CN120020255B - Genes that regulate soybean density tolerance and their applications - Google Patents

Abstract

The invention provides isolated genomic sequences regulating the compact-tolerant type of leguminous plants, preferably plants of the genus Glycine, pisum, cinnamomum, e.g.soybean, pea, mung bean, fava bean, black bean, cinnamomum, and the use thereof, said genomic sequences being shown in SEQ ID NO. 3. The invention has important application value for developing close-planted soybeans, intercropped soybeans and the like, and has important value for optimizing soybean plant type architecture and improving the theory and practical research related to soybean yield in unit area.

Description

Gene for regulating soybean compact-tolerant strain and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a gene for regulating and controlling soybean compact-tolerant strain and application thereof.

Background

The soybean can provide abundant vegetable proteins and grease for human beings, and is an important grain and oil economic crop. However, the soybean in China is not changed in long-term dependence on imported situation, the soybean production gap in China is increased year by year, and the single soybean yield is greatly different from the international average level. Therefore, under the actual condition that the available cultivated area of China is gradually reduced, how to avoid competing with main grain crops for land is a concern for improving the soybean yield per unit area of China under the condition that the population of China is changed in dietary structure and the demand of vegetable protein is met. By means of the design of ideal soybean plant type, the soybean planting density in unit area is optimized, and the reduction of field illumination loss is the biological basis for solving the problem.

As early as 1968, donald proposed ideal plant type (ideotype) for crop to guide breeding (Donald, 1968), namely, the molecular regulation mechanism of the existing high-yield plant character is deeply understood, and various excellent characters are optimally combined on the basis, so that crops with ideal plant type are designed, and higher grain yield is expected to be obtained. In the "green revolution", by planting semi-dwarf wheat and rice, the stalk biomass is sacrificed, not only is the grain yield improved, but also excellent lodging resistance is exhibited (Peng et al, 1999). Rice IPA1 (IDEAL PLANT Architecture 1) can reduce tillering number, increase spikelet branching number, and further increase yield (Jiao et al.,2010; miura et al.,2010; wang et al., 2018). Wheat TaCOL-B5 increased yield by 11.9% by increasing the number of branches and nodes of the scion (Zhang et al 2022).

In order to realize the green revolution of the soybean industry, ideal plant types of soybeans, i.e., suitable plant heights, shorter internode lengths, more knots, fewer or no branches, moderate number of pods per knot, high pod yield, high four pod ratio, moderate hundred grain weight, small petiole angle, short petiole, etc. are proposed (Liu et al 2020). In the past, the research of soybean yield improvement mainly focuses on higher grain weight and more pod numbers, which often results in small source and insufficient warehouse, and breakthrough improvement is difficult to realize. Therefore, under the balance of a source and a library, the lateral organs are enabled to have reasonable space positions, the plant type architecture is optimized, the utilization efficiency of the field overall plants on light energy is improved, the soybean yield is improved on the whole, and the soybean branch plant type design becomes an excellent thought for solving the problem.

Therefore, the field light energy loss is reduced, the soybean planting density is improved, and a proper branch structure is considered first. Among the various traits affecting the soybean yield, the branching traits including the length of branches, the included angle between the branches and main stems and the like affect the ventilation and light energy utilization of soybean groups, and are important factors for constructing ideal soybean plant types and affecting the soybean yield. Among these, branch number is an important agronomic trait affecting crop yield. In the gramineous rice, the tillering number is reduced and the scion branching number is increased, so that the yield is finally improved. In dicotyledonous plant soybean, the branch number character belonging to quantitative character is controlled by multiple genes of main effect and micro effect, and is influenced by environmental factors such as planting density, illumination condition and the like, which makes soybean branch number character research particularly complex. At present, no soybean compact-tolerant strain type modeling related genes are reported.

Disclosure of Invention

According to the invention, through functional research on soybean Ln gene, natural variation Ln of Ln obviously reduces branch number, and through transgenic functional verification, ln can shorten petiole, shorten internode, shorten plant height and obviously reduce branch number to single stem, thus being a good close-planting-resistant plant type. The method lays a genetic foundation for molecular mechanism analysis of soybean strain modeling, and provides gene resources for the improvement of the unit area yield of the subsequent soybean.

Specifically, the invention provides the following technical scheme:

in one aspect, the invention provides an isolated genomic sequence of a regulated leguminous plant, preferably a plant of the genus glycine, pea, chickpea, such as soybean, pea, mung bean, broad bean, black bean, chickpea compact-tolerant type, characterized in that said genomic sequence is as shown in SEQ ID NO. 3.

In a further aspect, the invention provides the use of ln for regulating the compact-tolerant plant type of leguminous plants, preferably plants of the genus Glycine, pisum, cinnamomum, such as soybean, pea, mung bean, broad bean, black Bean, cinnamomum, characterized in that the coding sequence of ln is as shown in SEQ ID NO. 1.

In some embodiments, the amino acid sequence of ln is shown in SEQ ID NO. 2.

In some embodiments, the genomic sequence of ln is shown in SEQ ID NO. 3.

In some embodiments, the use is manifested by one or more of the following:

a. Reducing the number of branches;

b. Reducing plant height;

c. reducing the number of the nodes;

d. shortening the internode length;

e. Shortening the length of the leaf stalk.

In another aspect, the invention provides an expression vector, characterized in that the expression vector comprises a genomic sequence as described above.

In some embodiments, the expression vector has an antibiotic marker and/or an anti-chemical agent marker.

In another aspect, the invention provides a host cell, characterized in that the host cell comprises a genomic sequence or an expression vector as described above.

In a further aspect, the present invention provides a method of modulating a compact-tolerant strain of leguminous plants, preferably of the genus glycine, of the genus pea, of the genus chickpea, such as soybean, pea, mung bean, fava bean, black bean, chickpea, characterized in that the method comprises the step of introducing an expression vector or host cell as described above into a leguminous plant, preferably of the genus glycine, of the genus chickpea, such as soybean, pea, mung bean, fava bean, black bean, chickpea, or cell or tissue.

In some embodiments, the compact tolerant plant type exhibits one or more of a reduced number of branches, b reduced plant height, c reduced number of nodes, d reduced internode length, e reduced petiole length, and still grows well when planted at high density relative to wild type or control plants.

Definition of the definition

High-density planting, namely, defining that 50 plants per square meter of Dongnong are planted at a high density of 80-120 plants per square meter, for example, the planting density is 5cm plant spacing and 50cm row spacing.

Low-density planting, namely, defining that 50 plants per square meter of Dongnong are planted at a low density of 26-40 plants per square meter, for example, the planting density is 15cm plant spacing and 50cm row spacing.

Advantageous effects

The soybean plant type modeling related protein, the coding nucleic acid sequence and the genome nucleic acid sequence (including the promoter) provided by the invention are all found for the first time by the applicant, and the phenotypic analysis verification of transgenic plants and wild plants shows that the expression of the soybean plant type modeling protein can obviously influence the soybean plant type.

The invention has great theoretical and practical values for the construction of ideal plant types of soybean and the breeding and creation of close-tolerance varieties and the related basic and application researches thereof.

Drawings

Fig. 1 shows that the ln plant type is compact at low density, indicating that ln can shape the soybean compact-tolerant plant type. Wherein, A shows ln to make the plant compact, B shows ln to obviously reduce the branch number, plant height and internode distance, C shows ln to shorten the petiole, D shows the relative expression quantity of ln over-expression plant in the leaf.

FIG. 2 shows the behavior of transgenic material at different planting densities. Wherein A shows the behavior of DN50 planted at low density (15 cm plant spacing; 50cm line spacing; B shows the behavior of DN50 planted at high density (5 cm plant spacing; 50cm line spacing), C shows the behavior of ln plant planted at low density (15 cm plant spacing; 50cm line spacing), D shows the behavior of ln plant planted at high density (5 cm plant spacing; 50cm line spacing).

FIG. 3 shows an ln genomic expression vector construction profile.

FIG. 4 shows statistics of plant height (n≥30), number of branches, number of nodes, petiole length, and internode length between ln transgenic material and wild type Dongnong 50 material at low density. The method comprises the steps of sample number, sample mean value, sample standard deviation, sample extremum and difference significance level of ln transgenic material and wild type dongnong 50 material, wherein single tail T test is adopted for significance test, P is less than 0.01, and n=30.

FIG. 5 shows the yield test of ln transgenic wild type Dongnong 50 material at different planting densities (LD: low density, 15cm row spacing, 50cm row spacing; HD: high density, 5cm row spacing, 50cm row spacing). Wherein, A shows the single plant yield (n=30) under different planting densities, and B shows the cell reduced mu yield (n=3) under different planting densities. Tukey's multiple comparisons, P <0.05 or P <0.01, were performed using one-way anova.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The quantitative tests in the following examples were all performed in triplicate, and the results were averaged and statistically significant.

In the examples described below, the transformation recipient is dongnong 50 (DN 50, black bean 2007022), which is commercially available. The vector pTF101.1 and Agrobacterium strain EHA101 were purchased from China center for plasmid vector strain cell gene collection (Biovector Science Lab, inc).

Consumable materials such as homologous recombination kit are purchased from Novain Biotechnology Co., ltd and Tiangen Biotechnology (Beijing) Co., ltd.

Example 1ln protein and discovery of the Gene encoding it

The gene ln (Fang et al, 2013) for controlling the soybean leaf tip and four pod traits is identified by a map cloning method in the earlier stage of the subject group, the CDS sequence of the gene is shown as SEQ ID NO. 1, and the coding amino acid sequence of the gene is shown as SEQ ID NO. 2. To further investigate the gene function of ln, we amplified from soybean cultivar DN50 (ln/ln) an approximately 8kb full length of the ln gene (SEQ ID NO: 3), including an upstream promoter of 2.6kb, an ln genomic DNA sequence of 1.8kb, and an ln downstream of 3.2kb (the genomic DNA sequence present in the present invention was chosen to be introduced relatively more closely to the true transcriptional translation in vivo), and transferred it into DN50, found that it significantly improved the soybean plant architecture, which exhibited compact plant type, shorter petioles, NO branching, and internode shortening suggested that it was suitable for close planting, contributing to increased yield per unit area.

Example 2 functional verification of ln protein

1. Construction of recombinant plasmids

Genomic DNA was extracted from Dongnong 50 (DN 50), and the ln gene was subjected to KOD high-fidelity PCR amplification using it as a template, and then homologous recombination was performed to pTF101.1 vector, obtaining recombinant plasmid pTF101.1-pGmln-gGmln. The specific operation is as follows:

1. extracting leaf DNA of soybean variety DN 50.

2. And (3) taking the total DNA obtained in the step (1) as a template, and carrying out PCR amplification by using a primer pair consisting of F1 and R1 to obtain a PCR amplification product.

F1:

5’-ATGTTGACCTGCAGGCATGCAAGCTTGGCGTCAAACTTTACCGAT-3’(SEQ ID NO:4)

R1:

5’-AACAGCTATGACATGATTACGAATTCACTTTTAAGCACACCTGACC-3’(SEQ ID NO:5)

3. The pTF101.1 vector (purchased from China plasmid vector strain cell gene collection center) was digested with restriction enzymes HindIII and EcoRI, and subjected to 1% agarose gel electrophoresis, and an about 8kb linearized vector was recovered.

4. The PCR product of step 2 and the vector backbone of step 3 were joined by homologous recombination to give recombinant plasmid pTF101.1-pGmln-gGmln (see FIG. 3 for a map).

2. Acquisition of ln-overexpressing transgenic plants

1. Recombinant plasmid pTF101.1-pGmln-gGmln was introduced into Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain EHA101 (purchased from China center for type culture Collection of plasmid vector strain cell genes) to obtain recombinant Agrobacterium.

2. And (3) carrying out soybean genetic transformation (Li et al, 2017) on the recombinant agrobacterium obtained in the step (1) by adopting an agrobacterium infection transformation method, and transforming a receptor plant DN50.

(1) Soybean seeds are selected for sterilization, the soybean seeds with big and full grain, smooth surface, no disease spots and no scars are subpackaged and placed in a culture dish, the culture dish is opened and placed in a dryer, a chlorine fumigation sterilization method is adopted for seed sterilization, a small beaker is placed in the dryer, 100mL sodium hypochlorite is added, then 5mL concentrated hydrochloric acid is added, a cover is quickly covered, sterilization is carried out for 10-14h, after sterilization is completed, the soybean is taken out from the dryer, and air is blown for more than 30min in an ultra clean bench, so that redundant chlorine is eliminated.

(2) The soybean seeds are swelled and germinated, namely, the sterilized seed umbilicus is downwards inserted into a germination culture medium, and the soybean seeds are placed in a dark environment for 16-18h at the temperature of 23 ℃.

(3) Agrobacteria preparation, in which the bacteria stored at-80 ℃ are taken out, added into 5mL of YEB culture medium containing rifampicin and kanamycin resistance, cultured at 28 ℃ and 250rmp for 24 hours for resuscitation, then 200 mu L of resuscitated bacteria liquid is sucked and added into 200mL of YEB culture medium containing rifampicin and kanamycin resistance, and the bacteria liquid is collected after culturing at 28 ℃ and 250rmp until OD ₆₀₀ = 0.6. Centrifugation was performed at 22 ℃,5,000rmp for 10min, and then the cells were resuspended in liquid-counterstain medium to OD ₆₀₀ = 0.5,22 ℃, and cultured at 70rmp for 0.5 h.

(4) The preparation of the explant comprises taking out the seed coat of the swelled seed, cutting off the part of the hypocotyl away from cotyledon with a surgical knife to make the residual hypocotyl about 2-3mm, dividing soybean seed into two parts, and removing embryo with small iron brush to obtain the prepared explant.

(5) Infection of the explant, namely placing the prepared explant into an agrobacterium suspension, enabling the heavy suspension to permeate the explant, and culturing for 0.5h by gently shaking.

(6) The explant is co-cultured with agrobacterium, the infected explant is sucked off excessive bacterial liquid on sterile filter paper, a piece of sterile filter paper is placed on a co-culture medium, the sucked-off explant is placed on the medium at 23 ℃ and is cultivated in darkness for 3-5d.

(7) And (3) inducing and culturing the callus, namely transferring the explant into a callus induction culture medium after the co-culture of the explant and the agrobacterium, slightly inserting the explant into the culture medium at an inclination of 45 degrees at the moment, culturing for 14 days at 23 ℃ under 16h illumination/8 h darkness.

(8) Bud induction culture, namely cutting the induced callus from an explant, putting the explant into a bud induction culture medium, culturing for 14d at 23 ℃ in 16h illumination/8 h darkness, and transferring the callus into a new bud induction culture medium every 14 d;

(9) Bud elongation culture, namely transferring the explant with the induced new buds into a bud elongation culture medium, and transferring the explant into the new bud elongation culture medium every 2 times until the buds are elongated to more than 3 cm.

(10) Rooting culture, namely cutting off elongated buds, inserting the buds into a rooting culture medium until the length of roots exceeds 3cm, then hardening seedlings, transplanting the seedlings into a greenhouse, irradiating for 16 hours at 24 ℃ or darkness for 8 hours until the seedlings are mature, spraying Basta and performing PCR (polymerase chain reaction) identification on positive lines.

TABLE 1 formulation of 1L germination medium

TABLE 2 formulation of 1L YEB Medium

TABLE 3 formulation of 1L aggressive baths

TABLE 4 formulation of 1L Co-culture Medium

TABLE 5 formulation of 1L callus induction medium

TABLE 6 formulation of 1L bud induction Medium

TABLE 7 formulation of 1L bud elongation Medium

TABLE 8 formulation of 1L rooting medium

Soybean leaves were coated with 0.1% Basta herbicide (Cool pulse, CB2471-100 mL) and no yellowing response was observed after 3 days as transgenic positive plants. Seeds of the T0 generation were harvested. And the subsequent T1 generation and subsequent passage transgenic lines are sprayed with 0.1% Basta herbicide for screening, and after Basta is resisted, successful ln over-expression transgenic plants are obtained, and the total number of the transgenic lines ln-1, ln-2 and ln-3 is 3 (figure 1).

The ln transcript level expression level was detected by fluorescence quantitative PCR by extracting total RNA and reverse transcribing cDNA. The RT-qPCR primer was designed as follows:

ln gene

F2:5’-GTGTTTGCCTCACATCATTTTTCC-3’(SEQ ID NO:6)

R2:5’-TTGTGCAGCAATGTTATGATCACA-3’(SEQ ID NO:7)

Reference gene ACTIN

F3:5’-CGGTGGTTCTATCTTGGCATC-3’(SEQ ID NO:8)

R3:5’-GTCTTTCGCTTCAATAACCCTA-3’(SEQ ID NO:9)

The instrument model is Roche LIGHT CYCLER 480,480, and the reagent is480 SYBR GREEN I MASTER, the reaction system is as follows (20. Mu.L):

TABLE 9 RT-qPCR reaction System

The RT-qPCR parameters were set as follows:

A thermal start at 95℃for 5min, denaturation at 95℃for 10s, annealing at 59℃for 10s, and elongation at 72℃for 7s for 45 cycles, and a melting curve at 95℃for 5s, denaturation at 65℃for 1min, and rise of 65℃to 97℃at a temperature rise rate of 0.11℃/s were made after the amplification cycle was completed.

At least 3 technical replicates per 1 amplification reaction. The relative expression level was calculated by the method of 2 ^-ΔΔCt.

The results showed that in the over-expressed strain, the ln expression level was increased by a hundred-fold, and the expression level was significantly up-regulated (see fig. 1D).

Transgenic phenotype statistics of example 3 ln

This example demonstrates that transgenic plants over-expressing ln gene in DN50 background are compact in plant type, significantly reduced in branch number as single stalk, significantly reduced in plant height (plant height reduced to 56.8%,49.24% and 46.04%), reduced in node number by 7.01%,11.06% and 11.37%, significantly reduced in node length (node length reduced to 39.94%,38.84% and 37.71%), and significantly reduced in leaf stalks (leaf stalk length reduced to 13.3%,10.35% and 6.75%) (fig. 1A-C, fig. 4). And, compared with the low-density planting condition (15 cm plant spacing; 50cm line spacing), the high-density planting mode (5 cm plant spacing; 50cm line spacing) still shows good ln transgenic plants (see FIG. 2). The embodiment shows that the ln gene overexpression can significantly improve the plant type, so that the plant architecture is more compact, and plays an important role in dense planting resistance.

For this reason we further compared the yields at different densities. The results showed that high density planting resulted in 68.2% yield reduction for DN50 individuals, whereas the single plant yield was only 22.9% lower for ln transgenic plants (fig. 5A), suggesting that ln transgenic plants can increase overall yield by increasing the number of plants per unit area. Furthermore, the cell yield under different planting densities is identified, and the ln transgenic plants can be obviously increased by comparing the yield per mu. Ln transgenic plants were increased by 38.5% compared to DN50 under high density planting conditions and by 27.9% compared to DN50 under low density planting conditions (fig. 5B). The result again proves that the ln transgenic plants can reduce the field illumination loss and increase the light energy utilization of unit area by increasing the planting density, and finally, the unit yield of the soybeans is improved.

Sequence(s)

SEQ ID NO 1ln protein CDS sequence

ATGAGACCAGAACGAAACCCCTTACATCTTAACAATTTGCCCGATGAGTACTCTAGAGATGGCAAACAAGTCCTCGAAGACCATACCTCTTCATCCGGTTGCAGGAAAAAGAAAAGCGGCGGGAAGGATGGAAAAGACGAGTGTGGGAAGGTCTACGAGTGTAGATTTTGTTCCCTCAAGTTCTGCAAGTCTCAGGCTCTTGGGGGACACATGAACCGCCACCGCCAAGAGAGGGAAACGGAGACGCTGAACCAGGCTCGTCAACTGGTCTTTCGTTGTGATCATAACATTGCTGCACAAGGTGCCCCTCACTTAGGATGCTGCCAAACAATAGGAACGGGGGGTTATCATCCCTCAGGAGACCCAACAGTGCCTCTAAGATTCCCAAGATACTTCTCAGGTTCATCCTCAACTCACATGCCACCATCCCCGCCACCGCCGCCGCCACCGCAACGACCATACCTATACCCTTCACCTACGAGGCCAGTGTCATTTGGGTCATCACACTTCCCTCTCCAGCATGCAGTGAACGATTACTATGTGGGCCACGTGATGAGTGGTGGCAGCCACGGACACTATGTTGGAGGAGAGAGCACAAGGAGTTACACGTGCATTGGTGCACCGGTGGGGCAAGGTGGCGGATTCGCTGGTGGTAAGGAGGGGTCTGCAGTGCAGGAGGAAGGGTTGAGTACTTGGGGAAGGGGCTATTCAGGTGCACAGGATCGTTTGGATCCTCCCTCAGCGATCAATCGGTTTCAAGATGGTTTCTAA

SEQ ID NO 2ln protein amino acid sequence

MRPERNPLHLNNLPDEYSRDGKQVLEDHTSSSGCRKKKSGGKDGKDECGKVYECRFCSLKFCKSQALGGHMNRHRQERETETLNQARQLVFRCDHNIAAQGAPHLGCCQTIGTGGYHPSGDPTVPLRFPRYFSGSSSTHMPPSPPPPPPPQRPYLYPSPTRPVSFGSSHFPLQHAVNDYYVGHVMSGGSHGHYVGGESTRSYTCIGAPVGQGGGFAGGKEGSAVQEEGLSTWGRGYSGAQDRLDPPSAINRFQDGF

SEQ ID NO. 3ln genomic sequence (including promoter, 5'-UTR (underlined), exon, intron, 3' -UTR sequence, downstream sequence)

TGGCGTCAAACTTTACCGATAAAAGAAGAAACTTTTGAATTGTTTATAAATAGTTAACTATACAATTATAGACAGGGTAATTCGGTTTGGTTTCAAAGAATGATTTGATTTAATTTGGTTTGAAAGTTTTGCTAGTGGTATATTTACTGTTGGTTCCCCGCCTCATGTATGTTTAGAGTTGAAAGTCGAAACCTCTTGAGATTGCATCATTTAAGCGCTTACAGTACCCCTCGGCTCAAATGAATTTGTCAGGAAACTTTTTTCCTCTAATACATATCACCATAATCTGATTTGTAGTACCACGATCTGGAACAGATATTCTCCTCCCCACGTTTTTATCAATAGAATCCAACACTCCTTGTAAATTTGAAATATGAAAGTTTTAATACTTGATGTTATTTTGGTATTTCTCATGGTCTAAAACTCGATCTCATGTAATTTACAATCTTTGGCTTTGTTTGTTTCATTTCTTTGCCTAAAATATAATTAATTGACGGGTTTGATTTCATGCACTTTCTGTCACAAGGAAAGGTAACATTTAAAAGTTTTCAATTTTAAATTTTGACTGCTTGAAGGGAAGCTAGGTAGGAAGGCAACATGATGATGAAGACAATTTCCTCTACTGGTTTGTAAGTTTGCCTTGCTAGAGTTCTGAACATGGAAGAAATGTTTGTTGGATTTACTGCTTAAATTCGTGAAGGACCACTTGAAAAGTAATGATATATATACACACACTTGCAAGAAAAATTTCAGATAGGTGGGGCGCGGTCATTGCATGAATCTTTGGGTCTAGGTTTTGAAAATATCGAATGTGTCTCATATCATTTCCTTCATAATACCAACCAGCTAGTCAAGGCCAGCTGCAGTAGTGTGGTCCATGAATATCACCTGATTTTAATGCCTAGCTAGCCCTCTTATTAGGTTGTGTCTTCTTTCTCCTTTATATCCATTTCAGGTGTGTGGTGGGGGAAGCACTGATCAGCTTGTGGACAACATCAGCCCCTCTAACTATTGTTTCTTTTTGCCATTTACACATATATGCTGCTCCAAATCAAGACCCAGCCCTTAATTACCAAGTAATTAAAACAGCTGGTAATTATTATTATTATTATTATTATTTCAACCGTTAAAGCTAGCTAGCTTTTTGACCTGTACTAGTAGTATGTATATATACTTTTCTCTCAGCTTTAAACTAGATAGTAATCAATAACCCCCACCACTATTTTTTAAAAAAAAAATGTAAAACAGTTAATTGCTTCTGCATATTAATTTGGAGAACACTTTGTTTTACCTAGCTAACTAGTTAACTATCTTATAAAGGAATCACTCATTAGAGATGGAGCTATAGTGTTTGGATTCAAAGAAAACGCATCTAAAGCCAACAGAGAATATAATGATGAATGTGAGACATACATTAAGAAAATTCAAGTTCATATATTTCTCCACTTTTTTCATGAAAAGGACTATGTATATGTAATCTATGAGATATAGCAACTTTCAAAGAATAAAACAACAAGTGGGGGGTCGAGTCTATCTCAGCTTAGATTGCAAAAAACCCTAATGATAGATACTGATAGCAACCCTTCCTTGATGCAAGTCTAAGTCCCCTGGGTAGAATTTTACCCCCAGAAGGAGGCATTGAGCTATTGTGGACCATACACGAACTGTTCTCAAACATGCCCCCCCTCTTTAAGCACAATAACCACGCAAAATGTCTACGTACCCTATGCCTTGTCTTGCTTTCTTTCTGGTATAAATGTCATCTTTCATTCTTAGGGCAACCTTTAATGCTCAAAGATTTGTACTGTACATTTGATGGACATCCTTTGTTGGTAAAGATTTTCTTTTGAGATATTAATTAATTATTTTTTTCAGTGTATTTGACTGATATTGTTTGAATATTTTTGGACCCTTTGTTGATAGCTCTAGCTAGGGCAAAACAAAAAAAAACTAGATGCAGAAGTATCATATATCTATGCAACATCATATTTTTGCAATAAAATTATATAATAACTAGCATAAATGAAATAGTCATTTTTTTTTATATATCTCTTCATCATATAATTCACTTTATCACATGTTTCTTTTTATCTCTTATTTGTTGTATATATTTATTATATTAATATAAATTCTCTATAGATTACTAAATAGGAATAATTAATACAAAAGCAACATGTTTCCGAATCACATATCTTTGCCCTTTTCTAATTCTGTCCTATCATTTCCCAAGTCTCAACCAAACTAGATTAAGTTAAGCCCCCCCCCCGCCCCCCCCCTCCACACTCACTCTCACACTCTCTTTTTTTAAAGTCACGAGACCCACTGTCCAATTATTCATTCAATTTTTGTGTGGGGGGGCGGGGGGAGATAAGAGAGAGAGAGAGAGTGGCAGAGGAACTGATAGAGAACTTTCAAAGCTCTTTATCTTCTACCATCACTCAACCAGCCTCTATATTGCAGTCTCAAACTGAAAGTCTAAAATTTTTTGTAAAAAGCTTTAGTTTTATCCCTACCCCCACCCCATCTGAAAGAAAGAGTGTTTGCCTCACATCATTTTTCCCCTTTCTGTCTCTCTCTCTGTCGGTACCATGTAGGTCTTCCCCCACTACTACACCTTCACACCCTTCTTTTCTTCCTCCTCTTTCTCCTTCTCTAAACCCTTTAACTTTCTCTCTCTTATGACTTTGTTGTTCCTTTACAGGAGACCAGAACGAAACCCCTTACATCTTAACAATTTGCCCGATGAGTACTCTAGAGATGGCAAACAAGTCCTCGAAGACCATACCTCTTCATCCGGTAAACTTCATGATCATACCAATATATATATATATGCACGCTGAAATATCAAAGGAACACTTTTTTTTTCTTTCTATTTACCGAATTCTATTTCACAATCACACACCACCATGGGAAGCCTTTCACTTTTCACAGGAAACAAAGTTGAACACTGCCCGTGGTTTGTTACCCATCCCTGTGGTATATTCCTTGACCATGAAACAGTGTTCACCACACTTTTTTTTCCTCATTTTTTTATTCTTTTTGTTTTGTCCTTGTATACGTTTCTTTTCTTTTTTTGTTTCTTCTCCTTTTCCTCTTCTACTTCGTTATAGTATCTCTTAAAAGTTATGGAAAAAATAAAATAAGAAAAACATAAAAGTAGTTAGGTGATAGAATAGAACTTAAAAAAAAGAGAGTATTTGTGTGCTGGCGAGCGGTAAGTTAATTAATTGAATTGATTTGATGGAGGATTTGGGTAATCGAGAATGGGGAGTTAAAGGCAAAGAAGGGTGTCATGGAAGTTGCTTTAATTGGATGGTTATAGTTGCAGGTTGCAGGAAAAAGAAAAGCGGCGGGAAGGATGGAAAAGACGAGTGTGGGAAGGTCTACGAGTGTAGATTTTGTTCCCTCAAGTTCTGCAAGTCTCAGGCTCTTGGGGGACACATGAACCGCCACCGCCAAGGCTAGTACCACCTATATTATTATACTTTATATTTTCTTCCTCTTTTCTTTTACCACTCCTTCTCACAAGAACCAAATCTCTTCTGGCCGTTAATGATTTGGCCATGAAAGCTTGTCATTTCAATCACAAAATGTTGTGTTGTTGGATTGCTGAAATTCGATCATCGAGAGGGCCCCTTCAGCTGTTTTTTTTTTTTCTTTTTGCTTTGCTTTATTATTTGTTTTTGTGATTGTTTTGAGCAGAGAGGGAAACGGAGACGCTGAACCAGGCTCGTCAACTGGTCTTTCGTTGTGATCATAACATTGCTGCACAAGGTGCCCCTCACTTAGG

GTATGCACCCATTACTTCCATGCAGCGATTCTCTTCCTATTTCTTCTTC

TTGTTCAACATTTATATATTTCATTTCTCAAATACTTGTTTTTCTGTGGG

TTCTTTAGCGTTTGTTAACGTTGTTTTTTCAAGGTATAACACATAATAT

TTGGGTACTCAGCATGACACTGTTTGATACTGCGATTTATTTGTGATA

ATATTCAATTCAGATGCTGCCAAACAATAGGAACGGGGGGTTATCATC

CCTCAGGAGACCCAACAGTGCCTCTAAGATTCCCAAGATACTTCTCA

GGTTCATCCTCAACTCACATGCCACCATCCCCGCCACCGCCGCCGCC

ACCGCAACGACCATACCTATACCCTTCACCTACGAGGCCAGTGTCAT

TTGGGTCATCACACTTCCCTCTCCAGCATGCAGTGAACGATTACTATG

TGGGCCACGTGATGAGTGGTGGCAGCCACGGACACTATGTTGGAGG

AGAGAGCACAAGGAGTTACACGTGCATTGGTGCACCGGTGGGGCAA

GGTGGCGGATTCGCTGGTGGTAAGGAGGGGTCTGCAGTGCAGGAGG

AAGGGTTGAGTACTTGGGGAAGGGGCTATTCAGGTGCACAGGATCG

TTTGGATCCTCCCTCAGCGATCAATCGGTTTCAAGATGGTTTCTAAAG

AGATGAGAGATTCTTTGTTTGAGTGGTTTTTGGTTTGTGTTATGTTTC

TGTTATGTTATGTGGTATCATCATAAAGACACGCAATCCAGAGAGAGA

GAGAGGTGGTTTTTGGTTTATCTACAGTAACCAGGGACAGCTTTTGA

GCTCATGGACACGCTCAGCTACTTTGGCTTGGAGTTGTGGGTGGGAT

TTCTTCTCAACATCGCCTTTTGTATTGGTAGCTAGCTAGCTAGCTTGG

ACAACCAGTTAGATTTGAACTGAAACTTTCCAAACTCCTTTTCCTTT

GCATGCCAAATTACAACCATTTTCCTTCTCAGTAATTTGTTCTTTCAAT

ATCCTTCTATTATATTACACTAGTAACTAGTAAGTAATATCTATCTCTGT

TACTATTTTATATATACATCCTTCTATTTTATATATGCCTGTTTTTGTGTT

TAATTTGCTATTCCAAAATCTCTAAAACCCCCCGTCACTCTCGATCGC

TCTCACTCTCTCTATCCAAACCAAAATTGGGATCTTCTAGTTTCTTGA

AATGTTTTATGGTCTCACTGTCACACTATGCTAGCACAATTAGGGTAA

TGACACAGAATTTTTGGAGTCCTCTTGCGAAAAAACCATCTTGCTGA

ATTAATGCTTCCGGAATAGGATATTCAAACAATCAAATTTGCCAAAAT

ATCTAATTTCTTCTTAAAGAAAAGCTTTATCCTCCCTCCCTATTTCTAC

GATATTGTAAATAATTATTTTAAAATGTTAAAATACAGATCGAGATTAT

TTTTAGCAGCAGACAGTGAATGCAGCTATATTTAATTTGCTTGGTACA

TACCGAACATAACTACTATCTAAGGAATAAAAACACTCTTGCTGTCA

GCTCCAAGAGCTTCTACCAAATGCAGCAGCTTAGAGAAATATATACA

CAGCCATAACTAGCTCATGATCGTTCTTGAAACAGTAAAACACGTGT

AAATAATGAAATAATGAAACCACATTATTAGCATGATTTTTCTTTTCTT

TTCTCTTGTTTTCAATACTTTTATTTACCGACTAGTTCTCTCCTCTCTG

TTGGAAATGGATATATTTTTCTTTGTATATAGGTTGTGCTTCCTTTACG

CACAGTAAGTGAGGATTTTATTATAAGTAAATTATCCTCATAAAAAATA

TTTTAAAAAGGTATATTCTGGCTTGGCTTCTACTATTCCTATATATTTTT

GGTTCGTGTTATATAGTTTAGTTCGTGTTTTCACTCAAGGAGCATTAA

TTCCTTGACTCAACAAAGGCAAATCTGCTAAACCATTAATAAGTGAT

GAAATTTATTTTTTCTAGGGATGGTAAAAATGGTTTGTATCGTATGAA

AAGGAAAGGCGATGATTAGTGACTTAGTGGCAGTGTTTATGGTAGAT

CCAAATATTCAATGGCAATAGCAACATTGGCTGATGCGGCACGAATT

CAGTGGCATGGTGATGATGGCAAGTAGTGACATCAGCAATTAAAGAT

GAAGATGGAAGAAGGTGGTGGTAGTAAATGGCTGCAATAACAATTG

CTATACAGCAAAAGAATGTACGTAAATAATTTTTTTTAATAGAATACAT

GTATTCTTTTCATTATATAATTATGCAAGAGTGATAATTATTATGGGCGA

TATATTTTTTCTTTAAAACAATTATATATAAGTTGATTTACAAGTTTAGG

AGAAGAATATAATGAGTACTTAGAATTTAATTAGGATTGATTTATGAA

AAATAGGTGTCTAAATTAGAATTATTTGTCAAGAGTGAAATTTGAATT

TACATTTTGGCTTGGCTGGTAAAAGTAAAAAAGACACTACAAATTTT

GAATTTAACTCATTTAGTTTATTATATGACTTAATTGTATTTTAATGTTT

AATTTCTATTTAAATAATTTTTTATATTATAAATTCAATATATTTAACAA

ATTCATGTTTGAAGAATAAAGTGAAATAGTGCCACAAGACCTATGGT

TCAGCATCTTTCTTTTCACTTGATCCATCTCGCTTTAATTAGAGTCATA

ATATATCTCTTCTGATCTTCTACTGCCTGCGGTACAAGTCAACACATA

GTTCAATAATTGTCATCTTAATGTTGGTTGACAAGTGAAGCATATATAT

GTCGAAATAGTTGGACTTATATTTTGCATAAGATCACAGCTTGTGGGA

TTTGTTTCAGATTTTAATCAATTTTGTTTGCTCACAAAAATGAGTTTAAATAATCAAACATTTAATGAGTCAACAACTTAGAATGAAACACACATAATTTAACTCCTTACAACTGTTAAAAAAAAAAAAAAAAGCATGATCCAACGTGGTAATGTTCAGCAAGGTGGGATGTACTATGTACCTCATGCTTTTTATCCCCTAAGATTTTGGGAGCAATGGATCCAAAAATTTGAGTCATTTTGGAGTCATAATATTTTATAGAATCCAAAAAACAAAGTTGTGCTAATAAAATAAAAATTAAATAATAACTTTATATTCTAACAATATATTAAAAAATTAAGGGGGCACGTGCCCCCATAATATAAATGTAGGACCATCCTGCTCGCGGGGTCTAGGTTCACAAAGAAATTAGAAGGCTTTATTTAATTAAATGATTGTTATATTTAACATGCTTGTAAAAAGCATATTTATGTATGGATAAAAAAGGCATATTTATGTTATTTCTAAATCCGGTAAAAGACATAGAAACATCACTGTGTCTTTTAAACGAATATAAACTAAATGATCGTTATTGATAAATTTTTGTCTATGCGGGTTTATTTGACTTGAATCAAATTAAAGCAAAACTGTAACTTTAGTCAGGCTAGTTACTTCTGTCTGATTCAAAAGTAATGATCGCACTCAGTTTCAATACTTGACCAGATAATTGAGGTTTGAGTTTCCAACCTCACAAATAATTAGAAACCTGTTTAGTCCTGTTTCTCCATGGAGTATTAATTAATTCAAATTCAAATTAGAGGAGTGGCATATATAGAATAGCTTAGCTAGCTAAGTAAGCTAACCTTATATGCCTACACTGAGGTAGCTCTATCATATATAGGTGTAGCTGAGGTGACTAGATAGAACCCAAAGATTACTGAGACATTTCAGGCGGCCTAAGAAGCATAGGCTTGAATTATAGACCAAATCCATTTTTGGGCAAGCCATGGACATGCATGAATGTGGGGACATATATATATTAGGGTTCCAGGTCAGGTGTGCTTAAAAGTG

SEQ ID NO. 4ln primer amplified sequence F1

ATGTTGACCTGCAGGCATGCAAGCTTGGCGTCAAACTTTACCGAT

SEQ ID NO. 5ln primer amplified sequence R1

AACAGCTATGACATGATTACGAATTCACTTTTAAGCACACCTGACC

SEQ ID NO. 6ln fluorescent quantitative PCR primer F2

GTGTTTGCCTCACATCATTTTTCC

7Ln fluorescent quantitative PCR primer R2 of SEQ ID NO

TTGTGCAGCAATGTTATGATCACA

SEQ ID NO. 8 internal reference gene ACTIN fluorescent quantitative PCR primer F3

CGGTGGTTCTATCTTGGCATC

Fluorescent quantitative PCR primer R3 for reference gene ACTIN of SEQ ID NO. 9

GTCTTTCGCTTCAATAACCCTA

Reference to the literature

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The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (7)

1. The use of overexpression of the ln gene in regulating the dense-planting tolerance of soybean, characterized in that the amino acid sequence encoded by the ln gene is as shown in SEQ ID NO:2, and the dense-planting tolerance is manifested in one or more of the following: a. Reduce the number of branches; b. Reduce plant height; c. Reduce the number of sections; d. Shorten the intersegmental length; e. Shorten the petiole length. 2. The use according to claim 1, wherein the coding sequence of the ln gene is as shown in SEQ ID NO:1. 3. The use according to claim 1, wherein the nucleotide sequence of the ln gene is as shown in SEQ ID NO:3. 4. The use of an expression vector containing the nucleotide sequence shown in SEQ ID NO:3 in regulating the dense-planting tolerance type of soybean, characterized in that the dense-planting tolerance type exhibits one or more of the following: a. Reduce the number of branches; b. Reduce plant height; c. Reduce the number of sections; d. Shorten the intersegmental length; e. Shorten the petiole length. 5. The use according to claim 4, wherein the expression vector is labeled with antibiotics and/or with anti-chemical reagents. 6. Use of a host cell comprising the expression vector of claim 4 or 5 in regulating soybean density-tolerant plant type, characterized in that the density-tolerant plant type exhibits one or more of the following: a. Reduce the number of branches; b. Reduce plant height; c. Reduce the number of sections; d. Shorten the intersegmental length; e. Shorten the petiole length. 7. A method for regulating the dense-planting tolerance of soybean, characterized in that the method includes the step of introducing the expression vector as described in claim 4 or 5 or the host cell as described in claim 6 into a soybean plant, cell or tissue, wherein the dense-planting tolerance is characterized by one or more of the following: compared to the control or wild-type plant, a. reduced number of branches; b. reduced plant height; c. reduced number of nodes; d. shortened internode length; e. shortened petiole length.