Application of soybean GmABCG5L gene in improving plant seed setting rate

Abstract

The invention discloses application of soybean GmABCG5L gene in improving plant seed setting rate, and belongs to the technical field of plant breeding. In order to solve the problem of how to promote plants to improve seed setting rate, the invention provides a method for promoting plants to improve seed setting rate, wherein a recombinant vector is obtained by constructing a GmABCG5L gene and connecting it with a pROK vector, the recombinant vector is transferred into Agrobacterium to prepare Agrobacterium carrying the GmABCG5L gene, and then a transgenic plant carrying the GmABCG5L gene is obtained by using an Agrobacterium-mediated leaf disc method, and by culturing the transgenic plant carrying the GmABCG5L gene, it is found that the transgenic plant carrying the GmABCG5L gene has a good growth state, an enlarged inflorescence compared with a wild type, an increased number of florets, and a significantly improved total seed amount compared with a wild type, indicating that the GmABCG5L gene can improve the plant seed setting rate.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and particularly relates to application of soybean GmABCG5L gene in improving plant seed setting rate. Background Art

[0002] Soybean (Glycine max L.) is one of China's major oil crops, and is also an important food crop and sideline crop. With the rapid development of China's economy and the annual reduction in soybean planting area, domestic soybean production has been declining year by year. The amount of seed set seriously affects the yield and quality of crops. Discovering beneficial gene resources in plants is one of the key ways to increase the seed set rate of soybeans and other crops.

[0003] ATP-binding cassette transporters (ABC transporters) are one of the oldest gene families and one of the largest transporter families to date. They can participate in the transport of metals, hormones, and various metabolites in plants. The results of soybean whole genome sequencing showed that soybean contains 261 ABC transporters, more than twice the number of other model plants. Among the 261 soybean ABC proteins, members of the ABCG subfamily are the most abundant, which is the main reason for the expansion of this gene family. ABCG5L is a semi-molecular transporter of the ABCG transporter family, which was found to be associated with iron deficiency stress response in soybean. Studies have found that this protein can also participate in the transport of wax and lipid molecules in other species, but there are still no reports of ABCG family members participating in grain development. Therefore, studying the function of soybean GmABCG5L gene in enhancing plant seed setting rate and exploring favorable gene resources are of great importance to improving crop yield and quality. Summary of the invention

[0004] The invention provides application of soybean GmABCGL protein in promoting plant seed setting.

[0005] Further, the sequence of the GmABCGL gene is shown as SEQ ID NO. 3. The amino acid sequence of the GmABCGL protein is shown as SEQ ID NO. 4.

[0006] It is further defined that the plant is tobacco.

[0007] The present invention provides a breeding method for transgenic plants that promote seed setting, and the steps of the breeding method are as follows:

[0008] Step 1: Connect the gene sequence shown in SEQ ID NO.3 to the pROK vector to obtain a recombinant vector;

[0009] Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0010] Step 3: Infect plants with the recombinant Agrobacterium obtained in step 2 to obtain transgenic plants.

[0011] It is further defined that the primers for cloning the gene sequence shown in SEQ ID NO.3 in step 1 are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] Further defined, the steps of infecting the plant with Agrobacterium in step 2 are as follows:

[0013] 1) Spread the Agrobacterium carrying the gene sequence shown in SEQ ID NO.3 on YEB solid medium containing kanamycin, streptomycin and rifampicin, and culture at 28°C for 48 hours;

[0014] 2) resuspending the Agrobacterium colonies obtained in step 1) using 1 / 2MS liquid culture medium to obtain Agrobacterium bacterial solution, and then diluting the bacterial solution with 1 / 2MS to an OD600 value of 0.4-0.6;

[0015] 3) Take plant leaves, infect them in the Agrobacterium bacterial solution obtained in step 2) for 6-7 minutes, take out the plant leaves and place them on sterile filter paper, remove the attached bacterial solution, place them on MS1 ​​culture medium, and culture them in the dark for 2 days.

[0016] The present invention provides a recombinant vector, which carries the gene sequence shown in SEQ ID NO.3.

[0017] It is further defined that the starting vector of the recombinant vector is xx.

[0018] The present invention provides a method for constructing the above-mentioned recombinant vector, characterized in that the specific steps of the construction method are as follows:

[0019] Step 1: The plasmid DNA of pROK was digested with BamH I and Sac I to obtain a linear vector, and the pMD18T-GmABCG5L vector was digested with BamH I and Sac I to obtain a gene fragment;

[0020] Step 2: Connect the linear vector obtained in step 1 to the gene fragment to obtain a recombinant vector.

[0021] It is further defined that the system of the ligation reaction in step 2 is: 5 μL of linear vector, 3 μL of gene fragment, 1 μL of 10×T4 DNA ligation buffer and 1 μL of T4 DNA ligase, the reaction temperature is 16° C., and the reaction time is 12 h.

[0022] Beneficial effects: The transgenic tobacco obtained by the Agrobacterium-mediated leaf disc method of the present invention has a larger capsule volume and a significantly increased seed number compared with wild-type plants under the same conditions, indicating that the GmABCG5L gene can increase the seed setting rate of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a map of the PROKII vector;

[0024] Figure 2 This is an evolutionary tree diagram.

[0025] Figure 3 The PCR result diagram for verifying positive transgenic tobacco, where M is DL2000, 0 is blank control, - is negative control, + is positive control, and 1-8 are transgenic tobacco tested;

[0026] Figure 4 is the identification result of the recombinant plasmid;

[0027] Figure 5 These are pictures of positive transgenic tobacco tissues, where A is callus tissue, B is Km-resistant buds, C is rooting culture of resistant tobacco buds, and D is rooted tobacco.

[0028] Figure 6 The figures are of wild-type and transgenic tobacco capsules, where A is the control and B is the transgenic tobacco.

[0029] Figure 7 These are pictures of the seed setting of transgenic tobacco, where A is the control, and B and C are transgenic tobacco. DETAILED DESCRIPTION

[0030] The brand and full name of the pROKⅡ vector, the vector map is as follows Figure 1 shown.

[0031] Example 1. Construction of recombinant vector

[0032] 1. Using PCR technology to clone the soybean GmABCG5L gene from soybean:

[0033] 1. Extraction of soybean RNA:

[0034] (1) Place 0.1 g of soybean in a mortar pre-cooled with liquid nitrogen and grind into fine powder.

[0035] (2) Transfer the powder to a 1.5 mL sterile centrifuge tube and add 450 μL RL (confirm whether β-mercaptoethanol is added).

[0036] (3) Transfer all the liquid to the filter column, centrifuge at 12000 rpm for 5 min, and carefully pipette the supernatant into a new RNase-Free centrifuge tube.

[0037] (4) Slowly add 0.5 times the volume of the supernatant anhydrous ethanol, mix well, transfer the resulting solution and precipitate into the adsorption column, centrifuge at 12000 rpm for 60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0038] (5) Add 350 μL of deproteinized solution to the adsorption column, centrifuge at 12,000 rpm for 60 s, discard the filtrate, and return the adsorption column to the collection tube.

[0039] (6) Prepare DNase working solution: add 70 μL RDD solution to 10 μL DNase I reaction solution and mix gently.

[0040] (7) Add 80 μL of DNase working solution to the adsorption column filter membrane and let it stand for 15 min.

[0041] (8) Add 350 μL of deproteinized solution to the centrifugal adsorption column, centrifuge at 12,000 rpm for 60 s, discard the filtrate, and return the adsorption column to the collection tube.

[0042] (9) Add 300 μL of rinse solution to the centrifugal adsorption column, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 60 s, discard the filtrate, and return the adsorption column to the collection tube.

[0043] (10) Repeat step 9.

[0044] (11) Centrifuge at 12000 rpm for 2 min, discard the filtrate, and place the adsorption column at room temperature for 5 min to completely dry the rinsing solution remaining in the adsorption column.

[0045] (12) Transfer the centrifugal adsorption column to an RNase-free collection tube, add 30 μL RNA elution buffer and let stand for 1-2 min. Centrifuge at 12000 rpm for 2 min. The collection tube contains the RNA sample and is stored at -80°C.

[0046] 2. Synthesis of cDNA

[0047] Using the extracted RNA as a template, cDNA was obtained using the reverse transcription PCR method. The reaction process is as follows:

[0048] (1) Preparation of reaction solution I:

[0049]

[0050] (2) Mix the reaction solution I and place it in a 65°C water bath for 5 min. After the water bath, quickly place it in ice.

[0051] (3) Preparation of reaction solution II:

[0052]

[0053] Reaction procedure:

[0054] 30℃10min

[0055] 42℃20min

[0056] 99℃5min

[0057] 4℃5min

[0058] 3. Amplify target gene: Primer sequence:

[0059] GmABCG5L F:5'-CG GGATCC ATGATGCAAGGCTGTGAG-3' (SEQ ID NO. 1);

[0060] GmABCG5L R:5'-C GAGCTC TTAGTTGGTGACAACCCTG-3' (SEQ ID NO. 2);

[0061] GmABCG5L gene PCR amplification program: 94℃5min, 30 cycles of 94℃30s, 60.5℃30s, 72℃2min, and finally 72℃10min; 1% agarose gel electrophoresis detection; gel recovery of target gene bands: DNA gel recovery kit was used to recover the target gene fragment GmABCG5L obtained by PCR amplification;

[0062] GmABCG5L gene CDS sequence (target gene fragment GmABCG5L): (SEQ ID NO.3)

[0063]

[0064]

[0065] The amino acid sequence of the GmABCG5L protein is shown in SEQ ID NO.4.

[0066]

[0067] Construction of cloning vector pMD18T-GmABCG5L: Connect the GmABCG5L target gene fragment to the cloning vector pMD18T. The reaction system is: 1 μL of pMD18-T vector, 3 μL of target gene fragment solution, 1 μL of ddH 2O, 5 μL of Solution I, reaction temperature 16°C, reaction time more than 12 h (overnight).

[0068] 4. The ligation product is transferred into competent E. coli using heat shock method:

[0069] (1) Take the competent E. coli out of the -80°C freezer and quickly place it on ice. After it melts, add 20 μL of the ligation product and place it on ice for 30 min.

[0070] (2) After taking out, quickly place in a 42°C water bath for heat shock for 1 min 15 s, then quickly insert into ice and place in an ice bath for 2 min. Add 800 μL of LLB liquid culture medium, place in a shaker at 200 rpm, 37°C, and shake for 1 h;

[0071] (3) Take out the centrifuge tube, centrifuge at 4000 rpm for 5 min, take out 800 μL of the supernatant, resuspend the remaining bacteria, and apply the remaining approximately 200 μL of the bacterial solution on two LB solid culture media containing ampicillin resistance at a ratio of 3:1;

[0072] (4) Incubate the culture medium upside down at 37°C overnight; pick a single colony on the culture medium for identification;

[0073] 5. Sequencing and bioinformatics analysis of GmABCG5L gene:

[0074] (1) Use the software ORF Finder to predict the open reading frame of the GmABCG5L gene.

[0075] (2) The predicted amino acid sequence of GmABCG5L was compared using NCBI protein BlAST to obtain the structural domain of GmABCG5L protein. The amino acid similarity with vacuolar iron transporter homolog 1-like (Abracium sibiricum, XP_027367276.1) was 85.59%. The GmABCG5L and ApABCG5L sequences were compared using DNAMAN software to find out the amino acid difference sites in the corresponding structural domains. The function of this gene in Abracium sibiricum has not been discovered yet, and no ABCG5 and ABCG5L genes have been found in tobacco.

[0076] (3) The theoretical isoelectric point of GmABCG5L protein was analyzed using ProtParam software. The transmembrane domain of GmABCG5L protein was analyzed using TMHMM 2.0.

[0077] (4) Use SOPMA software to predict and analyze the secondary structure domain of GmABCG5L protein.

[0078] (5) The subcellular localization of GmABCG5L protein was analyzed using ngLOC (http: / / genome.unmc.edu / ngLOC / index.html) software.

[0079] (6) Protein sequences of 16 species including wild soybean (Glycine soja), carob (Ceratonia siliqua), cowpea (Vigna unguiculata) and Arabidopsis thaliana were downloaded from NCBI and aligned using the software MEGA7.0. The phylogenetic tree was constructed. The results are as follows: Figure 2 shown.

[0080] 2. Construction of plant gene expression vector pROK-GmABCG5L plasmid:

[0081] 1. Extract the plasmids of pMD18T-GmABCG5L and pROK: Extract the plasmids of pMD18T-GmABCG5L and pROK using the AXYGEN kit;

[0082] 2. Enzyme digestion of pMD18T-GmABCG5L and pROK plasmids:

[0083] (1) The pROK plasmid DNA was double-digested with BamH I and Sac I. The double-digestion reaction system was: 65 μL of pROK plasmid, 10 μL of 10×M Buffer, 3.5 μL of BamH I, 3.5 μL of Sac I, and 10 μL of BSA;

[0084] (2) The plasmid DNA of pMD18T-GmABCG5L was double-digested with BamH I and Sac I. The double-digestion reaction system was: 50 μL of PCR product, 10 μL of 10×M Buffer, 2.5 μL of BamH I, 2.5 μL of Sac I, 5 μL of BSA, and 30 μL of ddHO. 2 O;

[0085] (3) Recover the enzyme digestion products of plasmid DNA of pMD18T-GmABCG5L and pROK using a DNA gel recovery kit;

[0086] (4) Ligation of the expression vector pROK and the target gene fragment GmABCG5L. The reaction system during ligation is: 5 μL of pROK plasmid DNA digestion product, 3 μL of GmABCG5L digestion product, 1 μL of 10×T4 DNA Ligase Buffer and 1 μL of T4 DNA Ligase; the reaction conditions are: reaction temperature 16°C, reaction time more than 12 h (overnight);

[0087] (5) Ligation product: pROK-GmABCG5L is transformed into competent E. coli;

[0088] (6) Identification of recombinant plasmids:

[0089] Colony PCR identification was performed using GmABCG5L gene primers containing BamH I and Sac I restriction sites. The results were as follows: Figure 4 As shown, the reaction system is: ddH 2 O 7.7μL, 10×PCR Buffer 1μL, dNTP Mix (2.5mmol / L) 0.8μL, GmABCG5L-BamH I (10μmol / L) 0.4μL, GmABCG5L-Sac I (10μmol / L) 0.4μL, rTaq DNApolymerase (5μmol / L) 0.1μL, Template DNA

[0090] Single colony reaction program: (30 cycles)

[0091]

[0092] The recombinant pROK-GmABCG5L plasmid DNA was extracted for enzyme digestion verification. The reaction system for verification was: 6.6 μL of pROK-GmABCG5L recombinant plasmid, 1 μL of 10×T Buffer, 1 μL of BSA, 0.3 μL of BamH I, 0.3 μL of SacI, 0.8 μL of ddH 2 O;

[0093] Example 2. A method for breeding transgenic plants that promote seed setting

[0094] The pROK-GmABCG5L plasmid obtained in Example 1 was transformed into Agrobacterium:

[0095] Plant seedlings were co-cultured with the pROK-GmABCG5L plasmid, and bud induction and growth were performed after cultivation.

[0096] Aseptic culture of plant seedlings:

[0097] (1) Place wild-type tobacco seeds in a sterilized centrifuge tube, disinfect with 75% alcohol for 3 minutes, and rinse with sterile water 6-8 times.

[0098] (2) Treat with 1 mL of 10% sodium hypochlorite for 5 min and rinse with sterile water 6-8 times.

[0099] (3) Spread the treated seeds evenly on MS solid culture medium and culture them until seedlings grow.

[0100] (4) The seedlings were transferred into bottles containing MS solid culture medium and continued to be cultured into large seedlings. The leaves were used for infection experiments:

[0101] 1) Cut tobacco leaves into squares of about 1 cm × 1 cm and pre-culture for 48 hours.

[0102] 2) Activating the Agrobacterium:

[0103] In a clean bench, pROK-GmABCG5L positive bacteria were spread on YEB solid medium containing Km, Str, and Rif, and incubated at 28°C for 48 hours, then the bacterial suspension was obtained by resuspending the colony with 1 / 2MS liquid medium, and then diluted with YEB to an OD600 of about 0.4-0.6. The above bacterial suspension was transferred to a 50mL conical flask for infecting tobacco.

[0104] 3) Take out the pre-cultured leaves and infect them in the bacterial solution for 6 minutes, turning them upside down to ensure that the leaves are fully in contact with the bacterial solution. Take out the leaves and place them on sterile filter paper, absorb the attached bacterial solution, place the leaves with the back facing up on the MS1 culture medium, and culture them in the dark for 2 days.

[0105] 4) Bud induction:

[0106] The leaves were transferred to MS2 medium for bud induction, and the medium was changed every 3 days. After 3 consecutive times, the medium was changed every 2 weeks.

[0107] 5) Rooting induction:

[0108] When the differentiated buds grow to about 2 cm, they are cut off and transferred to MS3 medium for rooting culture.

[0109] 6) Transplantation and domestication:

[0110] The rooted tobacco seedlings were transferred to soil for cultivation.

[0111] The tobacco plants obtained after the above transformation are GmABCG5L gene-transformed tobacco resistant plants.

[0112] PCR identification was performed on Km-resistant plants. Using pROK-GmABCG5L plasmid as a positive control, 8 of them were able to amplify a fragment of about 600 bp that was consistent with the positive control fragment, which preliminarily proved that GmABCG5L had been transferred into tobacco. Some PCR identification results are shown in Figure 2. Figure 3 shown.

[0113] like Figure 5 As shown, pROK-GmABCG5L positive bacteria were spread on YEB solid medium containing Km and Rif, and the bacterial solution was obtained after the colony was resuspended with liquid YEB, and then the bacterial solution was diluted with YEB to an OD600 of about 0.4-0.6. The GmABCG5L gene was genetically transformed into tobacco by the Agrobacterium-mediated leaf disc method, and resistant plants of the GmABCG5L gene were obtained. The infected leaves were first cultured in the dark on MS1 ​​for 2-3 days, and then moved to MS2 for screening. Callus tissue would grow around the leaves that were successfully infected, and then small buds would differentiate, while callus tissue and small buds would not grow around the leaves that were not successfully infected. The resistant buds were cut off and moved to MS3 medium for rooting culture. A total of 8 kanamycin-resistant plants were obtained, and 8 positive plants were found after PCR identification. The grown resistant plants were transplanted into pots containing soil: vermiculite at a ratio of 1:1.

[0114] Figure 6 Figure 1 shows the capsules of tobacco transgenic with the GmABCG5L gene. Figure A: WT tobacco capsule; Figure B: GmABCG5L tobacco capsule. Compared with wild-type tobacco, the capsules of tobacco transgenic with the GmABCG5L gene are larger.

[0115] The statistical analysis results are shown in Table 1 and Figure 7 As shown, Figure 7 The figure is a diagram of the seed setting of tobacco transgenic with GmABCG5L gene, A: WT tobacco seeds; B, C: GmABCG5L transgenic tobacco seeds. The results show that the seed setting of tobacco transgenic with GmABCG5L gene is more than that of wild type. The method of transgenic with GmABCG5L gene can increase the seed setting rate of plants by about 2 times. The statistical analysis shows significant difference (P < 0.05).

[0116] Table 1 Seed setting rate statistics

[0117]

[0118]

[0119] The weight of all seeds per capsule is in grams. The average of 30 capsules is used in the table. SEQUENCE LISTING <110> Harbin Normal University <120> Application of soybean GmABCG5L gene in improving plant seed setting rate <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 26 <212> DNA <213> Synthesis <400> 1 cgggatccat gatgcaaggc tgtgag 26 <210> 2 <211> 26 <212> DNA <213> Synthesis <400> 2 cgagctctta gttggtgaca accctg 26 <210> 3 <211> 1950 <212> DNA <213> Synthesis <400> 3 atgatgcaag gctgtgaggt tgatgccatt ggcataaact acacgatcca cacacacaaa 60 tcggagcacc cttttaaaat cttcagcaac aaatctgcac atcttgacac tgaacaagat 120 ggtaaagagc cagaagagga agcagaagtt gaacaaagtt gcagtggagt aaggcatgtg 180 ctgaagaatg tgagtttcca agccaagcca tgggagatcc tagcaattgt tgggccaagt 240 ggagctggca aatcatcact gcttgagatt cttgctggta aacacagccc tcaaagtgga 300 actgtgtttc tgaaccacaa gcctgtagac aaagctcagt tcaggaagct ttcagggtat 360 gttacacaaa aggacacttt gtttccttta cttacagttg aagaaaccct catgtttagt 420 gcaaagctga ggctgaagct ttctcaagag caactttgct ccagggtcaa gtcactgatt 480 caggagcttg gacttgacaa tgttgctggc actagaattg gtgatgatag ggtccgaggc 540 atatctggtg gtgagaggcg cagagtctcc attggtgttg aagtcattca tgatccaaaa 600 gtgctgattc ttgatgaacc aacttcaggg cttgacagta cttcagcttt gcaaataatt 660 gacatgctta aggtcatggc cgacacaaga gggagaacca taatccttag tattcaccaa 720 cccgggttca gaatagtgaa gctgttcaat tcattgttgc tgttggccaa tggaagtgtt 780 ttgcaccacg gaacagctga tttgcttagt gtgaatctaa ggctgatggg tctggagctt 840 cctcttcatg tccatgtagt tgaatttgcc attgagtcca ttgataccat tcagcagcaa 900 cagaaatgtg tgccagttca ggtagaaact ccaaggcaac tgccagggac aatactacag 960 aagaaaggtg gtgatggtga agcaggtgag ggtaggaatg gtaagctcac tttgcaacag 1020 ctctttcagc aatccaaggt aattgatgaa caaaccatgt atgctggaat ggatttcact 1080 agtgagtttg ctaattctag attgagagaa actatgattc tcagtcatag gttctccatg 1140 aacatattcc gtacaaaaga gctatttgca tgcaggacag ttcagatgct ggtttctggg 1200 ctggttgtgg ggtccatcttt ttgtaacctt aaggatgatc tagagggagc ttttgaaagg 1260 gtgggtctat ttgctttcat attaacgttt ttattgtcaa gcagcataga agctctacca 1320 atttttctgc aagaaaggga gattctaatg aaggagacat cttgtggaag ctatcgtgtg 1380 tcatcctatg ctattgcaaa tggcctagtt tacttgccat ttcttctcat cctagccatt 1440 ttgttctcaa tgcccttgta ctggcttgta gggctcaacc ggaacttctt ggctttttg 1500 cactttctgt tgctgatatg gctgattctt tacacggcca attcagttgt ggtgtgtttc 1560 agtgctctgg tgcctaattt tattgttggg aactcagtga ttgctggtgt tatagggtca 1620 ttcttcttat tctctggata cttcatatca aaacaggaaa ttccaaatta ctggattttc 1680 atgcattata tatccttgtt taagtatccc tttgaagggt tcctgatcaa tgagttctcc 1740 aactcgggga agtgcttgga atacatgttt ggggcatgta taaagagcgg agaggatgtg 1800 cttaaagaag aagggtacgg gggagagagt aatagatgga agaatgttgg ggtcactgtg 1860 tgcttcatct tggtttacag gttcatttct tatgttattc ttagatacag atgttcccag 1920 aggggatttg gcagggttgt caccaactaa 1950 <210> 4 <211> 649 <212> PRT <213> Synthetic <400> 4 Met Met Gln Gly Cys Glu Val Asp Ala Ile Gly Ile Asn Tyr Thr Ile 1 5 10 15 His Thr His Lys Ser Glu His Pro Phe Lys Ile Phe Ser Asn Lys Ser 20 25 30 Ala His Leu Asp Thr Glu Gln Asp Gly Lys Glu Pro Glu Glu Glu Ala 35 40 45 Glu Val Glu Gln Ser Cys Ser Gly Val Arg His Val Leu Lys Asn Val 50 55 60 Ser Phe Gln Ala Lys Pro Trp Glu Ile Leu Ala Ile Val Gly Pro Ser 65 70 75 80 Gly Ala Gly Lys Ser Ser Leu Leu Glu Ile Leu Ala Gly Lys His Ser 85 90 95 Pro Gln Ser Gly Thr Val Phe Leu Asn His Lys Pro Val Asp Lys Ala 100 105 110 Gln Phe Arg Lys Leu Ser Gly Tyr Val Thr Gln Lys Asp Thr Leu Phe 115 120 125 Pro Leu Leu Thr Val Glu Glu Thr Leu Met Phe Ser Ala Lys Leu Arg 130 135 140 Leu Lys Leu Ser Gln Glu Gln Leu Cys Ser Arg Val Lys Ser Leu Ile 145 150 155 160 Gln Glu Leu Gly Leu Asp Asn Val Ala Gly Thr Arg Ile Gly Asp Asp 165 170 175 Arg Val Arg Gly Ile Ser Gly Gly Glu Arg Arg Arg Val Ser Ile Gly 180 185 190 Val Glu Val Ile His Asp Pro Lys Val Leu Ile Leu Asp Glu Pro Thr 195 200 205 Ser Gly Leu Asp Ser Thr Ser Ala Leu Gln Ile Ile Asp Met Leu Lys 210 215 220 Val Met Ala Asp Thr Arg Gly Arg Thr Ile Ile Leu Ser Ile His Gln 225 230 235 240 Pro Gly Phe Arg Ile Val Lys Leu Phe Asn Ser Leu Leu Leu Leu Ala 245 250 255 Asn Gly Ser Val Leu His His Gly Thr Ala Asp Leu Leu Ser Val Asn 260 265 270 Leu Arg Leu Met Gly Leu Glu Leu Pro Leu His Val His Val Val Glu 275 280 285 Phe Ala Ile Glu Ser Ile Asp Thr Ile Gln Gln Gln Gln Lys Cys Val 290 295 300 Pro Val Gln Val Glu Thr Pro Arg Gln Leu Pro Gly Thr Ile Leu Gln 305 310 315 320 Lys Lys Gly Gly Asp Gly Glu Ala Gly Glu Gly Arg Asn Gly Lys Leu 325 330 335 Thr Leu Gln Gln Leu Phe Gln Gln Ser Lys Val Ile Asp Glu Gln Thr 340 345 350 Met Tyr Ala Gly Met Asp Phe Thr Ser Glu Phe Ala Asn Ser Arg Leu 355 360 365 Arg Glu Thr Met Ile Leu Ser His Arg Phe Ser Met Asn Ile Phe Arg 370 375 380 Thr Lys Glu Leu Phe Ala Cys Arg Thr Val Gln Met Leu Val Ser Gly 385 390 395 400 Leu Val Val Gly Ser Ile Phe Cys Asn Leu Lys Asp Asp Leu Glu Gly 405 410 415 Ala Phe Glu Arg Val Gly Leu Phe Ala Phe Ile Leu Thr Phe Leu Leu 420 425 430 Ser Ser Ser Ile Glu Ala Leu Pro Ile Phe Leu Gln Glu Arg Glu Ile 435 440 445 Leu Met Lys Glu Thr Ser Cys Gly Ser Tyr Arg Val Ser Ser Tyr Ala 450 455 460 Ile Ala Asn Gly Leu Val Tyr Leu Pro Phe Leu Leu Ile Leu Ala Ile 465 470 475 480 Leu Phe Ser Met Pro Leu Tyr Trp Leu Val Gly Leu Asn Arg Asn Phe 485 490 495 Leu Ala Phe Leu His Phe Leu Leu Leu Ile Trp Leu Ile Leu Tyr Thr 500 505 510 Ala Asn Ser Val Val Val Cys Phe Ser Ala Leu Val Pro Asn Phe Ile 515 520 525 Val Gly Asn Ser Val Ile Ala Gly Val Ile Gly Ser Phe Phe Leu Phe 530 535 540 Ser Gly Tyr Phe Ile Ser Lys Gln Glu Ile Pro Asn Tyr Trp Ile Phe 545 550 555 560 Met His Tyr Ile Ser Leu Phe Lys Tyr Pro Phe Glu Gly Phe Leu Ile 565 570 575 Asn Glu Phe Ser Asn Ser Gly Lys Cys Leu Glu Tyr Met Phe Gly Ala 580 585 590 Cys Ile Lys Ser Gly Glu Asp Val Leu Lys Glu Glu Gly Tyr Gly Gly 595 600 605 Glu Ser Asn Arg Trp Lys Asn Val Gly Val Thr Val Cys Phe Ile Leu 610 615 620 Val Tyr Arg Phe Ile Ser Tyr Val Ile Leu Arg Tyr Arg Cys Ser Gln 625 630 635 640 Arg Gly Phe Gly Arg Val Val Thr Asn 645

Claims

1. Use of soybean GmABCGL protein or a gene encoding GmABCGL protein in promoting plant seed setting, characterized in that: The amino acid sequence of the GmABCGL protein is shown in SEQ ID NO.4; the gene sequence encoding the GmABCGL protein is shown in SEQ ID NO.3; and the plant is tobacco.

2. A method for breeding transgenic tobacco that promotes seed setting, characterized in that: The steps of the breeding method are as follows: Step 1: Connect the gene sequence shown in SEQ ID NO.3 to the pROK vector to obtain a recombinant vector; Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Infect tobacco with the recombinant Agrobacterium obtained in step 2 to obtain transgenic tobacco.

3. The breeding method according to claim 2, characterized in that The primers for cloning the gene sequence shown in SEQ ID NO.3 in step 1 are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. The breeding method according to claim 2, characterized in that: The steps of infecting plants with Agrobacterium in step 2 are as follows: 1) Spread the Agrobacterium carrying the gene sequence shown in SEQ ID NO.3 on YEB solid medium containing kanamycin, streptomycin and rifampicin, and culture at 28°C for 48 hours; 2) Resuspend the Agrobacterium colonies obtained in step 1) in 1 / 2MS liquid medium to obtain Agrobacterium bacterial solution, and then dilute the bacterial solution with 1 / 2MS to OD 600 The value is 0.4-0.6; 3) Take plant leaves, infect them in the Agrobacterium bacterial solution obtained in step 2) for 6-7 minutes, take out the plant leaves and place them on sterile filter paper, remove the attached bacterial solution, place them on MS1 ​​culture medium, and culture them in the dark for 2 days.

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