CN112501179A

CN112501179A - Method for regulating soybean nodulation and close planting by GmPIN1 gene mutation and application thereof

Info

Publication number: CN112501179A
Application number: CN202011261356.2A
Authority: CN
Inventors: 陈栩; 崔原原; 柯美玉; 高震; 黄来妹; 陈娇梅
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2021-03-16
Anticipated expiration: 2040-11-12
Also published as: CN112501179B

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 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 soybean symbiotic nodulation, and the nodulation number of soybeans is reduced and the side branch included angle 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

Method for regulating soybean nodulation and close planting by GmPIN1 gene mutation and application thereof

Technical Field

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

Background

The soybean is one of the important economic crops in China, is rich in protein and grease, and has very high nutritional value. However, with the increasing living standard of residents in China, the soybean yield in China cannot meet the requirements of the residents, so that a large amount of soybeans need to be imported from foreign countries every year in China. According to statistics, the import quantity of soybeans in China is rapidly increased from 1394 ten thousand tons in 2001 to 8806 ten thousand tons in 2018^[1]. Further analysis finds that the yield per unit area of the soybeans in China is almost unchanged in the period of 20 years, and the yield per unit area of the soybeans in China is far lower than that of the soybeans in the United states, Brazil and Argentina at the same period^[1]. Therefore, it is very necessary to increase the yield per unit area of soybean in our country.

Agricultural production practices over the past decades have shown that the application of fertilizers can be effective in increasing the yield of crops. The statistical data of the food and agriculture organization of the United nations show that the contribution rate of the application of the fertilizer to the crop yield increase reaches 40 to 60 percent^[2]. During the period from 1952 to 1992, the correlation coefficient between the fertilizer application amount and the grain yield in China is as high as more than 92 percent^[2]. However, the absorption and utilization of fertilizer by crops is not efficient. The statistical data shows that the utilization rate of nitrogen for crops is 30-60%, the utilization rate of phosphorus is 2-25%, and the utilization rate of potassium is 30-60%^[3]. The large amount of fertilizer application not only causes waste, but also brings harm to the surrounding ecological environment and human health. Nitrogen-fixing microorganisms in nature can convert nitrogen in the atmosphere into ammonia which can be absorbed and utilized by plants. Wherein the nitrogen fixed by the root nodule formed by the leguminous plant and the rhizobium accounts for about 60 percent of the total nitrogen fixed amount of the biological nitrogen fixation^[4]. It has been shown that under the same planting conditions, soybeans areLegume crop with maximum nitrogen fixation^[5]. The nitrogen fixation amount of the soybean accounts for more than 50 percent of the total nitrogen absorbed by the soybean^[5]. Further research shows that the symbiotic nitrogen fixation of soybean root nodules plays an important role in the development of seeds^[6]. Researchers have also found that inoculation with rhizobia can result in a dramatic increase in soybean yield^[7]. In addition, the yield of soybean can be obviously improved by increasing the output of nitrogen in the root nodule^[8]. In conclusion, reasonable utilization of nodulation and nitrogen fixation of soybeans is beneficial to improving the yield of soybeans, and reasonable utilization of biological nitrogen fixation can effectively reduce application of fertilizers. Therefore, it is very necessary to understand the growth and development of soybean nodules. Auxin as an important hormone for regulating plant growth and development has been reported to play a crucial role in the root nodule development process of leguminous plants such as Lotus corniculatus and alfalfa^[9-12]. However, the research on the PIN gene in the root nodule development is still relatively few, and no relevant report is found in soybean^[13]. Therefore, the development of PIN gene-related studies in soybean is essential for an in-depth understanding of the nodule formation mechanism. Meanwhile, the method provides a necessary theoretical basis for obtaining the soybean plants with high nodulation efficiency.

In addition, the plant type is an important character determining the high yield of the soybean, and the cultivation of the high-yield soybean with an ideal plant type by optimizing the plant type of the soybean is always the target of breeders and researchers for decades, however, the reports of major genes for regulating and controlling the ideal plant type of the soybean are less^[14]. An important index of an ideal plant type is an included angle between a lateral branch and a main stem, and the size of the included angle influences the canopy structure and the whole light receiving area of the overground part of a plant and finally influences photosynthesis and yield. The smaller the included angle is, the leaf surface can be extended upwards, the occupied space area of the whole plant is small, thus being beneficial to reasonably utilizing the light source and having good ventilation, and promoting the average acre yield to be improved by close planting. It is known that auxin plays a key role in controlling the apical dominance and lateral shoot growth of plants, and a large amount of auxin produced by the apical buds is transported down to the lateral buds, resulting in the inhibition of the growth of the lateral buds^[15,16]. Thus, auxin transport control is utilizedThe included angle of the lateral branches has important application significance in the close planting of plants^[17]。

Disclosure of Invention

The invention aims to create a gene-edited soybean Gmpin1abc triple mutant without GmPIN1a, GmPIN1b and GmPIN1c proteins capable of normally transporting auxin, and provide good germplasm resource reserve for establishment and application of ideal plant types of soybeans.

In order to solve the technical problems, the invention adopts the following technical scheme:

a 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;

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

Furthermore, three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in the GmPIN1 are subjected to base deletion simultaneously in a gene knockout mode, so that the protein is terminated early.

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

s1: designing and constructing sgRNA simultaneously targeting three genes of GmPIN1a, GmPIN1b and GmPIN1c by using a CRISPR-GE (http:// skl.scau.edu.cn/home /);

s2, obtaining double-stranded oligonucleotides by using an 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 (Basta) screening on the tissue culture seedlings obtained from the S7, thereby obtaining the stable transgenic soybean without the CRISPR/Cas9 vector fragment.

The method for regulating the nodulation and close planting of the soybeans by the GmPIN1 gene mutation can be applied to regulation of the nodulation number and side branch included angle in the soybeans and related plant types.

Has the advantages that:

the invention creates a gene-edited soybean triple mutant without Gmpin1abc capable of normally transporting auxin, soybean protein GmPIN1a, GmPIN1b, GmPIN1c and coding genes thereof for the first time, which are closely related to the occurrence of symbiotic nodulation of soybeans, and after gene knockout, the nodulation number of the soybeans is reduced and the side branch included angle of the overground part is obviously weakened. 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.

Drawings

FIG. 1 is a nucleotide sequence of Gmpin1abc in which gene 1 edits soybean homozygous triple mutant.

FIG. 2 is the nodulation phenotype of the gene-edited soybean triple mutant of wild type soybean and Gmpin1abc in example 1;

FIG. 3 shows the plant phenotype of the soybean triple mutant edited by the genes of wild type soybean and Gmpin1abc in example 2;

FIG. 4 is the lateral shoot phenotype of the homozygous triple mutant soybean with the gene editing of Gmpin1abc-L1 in wild type soybean in example 2.

Detailed description of the invention

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental methods and apparatuses in the following examples are conventional methods and apparatuses unless otherwise specified. The test materials used in the following examples were all purchased from conventional biochemical laboratories unless otherwise specified. The determination of the mutant mutation sites in the following examples was determined by sequencing by conventional sequencing companies. In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention is provided in connection with the specific embodiments. Examples of these preferred embodiments are illustrated in the specific examples.

It should be noted that, in order to avoid obscuring the technical solutions of the present invention with unnecessary details, only the technical solutions and/or processing steps closely related to the technical solutions of the present invention are shown in the embodiments, and other details that are not relevant are omitted.

In the examples, No. 6 Huachun was selected as the soybean cultivar. Huachun No. 6 is a soybean variety which is bred by agricultural college of southern China agricultural university and is bred by article Guizao No.1 multiplied by Brazil No. 8. The third meeting of the second national crop variety approval committee passes by 7 and 28 days in 2009, and the approval number is national approval bean 2009012.

Soybean CRISPR/Cas9 vector pGES 201: the description is as follows: bai M, Yuan J, Kuang H, et al.Generation of a multiple Mutagenesis ia poled CRISPR-Cas9 in Soybean [ J ]. Plant Biotechnology Journal,2019,18(3) ]: the public is available from horticulture center, the strait joint research institute, university of agriculture and forestry, fujian.

referring to the attached figure 2, the sgRNA sequence of the gene editing site of CRISPR-Cas9 targets the first exon positions of GmPIN1a, GmPIN1b and GmPIN1c, the boxed region is the targeting site, and the nucleotide marked by an ellipsis is the deletion condition after editing of the gene editing soybean triple mutant of GmPIN1 abc.

S2, obtaining double-stranded oligonucleotides by using an 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.

Example 1

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

The duration of the light during the growth of the soybeans was 14 hours and the temperature was 25 ℃. The soybean seeds were germinated on vermiculite. On the 7 th day after seed germination, rhizobium BXYD3 with an OD value of 0.15 was poured uniformly from the vermiculite surface to the soybean roots, followed by pouring 1/10 nitrogen hydroponic solution for moisture retention and continued cultivation for 14 days.

1/10 Nitrogen hydroponic fluid formulation is shown in the following table:

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

The nodulation phenotype of wild soybean (WT) and Gmpin1abc gene editing soybean triple mutant is shown in figure 2; gmpin1abc shows that the number of nodules number decreased overall and the nodule density (number of nodules divided by root area) decreased, but the average diameter of nodules size profile did not change significantly. The roots of the 7-day seedlings were counted for nodule number, density and diameter 14 days after rhizobium infestation. P-values were tested by two-tailed Student's t-test (. about.p < 0.001).

Example 2

The duration of the light during the growth of the soybeans was 14 hours and the temperature was 25 ℃. The soybean seeds were germinated on vermiculite. On the 10 th day after the seeds germinate, the seeds are transplanted into soil mixed with nutrient soil and loess and planted outdoors for 45 days.

The plant type phenotype of the wild soybean (WT) and the Gmpin1abc gene editing soybean triple mutant is shown in figure 3; after the seedlings in 10 days are transplanted into soil and grow outdoors for 45 days, the whole plant type is short and small, and the included angle of lateral branches is reduced.

The lateral shoot phenotype of the homozygous soybean mutant of wild soybean and Gmpin1abc-L1 gene editing is shown in figure 4; compared with wild WT, the side branch angle (branch angle) of the homozygous triple mutant soybean Gmpin1abc-L1 without the CRISPR/Cas9 vector fragment is remarkably reduced, and the number of side branches (branch number) is remarkably increased. P-values were tested by two-labeled Student's t-test (. sup.

Reference documents:

[1] xiaoweidong, male Ledum, Chinese Soybean industry development, Main problems, reasons and countermeasures suggestion [ J ]. globalization, 2019,05): 105-18+36.

[2] Wangzhong, gold delivery, research progress of precise management of agricultural nutrient resources [ J ] Shanxi agricultural science, 2005,01): 68-72.

[3] Zhengjiangyong, Duliqing, agricultural chemical fertilizer pollution and environmental protection countermeasure in China [ J ] tropical agriculture in China, 2013,02): 76-8.

[4] Lianxin, Shaoxing, Liaohong, contribution and application potential of soybean symbiotic nitrogen fixation in agricultural weight loss synergy [ J ]. Soy science, 2016,35(04):531-5.

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

[6] 338-42 parts of Guohilong, Machunmi, Dukun, et al, absorption and utilization of different nitrogen sources in the growth of spring soybeans [ J ]. Nuclear agriculture bulletin, 2008,03 ].

[7] Role of leguminous plants in the development of ecological agriculture [ J ] agronomy report of Anhui, 2007,07):150-1+06.

[8]CARTER A M,TEGEDER M.Increasing Nitrogen Fixation and Seed Development in Soybean Requires Complex Adjustments of Nodule Nitrogen Metabolism 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 Medicago truncatula 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 is accompanied 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 truncatula PIN 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 bean[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> method for regulating soybean nodulation and close planting by GmPIN1 gene mutation and application thereof

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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 tttttttaaa 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 tttttttaca ctctcatgct tgatgaaagg tttaagtaaa 900

aaaatgttta ttgtaaagct aaattaggtc catgaataat tttaagggag aattacaatt 960

gcaatgacac ttttaatttg taaaaaaata ctcacctcct ttggaatgag tttttttaag 1020

gttatgattt tttaaatgat caacaaactt ttattataat ttgtaatttg atgtcattgc 1080

atatattaat tgacattaat attttagtat gaaaaattta taaaaaaaca attaaataaa 1140

tagtatttga ttaaatagaa ataataattt tcttatcatg acatttattc aagagttaac 1200

aactaaaagg attgattttt 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

attttttata 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 ctactagcat 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 tttttatgat 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 gttatttttg 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 tttttaaatt tatttatttg ttttcttttt cttttttgaa 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 tttattttaa 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 tttttttctt 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 ccagtttctg 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

tttttttgcc 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 tttatttgcc 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

tttttgggat 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

tttttctttt 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

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;

wherein the GmPIN1a gene nucleic acid sequence is shown in SEQ ID No. 1; the GmPIN1b gene has a nucleic acid sequence shown as SEQ ID No. 2; the nucleic acid sequence of the GmPIN1c gene is shown in SEQ ID No. 3.
2. The method for regulating soybean nodulation and close planting by GmPIN1 gene variation as claimed in claim 1, wherein a gene knockout mode is adopted to cause three homologous genes GmPIN1a, GmPIN1b and GmPIN1c in GmPIN1 to have base deletion simultaneously so as to terminate protein prematurely.
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. 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.