CN112501179B - Method for regulating soybean nodulation and close planting by GmPIN1 gene mutation and application thereof - Google Patents

Abstract

The invention discloses a method for regulating soybean nodulation and close planting by GmPIN1 gene mutation and application thereof. The application provided by the invention is specifically the application of three homologous genes GmPIN1a, GmPIN1b and GmPIN1c coded by GmPIN1 in regulation and control of soybean root nodule formation and plant type establishment. Experiments prove that the soybean protein GmPIN1a, GmPIN1b, GmPIN1c and coding genes thereof are closely related to the occurrence of symbiotic nodulation of soybeans, and the nodulation number of the soybeans is reduced and the included angle of side branches of the overground part is obviously weakened after gene knockout. And moreover, plants without CRISPR/Cas9 vector fragments are screened from homozygous triple mutant descendants of the transgenic soybean Gmpin1abc-L1, and excellent germplasm resource storage is provided for adjusting symbiotic nitrogen fixation and close planting of soybeans and high-yield breeding.

Description

A method for regulating soybean nodulation and dense planting by GmPIN1 gene mutation and its application

technical field

The invention relates to the field of plant genetic engineering, in particular to a method for regulating soybean nodulation and dense planting by GmPIN1 gene mutation and its application.

Background technique

As one of the important economic crops in my country, soybean is rich in protein and oil and has very high nutritional value. However, with the continuous improvement of the living standards of our residents, my country's soybean production has been unable to meet its own needs, so that my country needs to import a large amount of soybeans from abroad every year. According to statistics, China's soybean imports have rapidly increased from 13.94 million tons in 2001 to 88.06 million tons in 2018 ^[1] . Further analysis found that in the past 20 years, the yield per unit area of soybeans in China has hardly changed, and the yield per unit area of soybeans in China is much lower than that of the United States, Brazil and Argentina in the same period ^[1] . Therefore, it is very necessary to improve the yield per unit area of soybean in my country.

Agricultural production practices over the past few decades have shown that the application of fertilizers can effectively improve crop yields. Statistics from the United Nations Food and Agriculture Organization show that the application of chemical fertilizers contributes 40% to 60% to the increase in crop yield ^[2] . During the period from 1952 to 1992, the correlation coefficient between fertilizer application and grain yield in China was as high as 92% ^[2] . However, the uptake and utilization of fertilizers by crops is not efficient. Statistics show that the utilization rate of nitrogen by crops is 30%-60%, the utilization rate of phosphorus is 2%-25%, and the utilization rate of potassium is 30%-60% ^[3] . The large-scale application of fertilizers not only causes waste but also harms the surrounding ecological environment and human health. Nitrogen-fixing microorganisms in nature can convert nitrogen in the atmosphere into ammonia that plants can absorb and utilize. Among them, the nitrogen fixed by the root nodules formed by legumes and rhizobia accounts for about 60% of the total nitrogen fixation of biological nitrogen ^[4] . Studies have shown that under the same planting conditions, soybean is the legume crop with the most nitrogen fixation ^[5] . The nitrogen fixation of soybean accounts for more than 50% of the total nitrogen absorbed by soybean ^[5] . Further research shows that the symbiotic nitrogen fixation of soybean root nodules plays an important role in the development of seeds ^[6] . The researchers also found that the inoculation of rhizobia can significantly increase the yield of soybean ^[7] . In addition, increasing the nitrogen output in the root nodules can also significantly increase the yield of soybean ^[8] . To sum up, the rational use of soybean nodule nitrogen fixation is beneficial to improve soybean yield, and the rational use of biological nitrogen fixation can effectively reduce the application of fertilizers. Therefore, it is very necessary to deeply understand the growth and development of soybean root nodules. As an important hormone regulating plant growth and development, auxin has been reported to play a crucial role in the nodule development of leguminous plants such as L. japonicus and alfalfa ^[9-12] . However, the research on the role of PIN gene in root nodule development is still relatively small, and there is no relevant report in soybean ^[13] . Therefore, it is necessary to carry out PIN gene-related research in soybean to deeply understand the mechanism of root nodule formation. At the same time, it also provides the necessary theoretical basis for obtaining high-efficiency nodulating soybean plants.

In addition, plant type is an important feature that determines the high yield of soybean. For decades, it has been the goal of breeders and researchers to cultivate high-yield soybeans with ideal plant type by optimizing soybean plant type. However, the main effect of regulating the ideal plant type of soybean is Gene reports are less ^[14] . An important indicator of the ideal plant type is the angle between the lateral branch and the main stem. The angle of the angle affects the canopy structure and overall light-receiving area of the above-ground part of the plant, and ultimately affects photosynthesis and yield. The smaller the included angle is, the leaf surface will be able to stretch upwards, and the whole plant will occupy a small space, which not only facilitates the rational use of light sources and good ventilation, but also promotes the increase of the average yield per mu through dense planting. It is well known that auxin plays a key role in the control of apical dominance and lateral shoot growth in plants, and a large amount of auxin produced by apical buds is transported down to lateral buds, resulting in the inhibition of lateral bud growth ^[15,16] . Therefore, the use of auxin transport to control the angle of lateral branches has important application significance in plant dense planting ^[17] .

SUMMARY OF THE INVENTION

The purpose of the present invention is to create a gene-edited soybean Gmpin1abc triple mutant that does not contain GmPIN1a, GmPIN1b and GmPIN1c proteins that can normally transport auxin, and provides a good germplasm resource reserve for the establishment and application of an ideal soybean plant type.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions:

The method for regulating soybean nodulation and dense planting by GmPIN1 gene mutation is characterized in that three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in soybean plant GmPIN1 are mutated to obtain soybean triple mutant Gmpin1abc;

The nucleic acid sequence of GmPIN1a gene is shown in SEQ ID No.1; the nucleic acid sequence of GmPIN1b gene is shown in SEQ ID No.2; the nucleic acid sequence of GmPIN1c gene is shown in SEQ ID No.3.

Further, the three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in GmPIN1 were simultaneously deleted by gene knockout, and the protein was terminated early.

Further, soybean triple mutant Gmpin1abc is obtained by the following method:

S1: Use CRISPR-GE (http://skl.scau.edu.cn/home/) online tool to design and construct sgRNAs targeting GmPIN1a, GmPIN1b, and GmPIN1c at the same time;

S2, using an annealing reaction to obtain a double-stranded oligonucleotide;

S3: Linearize pEF1A2 vector with BsaI endonuclease;

S4: The products obtained from S2 and S3 are linked together by T4 ligase;

S5: introduce the plasmid vector obtained in S4 into GV3101 Agrobacterium;

S6: the Agrobacterium obtained by using S5 is used to infect soybean;

S7: the product obtained in step S6 is subjected to plant tissue culture;

S8: Perform plant resistance (Basta) screening on the tissue culture seedlings obtained in S7 to obtain stable transgenic soybeans without CRISPR/Cas9 vector fragments.

The above-mentioned method of regulating soybean nodulation and dense planting by GmPIN1 gene mutation can be applied to the regulation of nodulation number and collateral angle in soybean and its related plant types.

Beneficial effects:

The present invention is the first to create a gene-edited soybean triple mutant that does not contain Gmpin1abc that can normally transport auxin. The soybean proteins GmPIN1a, GmPIN1b, GmPIN1c and their encoding genes are closely related to the occurrence of soybean symbiotic nodule, and the gene knockout reduces soybean the number of nodules and significantly weakened the lateral branch angle of the aerial part. In addition, plants without the CRISPR/Cas9 vector fragment were screened from the offspring of homozygous triple mutants of transgenic soybean Gmpin1abc-L1, providing excellent germplasm resources for regulating soybean symbiotic nitrogen fixation, dense planting, and high-yield breeding.

Description of drawings

Figure 1 shows the edited nucleotide sequence of the Gmpin1abc gene-edited soybean homozygous triple mutant.

Figure 2 is the nodulation phenotype of wild-type soybean and gene-edited soybean triple mutants of Gmpin1abc in Example 1;

Fig. 3 is the plant phenotype of the gene-edited soybean triple mutants of wild-type soybean and Gmpin1abc in Example 2;

Figure 4 shows the collateral phenotype of the wild-type soybean and the gene-edited soybean homozygous triple mutant of Gmpin1abc-L1 in Example 2.

Specific implementation method

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods and devices in the following embodiments are conventional methods and devices unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The mutation sites of mutants in the following examples were all determined by conventional sequencing companies. In order to make the purpose, technical solutions and advantages of the patent of the present invention clearer, the specific implementation of the patent of the present invention will be described in detail below with reference to specific embodiments. Examples of these preferred embodiments are illustrated in the specific examples.

Here, it should also be noted that, in order to avoid obscuring the technical solutions of the patent of the present invention due to unnecessary details, only the technical solutions and/or processes closely related to the solutions according to the patent of the present invention are shown in the embodiments. steps, while omitting other less relevant details.

In the embodiment, the soybean cultivar is Huachun No. 6. Huachun No. 6 is a soybean variety bred by the Agricultural College of South China Agricultural University, using the variety Guizao No. 1 × Brazil No. 8. On July 28, 2009, it was approved by the third meeting of the Second National Crop Variety Approval Committee, and the approval number was 2009012.

Soybean CRISPR/Cas9 vector pGES201: described in: Bai M, Yuan J, Kuang H, et al. Generation of a Multiplex Mutagenesis Population via Pooled CRISPR-Cas9 in Soybean[J]. Plant Biotechnology Journal, 2019, 18(3).: Public It can be obtained from the Horticultural Center of the Straits Joint Research Institute of Fujian Agriculture and Forestry University.

The method for regulating soybean nodulation and dense planting by GmPIN1 gene mutation is characterized in that three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in soybean plant GmPIN1 are mutated to obtain soybean triple mutant Gmpin1abc;

The nucleic acid sequence of GmPIN1a gene is shown in SEQ ID No.1; the nucleic acid sequence of GmPIN1b gene is shown in SEQ ID No.2; the nucleic acid sequence of GmPIN1c gene is shown in SEQ ID No.3.

Further, the three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in GmPIN1 were simultaneously deleted by gene knockout, and the protein was terminated early.

Further, soybean triple mutant Gmpin1abc is obtained by the following method:

S1: Use CRISPR-GE (http://skl.scau.edu.cn/home/) online tool to design and construct sgRNAs targeting GmPIN1a, GmPIN1b, and GmPIN1c at the same time;

Referring to Figure 2, the sgRNA sequence of the gene editing site of CRISPR-Cas9 targets the first exon position of GmPIN1a, GmPIN1b and GmPIN1c, the boxed area is the targeting site, and the nucleotides marked with ellipses are Gmpin1abc. Gene-edited soybean triple mutant deletions after editing.

S2, using an annealing reaction to obtain a double-stranded oligonucleotide;

S3: Linearize pEF1A2 vector with BsaI endonuclease;

S4: The products obtained from S2 and S3 are linked together by T4 ligase;

S5: introduce the plasmid vector obtained in S4 into GV3101 Agrobacterium;

S6: the Agrobacterium obtained by using S5 is used to infect soybean;

S7: the product obtained in step S6 is subjected to plant tissue culture;

S8: Perform plant resistance screening on the tissue culture seedlings obtained in S7 to obtain stable transgenic soybeans without CRISPR/Cas9 vector fragments.

Example 1

Wild-type soybean (WT) and soybean triple mutant Gmpin1abc were bred in the following manner.

The light duration between soybean growth was 14 hours and the temperature was 25°C. Soybean seeds were germinated on vermiculite. On the 7th day after seed germination, the rhizobia BXYD3 with an OD value of 0.15 was evenly watered from the vermiculite surface to the soybean roots, followed by 1/10 nitrogen hydroponic solution for moisturizing and continued cultivation for 14 days.

The formula of 1/10 nitrogen hydroponic solution is shown in the following table:

Element KNO₃ NH₄NO₃ Ca(NO₃)₂·4H₂O MgSO₄·7H₂O Concentration (mmol/L) 0.15 0.04 0.12 0.5 Element K₂SO4 ZnSO₄·7H₂O MnSO₄·H₂O CuSO₄·5H₂O Concentration (mmol/L) 1.05 1.5*10^-3 0.5*10^-3 0.5*10^-3 Element (NH₄)₆M₉O₂₄ NaB⁴O⁷·10H₂O KH₂PO₄ Fe-EDTA Concentration (mmol/L) 0.15*10^-3 2.5*10^-3 0.25 0.04 Element (NH₄)₂SO₄ MgCl₂·6H₂O CoCl₂·6H₂O CaCl₂ Concentration (mmol/L) 0.03 0.025 0.0001 1.2

The nodulation phenotypes of wild-type soybean (WT) and Gmpin1abc gene-edited soybean triple mutants are shown in Figure 2; Gmpin1abc showed an overall decrease in nodule number and nodule density (nodule number divided by root area) decreased, but the mean nodule size profile of nodules did not change significantly. Number, density and diameter of root nodules 14 days after rhizobium infection of roots of 7-day-old seedlings. P-values were tested by two-tailedStudent’s t-test (***p<0.001).

Example 2

Wild-type soybean (WT) and soybean triple mutant Gmpin1abc were bred in the following manner.

The light duration between soybean growth was 14 hours and the temperature was 25°C. Soybean seeds were germinated on vermiculite. On the 10th day after the germination of the seeds, they were transferred into soil mixed with nutrient soil and loess and planted outdoors for 45 days.

The plant phenotypes of wild-type soybean (WT) and Gmpin1abc gene-edited soybean triple mutants are shown in Figure 3; 10-day-old seedlings were transplanted into soil and grown outdoors for 45 days. The overall plant type was short, and the angle of lateral branches was reduced.

The lateral branch phenotypes of wild-type soybean and Gmpin1abc-L1 gene-edited soybean homozygous triple mutants are shown in Figure 4; compared with wild-type WT, homozygous triple mutant soybean Gmpin1abc-L1 without the CRISPR/Cas9 vector fragment The branch angle was significantly reduced and the branch number was significantly increased. P-values were tested by two-tailed Student's t-test (**p<0.01, ****p<0.0001).

references:

[1] Xiao Weidong, Du Zhixiong. Development of China's soybean industry: main problems, reasons and countermeasures and suggestions [J]. Globalization, 2019, 05): 105-18+36.

[2] Wang Hongting, Jin Jiyun. Research progress on precise management of agricultural nutrient resources [J]. Shanxi Agricultural Sciences, 2005, 01): 68-72.

[3] Zheng Liangyong, Du Liqing. Agricultural chemical fertilizer pollution and environmental protection countermeasures in my country [J]. China Tropical Agriculture, 2013, 02): 76-8.

[4] Li Xinxin, Xu Ruineng, Liao Hong. Contribution and application potential of soybean symbiotic nitrogen fixation in agricultural weight loss and efficiency enhancement [J]. Soybean Science, 2016, 35(04): 531-5.

[5]HERRIDGE D F, PEOPLES M B, BODDEY R M.Global inputs of biologicalnitrogen fixation in agricultural systems[J].Plant&Soil,2008,311(1-2):1-18.

[6] Guo Hailong, Ma Chunmei, Dong Shoukun, et al. Absorption and utilization of different nitrogen sources in spring soybean growth [J]. Journal of Nuclear Agriculture, 2008, 03): 338-42.

[7] Xie Yuying. The role of legumes in the development of ecological agriculture [J]. Anhui Agricultural Science Bulletin, 2007, 07): 150-1+06.

[8]CARTER A M,TEGEDER M.Increasing Nitrogen Fixation and SeedDevelopment in Soybean Requires Complex Adjustments of Nodule NitrogenMetabolism and Partitioning Processes [J].Curr Biol,2016,26(15):2044-51.

[9]BREAKSPEAR A,LIU C,ROY S,et al.The root hair"infectome"of Medicagotruncatula uncovers changes in cell cycle genes and reveals a requirement for Auxin signaling in rhizobial infection[J].Plant Cell,2014,26(12 ): 4680-701.

[10] PACIOS-BRAS C, SCHLAMAN H R, BOOT K, et al. Auxin distribution in Lotus japonicus during root nodule development [J]. Plant Mol Biol, 2003, 52(6): 1169-80.

[11]SUZAKI T,YANO K,ITO M,et al.Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus isaccompanied by auxin response[J]. Development,2012,139(21):3997-4006.

[12]TAKANASHI K,SUGIYAMA A,YAZAKI K.Involvement of auxin distribution in root nodule development of Lotus japonicus[J].Planta,2011,234(1):73-81.

[13]HUO X, SCHNABEL E, HUGHES K, et al. RNAi Phenotypes and the Localization of a Protein::GUS Fusion Imply a Role for Medicago truncatulaPIN Genes in Nodulation[J].J Plant Growth Regul,2006,25(2) :156-65.

[14] LIU S, ZHANG M, FENG F, et al. Toward a "Green Revolution" for Soybean [J]. Mol Plant, 2020, 13(5): 688-97.

[15] ROYCHOUDHRY S, KIEFFER M, DEL BIANCO M, et al. The developmental and environmental regulation of gravitropic setpoint angle in Arabidopsis and beans[J]. Sci Rep, 2017, 7(42664.

[16] YOSHIHARA T, SPALDING E P.LAZY Genes Mediate the Effects of Gravity on Auxin Gradients and Plant Architecture[J].Plant Physiol,2017,175(2):959-69.

[17] WANG B, SMITH S M, LI J. Genetic Regulation of Shoot Architecture [J]. Annu Rev Plant Biol, 2018, 69 (437-68.

<110> Fujian Agriculture and Forestry University

<120> A method for regulating soybean nodulation and dense planting by GmPIN1 gene mutation and its application

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tttgagactc cttagagcac gacaacaaat gttcaatgaa atacaaaagc atcaccataa 5700

ttgaatagga ggaatcgatc aacggatgag ttttcatttt tcttcttctt ttttttttaa 5760

tgaattgtcc ttgctcagtg aaaatgtaaa atcatgtttg tagctaattt ataaaatggc 5820

tatctcgtta aatttcaaat taaaaattct cccgataccg tatcactcat caagcgaaga 5880

aggaatagag aggc 5894

<210> 2

<211> 6113

<212> DNA

<213> Soybean (Glycine max (L.) Merr.)

<400> 2

tacacatcat caaataataa attgtcacat aaataaaaaa aatgtagact tttataataa 60

tgattttgag agttgtgact ggtttttaat tagttagcag tgtaatgatc tttatactga 120

gaatatatca aaattaaact tatttaataa acatgcaaaa aaaaaatcca ttttttttaa 180

ttccaaattt catcattgaa aatatatatc aacattgcaa attccaaatt ccaaatcaat 240

ttcttttttg cctaactaga aaaacaaacc aaaaaaaaca gcagatttga atctttcaac 300

tagacataat tattcaaacg gacatgttat gctcaatggt ttgctcaact agtatgatta 360

aaaagcacat ttttttaaa tagtcatttt gtcccttaat gtataatggg cttataaatg 420

tgtccctgaa agatgaaaat acaaaattta gtcctcaaaa gtgtaaaaag tactataaat 480

atattcaact gttaactttc atccatcact gttaataaaa tagtctatgt gacacagagg 540

aaaaaatttg tcactgaaat gatggtgaac gtagtcatct ctaattacca gcatagggac 600

atatttgtca tataatattt tttttacttt tcgtcttctc actccactgg caaatgtgtc 660

cctgaaagat gaaaatacaa aatttagtcc tcaaaagtgt aaaatgcaca acaaatatat 720

ccaaacgtta ataatacatt atgactaatt attataattt gaaaaaaatt ataatgattg 780

tgacaatttt ttttaataaa ctcgtaagtt aaattgagtc taaaagaaaa aagaatactt 840

attttataaa ctataaataa ttttttttaca ctctcatgct tgatgaaagg tttaagtaaa 900

aaaatgttta ttgtaaagct aaattaggtc catgaataat tttaagggag aattacaatt 960

gcaatgacac ttttaatttg taaaaaaata ctcacctcct ttggaatgag ttttttttaag 1020

gttatgattt tttaaatgat caacaaactt ttattataat ttgtaatttg atgtcattgc 1080

atatattaat tgacattaat attttagtat gaaaaattta taaaaaaaca attaaataaa 1140

tagtatttga ttaaatagaa ataataattt tcttatcatg acatttattc aagagttaac 1200

aactaaaagg attgatttttt ttgtaatgga agaaaagtaa gaagaattca tcaaagcaat 1260

acaaattcac ttctattggt aatattttat gcatgtctca accgttgatc aagcttaaaa 1320

attatataac ggaaacaaac acttaccggt gtgatatagc tctcccaaaa attttataaa 1380

tttttcaaaa ttataataat tagtcacgat ctattattaa cgtccgaata tatttgtcgc 1440

atttttttata ctttccggga cgcgtttgtc agcggaatgg aaaaacgaaa agtaaaaaaa 1500

atattatgac aaatatgctt ctatgctggc aattagagat gttcacattg acaatgattt 1560

cagtgataaa tttgttcatc tgtgccacgt aagctatttt attaacggtg acggacgaaa 1620

attaatgacc ggatatattt gtcgtacttt ttacactttg aggaactaga ttttgtattt 1680

ttatctttga gagatacatt tgtcaataaa ttacacattg agagacaaaa gtgactattt 1740

atcttttttt ctataaatgt tttttttttc ttgaaaaaac ccttcataaa gaaaccgtgg 1800

atctagaaag agtcccttta caaatcctaa agcactcccc caaaacaagt actttagaca 1860

ttcccacctc aacacaaaat gcttttccta agctttctgt gacaaattta ggacccatag 1920

aatagaaggt agggagggag agcctcacat ccactcacca aagtttccaa agccatcaaa 1980

taccccccca aaccaggcac taccacctcc catcccttcc caaaccccaa accccaaatc 2040

caaaccaaac acacttcaaa acaaaaagca aaacaacaaa gtggcaatac aaaaaaaaaa 2100

aaaaactaaa taaaaaggca acaaaccccc ccactctgcc ttgtgctttg gagactgcga 2160

gtgcaacctt tcttgcagct cgcaaagctg aaaaaatatt tgctgtattc tgcagcatat 2220

taccaccacc cactcactca cttccccaaa aacacatgct cttccacatc cctatatatc 2280

ttttcaatct tcatcatcat catcaccacc aactccaact ctccaaaact tgccacttca 2340

accttcctat atattccttc ctccactctc tttctttact tcttctgcta tctttctgag 2400

agacctcaca aacccacaaa tatgatcacc ttaacagact tctaccatgt catgactgca 2460

atggtgccac tctatgtggc catgatacta gcctatggct cagtgaagtg gtggaagatt 2520

ttctcccctg accaatgctc tggcatcaac cgttttgtgg cactctttgc agtgcctctt 2580

ctctccttcc acttcatagc ctccaacaac ccttacgaaa tgaacctcag gttcctagct 2640

gctgacaccc ttcaaaagat cataatacta gtcctccttg cagtttggag caacatcgcc 2700

aaaaggggtt gcttggaatg ggccataacc ttgttctctc tctccaccct ccctaacact 2760

ttggtcatgg gcatcccttt gctcaaaggg atgtatggtg acttctcagg gtcccttatg 2820

gtgcaaattg tggtcctcca gtgtatcatt tggtacacct tgatgctgtt cttgtttgag 2880

tttagaggtg ccagaatgct tatctctgag cagttccctg acactgctgg ctccattgtc 2940

tccatccatg ttgactctga tgtcatgtca ttggatggaa ggcaaccact tgagactgaa 3000

gctgagatca aggaagatgg taaactccat gtcactgtga ggaagtccaa tgcctcaaga 3060

tcagacatct tctcaagaag gtctcagggt ctctcttcca ccactccacg cccttctaac 3120

ctcactaatg ctgagatata ttctttgcaa tcctctagga accctacacc gagaggttcc 3180

agcttcaacc acactgattt ctactctatg atggctgctg gtggcaggaa ctccaacttt 3240

ggtgcctctg atgtttatgg cctttcagcc tcaagagggc caactccaag gccttctaac 3300

tatgatgaag atggtgggaa gccaaagttc cattaccatg ctggtggaac tggacactac 3360

cctgcaccaa accctggcat gttctctccc tcaaatgggt ccaaaagtgt tgctgctgct 3420

aatgctaatg ctaatgccaa aaggcctaat gggcaggctc agctgaagcc tgaggatggg 3480

aatagggacc ttcatatgtt tgtttggagt tcaagtgctt caccagtctc tgacgtgttt 3540

ggtgcccatg agtatggagg tcatgatcag aaagaagtca aattgaatgt atctccaggg 3600

aaaggtctta tttttgtttt gttctttcca agttggattt ttttgtctta atacgtgttt 3660

gaaattatgt tcagaagcat gaaattacat ccggcatgtg gaagttaaaa cactagcat 3720

tcttagtaaa tgtgaaaaat ttatatttaa gatgcagaac caaacacgat aatattagtc 3780

ttttatttgc ttgtttgttt gttttcctta tgtgggtgct aacttgtggt gtttggtaac 3840

cagtggagaa tcatagggac actcaagaag actacctaga gaaagatgag ttcagctttg 3900

ggaatagagg aatggatagg gagatgaatc agcttgaagg tgagaaggtt ggagatggga 3960

agccaaaaac catgcctcca gcaagtgtga tgacaaggct tatattgatt atggtgtgga 4020

gaaaactcat cagaaacccc aacacctact ctagcctaat tggcctcact tggtctcttg 4080

tttcattcaa gtagggattt tttttttctt tctccatgtt tattatgcac atttcatatg 4140

tagttgtacc actcattctt gtttcttttt gcctttccat ttttattgat tgtggttgag 4200

tgttgtgttg ttccttgcag gtggaatgtg gagatgcctg ccataatagc aaagtctatc 4260

tccatattgt cagatgcagg gcttggcatg gccatgttca gtcttggtca ggacaattct 4320

ctccttcttt ttatcttttt agctcagaaa ataacttgtt ctgacatcat ctttgctttg 4380

cttgagagag aaaaaagaaa aaagcagaaa atcttttctg attccatgtt gatgtttact 4440

ttgattaatg attatttatt ttaaattaaa caagcaacta ggaggaggcg tgataactag 4500

agaaaaagca aagaaagcaa acagtataat ttatgataat gtgtgcactc ttttttatgat 4560

aacttttttt gccaagtttg tgatgatgcg gggttggtgg tggtggtggt gatgatgaaa 4620

gttcattcat cccaaagggg gtgattgact gagagatttg tagaagtggt ggtggtgata 4680

ctgatggaca tgctttgctt ttttcttttt ttttatatga atgtgtgaca ggtctcttca 4740

tggctttgca accgagggtc atagcatgtg gaaattccac cgcagctttt gccatggctg 4800

tgagattcct tacaggtcca gctgtcatgg cagctgcttc cgttgctgtt ggactcaaag 4860

gtgttctcct acatgttgcc attgttcagg tatctctctc tctctctctc tctctctctc 4920

ttctgctccc tcctcacaaa gccaatagta caatacaaca ttcattgcat aagctcctaa 4980

aactacttta ttttaaattt gcaacaccat ttacacccac ctgtgtctag ggcccaagat 5040

agtcttttgt attcatgtca ttcaattata aatcatcaat tataaatcat cattgttaac 5100

gacggtatct gatgataaat aattgatcta actaaattct aattggttga atttaagttt 5160

actctagtac aaccataaca taaaaatgtt acatagaaaa gttgttttat actagagtgg 5220

aaaaagtaac attaattggt tatggacact gaaagataaa accccaaaaa aatgatagta 5280

agaattaaca aactcactac aagtcctgtt tgaaaagaat aatatgaaaa ctgaagatct 5340

tagtactaat agtgaggatt catatgatca tttgtgattg tacaacaaag atttgaaact 5400

gcagttgaca tgtttgtttt gtttgctttg tcctgtttgg catctgaatg tcaaaaaaat 5460

ataggcagct cttccccaag gaattgtccc atttgtcttt gctaaggaat ataatgtaca 5520

tcctgatatt ctcagcacag cgtgagtatt caaatctaat ctttatatgg ctcaattagg 5580

actttctctc cccaccattc ctttttcatt ttcattcaat tactttctct tttaatccta 5640

accaatggac cttctttaac ccaatgcagt gttattttttg ggatgttgat tgctttgccc 5700

ataactctag tgtactacat cttgttgggg ttgtgaatga aagaaatgat ggatgataca 5760

gaagattcac gtgtggcatc catgcaaagc tcggttgagg cttgttgagc atcagacaaa 5820

aaaaaggggg tcataaagca acaatagaaa agaagcatca cgagaatttg gataggaaga 5880

accccaggat cagttttttt ttttttaaatt tatttatttg ttttcttttt ctttttttgaa 5940

tgaattgccc tttgttagtg aaaattagtg taaaatcatg atgtagctaa tttacaaaat 6000

gattatctcg ttaaattttt atattataat gtcctcggat tccatgtcac tcatcaattg 6060

aaggataaga aagcatggga aactttgttg atgaaaatgg tagcggtatg atc 6113

<210> 3

<211> 5753

<212> DNA

<213> Soybean (Glycine max (L.) Merr.)

<400> 3

ttcgcaatca agaagatagt gatgccttgt cttcagctgc tttattttcc atactatttt 60

tatttcctac aatatattga tattgattgt ttctactttc tcctggaaca tggaacacag 120

tttagttagt ctgtatttca attgttttat ttattttaa ctaaacctag cattgtggat 180

ttgtggtgtg gtcccaccta acaaaatgaa cccgaagctc ttggaagtgc aagccttatt 240

agatatcgtt gggaaaagga caatatgtct atgtgttggt tttcaccctc ttcatctctt 300

ctatttgtcc tctgttttct gtatcctcca ctccttggaa gtcatgtata gccttatcta 360

tctatcttct tagatcgagg agtttcccat cttaattcat ctaaccctat tgaaatgttt 420

ctctaaccac ttatttgatt ttaataatat tatactagta ggaataaatc aattatcttg 480

tataggtaat aagaatataa tatgaaaagt tgttgaaata agtgatttga aatttgggtt 540

ttttaattaa gatttcaggt tttccaactt aagcgtatgt agtcgcaatt agacatataa 600

ttttacctcg aaatgggtta aacaaaaaaa agaatataat tggaaagcga ctttaattga 660

agagtttttt tatattttta atatattata aattattttt atataaaatt tagtcattat 720

ttttttataa ctaagatgtg tattcttcct cccacctggt caattcaatt ttggagtaaa 780

gtggtgtctt cggttgtcac tccatgctca ggtaaaaaaa attgttataa aaattaataa 840

attaacttac aatacatata gtttgtaatt aaaaaatggt gtaaaagact ttgttacatt 900

gtcctcatta catgttaaat ctaaaattta ataagaatat atttagaata ttttataaga 960

aaatttacac tatcatcaaa taataaattg tcatataatt taaattgttg acaatataat 1020

gatctttata cagagaatat atcaaaacta aacttattta ataaatatgc aagttaaatc 1080

tatttttttt caattccaaa tttcaacatc gcaaatttca aattccaaat ctatttgtta 1140

ttgctaacta gaaaaacaaa ggaaaaaaaa acccagtaga tttgaatctt tcaacagaca 1200

caatattgaa ctagacataa ttattcaaac agacatatta tgctcaacta gtatgattaa 1260

acaacacaat ttttttttctt ctatatatgt ttctttttat cttaaaaccc ttcataaaga 1320

aaccgtggat ccagagagaa agagtccctt tacaaagctt aaagcactcc cccaaaacaa 1380

gtgttcttgg agacattccc acctcaaccc aaaatacttt tcctaagctt tttgtgacaa 1440

atttaggacc catagaatag aaggtaggga gggagagagc ctcacacacc cacccaccca 1500

agtttccaaa gccatcaaat cccttcccaa accccaaatc caaacacact tcaaaaaaag 1560

aaaaaaaaaa caaaactacg aaaaaaaaac taaataaaaa ggcaacaaaa ctccccccac 1620

tctgccttgt gctttggaga ctgcaagtgc aaccttgctt gcagctctca aagctgaaaa 1680

aatatttgct gtattctctg ctgcacatta gcaccattca ctcactcact gccccaaaac 1740

cacatgctct tccacatccc tatataaaat cttttcaatc ttcataatca tcatcatcac 1800

caccaactcc aactcaaact ctccaaaacc tgccacttca accttcctat atattccttc 1860

cctcactctc ttctgcttct atcatctttc tgagaggctt gttgacacac aaaaaatgat 1920

caccttaaca gacttctacc atgtgatgac tgcaatggtg ccactctatg tggccatgat 1980

actagcctat ggctcagtga agtggtggaa gattttctcc cctgatcaat gctctggcat 2040

caaccgtttt gtggcactct ttgcagtgcc tcttctctcc ttccacttca tagcctccaa 2100

caacccttat gagatgaacc tgaggttcct agctgctgac acccttcaaa agatcataat 2160

actagtcctc cttgcagttt ggagcaacat caccaaaagg ggttgtttgg aatgggccat 2220

aaccttgttc tctctctcca ccctcccaaa cactttggtt atgggcatcc ctttgctcaa 2280

agggatgtat ggtgacttct cagggagcct catggtgcaa attgtggtcc tccagtgcat 2340

catttggtac accttgatgc tcttcttgtt tgagtttaga ggtgccagaa tgctcatctc 2400

tgagcagttc cctgacactg ctgcctccat tgtctccatc catgtggact ctgatgtcat 2460

gtcattggat ggaagacaac cacttgagac tgaagctgag atcaaggaag atggtaaact 2520

ccatgtcact gtgaggaaat ccaatgcttc aagatcagac atcttctcta gaaggtctca 2580

gggtctctct tccaccactc cacgcccttc caaccttacc aatgctgaga tatactcttt 2640

gcaatcctct aggaacccta cgccgagagg ctctagtttc aaccacactg atttctactc 2700

catgatggct gctggtggca ggaactcaaa ctttggtgcc tctgatgttt atggcctttc 2760

agcttcaaga gggccaactc caaggccttc taactatgat gaagatggtg ggaagccaaa 2820

gtttcattac catgctgctg gtggaactgg gcactaccct gcaccaaacc ctggcatgtt 2880

ctctccctct aatgggtcca aaagtgttgc tgctaatgct aatgccaaga ggcctaatgg 2940

gcaggctcag ctgaagcctg aggatgggaa tagggacctt catatgtttg tttggagttc 3000

aagtgcttca ccagttttctg atgtgtttgg tgcccatgag tatggaggag gtcatgatca 3060

gaaagaagtc aaattgaatg tatctccagg aaaaggtttt atttttgttt ttgttctttc 3120

taggttggat tttttgagaa atctgttcag aagtaagtta aaactactag cttcttagta 3180

aatgtgaaaa attatatcta agatgtagaa ccaaacacaa taatattagt ctttatttgt 3240

ttgtttactt gtggtgtttg gtaaccagtg gagaataatc atagagacac tcaagaagac 3300

tacctagaga aagatgagtt cagctttggg aatagagaaa tggacaggga gatgaatcag 3360

cttgaaggtg agaaggttgg agatgggaaa ccaaaaacca tgcctccagc aagtgtgatg 3420

acaaggctta tattgattat ggtgtggaga aaactcatca gaaaccccaa cacctactct 3480

agcctaattg gtctcacttg gtctcttgtt tcattcaagt aggaaaataa ataaattctt 3540

tctccatgtt ttattatgca acatttcata tgtagttgta ccactctttg ttgttttttt 3600

ttttttttgcc tttccatttt gattggtttg tggttgagtg ttgtgttgtt gttccttgtg 3660

tgcaggtgga atgttgagat gcctgccata atagcaaagt ctatctccat attgtcagac 3720

gcagggcttg gcatggccat gttcagtctt ggtcaggaca attctctcct tgttacctta 3780

caaaaaaaaa tgcttttttt agctcagaaa ataacttgtt ctgacatctt ctttgcttac 3840

ttgagagaga aaaaaagaaa aagcagaaaa acttttgtga ttccatgttg ctgttcttta 3900

ctttgtttaa tgattattat tttaaattaa acaagcaaca aggaagagga ggcgttataa 3960

ctagagaaaa aaagcaaaga aagcaaactg tgtaatgtta tgataatgtc tgcactcttt 4020

ttatgataac ttattttgcc aagttttgtg atgatgtggg gttggtggtg gtgatgatga 4080

atggtcattc atcacaaagg ggtgtgattg actgagagat tagtagtagt agtagtggtg 4140

atactgatgg gggcatgctt tgcttttttt atatgaattt gtgacaggtc tcttcatggc 4200

tttgcaaccg agggtcatag catgtggaaa ttccacagca gcttttgcca tggctgtgag 4260

attccttaca ggtccagctg tcatggcagc tgcttccatt gctgttggac tcaaaggtgt 4320

tctcctacac gttgccattg ttcaggtatg tctctctctt tgttcccttc tcacaaagcc 4380

actcactagt acaattacaa tacaacatca ttcattgcat aagttccaaa aactacttta 4440

ttttaaattt acaacaacat ttacatccac ctatgcctag ggcccaagat agtcttttgc 4500

acttatgtcg tttaattata aattattatt tttaatttat aaaagaaaaa tggtacatgc 4560

acatttaggg ggctatttaa cacctagaga ggaagaaaaa ggagaaaaaa aatataaaca 4620

agatggaatt tatgatggat aagaagaaaa agataaagag aaatgaaaat gtttgaaaag 4680

atttaaaata atgagatgtt tgtatatcat tattttttat aaaaataatt acactaaaaa 4740

tcatattaag gatgatgtct agttgatgaa taattgatct aactaaattc taattgggtg 4800

aatttaagtt ttactagtac aaccataata aaaatgtttc atagagtgga aaaggaagca 4860

ttaaattggc tatgaacaca gaattaacaa actcactata agtcctgttt gaaaagaata 4920

atataaaaat tgaagatctt agtactagta gtgaggattc aaatgatcat gtttgtgatt 4980

gtacaacaag gatttgaaac tgcagttgac atgtttgttt gtttgcttgt cctgtttgtt 5040

tggcaactga atgtcaaaaa aatataggca gctcttcccc aaggaattgt cccatttgtc 5100

tttgctaagg aatataatgt acatcctgat attctcagca cagcgtgagt attcaaatct 5160

aattcttata tggctcaatt agaactttct ctcccctcca ttcctttttc cttttcattc 5220

aattactttc tcttttaatc ctaaccaatg gagcttcttc tttaacccaa tgcagtgtta 5280

ttttttgggat gctgattgct ttgcccataa ctctagtgta ctacatcttg ttggggttgt 5340

gaatgaaaga aatgatggat gatacagaag attcacgtgt ggcatccatg caaagcttgg 5400

ttgaggttgt tgagaatgag agaaaaaaaa ggtcataaag caacaataga aaagaagcat 5460

cacgagaatt tggataggaa gaagaacccc aggatcagtt ttttttattt atttgttttc 5520

ttttttctttt ttgaatgaat tgccctttct tagtgaaaat taatgtaaaa tcatgatgta 5580

gctaatttac aaaatgatta tctcgttaaa attttatatt ataatgacct cggattccat 5640

gtcactcatc aattgaagga taagaaagca tgagaaactt agttgatgaa aatggtagtg 5700

ctatgatcat ttttcccaat ataatattaa acttcatatt gctttttctt tca 5753

Claims (4)

1. The method for regulating soybean nodulation and close planting by GmPIN1 gene mutation is characterized in that three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in a soybean plant GmPIN1 are mutated to obtain a soybean triple mutant GmPIN1 abc; the method specifically comprises the following steps:

   the nucleic acid sequence of the GmPIN1a gene is shown as SEQ ID No.1, so that the nucleotide sequence between 2513-2541 nucleotides of GmPIN1a is deleted; the nucleic acid sequence of the GmPIN1b gene is shown as SEQ ID No.2, so that the nucleotide sequence between 2589-2617 nucleotides of GmPIN1b is deleted; the nucleic acid sequence of the GmPIN1c gene is shown in SEQ ID No.3, and the nucleotide sequence between 3254-3282 nucleotides of GmPIN1c is deleted.
2. 2. The method for regulating soybean nodulation and close planting by GmPIN1 gene variation as claimed in claim 1, wherein the gene knockout mode is adopted to cause three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in GmPIN1 to have base deletion simultaneously, so that the protein is terminated early.
3. 3. The method for regulating soybean nodulation and close planting by GmPIN1 gene variation according to claim 2, wherein the soybean triple mutant Gmpin1abc is obtained by the following method:

   s1: designing and constructing sgRNA simultaneously targeting three genes of GmPIN1a, GmPIN1b and GmPIN1 c;

   s2: obtaining double-stranded oligonucleotides by annealing reaction;

   s3: linearizing pEF1A2 vector using BsaI endonuclease; s4: the products obtained from S2 and S3 were ligated together using T4 ligase; s5: introducing the plasmid vector obtained in S4 into Agrobacterium GV 3101; s6: using the agrobacterium obtained in S5 to infect soybean; s7: performing plant tissue culture on the product obtained in the step S6;

   s8: and (3) performing plant resistance screening on the tissue culture seedlings obtained in the S7 to obtain the stable transgenic soybean without the CRISPR/Cas9 vector fragment.
4. 4. The application of the method for regulating the nodulation and close planting of soybeans by gene mutation of GmPIN1 according to any one of claims 1 to 3 is characterized by being applied to the regulation of the included angle of the side branches of the nodulation number in the soybeans and related plant types.

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