstream primer 5'-ACGTCATGCCAGTTCCCGT-3". Eight independent transgenic lines of To generation were finally obtained.

The harvested T1 generation seeds of transgenic materials were potted in sterilized mixed nutrient soil (nutrient soil: vermiculite = 2:1) and cultured in greenhouse at 24°C under 18/6 illumination intensity of 140 u moles/m’/sec.

The leaves of T1 generation soybean transformed plants were coated with cotton swabs with the combination of 200 mg/L glufosinate+0.1% Tween-20. The leaves were coated on half of soybean leaves with the main vein as the boundary.

The other half leaves were marked with a marker pen to indicate the control without herbicide.

After 3-5 days, the leaves were observed.

As shown in Figure 2A, if half of the leaves coated with herbicide have obvious withering phenomenon compared with the control, it means that the sample is negative.

If the leaves are normal, the sample is positive.

DNA was extracted from the leaves of the

13 LU5S00576 positive plants detected by glufosinate, and the positive materials were further screened by PCR amplification of bar gene fragment. As shown in Figure 2B, bar gene can be detected by transgenic materials. PAT/bar protein was detected by LibertyLink® strip (EnviroLogix Inc, USA) from the leaves of soybean transgenic plants which were positive by the above two methods. The method is as follows.

(1) Place a sample leaf tissue between a cover and a tube body of a disposable tissue extraction tube, quickly cover the cover to obtain a circular leaf tissue, and place the leaf at the bottom of the extraction tube with a pestle.

(2) Insert the pestle into the tube, rotate the pestle to crush the leaf, press continuously for 20-30 seconds, and add 0.5 mL of extraction buffer.

(3) Repeat the grinding step to make the sample fully contact and mix with the buffer, and remove the pestle.

(4) Keep the reaction tube upright, insert the test paper into the reaction tube, the sample liquid will rise along the test paper, and read the result after 10 minutes of reaction.

As shown in Figure 2C, if two red lines are displayed on a test strip in a sample, it means that the coding product (PAT) of the bar gene can be detected at the translation level, and it also means that the sample is a positive transgenic material.

Embodiment 4 Southern blot hybridization analysis of transgenic materials The DNA of four T1 generation plants (showing glufosinate resistance, positive by PCR verification and LibertyLink® strip detection) was used for Southern analysis to detect the copy number of target gene inserted into genome. Specific methods are as follows.

(1) Preparation of probe The primer sequence for preparing GmOLEO1 gene probe is as follows:

Seq ID NO.9: GMOLEO1-PF 5°-GCTACTCCACTCAGGTCGTC-3’; Seq ID NO.10: GMOLEO1-PR S’-CACCCCACTGATTTGCTG-3". Step 1: The probe template was prepared by PCR, and the PCR reaction solution (50 ul system) was prepared according to the following components: 10xbuffer (plus Mg**) (5 ul), dUTP labeling mixture (5 ul), GmOLEOI-PF (1 ul), GmOLEOI-PR (1 ul), Taq enzyme (1 ul), DNA (1 ul), and ddH>O (36 ul). Step 2: The reaction was carried out on BIO-RAD PTC-200 PCR instrument, and the reaction procedure was set as follows.

Pre-denaturation at 94°C for 5 min.

Denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec, a total of 35 cycles.

Then renaturation at 72°C for 7 min.

Preservation at 4°C.

Step 3: 1% agarose gel electrophoresis was performed on PCR products.

The control DNA was recovered by agarose gel purification and recovery kit (Zhong Ding Company, item number DROI1-50 T), and then it was sequenced correctly for later use. (2) Extraction and enzyme digestion of genomic DNA Genomic DNA was extracted from tissue samples, and the genome was digested after 1% agarose gel electrophoresis detection.

The digestion system was prepared according to the following components: 10xM buffer (60 ul), Hind III (30 pl), genome (10 ug) and sterilized water (up to 600 ul). The endonuclease was added twice, with 10 ul added at the first time, gently stirred, digested at 37°C for 2 h, then added with 10 pl, gently stirred, and digested at 37°C overnight (about 16 h), and then added with 10 pl the next day, and continued to react for 4 h.

Sul of each sample was detected by 1% agarose gel electrophoresis.

The digested DNA was extracted with equal volume of phenol: chloroform, and the product was dissolved in 50 ul deionized water.

LU5S00576 (3) Electrophoresis The prepared samples were subjected to 0.7% agarose gel electrophoresis at 25 V constant pressure and low temperature overnight. (4) Film transfer Step 1: The gel was placed in a plate, and several times of denature solution was added after the gel was rinsed with distilled water once for shaking at room temperature for 45 min.

Step 2: The gel was rinsed twice with distilled water and shaken at room temperature for 2x15 min after the neutralized solution was added.

Step 3: The gel was rinsed twice with distilled water, and 2xSSC was added to balance gel and positively charged nylon membrane for 5 min.

Step 4: After the membrane was transferred by upward capillary method for 20 h, it was taken off and marked, rinsed once in 2xSSC, and then baked and fixed at 80°C for 2h. (5) Hybridization Step 1: Pre-hybridization: 10.0ml of hyb-100 was added into the hybridization tube and pre-hybridized at 37°C for 2h.

Step 2: Probe denaturation: the probe was denatured in a PCR instrument at 100°C for 10 min, and immediately cooled in an ice water bath for 5 min.

Step 3: Hybridization: the prehybridization solution was drained, and 20 ul of newly denatured probe was added to 10.0 ml Hyb-100. Mixed them well and hybridized overnight at 37°C. (6) Membrane washing and signal detection

16 LU5S00576 Step 1: After hybridization, the membrane was washed at room temperature with 20 ml 2xSSC/0.1% SDS for 2x5 min.

Step 2: The membrane was washed with 20 ml 1xSSC/0.1% SDS at 65°C for 2x15 min.

Step 3: The membrane was put in 20 ml washing buffer to balance for 2-5 min.

Step 4: The membrane was blocked in 10 ml blocking solution for 30 min (shaken gently on a shaker). Step 5: Anti-Dig-AP was centrifuged at 10000 rpm for 5 min.

Anti-Dig-AP was diluted with blocking solution (1: 5000) after centrifugation, and then 2.0 ul Anti-Dig-AP was added into 10 ml blocking solution to mix evenly.

Step 6: After blocking, the blocking solution was poured out, diluted 10 ml antibody solution was added, and then the membrane was soaked for at least 30 min.

Step 7: The antibody solution was removed, and the membrane was slowly washed twice with 20 ml washing buffer for 15 min each time.

Step 8: Iml of CSPD was dripped on the front side (nucleic acid side) of the membrane, isolated from air, reacted at 15-25°C for Smin.

Excess liquid was removed, and the membrane was incubated at 37°C for 10min.

Step 9: X-ray film was used to expose, develop, fix and wash in the darkroom and the results of exposure was recorded.

As shown in Figure 3, non-transgenic soybean has four copies.

Because the target gene is the endogenous gene of soybean and soybean is an ancient tetraploid plant, compared with non-transgenic soybean, T1 generation transgenic plants show the same T-DNA integration mode, and the number of transgenic copies is very low, and each transgenic line only contains one copy of insertion.

17 LU5S00576 Embodiment 5: Determination of Oil Content in Soybean Seeds The oil content of non-transgenic soybean and GmOLEQ1 overexpression soybean seeds was determined by near infrared spectrum grain analyzer (wave pass model DA7200, Sweden). The calibration curve was produced by the Grain and its Products Quality Inspection and Testing Center of the Ministry of Agriculture. The curve is based on 900 soybean samples with different oil content in grains. The determination coefficient R2 of the curve equation is 0.96, and the standard error is 0.50. Each sample was put into a 60 mm cup for rotary scanning, and each sample was scanned 3 times. The results showed that the oil content of seeds increased significantly after overexpression of oleosin gene GmOLEOI in soybean. As shown in Figure 4, the oil content of non-transgenic soybean is about 17.84%, while the oil content of four overexpressed strains is about 20.13%,

20.17%, 20.07% and 19.76% respectively, which is 12.86%, 13.06%, 12.52% and

10.79% higher than that of non-transgenic soybean. < 110 > Henan agricultural university < 120 > Soybean oleosin gene GmOLEQ and its coding protein and application <160> 10 <210> 1 <211> 444 <212> DNA <213 > Soybean (Glycine max L.) <220> < 223 > Soybean oleosin gene GmOLEOI <400> 1 atggctgagc ttcactacca acaacaacac caataccctc accgataccce taatgatcca 60 tacgaacaac aaactagcta ctccactcag gtegtcaagg cogccaccec cotcaccecg 120 ggcggctece tettgatect cgectegtte atccttgccg geaccatcat cgecctcace 180 atcgtcacac caccectcet catcttcagt ccggttcteg tececcgeggt gatcaccgte 240 gcgetgetga gectggggtt cettgectee ggcgggtteg gtotggcgec gatcacggtg 300 ctggcgtega tetacaggta cgtcaccggg aagtacccac ctggcgcgea tcagttggac 360 agcgcgccte acaagatcat ggacaaggcg cgtgagatca aggactatge acagcagcaa 420 atcagtgggg tgcaggcttc ttaa 444 <210>2 <211> 147 <212> PRT < 213 > Soybean (Glycine max L.) <220> < 223 > Protein encoded by soybean oleosin gene GmOLEO] <400> 2 Met Ala Glu Leu His Tyr Gln Gln Gln His Gln Tyr Pro His Arg Tyr 151015 Pro Asn Asp Pro Tyr Glu Gln Gln Thr Ser Tyr Ser Thr Gln Val Val

Lys Ala Ala Thr Ala Val Thr Ala Gly Gly Ser Leu Leu Ile Leu Ala 40 45 Ser Leu Ile Leu Ala Gly Thr Ile Ile Ala Leu Thr Ile Val Thr Pro 50 55 60

Pro Leu Val Ile Phe Ser Pro Val Leu Val Pro Ala Val Ile Thr Val 65 70 75 80 Ala Leu Leu Ser Leu Gly Phe Leu Ala Ser Gly Gly Phe Gly Val Ala 85 90 95 Ala Ile Thr Val Leu Ala Trp Ile Tyr Arg Tyr Val Thr Gly Lys Tyr 100 105 110 Pro Pro Gly Ala Asp Gln Leu Asp Ser Ala Pro His Lys Ile Met Asp 115 120 125 Lys Ala Arg Glu Ile Lys Asp Tyr Gly Gln Gln Gln Ile Ser Gly Val 130 135 140 Gln Ala Ser 145 <210>3 <211>31 <212> DNA < 213 > Artificial synthesis <220> < 223 > Upstream primer for gene GmOLEO1 amplification <400> 3 cctaccacat taattactca ctettcactc a 31 <210> 4 <211> 29 <212> DNA

LU5S00576 < 213 > Artificial synthesis <220> < 223 > Downstream primer for gene GMOLEOI amplification <400> 4 tcaactttaa coctcattec tgcattcat 29 <210> 5 <211> 38 <212> DNA < 213 > Artificial synthesis <220> < 223 > Seamless cloning of upstream primers <400> 5 tttggagaga acacgtatgg ctgagcttca ctaccaac 38 <210> 6 <211> 38 <212> DNA < 213 > Artificial synthesis <220> < 223 > Seamless cloning of downstream primers <400> 6 tcggggaaat tcggegttaa gaagcctoca ceccacte 38 <210> 7 <211> 18

21 LU5S00576 <212> DNA < 213 > Artificial synthesis <220> < 223> Upstream primer for bar gene amplification <400> 7 atgagcccag aacgacec 18 <210> 8 <211> 19 <212> DNA < 213 > Artificial synthesis <220> < 223 > Downstream primer for bar gene amplification <400> 8 acgtcatgcc agttcecgt 19 <210> 9 <211> 20 <212> DNA < 213 > Artificial synthesis <220> < 223 > Upstream primer for preparation of GMOLEO1 gene probe <400> 9 gctactccac tcaggtcgtc 20 <210> 10

22 LU5S00576 <211> 18 <212> DNA < 213 > Artificial synthesis <220> < 223 > Downstream primer for preparation of GMOLEOI1 gene probe <400> 10 caccccactg atttgctg 18

20210825 PT1390 2021-6303 Sequence.txt LU500576 1 SEQUENCE LISTING 2 3<110> Henan Agricultural University 4 5<120> Soybean Oleosin Gene GmOLEO1 and Its Coding Protein and Application 6 7<130> PT1390LU 8 9<160> 10

11<170> BiSSAP 1.3.6 12 13<210> 1 14 <211> 444 <212> DNA 16 <213> Artificial Sequence 17 18 19 <220> 20<223> Soybean oleosin gene GmOLEO1 21 22<400> 1 23 atggctgagc ttcactacca acaacaacac caataccctc accgataccc taatgatcca 60 24 tacgaacaac aaactagcta ctccactcag gtcgtcaagg cggccaccge cgtcaccgeg 120 26 27 ggcggctcee tettgatact cgecctegttg atcottgccg gcaccatcat cgecctcecacce 180 28 29 atcgtcacac caccgctcgt catcttcagt ccggttcteg tccececgeggt gatcaccgte 240

31 gcgetgetga gecctggggtt ccttgecctce ggegggtteg gtgtggegge gatcacggtg 300 32 33 ctggcgtgga tctacaggta cgtcaccggg aagtacccac ctggcgcgga tcagttggac 360 34 agcgcgectc acaagatcat ggacaaggcg cgtgagatca aggactatgg acagcagcaa 420 36 37 atcagtgggg tgcaggcttc ttaa 444 38 39 40<210> 2 41<211> 147 42 <212> PRT 43 <213> Artificial Sequence 44 45 46<220> 47 <223> Protein encoded by soybean oleosin gene GmOLEO1 48 49<400> 2 50Met Ala Glu Leu His Tyr Gln Gln Gln His Gln Tyr Pro His Arg Tyr 511 5 10 15 52 Pro Asn Asp Pro Tyr Glu Gln Gln Thr Ser Tyr Ser Thr Gln Val Val 53 20 25 30 54 Lys Ala Ala Thr Ala Val Thr Ala Gly Gly Ser Leu Leu Ile Leu Ala 55 35 40 45 56 Ser Leu Ile Leu Ala Gly Thr Ile Ile Ala Leu Thr Ile Val Thr Pro 57 50 55 60 58 Pro Leu Val Ile Phe Ser Pro Val Leu Val Pro Ala Val Ile Thr Val 59 65 70 75 80 60Ala Leu Leu Ser Leu Gly Phe Leu Ala Ser Gly Gly Phe Gly Val Ala Page 1

20210825 PT1390 2021-6303 Sequence.txt LU500576 61 85 90 95 62 Ala Ile Thr Val Leu Ala Trp Ile Tyr Arg Tyr Val Thr Gly Lys Tyr 63 100 105 110 64 Pro Pro Gly Ala Asp Gln Leu Asp Ser Ala Pro His Lys Ile Met Asp 65 115 120 125 66 Lys Ala Arg Glu Ile Lys Asp Tyr Gly Gln Gln Gln Ile Ser Gly Val 67 130 135 140 68 Gln Ala Ser 69 145 70 71<210> 3 72<211> 31 73<212> DNA 74 <213> Artificial Sequence 75 76 77 <220> 78 <223> Upstream primer for gene GmOLEOI1 amplification 79 80 <400> 3 81 cctaccacat taattactca ctcttcactc a 31 82 83 84 <210> 4 85 <211> 29 86 <212> DNA 87 <213> Artificial Sequence 88 89 90 <220> 91 <223> Downstream primer for gene GmOLEOl amplification 92 93 <400> 4 94 tcaactttaa cgctcattcc tgcattcat 29 95 96 97 <210> 5 98 <211> 38 99<212> DNA 100 <213> Artificial Sequence 101 102 103 <220> 104 <223> Seamless cloning of upstream primers 105 106 <400> 5 107 tttggagaga acacgtatgg ctgagcttca ctaccaac 38 108 109 110 <210> 6 111 <211> 38 112 <212> DNA 113 <213> Artificial Sequence 114 115 116 <220> 117 <223> Seamless cloning of downstream primers 118 119 <400> 6 120 tcggggaaat tcggggttaa gaagcctgca ccccactg 38 Page 2

20210825 PT1390 2021-6303 Sequence.txt LU500576 121 122 123<210> 7 124 <211> 18 125 <212> DNA 126 <213> Artificial Sequence 127 128 129 <220> 130 <223> Upstream primer for bar gene amplification 131 132 <400> 7 133 atgagcccag aacgacgc 18 134 135 136<210> 8 137 <211> 19 138 <212> DNA 139 <213> Artificial Sequence 140 141 142 <220> 143 <223> Downstream primer for bar gene amplification 144 145 <400> 8 146 acgtcatgec agttccegt 19 147 148 149 <210> 9 150 <211> 20 151 <212> DNA 152 <213> Artificial Sequence 153 154 155 <220> 156 <223> Upstream primer for preparation of GMOLEOl gene probe 157 158 <400> 9 159 gctactccac tcaggtcgtce 20 160 161 162 <210> 10 163 <211> 18 164 <212> DNA 165 <213> Artificial Sequence 166 167 168 <220> 169 <223> Downstream primer for preparation of GMOLEO1 gene probe 170 171 <400> 10 172 caccccactg atttgctg 18 173 174 175

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Claims (6)

23 LU5S00576 CLAIMS:

1. A soybean oleosin gene GmOLEQ]I, characterized in that the nucleotide sequence of the soybean oleosin gene GmOLEQ 1 is shown in the sequence table Seq ID NO. 1.

2. The protein encoded by soybean oleosin gene GmOLEOI according to claim 1, characterized in that the amino acid sequence of the protein is shown in sequence table Seq ID NO.2.

3. A vector containing the soybean oleosin gene GmMOLEO1 according to claim 1.

4. Application of soybean oleosin gene GMOLKOI according to claim 1 in cultivating high-fat soybean varieties.

5. Application of the vector according to claim 3 in cultivating high-oil soybean varieties.

6. A method for transforming soybean by using the soybean oleosin gene GMOLEOI of claim 1, characterized in that the method is to use the recombinant expression vector pCAMBIA3300-GmOLEOI to transform the gene of claim 1 into soybean cotyledon nodes, and then cultivate the transformed soybean cells into transgenic plants.