Use of gmidd5a protein and / or its encoding gene in regulating soybean seed oil content

Abstract

The application belongs to the technical field of genetic engineering and soybean breeding, and particularly relates to application of GmIDD5a protein and / or its coding gene in regulation of soybean seed oil content. Overexpression of the GmIDD5a protein and / or its coding gene in soybean can promote expression of a sucrose transporter family gene GmSWEET10a / b, thereby increasing the soybean seed oil content, and the seed oil content of the obtained transgenic soybean is significantly higher than that of wild-type soybean, which is of great significance for improving the soybean seed quality.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering and soybean breeding, and particularly relates to application of GmIDD5a protein and / or its coding gene in regulating soybean seed oil content. BACKGROUND

[0002] Soybean (Glycine max) is one of the most important crops in the world, providing about 69% of the world's protein and 28% of the world's vegetable oil (http: / / soystats.com). However, with the rapid growth of the global population and the continuous improvement of living standards, the demand for soybeans is also increasing. Genetic improvement of high-yield and high-quality soybeans is a very important breeding goal, and has far-reaching significance for ensuring global food security and promoting sustainable agricultural development.

[0003] Soybean seed oil content is a quantitative trait, which is controlled by multiple genes. Through linkage analysis and genome-wide association analysis, a large number of quantitative trait loci (QTL) associated with soybean seed oil content have been identified. These QTL discoveries have greatly helped identify genes associated with seed oil content, such as GmSWEET10a, which encodes a sugar transporter protein of the SWEET family, controls the distribution of sugar from the seed coat to the embryo, thereby affecting the seed oil and protein content of soybeans; POWR1, which encodes a CCT motif protein, has a pleiotropic effect on soybean seed oil, protein content, and hundred-seed weight; GmMFT, which encodes a phosphatidylethanolamine binding protein, simultaneously regulates soybean seed oil and protein content; GmFA9, which encodes a SEIPIN homolog, knocking out GmFA9 reduces the fatty acid content of soybean seeds and increases the seed storage protein content. However, the specific molecular mechanisms of these genes in regulating soybean seed traits still need to be further elucidated, and the correlation between these genes is largely unknown. Therefore, mining new soybean seed quality genes and elucidating their molecular mechanisms will help build a molecular regulatory network for regulating soybean seed quality. SUMMARY

[0004] The purpose of the present application is to provide the application of GmIDD5a protein and / or its coding gene in regulating soybean seed oil content, mine new soybean seed oil content regulatory genes, regulate soybean seed oil content, and breed soybeans with high seed oil content.

[0005] The application provides the application of GmIDD5a protein and / or its coding gene in regulating soybean seed oil content.

[0006] The amino acid sequence of the GmIDD5a protein is shown as SEQ ID NO. 1.

[0007] Preferably, the regulation comprises positively regulating the expression of the GmIDD5a protein and / or the gene encoding same, so as to increase the oil content of the soybean seeds.

[0008] Preferably, the CDS region of the gene encoding the GmIDD5a protein has the nucleotide sequence shown in SEQ ID NO. 2.

[0009] The application further provides application of the GmIDD5a protein and / or the gene encoding same in soybean breeding, wherein the soybean breeding comprises breeding soybean with high seed oil content.

[0010] The amino acid sequence of the GmIDD5a protein is shown in SEQ ID NO. 1.

[0011] The application further provides a method for increasing the oil content of soybean seeds, comprising overexpressing the GmIDD5a protein and / or the gene encoding same in a recipient soybean.

[0012] The amino acid sequence of the GmIDD5a protein is shown in SEQ ID NO. 1.

[0013] Preferably, the method for overexpressing the GmIDD5a protein and / or the gene encoding same comprises Agrobacterium transformation.

[0014] Preferably, the Agrobacterium transformation comprises the following steps:

[0015] The Agrobacterium containing the recombinant plasmid is used to infect the cotyledon node explants of the recipient soybean, and then the explants are cultured.

[0016] The recombinant plasmid comprises a basic vector and a GmIDD5a protein coding gene introduced into the basic vector.

[0017] Preferably, the culturing comprises dark culturing, bud induction culturing and rooting culturing in sequence.

[0018] Preferably, the dark culturing is performed for 3-5 days.

[0019] Preferably, the light / dark ratio of the bud induction culturing and the rooting culturing is 16h:8h, respectively.

[0020] Beneficial effects:

[0021] The application provides application of the GmIDD5a protein and / or the gene encoding same in regulating the oil content of soybean seeds, wherein the amino acid sequence of the GmIDD5a protein is shown in SEQ ID NO. 1. Overexpression of the GmIDD5a protein and / or the gene encoding same in soybean can promote the expression of the sucrose transporter family gene GmSWEET10a / b, significantly increase the oil content of soybean seeds, and is of great significance for improving the quality of soybean seeds. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows.

[0023] Figure 1 Agarose gel electrophoresis detection results of soybean IDD transcription factor GmIDD5a;

[0024] Figure 2 Identification results of bar protein in wild-type soybean and transgenic soybean lines; wherein, the arrow position represents the transgenic Bar protein;

[0025] Figure 3 Expression of soybean IDD transcription factor GmIDD5a in wild-type soybean and transgenic soybean lines; wherein, **P<0.01;

[0026] Figure 4 Expression analysis results of soybean IDD transcription factor GmIDD5a in different tissues of soybean and different development periods of seeds;

[0027] Figure 5 Expression analysis results of soybean IDD transcription factor GmIDD5a in different parts of seeds of soybean at 35 days after flowering;

[0028] Figure 6 Expression analysis results of soybean IDD transcription factor GmIDD5a in sucrose transporter family gene GmSWEET10a / b in seeds of soybean at 35 days after flowering; wherein, **P<0.01;

[0029] Figure 7 Analysis results of oil content in seeds of wild-type soybean and transgenic soybean lines; wherein, **P<0.01. DETAILED DESCRIPTION

[0030] The application provides application of GmIDD5a protein and / or its coding gene in regulation of soybean seed oil content; the amino acid sequence of the GmIDD5a protein is shown as SEQ ID NO. 1, and specifically, MAAPFSAASLFAIREEDQNQMKQQHSSTPSSSTTPAAPPPQKKRRNQPGTPYPDAEVIALSPKTLMATNRFICEVCNKGFQREQNLQLHRRGHNLPWKLKQKTNKEPKRKVYLCPEPTCVHHDPSRALGDLTGIKKHYSRKHGEKKWKCDKCSKKYAVQSDWKAHSKTCGTREYRCDCGTLFSRRDSFITHRAFCDALAQESAREAPNLSSAIGNQLYGNSNNMSLGLSQIPSIHDQNPQPSELMRFSGAPRAGQFDHILPPNIASSSPFRHSMQTPPFFLQESNQTYHDSNKPFQGLIQLSDLNNNNPSASNLFNLPFLSNRAINSNNYSEEQQFNTAEGSNFFSEGTMNIGSTDHQTSSTTVPSLFSTNSLQNNHLSHMSATALLQKASQIGSASSSNSININNTTTTNNTSASSLLRSLASKSDHQRQLGGGGAAANYATIFNNSVQEMMNISGFEAYDHHGGMNKEQKLGGVGGSDRLTRDFLGVAQQQQREGFNLMSSLEAETNNNAAPSGQSFGSGGNFQ.

[0031] In the application, as an embodiment, the regulation comprises: positively regulating expression of the GmIDD5a protein and / or its coding gene, and increasing soybean seed oil content.

[0032]

[0033] The present invention also provides the application of GmIDD5a protein and / or its encoding gene in soybean breeding; the soybean breeding includes cultivating soybeans with high seed oil content; the amino acid sequence of the GmIDD5a protein is shown in SEQ ID NO.1.

[0034] The present invention also provides a method for increasing the oil content of soybean seeds by overexpressing the GmIDD5a protein and / or its encoding gene in recipient soybeans; the amino acid sequence of the GmIDD5a protein is shown in SEQ ID NO.1.

[0035] In this invention, as one embodiment, the overexpression of the GmIDD5a protein and / or its encoding gene includes Agrobacterium transformation. As one embodiment, the Agrobacterium includes Agrobacterium tumefaciens EHA105. As one embodiment, the Agrobacterium transformation method includes the steps of: infecting recipient soybean cotyledonary explants with Agrobacterium containing recombinant plasmids followed by culture.

[0036] In one embodiment, this invention utilizes Agrobacterium containing a recombinant plasmid to infect soybean cotyledonary node explants, obtaining infected explants. In one embodiment, the recombinant plasmid comprises a basic vector and a GmIDD5a protein-coding gene introduced into the basic vector; in one embodiment, the basic vector comprises the pBWA(V)BS vector, purchased from Wuhan Boyuan Biotechnology Co., Ltd. In one embodiment, the OD of the Agrobacterium containing the recombinant plasmid... 600 The value is 0.5. As one implementation method, the contamination is performed by immersion.

[0037] In one embodiment, after obtaining the infected explants, the present invention performs dark culture on the infected explants to obtain dark-cultured explants. In one embodiment, the dark culture time is 3-5 days; in another embodiment, the dark culture time is 4 days.

[0038] In one embodiment, after obtaining the explants cultured in the dark, the present invention performs bud induction culture on the explants cultured in the dark to obtain the first explant. In one embodiment, the light-dark ratio of the bud induction culture is 16h:8h. The present invention does not have strict requirements on the method of bud induction culture; conventional culture medium is used for induction culture until the buds grow to approximately 5cm.

[0039] As an implementation form, after obtaining the first explant, the application carries out rooting culture on the first explant to obtain transgenic soybean. As an implementation form, the light-dark ratio of the rooting culture is 16h:8h. The application does not have strict requirements on the rooting culture mode, and the first explant can be rooted by using conventional culture medium for culture. As an implementation form, the rooting culture time is 21d.

[0040] The GmIDD5a protein of the application is a protein encoded by the soybean IDD transcription factor GmIDD5a. Overexpression of the GmIDD5a protein and / or its encoding gene in a receptor soybean can improve the transcription of the sucrose transporter family gene GmSWEET10a / b, thereby increasing the oil content of soybean seeds, and the oil content of transgenic soybean seeds is significantly higher than that of wild-type receptor soybean. It has important significance for improving the quality of soybean seeds.

[0041] In order to further illustrate the application, the application of the GmIDD5a protein and / or its encoding gene in regulating the oil content of soybean seeds is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0042] Unless otherwise specified, the reagents used in the examples are purchased from conventional biochemical reagent companies. In the following examples, % is the mass percentage unless otherwise specified. In the quantitative test in the following examples, three repeated experiments are set, and the average value is taken as the result.

[0043] Example 1

[0044] Construction of soybean IDD transcription factor GmIDD5a overexpression vector

[0045] (1) Using soybean (Williams 82) cDNA as a template, PCR amplification was carried out by using the upstream primer GmIDD5a-F (5'-AAC ACGGGGGACTTTGCAACATGGCAGCACCTTTTTCTGCAGCATC-3', SEQ ID NO. 3) and the downstream primer GmIDD5a-R (5'-TGAAGACAGAGCTAGTTACATCACTGAAAGTTCCCACCACTTCCAAAAG-3', SEQ ID NO. 4);

[0046] PCR amplification system: ddH2O 8.5 μL, KOD high-fidelity enzyme 12.5 μL, upstream primer GmIDD5a-F 1 μL, downstream primer GmIDD5a-R 1 μL, and 100 ng / μL cDNA template 2 μL; PCR amplification procedure: 98℃ 5 min; 94℃ 10 sec, 52℃ 15 sec, 68℃ 20 sec, 36 cycles in total; 68℃ 5 min;

[0047] (2) The amplified product obtained in step (1) is sequenced to obtain a GmIDD5a CDS sequence with a total length of 1581 bp, the nucleotide sequence of which is shown as SEQ ID NO. 2, and the corresponding amino acid sequence is shown as SEQ ID NO. 1.

[0048] (3) The amplified product obtained in step (1) is subjected to 1% agarose gel electrophoresis detection, 5v / cm voltage, 20 min, and the PCR band (1581 bp) is observed in the gel imaging system to determine whether it is correct Figure 1 ), and the gel recovery kit is used for recovery, and the recovered product is named rDNA I.

[0049] (4) The pBWA(V)BS vector is subjected to enzyme digestion using Bsa I restriction endonuclease, and the enzyme digestion product is purified using a PCR purification kit, and the purified product is named pBWA(V)BS(D) and used for recombination reaction;

[0050] The enzyme digestion system is: 200 ng / μL pBWA(V)BS 4 μL, Buffer 2 μL, Bsa I 1 μL, and ddH2O is supplemented to 20 μL; the enzyme digestion condition is: 37℃, 1h.

[0051] The recombination reaction system is: EasyClone Mix 10 μL, rDNA I 5 μL, pBWA(V)BS(D) 5 μL; the recombination reaction condition is: 37℃, 30 min.

[0052] (5) 10 μL of the recombination product is transformed into E. coli competent cells, and 6 (or more) colonies are picked with a sterile toothpick on solid LB medium (10 g / L of protein peptone, 5 g / L of yeast extract, 10 g / L of NaCl, 16 g / L of agar, and the balance of water) containing kanamycin, and the colonies are cultured in 200 μL of liquid LB medium (10 g / L of protein peptone, 5 g / L of yeast extract, 10 g / L of NaCl, and the balance of water) with kanamycin, and the positive clones are screened by PCR detection and sent to a company for sequencing.

[0053] (6) Take 100 μL of the sequencing correct positive clone, inoculate in 10 mL liquid LB medium with kanamycin, shake culture for 12 h, use the plasmid extraction kit produced by ShangHai Genechem Co., Ltd. to extract the plasmid, and obtain the recombinant plasmid.

[0054] (7) Transform 2 μL of the recombinant plasmid into EHA105 Agrobacterium competent cells, inoculate on solid LB medium containing kanamycin and rifampicin antibiotics, invert culture at 28°C for 48 h, use a sterilized toothpick to pick 6 (or more) colonies, inoculate in 200 μL liquid YEP medium (10 g / L proteose peptone, 5 g / L yeast extract, 5 g / L NaCl and the rest water) with kanamycin, shake culture; finally, PCR detection is used to screen positive clones, and obtain Agrobacterium carrying the target gene plasmid.

[0055] Example 2

[0056] Obtaining of soybean overexpression GmIDD5a transgenic plants

[0057] (1) Select disease-free, crack-free, and full soybean seeds of Williams 82, use chlorine sterilization method for 16-20 h, and the experimental procedure is carried out in a fume hood. After sterilization, blow the seeds for 24 h in a clean bench to remove residual chlorine, seal the culture dish containing the sterilized soybeans, and store at 4°C for standby.

[0058] (2) Inoculate the sterilized seeds with the hypocotyls downward on the germination medium (3.2 g / L B5 powder, 30 g / L sucrose, 8 g / L agar and the rest water, pH 5.85), and place in a 25°C incubator for dark culture for 1 day.

[0059] (3) Spread the Agrobacterium carrying the target gene plasmid on the liquid YEP medium for primary activation, collect the bacteria after 48 h of culture, and activate again on a new medium. After 24 h of culture, collect the bacteria in the infection liquid (0.32 g / L B5 powder, 3.9 g / L MES, 30 g / L sucrose, 1.67 mg / L 6-BA, 8 g / L agar, 0.25 mg / L GA3, 39 mg / L acetosyringone and the rest water, pH 5.45), vortex mix, adjust the OD of the bacterial liquid to 0.5 by a spectrophotometer, obtain the Agrobacterium-containing infection liquid, and reserve. 600

[0060] (4) After cutting the cotyledon node explants, immerse them in the Agrobacterium-containing infection liquid; after infection, inoculate the explants on the co-culture medium (0.32 g / L B5 powder, 3.9 g / L MES, 30 g / L sucrose, 1.67 mg / L 6-BA, 5.4 g / L agar and the rest water, pH 5.45) with filter paper on the surface, and dark culture for 3-5 days.​

[0061] (5) After recovery culture, the explants with cluster buds were transferred to bud induction solid medium (B5 powder 0.32 g / L, sucrose 30 g / L, MES 0.6 g / L, 6-BA 1.67 mg / L, agar 8.4 g / L and the balance of water, pH 5.75) with the cut surface of explants upward, and cultured for 7-10 days.

[0062] (6) The explants with cluster buds after recovery culture were inoculated into screening medium (after sterilization of bud induction solid medium, add thiostrepton 150 mg / L, carbenicillin 250 mg / L, screening agent glufosinate 8 mg / L), 16h / 8h light / dark culture, and screened for 21 days.

[0063] (7) The cluster buds with good growth after screening were transferred to new elongation medium (MS powder 4.3 g / L, MES 0.6 g / L, sucrose 30 g / L, B5 organic 10 mL / L, aspartic acid 50 mg / L, glutamine 50 mg / L, agar 9.23 g / L and the balance of water, pH 5.7; after sterilization, add IAA 0.3 mg / L, GA3 0.5 mg / L), 16h / 8h light / dark culture, and screened for 21 days.

[0064] (8) When the sprouts grew to about 5 cm, they were transferred to rooting medium (MS powder 4.3 g / L, MES 0.6 g / L, sucrose 30 g / L, B5 organic 10 mL / L, aspartic acid 50 mg / L, glutamine 50 mg / L, agar 9.23 g / L and the balance of water, pH 5.7) for further screening. 16h / 8h light / dark culture, and screened for 21 days.

[0065] (9) The presence or absence of bar protein in transgenic soybeans was identified by Bar test paper method, and the expression of soybean IDD transcription factor GmIDD5a in transgenic soybean lines was detected by qRT-PCR. Two high-expression soybean IDD transcription factor GmIDD5a lines were obtained, marked as QE-1 and QE-2( Figure 2 and Figure 3 ).

[0066] Example 3

[0067] Effect of soybean IDD transcription factor GmIDD5a on oil content in soybean seeds

[0068] (1) The expression of soybean IDD transcription factor GmIDD5a in different tissues of williams 82 soybean was detected by qRT-PCR. The results showed that soybean IDD transcription factor GmIDD5a was highly expressed in seeds 35 days after flowering( Figure 4 ).

[0069] (2) The expression level of soybean IDD transcription factor GmIDD5a in different parts of the seeds (seed coat, embryo, cotyledon) of williams 82 soybean 35 days after flowering was detected by qRT-PCR, and the results showed that the soybean IDD transcription factor GmIDD5a was mainly expressed in the seed coat Figure 5 ).

[0070] (3) Since IDD family proteins are involved in the regulation of sugar transport and metabolism (Seo PJ, Ryu J, Kang SK, et al. 2011. Modulation of sugar metabolism by an INDETERMINATE DOMAIN transcription factor contributes to photoperiodic flowering in Arabidopsis. The Plant Journal. 65:418-29.), the inventors speculated that the soybean IDD transcription factor GmIDD5a might affect seed quality traits by regulating the transport of sugar from the soybean seed coat to the embryo. To verify this hypothesis, the inventors analyzed the transcription level of sucrose transporter family genes in wild-type williams 82 soybean and the transgenic soybean lines obtained in Example 2, and found that overexpression of soybean IDD transcription factor GmIDD5a could significantly promote the transcription of sucrose transporter family genes GmSWEET10a / b Figure 6 ).

[0071] (4) Since overexpression of GmSWEET10a / b can significantly increase the oil content of soybean seeds (Wang SD, Liu SL, Wang J, et al. 2020. Simultaneous changes in seed size, oil content and protein content driven by selection of SWEET homologues during soybean domestication. National Science Review, 7:1776-1786), based on the conclusion of step (3), the inventors detected the oil content of soybean seeds in wild-type williams 82 soybean and the transgenic soybean lines obtained in Example 2, and the results showed that the oil content of soybean seeds in the transgenic soybean lines obtained in Example 2 was significantly higher than that of wild-type soybean seeds, and overexpression of soybean IDD transcription factor GmIDD5a increased the oil content of soybean seeds from 22.4% to 23.9%, an increase of 1.5%Figure 7 ).

[0072] According to the above, it can be seen that the GmIDD5a protein and / or its coding gene can regulate the oil content of soybean seeds, and overexpression of the GmIDD5a protein and / or its coding gene can significantly increase the oil content of soybean seeds.

[0073] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. The application of GmIDD5a protein and / or its encoding gene in regulating soybean seed oil content, characterized in that, The regulation comprises positively regulating expression of the GmIDD5a protein and / or the gene coding the same to increase the soybean seed oil content. The amino acid sequence of the GmIDD5a protein is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The CDS region nucleotide sequence of the gene coding the GmIDD5a protein is shown as SEQ ID NO.

2.

3. Application of the GmIDD5a protein and / or the gene coding the same in soybean breeding; the soybean breeding comprises breeding soybean with high seed oil content. The amino acid sequence of the GmIDD5a protein is shown as SEQ ID NO.

1.

4. A method for increasing the oil content of soybean seeds, characterized in that, Overexpressing the GmIDD5a protein and / or the gene coding the same in a receptor soybean; The amino acid sequence of the GmIDD5a protein is shown as SEQ ID NO.

1.

5. The method of claim 4, wherein, The overexpression of the GmIDD5a protein and / or the gene coding the same comprises an Agrobacterium transformation method.

6. The method of claim 5, wherein, The steps of the Agrobacterium transformation method comprise: Infecting a receptor soybean cotyledon node explant with Agrobacterium containing a recombinant plasmid and then culturing; The recombinant plasmid comprises a basic vector and a GmIDD5a protein coding gene introduced into the basic vector.

7. The method of claim 6, wherein, The culturing comprises sequentially performing dark culture, bud induction culture and rooting culture.

8. The method of claim 7, wherein, The time of the dark culture is 3-5 days.

9. The method of claim 7, wherein, The light / dark ratio of the bud induction culture and the rooting culture is 16h:8h respectively; the time of the rooting culture is 21 days.

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