CN109504687B - Soybean lipid transporter gene GmABCA9 and application thereof - Google Patents

Abstract

The invention discloses a soybean lipid transporter gene GmABCA9 and application thereof, and the invention defines the characteristics of specific expression and endoplasmic reticulum localization of the soybean GmABCA9 gene in soybean seeds, the expression of the gene regulates the development of arabidopsis seeds, influences the content of fatty acid in the seeds, and shows that the gene participates in the endoplasmic reticulum lipid transport system to regulate the synthesis of seed oil.

Description

Soybean lipid transporter gene GmABCA9 and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a soybean lipid transporter gene and application thereof.

Background

Soybean is an important oil crop in China and also a main source of high-quality protein for human beings. Is one of the main crops in China and is also the agricultural product with the largest import quantity in China. In recent years, the oil yield of domestic soybean crops cannot meet the increasing demand of vegetable oil, and soybean oil is mainly imported oil in domestic markets. Therefore, the cultivation of domestic high-oil soybean varieties and the improvement of the yield of domestic soybean oil are urgent.

In order to cultivate high-yield high-oil soybean varieties, researchers regulate and control fatty acid synthetase and key transcription factors in oil metabolic pathways by means of genetic engineering, but the improvement of the oil content is only maintained to be about 10% -20%.

Lipid synthesis occurs in chloroplasts, followed by transport to the endoplasmic reticulum for processing. At present, few researches on the transportation system of oil and fat between chloroplast and endoplasmic reticulum are carried out, and no report is found in soybean.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to further improve the content of the soybean oil and fat.

The technical scheme of the invention is as follows: the nucleotide sequence of the soybean GmABCA9 gene is shown in SEQ ID No. 1.

The application of the soybean GmABCA9 gene shown in SEQ ID No.1 in regulating and controlling the oil content of plant seeds.

Further, the plant is a leguminous plant.

Further, the plant is soybean.

Further, the plant is arabidopsis thaliana.

The application of the expression vector containing the soybean GmABCA9 gene shown in SEQ ID No.1 in the regulation of plant seed lipid.

The amino acid sequence of the protein coded by the soybean GmABCA9 gene shown in SEQ ID No.1 is shown in SEQ ID No. 2.

GmABCA9 can be expressed in leaves, stems, roots and seeds of plants. The expression level of the gene is higher in the later period of soybean mature seed development.

After the GmABCA9 is transiently expressed in tobacco leaves, fluorescence signal observation is carried out on the transformed tobacco leaves and protoplasts by using a microscope, and the GmABCA9 is found to be positioned in the endoplasmic reticulum.

The Arabidopsis abca9 mutant has shriveled seeds and small grains. When GmABCA9 was overexpressed in Arabidopsis, the revertant seeds restored the same phenotype as the wild type. When grown on sucrose-free medium, abca9 mutant seedlings grew slowly and dysplastically, and when GmABCA9 was overexpressed in Arabidopsis, the revertant seedlings developed normally. These results indicate that GmABCA9 regulates the seed development process of transgenic arabidopsis thaliana.

The over-expression of GmABCA9 improves the contents of seed oil and C16:0, C18:1, C18:2 and C18:3 fatty acids. The GmABCA9 is shown to promote the accumulation of oil and fat by regulating the content of fatty acid in seeds.

Compared with the prior art, the invention has the following beneficial effects:

the invention defines the characteristics of specific expression and endoplasmic reticulum positioning of the soybean GmABCA9 gene in soybean seeds, the expression of the gene regulates the development of arabidopsis seeds, the fatty acid content of the seeds is influenced, and the gene is shown to participate in a transport system of endoplasmic reticulum lipid so as to regulate the synthesis of seed oil.

Drawings

FIG. 1: expression levels of GmABCA9 in different tissues of soybean; leaves: a blade; roots: a root; and (6) Seeds: seeds; pods: a fruit pod; and (5) Stems: a stem; the leaves, roots and stems are tissues of soybean seedling plants sowed for 30d, the seeds and pods are soybean tissues 30d after flowering, and the reference gene is soybean ACTIN.

FIG. 2: 35S, constructing a GmABCA9-GFP vector; in FIG. A, the vector construction is schematically shown. In the figure B, 1, 2 represent sample 1 and sample 2, respectively; m5: marker 5000. 35S fragment of GmABCA9-GFP after BamH I digestion: 1523bp and 840 bp.

FIG. 3: fluorescent signals of GmABCA9 in tobacco leaves; a, B, C, D is a tobacco leaf; e, F, G, H shows protoplasts of tobacco leaves.

FIG. 4: 35S, carrying out PCR identification on a GmABCA9-GFP transgenic arabidopsis plant; m5: marking a Marker 5000 by a molecule; and (2) preparing: by dH₂O is a negative control of the template; WT: a wild-type Arabidopsis plant; 1-15: transgenic arabidopsis plants.

FIG. 5: expression levels of GmABCA9 in different tissues of arabidopsis; roots: a root; siliques: carrying out silique; flowers: flower; and (5) Stems: a stem; rosette Leaves: lotus throne leaves; cauline Leaves: the stem grows leaves. The siliques are tissues of arabidopsis thaliana plants sowed for 45d, the roots, flowers, stems, rosette leaves and cauline leaves are arabidopsis thaliana sowed for 30d, and the internal reference gene is arabidopsis thaliana ACTIN 2.

FIG. 6: screening arabidopsis thaliana abca9 reversion mutant plants; m5: marking a Marker 5000 by a molecule; -1: by dH₂O is a negative control of the template; -2: negative control using abca9 mutant genomic DNA as template; WT: a wild-type Arabidopsis plant; 1-14: arabidopsis abca9 revertant plants.

FIG. 7: arabidopsis abca9 mutant and reversion mutant phenotypes; WT: a wild-type Arabidopsis plant; OE-2: overexpression strain 2; 2-C1: the abca9-1 reversion mutant strain 2; 2-C2: abca9-2 reversion mutant strain 2; 2-C3: the abca9-3 reversion mutant line 2.

FIG. 8: oil content of transgenic, mutant and revertant plant seeds; in the figure A, the oil content of the seeds of the over-expression plants; in the figure B, the oil content of the seeds of the mutant and revertant plants.

FIG. 9: fatty acid content of transgenic, mutant plant seeds; in the figure A, the fatty acid content of the transgenic plant seeds; in the figure B, the fatty acid content of seeds of the abca9 mutant plants.

FIG. 10: thousand kernel weight of seeds of over-expressed, mutant and revertant plants; in the figure, A, thousand kernel weight of transgenic seeds; in the figure B, mutant and revertant seeds are thousand kernel weight.

Detailed Description

Cloning of GmABCA9 Gene

Taking the RNA of the soybean Heilongnong 44 variety as a template, synthesizing cDNA, and performing PCR amplification, wherein an amplification primer pair comprises:

5'-TGCTCTAGAATGGCAACCACTCTCACC-3'

5'-TGCTCTAGACCACTGAAAAGCTACTGAACT-3'，

obtaining 2904bp gene sequence as shown in SEQ ID No.1 and named as soybean GmABCA9 gene; the total code number of 967 amino acids is shown in SEQ ID No. 2.

Expression analysis of GmABCA9 in Soybean tissue

The space-time expression of the gene provides an important basis for the verification of the function of the target gene to a certain extent. To analyze the expression level of GmABCA9 in different tissues of soybean, we extracted RNA from leaf, root, stem, 30d seeds and pod after flowering of black-sown 44 soybean sowed for 30d, and detected the expression level of GmABCA9 in different tissues of soybean by real-time quantitative PCR (see fig. 1).

After analyzing the real-time quantitative PCR result, the expression level of the GmABCA9 in soybean tissues sowed for 30d is lower than that in seeds and fruit pods sowed for 30 d. In the seeds and fruit pods which bloom for 30d, the expression level of the gene is higher in the seeds. The soybeans sowed for 30d are in vegetative growth period, while the expression of GmABCA9 in roots, stems and leaves can provide nutrient substances for vegetative growth of plants. The reproductive growth stage of soybean plants is from flowering to seed maturation, and compared with fruit pods, the expression level of the gene is higher in seeds 30d after flowering, which indicates that GmABCA9 can play a role in the maturation process of soybean seeds 30d after flowering and regulate the development process of the seeds.

Subcellular localization of GmABCA9

To analyze the subcellular localization of GmABCA9 protein, we fused the cloned GmABCA9 gene from soybean with the GFP tag of pCambia1300 after removal of the stop codon to construct a 35S GmABCA9-GFP vector. The restriction enzyme BamHI is used for enzyme digestion verification, and the result shows that target fragments of 1523bp and 840bp (shown in figure 2) appear, indicating that the vector construction is successful.

To analyze the subcellular localization of GmABCA9, we transferred the constructed 35S: GmABCA9-GFP vector into Agrobacterium GV3101, infected young tobacco leaves with Agrobacterium, and observed the fluorescent signal of GmABCA9 under a confocal laser microscope (see FIG. 3). In the micrographs of tobacco leaves, after the tobacco leaves are infected with the empty load, a strong green fluorescence signal can be observed in A in FIG. 3, and due to constitutive expression of a 35S strong promoter in plants, a plurality of fluorescence signals can be observed, and the fluorescence signals are mainly distributed in cell nuclei, endoplasmic reticulum, cell membranes and the like. When the expression of the GmABCA9 gene was promoted by a 35S strong promoter, a green fluorescence signal was found to surround the cell nucleus in 35S: GmABCA9-GFP infected tobacco leaves (B in FIG. 3), and a very clear filamentous signal was observed in the cytoplasm, consistent with the red fluorescence signal of endoplasmic reticulum marker in C in FIG. 3. After overlapping the gene green fluorescence signal with the endoplasmic reticulum marker red signal, a yellow signal was observed in D in fig. 3, indicating that GmABCA9 is localized to the endoplasmic reticulum.

In order to eliminate the interference of plant cell wall impurities and other substances on the fluorescence signal of a target gene, infected tobacco leaf protoplasts are extracted and observed under a microscope. Similar to the signals in tobacco lamina, the overlap of F and G in fig. 3 compared to the E in control fig. 3 indicates that GmABCA9 is located in the endoplasmic reticulum.

Expression of GmABCA9 in transgenic Arabidopsis thaliana

To verify the function of GmABCA9, we infected wild type Arabidopsis thaliana by the floral dip method and transferred the gene of interest into Arabidopsis thaliana. And screening the obtained T1 transgenic plant seeds on a culture medium of 1/2MS containing Hyg and Cef, and co-screening to obtain 25 positive seedlings. After the seedlings grow into robust seedlings, extracting leaf genome DNA for PCR verification. The target band is 672bp, and 23 positive plants are finally screened (the partial amplification result is shown in figure 4).

Expression of GmABCA9 in Arabidopsis thaliana

Higher expression of GmABCA9 in mature seeds has been demonstrated in soybean, suggesting that this gene may be involved in regulating the developmental process of seeds. To verify the expression pattern of the gene in arabidopsis plants, we analyzed the expression levels of gmaabca 9 in different tissues of transgenic arabidopsis. RNA of 30 d-sowed Arabidopsis thaliana rosette leaves, cauline leaves, roots, stems, flowers and 40 d-sowed siliques is extracted, and the expression level of GmABCA9 in different tissues of transgenic Arabidopsis thaliana is detected by real-time quantitative PCR (as shown in figure 5).

Analysis of the real-time quantitative PCR result shows that the GmABCA9 has the highest expression level in the pod of Arabidopsis thaliana. The gene expression level is lower in roots, stems, flowers, rosette leaves and cauline leaves, particularly the cauline leaves are most obvious, and the expression level is almost 0; the expression level of the gene is not obviously different in three tissues of root, stem and flower. During the development of arabidopsis seeds, the siliques continuously provide the seeds with the required nutrients. Therefore, the large expression of GmABCA9 in the silique suggests that the gene may be involved in the development process of transgenic seed maturation.

Screening of soybean GmABCA9 gene reversion Arabidopsis thaliana abca9 mutant plant

The constructed 35S: GmABCA9-GFP vector is transferred into agrobacterium GV3101, and the arabidopsis mutant plant is infected by the agrobacterium. Transferring a target gene GmABCA9 into an Arabidopsis thaliana abca9 mutant, and collecting T₁Seeds of the generation reversion mutant were screened on 1/2MS medium containing Hyg and Cef. After the seedlings are transplanted and grown into robust seedlings, extracting the leaf genome DNA of the revertant mutant plants to carry out PCR verification, and obtaining the strains with the target bands of 672bp as positive plants (partial amplification results are shown in figure 6). The results showed that, in the abca9-1-C plant, all the lines except line 11 were revertant plants. Strains 1-14 of abca9-2-C and abca9-3-C plants are all reversion mutant plants. The screening was continued according to this method until T₃Generation, 20 lines of each of the 3 revertant plants were screened in total.

Seed phenotype analysis of Arabidopsis abca9 mutant and revertant

The research reports that the expression of Arabidopsis AtABCA9 influences the yield of seed oil. To verify whether expression of this gene would affect the seed phenotype of arabidopsis thaliana, we observed the seed phenotype of arabidopsis thaliana abca9 mutant and reversion mutants.

The phenotype of the seeds of the three abca9 mutants was observed under a microscope (see figure 7), and it was found that the seeds of the abca9-1 and abca9-2 mutants were shriveled and small compared to WT, whereas the abca9-3 seed phenotype was almost identical to WT. When GmABCA9 was expressed in wild type and mutant plants, the transgenic plants were plump and large-grained compared to WT; the seeds of the abca9-1 and abca9-2 reversion mutants are obviously fuller than the seeds of the mutants, the seeds are large, and particularly, the phenotype of the seeds of the abca9-1 reversion mutants is most obvious; however, the phenotype of the seeds of the revertant abca9-3 was not significantly different from that of the mutant, probably because the T-DNA insertion site of the mutant was located in the intron region of the gene and did not completely inhibit the expression of the gene. The results indicate that mutations in abca9 affect the degree of fullness and seed size of seeds.

Determination of oil content in seeds of arabidopsis thaliana

After the seed phenotypes of the three Arabidopsis thaliana mutants and the reversion mutants and the growth conditions on a sucrose-free medium are researched and analyzed, the mutant abca9-1 and the reversion mutant abca9-1-C with more obvious phenotypes are selected for subsequent functional research. To verify the function of GmABCA9, we examined the seed oil content of GmABCA9 transgenic plants, mutants and reversion mutant plants (see fig. 8). Compared with WT plants, the oil content of seeds of transgenic plant lines OE-1, OE-2 and OE-4 is remarkably increased (as shown in A in figure 8), and is respectively increased by 15%, 17% and 11%. The oil content of the seeds of the abca9-1 mutant is reduced sharply compared with that of wild plants. When GmABCA9 was overexpressed in the abca9-1 mutant, the oil content of the seeds of reversion mutant lines 2-C1 and 3-C1 was significantly increased (as shown in B of FIG. 8) by 12% and 9%, respectively, compared to the mutant. The result shows that the expression of the GmABCA9 gene increases the content of grease in seeds of Arabidopsis plants.

Determination of fatty acid content in arabidopsis plant seed

To verify whether the increase in oil content of transgenic plant seeds is caused by an increase in certain fatty acids, we examined the relative amounts of different fatty acids in the seeds (see FIG. 9). Although the relative contents of C18:2 and C18:3 in the seeds of the mutant plants are increased compared with those of wild-type plants, the contents of C16:0 and C18:1 are not changed (as shown in B in FIG. 9). The total lipid content of the seeds of the abca9-1 mutant shows a sharp reduction trend, which indicates that the mutation of abca9 can up-regulate or down-regulate the levels of other fatty acids, so that the total lipid content is reduced integrally. In transgenic plants OE-1, OE-2, OE-3 and OE-4 seeds, the relative contents of C16:0, C18:1, C18:2 and C18:3 were all significantly increased (see A in FIG. 9). In OE-2 seeds, the relative amounts of C16:0, C18:1, C18:2, and C18:3 were increased by 3.4mg/g, 11.4mg/g, 15.4mg/g, and 10.2mg/g, respectively. The result shows that the GmABCA9 promotes the accumulation of seed oil by regulating the content of seed fatty acid.

Thousand seed weight analysis of Arabidopsis plants

The expression of the GmABCA9 gene influences the content of fatty acid and carbohydrate in seeds, and in order to verify whether the expression of the gene influences the weight of the seeds, 1000 seeds mature from transgenes, mutants and reversion mutants are selected and the weight of the seeds is detected. The results were found (see FIG. 10). The thousand kernel weight of transgenic OE-2 seeds was 21mg, a significant 18% increase compared to control 18mg, while the thousand kernel weight of the seeds of the three mutants and the revertant mutant was identical to the wild type. The results show that the weight of the seeds is increased by the overexpression of GmABCA9, and whether the weight of the seeds is influenced by regulating the contents of fatty acid and glucose in the seeds is still to be further researched.

The experiments prove that the soybean transport protein GmABCA9 regulates and controls the synthesis of grease. Fluorescent real-time quantitative PCR analysis shows that the expression level of the GmABCA9 is high in the mature period of soybean seeds, and the mature period of the seeds is an important period for oil accumulation of the seeds. The subcellular localization of GmABCA9 showed that the gene was localized to the endoplasmic reticulum, and that AtABCA9 has the same localization. The arabidopsis abca9 mutant has shriveled seeds and small grains, and when the arabidopsis abca9 mutant grows on a sucrose-free medium, seedlings yellow and develop abnormally. And when the seeds of the GmABCA9 reversion mutant plants grow in a sucrose-free culture medium, the phenotype is normal. The fat content of the arabidopsis thaliana abca9 mutant is reduced sharply, and the content of partial fatty acid components forming the fat is not obviously changed in seeds of the abca9 mutant, so that the additional fatty acid in the seeds of the mutant is probably not analyzed temporarily. The contents of oil and fatty acid components of the GmABCA9 transgenic seeds are remarkably increased, which shows that the expression of the GmABCA9 gene increases the synthesis of the oil by increasing the amount of fatty acid in the seeds.

Sequence listing

<110> university of the applicant's name southwest

<120> soybean lipid transporter gene GmABCA9 and application thereof

<130> 20181127

<160> 2

<170> SIPOSequenceListing 1.0

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<213> Soybean (Glycine max)

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ctcatattca ttttcctcat cttcgcaatc gacaaggcca tcaaggcaca gacctcgacc 180

tcctcctcct acaagagcgt cactgaccct cccatggaac cttctccccc catcactccc 240

tgcgaggaca agttcttcat caaactcccc tgctacgact tcgtctggag cggccacgcc 300

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cctttctctg ctgtggcttc aattggtcct gcatttttcc tcgcgatcgc catgtttaat 720

tttgttcttc agattagctc tttggtgacg gagaaagagc ttaaacttcg ccaggcaatg 780

aatatgatgg ggctttacga ctttgcttac tggttttcct ggctcatctg ggaggcagtt 840

gtcacaatcc tatcgtccct cctcatagtt ctctttggaa tgatgttcca gtttcgcttt 900

ttcttggata acagttttgt ggttctgttc gttctgttct tcctgtttga acttaatatg 960

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gggttctcta tatttattgt cggctttgtg actcagcttg tggtacaaca aggatttcct 1080

tacacagata gcttctccaa aactatacga aacgtgtggt cgttattccc tcccaatctt 1140

tttgctcaag gtataaaagt gctttcagat gcagttgcaa cttctgaaga caaaggcatt 1200

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gacatttata aatggcttgc ggctactttc tttctctggt ttgttctggc catttacttt 1320

gacaatataa tcccaaatgc atcaggtgtg aggaaatcaa tattgtactt tctaaatccc 1380

agttattgga tgggaaaagg aggtcaaaaa gtgaaagagg gtggggtttg tagctgtata 1440

ggttcagccc cacgtcaaga acaaagtaca ccagatgatg aggatgtcct tgaagaagaa 1500

aacaaagtga aacagcaact aacagaaggt cttcttgatg caaatgttgc tgttcagata 1560

cgtggccttg caaagacata tcctggcaca cgtagcattg gttgctgctt taaatgtaaa 1620

agaacttcac cttacaatgc agttaagggt ttgtgggtga actttgcaaa ggatcagtta 1680

ttttgtcttc taggacccaa tggagctgga aaaactacag ctattaattg cttgacaggg 1740

gtaactccag taactgatgg agatgcgttg atttatgggc attcaatcag aagttctact 1800

ggcatgtcaa acattcgaaa gcttatagga gtctgtccac agtttgatat cctatgggat 1860

gcactgtctg gtcaggaaca cctccaactc tttgctacta ttaaaggcct gtccccagct 1920

tccataaaat caattaccca gacttcatta gcagaggtaa gactcacgga tgcagccaaa 1980

gtgagagctg gaagttacag cggaggaatg aaacgccgtc tcagtgttgc aattgccctt 2040

attggtgacc ctaagttagt cattctggat gaaccgacta ctggtatgga tccaataaca 2100

agaaggcatg tgtgggacat aattgaaaat gcaaagagag ggcgtgccat tgttcttaca 2160

acgcactcta tggaagaagc tgacattcta agtgaccgta taggaatcat ggcaaaagga 2220

agtcttcgct gcattggcac ctcaatcaga ttgaagtccc gctttggtac tggttttatt 2280

gctaatatca gcttcaatgg aaacaatatc gaacatagtc ctgccaatgg agatgcaata 2340

tctacagaac gtcgtgaggc agtgaagaaa ttctttaaga atcatttaga tgtagtgcca 2400

aaagaggaga accataactt cctaactttt gtgattcctc atgacagaga ggcgctcatg 2460

acgaatttct tttcagagct ccaagataga gaagaagaat ttggcatatc tgatatccag 2520

cttggtctaa caacgctcga agaagttttc ttgaatattg ctagacaagc agagctagaa 2580

agtgctgcag ctgaagggag actagtgacc ctgaccttaa catctgggga atctgtgcag 2640

attcctatag gagctaggtt tgtgggaatt ccaggaacag agtctgctga aaaccctact 2700

gggtttatgg tagaagtata ctgggaacaa gatgatactg gtgccttatg cattgctggc 2760

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<213> Soybean (Glycine max)

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Arg Gln Ala Met Asn Met Met Gly Leu Tyr Asp Phe Ala Tyr Trp Phe

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Ser Trp Leu Ile Trp Glu Ala Val Val Thr Ile Leu Ser Ser Leu Leu

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Ile Val Leu Phe Gly Met Met Phe Gln Phe Arg Phe Phe Leu Asp Asn

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Ser Phe Val Val Leu Phe Val Leu Phe Phe Leu Phe Glu Leu Asn Met

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Gly Ser Ala Pro Arg Gln Glu Gln Ser Thr Pro Asp Asp Glu Asp Val

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Asp Ala Asn Val Ala Val Gln Ile Arg Gly Leu Ala Lys Thr Tyr Pro

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Phe Cys Leu Leu Gly Pro Asn Gly Ala Gly Lys Thr Thr Ala Ile Asn

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Asn Ile Glu His Ser Pro Ala Asn Gly Asp Ala Ile Ser Thr Glu Arg

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Arg Glu Ala Val Lys Lys Phe Phe Lys Asn His Leu Asp Val Val Pro

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Lys Glu Glu Asn His Asn Phe Leu Thr Phe Val Ile Pro His Asp Arg

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Glu Ala Leu Met Thr Asn Phe Phe Ser Glu Leu Gln Asp Arg Glu Glu

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Glu Phe Gly Ile Ser Asp Ile Gln Leu Gly Leu Thr Thr Leu Glu Glu

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Val Phe Leu Asn Ile Ala Arg Gln Ala Glu Leu Glu Ser Ala Ala Ala

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Glu Gly Arg Leu Val Thr Leu Thr Leu Thr Ser Gly Glu Ser Val Gln

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Ile Pro Ile Gly Ala Arg Phe Val Gly Ile Pro Gly Thr Glu Ser Ala

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Glu Asn Pro Thr Gly Phe Met Val Glu Val Tyr Trp Glu Gln Asp Asp

900 905 910

Thr Gly Ala Leu Cys Ile Ala Gly His Ser Gln Lys Val Pro Ile Pro

915 920 925

Asn Gly Val Gln Leu Ser Ser Ser Pro Ser Val Arg His Arg Arg Tyr

930 935 940

Leu Gly Arg Ser Gly Thr Val His Gly Val Val Ile Asp Pro Ser Gln

945 950 955 960

Val Ser Ser Val Ala Phe Gln

965

Claims (2)

1. The application of the soybean GmABCA9 gene shown in SEQ ID No.1 in regulating and controlling the oil content of plant seeds, wherein the plant is soybean or arabidopsis thaliana.

2. The application of the expression vector containing the soybean GmABCA9 gene shown in SEQ ID No.1 in the regulation of plant seed lipid is disclosed, wherein the plant is soybean or arabidopsis thaliana.

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