CN114316002A

CN114316002A - Soybean trichogenous-associated protein and coding gene and application thereof

Info

Publication number: CN114316002A
Application number: CN202011312656.9A
Authority: CN
Inventors: 田志喜; 刘书林; 张敏; 周国安; 潘毅; 樊磊; 杨霞; 刘智
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2020-09-30
Filing date: 2020-11-20
Publication date: 2022-04-12
Anticipated expiration: 2040-11-20
Also published as: CN114316002B

Abstract

The invention provides a protein separated from soybean and related to the property of no downy, a coding gene and application thereof, and also provides an expression cassette, a recombinant vector and a recombinant expression transformant thereof containing the gene, and a method for obtaining a corresponding gene engineering soybean plant.

Description

Soybean trichogenous-associated protein and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a protein related to a soybean trichogenous-free character, and a coding gene and application thereof.

Background

Plant epidermal hair (Trichome) develops from epidermal cells and is a specialized structure of epidermal tissues present in the aerial parts of most terrestrial plants. The classification can be single cell or multiple cell, branched or unbranched, and glandular or glandular according to the shape, location and self-attribute of the epidermal hair.

Plant epidermal hairs form a natural physical barrier between the epidermal layer and the environment, so that the reflectivity of leaves can be increased, the plant transpiration rate can be reduced, the tolerance of cold damage or ultraviolet radiation of plants can be enhanced, plant tissues can be protected from insect damage, and the like (Kang et al, 2010; Schimiller et al, 2009; Tingey, 1991; Wagner et al, 2004; Wang et al, 2008; Wester et al, 2009), and play an extremely important biological function in the growth and development process of plants. Plant epidermal hair is a model system for studying plant cell fate determination due to its simple structure and easy observation (Schellmann and Hulskamp, 2005).

Plants are subjected to various stresses from biotic or abiotic sources throughout their life cycle, and the epidermal hairs of plants, which serve as the outermost structures of plants, have the function of protecting against external environmental damage (Johnson, 1975; Loughner et al, 2008; Traw and Bergelson, 2003). The plant epidermal hair resists biotic stress and mainly plays a role in a physical barrier mode, and the damage of organisms to plant bodies is reduced. Studies in arabidopsis have found that epidermal hair-free is more susceptible to phytophagous insect damage (Loe et al, 2007).

In addition, the glandular hairs of plants can protect the plants themselves from attack by repelling, trapping or chemically poisoning insects by secreting large amounts of secondary metabolites (e.g., terpenes, phenols, alkaloids, sterols, etc.) (Valverde et al, 2001). In upland cotton, researchers have found that as the number of gland hairs increases with plant age, the amount of n-tridecan-2-one secreted also increases, and thus the insect resistance increases (Leite et al, 2001). The more secondary metabolites in tobacco, such as methyl jasmonate, the better the effect of insect resistance (Laue et al, 2000). In pepper, the material with epidermal hair has a better resistance to pepper spot disease than the non-hairy material (Kim et al, 2011). In tomato, secondary metabolites (zingiberene and acylsugars) secreted by type IV and type VI glandular hairs are well-protected against phytophagous insects such as bemisia tabaci, myzus persicae, red spiders, asparagus caterpillars and spider mites (Liedl et al, 1995; Maluf et al, 2001). In addition, the reduction of epidermal hairs and the change of forms in tomato mutants obviously cause the content of secondary metabolites to be reduced, such as terpenes, flavonoids, polyphenols and the like, and finally show the reduction of insect resistance (Kang et al, 2016; Kang et al, 2010).

In addition to the function of defending against the invasion of foreign organisms, the plant epidermal hair also has very important function in improving the response of plants to abiotic stress. For example, dense epidermal hair tomato material has a higher ability to resist drought and cold under the same conditions (Johnson,1975), and olive leaf epidermal hair can protect plant bodies from adverse conditions such as ultraviolet light and cold (Karabourniotis et al, 1992).

The soybean native product has 5000 years of cultivation history in China, and is an important economic grain crop. However, because the average yield of soybeans in China is low, the soybean production in China at present can not meet the soybean consumption requirement in China.

Various studies have been made on methods for imparting disease resistance and insect resistance to soybeans. For example, in the transgenic research of soybean aphid resistance, bacillus thuringiensis insecticidal crystal protein, Bt gene for short is used; insect-resistant genes derived from plants themselves such as protease inhibitor genes and lectin genes. Meanwhile, there are researches on mosaic virus-resistant transgenosis and soybean cyst nematode resistance against virus resistance, and introduction of other foreign DNA such as chickpea, saponin, etc., and an artificially synthesized insecticidal gene expression vector. However, the main researches of the current transgenic technology are Bt, chitinase, protease inhibitors and plant agglutinin genes, and the number and the types of available genes are limited, so that further screening and identification are needed. In addition, the method has the defects that the common selection markers, antibiotic genes and herbicide resistance genes can influence the human health and destroy the ecological environment.

Compared with the prior art, the self genes of the soybeans from various soybean variety resources are utilized for transgenosis, so that other exogenous genes cannot be introduced into the obtained plants, the gene function is easier to realize, and the safety is greatly improved. Furthermore, if natural characters which are easy to observe, such as root system density, epidermal hair and the like, are used as selection markers, the influence on human health and ecological environment can be avoided, and the method has incomparable advantages.

Compared with crops such as rice, corn and the like, the research of soybean is started later, only genes for individually regulating important agronomic traits including podding habit, flowering time, pod splitting and the like are reported at present, and few genes related to epidermal hair are reported in the reported soybean. For example, the application of soybean flavonoid 3' -hydroxylase, an antler hair color-related gene, to distinguishing gray and brown villous soybeans has been reported (2014), but the existing data on the regulation gene related to the antler hair density is less.

Disclosure of Invention

The invention aims to provide a soybean trichogenous-associated protein, and a coding gene and application thereof.

The inventor discovers a protein related to the downy character from a soybean germline PI 547739, and names the protein as GmP1 protein, and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

When the GmP1 protein is over-expressed by using a plant expression vector such as agrobacterium, the trichome character of the obtained plant is obviously changed to be absent compared with the original germ line. This effect was unexpected based on the prior art, and thus, the inventors have found an isolated protein which is a soybean trichogenous-associated protein, and have disclosed for the first time its naturally encoded gene sequence in soybean.

Phenotypic analysis and verification of transgenic plants and wild plants show that the soybean transgenic plants can show a trichogenous-free phenotype due to the over-expression (in the embodiment, the complementary expression of a self promoter is specifically used) of the soybean trichogenous-free related protein GmP 1.

The invention provides an isolated protein GmP1, which is a protein related to the property of controlling the absence of downy soybean, and comprises the following components:

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO; or

(b) A protein derived from the protein (a) by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of the protein (a) and having the activity of the protein (a); or

(c) The protein (a) or (b) is linked to a protein expressing a purification tag at the amino terminus or the carboxyl terminus.

Wherein, the expression purification tag can be various protein expression purification tags which are conventionally described in the field, and the amino acid types and sequences thereof can be listed but not limited to those shown in SEQ ID NO. 2-SEQ ID NO. 6, specifically see Table 1.

Table 1 sequences expressing purification tags

To achieve the above object, the present invention also provides an isolated gene encoding the protein as described above.

Preferably, the gene consists of the nucleotide sequence:

(1) the nucleotide sequence is shown as SEQ ID NO. 7; or

(2) The nucleotide sequence is shown as SEQ ID NO. 8; or

(3) A nucleotide sequence which is hybridized with the DNA sequence defined in (1) or (2) under strict conditions; or

(4) And (3) a nucleotide sequence having more than 90% homology with the DNA sequence defined in (1) or (2).

The above stringent conditions may be defined as those conventionally used in the art, and may be hybridized at 65 ℃ in a solution of 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate), followed by washing the membrane once with each of 2 XSSC, 0.1% SDS, and 1 XSSC, 0.1% SDS.

In order to achieve the above object, the present invention also provides an expression cassette comprising the aforementioned gene.

Preferably, the expression cassette further comprises operably linked regulatory sequences, such as promoters, terminators, and the like. The promoter may preferably be a Gmp1 promoter, a 35S promoter, more preferably a Gmp1 promoter of the nucleotide sequence shown as SEQ ID NO. 15, independently, the terminator preferably comprises the nucleotide sequence shown as SEQ ID NO. 16.

In order to achieve the above object, the present invention also provides a recombinant vector comprising the aforementioned gene or the aforementioned expression cassette.

The present invention includes the following.

1. A protein which is:

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO; or

(c) The protein (a) or (b) is connected with the protein expressing the purification label at the amino terminal or the carboxyl terminal, and the amino acid sequence of the expression purification label is preferably shown as any one of SEQ ID NO: 2-SEQ ID NO: 6.

2. A gene encoding the protein of claim 1.

3. The gene according to item 2, which comprises the following nucleotide sequence:

(1) the nucleotide sequence is shown as SEQ ID NO. 7; or

(2) The nucleotide sequence is shown as SEQ ID NO. 8; or

(4) A nucleotide sequence having more than 90% sequence identity with the DNA sequence defined in (1) or (2).

4. An expression cassette comprising the gene according to item 2 or 3, preferably further comprising a promoter, preferably a Gmp1 promoter having the nucleotide sequence shown in SEQ ID NO. 15, and independently a terminator, preferably having the nucleotide sequence shown in SEQ ID NO. 16.

5. A recombinant vector comprising the gene according to item 2 or 3 or the expression cassette according to item 4.

6. A recombinant expression transformant comprising the recombinant vector according to claim 5, which is preferably a plasmid, cosmid, phage or viral vector.

7. A method of genetically engineering a transgenic soybean plant comprising: regulating the expression of the protein of item 1, or the gene of item 2 or 3, in a soybean plant, thereby regulating the development process of soybean trichome, for example by transferring to the original soybean plant an expression cassette or a recombinant vector regulating the expression of the protein or gene, for example by transferring to the expression cassette of item 4, or the recombinant vector of item 5;

optionally, the method may comprise detecting expression of the protein or the gene;

optionally, the method may comprise selecting a soybean plant in which development of soybean trichome is regulated by confirming expression of the protein or the gene,

optionally, the detection is performed by PCR, preferably using primer pairs with sequences shown as SEQ ID NO 9 and SEQ ID NO 10,

optionally, the transfer is preferably performed using Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation.

8. Use of the protein according to item 1 or the gene according to item 2 or 3 in soybean genetic engineering.

9. Use of a molecular marker of the gene according to item 2 or 3 for screening of an antler-hair variety in breeding.

10. Use of the gene of item 2 or 3 as a target for gene editing for altering the density of soybean fuzz and/or creating soybean material of varying fuzz density.

Wherein the recombinant vector is obtainable by conventional methods in the art, such as: the gene or the expression cassette is ligated to various expression vectors to construct the gene or the expression cassette. Such expression vectors are conventional in the art and preferably include: various plasmids, cosmids, phages, viral vectors and the like, and the pTF101.1 vector is preferred in the present invention.

In the present invention, the recombinant vector containing a target gene can be constructed using an existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like.

The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor.

When the gene is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation gene, and the enhanced promoter or the constitutive promoter can be used independently or combined with other plant promoters; in addition, when using the genes of the present invention to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may also be used, but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence.

The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In order to achieve the above object, the present invention also provides a recombinant expression transformant comprising the above recombinant vector.

Wherein the recombinant expression transformant can be obtained by a conventional method in the art, such as: the above recombinant vector is transformed into a host microorganism (e.g., E.coli DH5 a). The host microorganism may be any of various host microorganisms conventionally used in the art, as long as it is sufficient to allow the above recombinant vector to stably self-replicate and allow the aforementioned gene or the aforementioned expression cassette carried thereby to be efficiently expressed.

In order to achieve the above object, the present invention also provides a method for obtaining a transgenic soybean plant, which comprises introducing the gene or the expression cassette into a target soybean to obtain a transgenic soybean plant exhibiting a trichogenous-free phenotype compared to the target soybean.

Among them, the method for introducing the objective soybean can be carried out by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and culturing the transformed soybean cells or tissues into plants.

In order to achieve the above object, the present invention also provides a use of the protein or the gene in soybean genetic engineering.

Wherein, the soybean genetic engineering is preferably the soybean genetic engineering aiming at regulating the development process of the soybean downy feathers.

Drawings

FIG. 1 shows the results of gene expression analysis of wild type and transgenic plants in example 3, in which the ordinate shows the expression amount of GmP1 gene and the abscissa shows the line number.

FIG. 2 shows the results of experiments on the number of fuzz of wild type and transgenic plants in example 3, wherein the ordinate shows the number of fuzz and the abscissa shows the number of lines.

FIG. 3 shows the corresponding photographs of the number of hairs of the wild type and transgenic plants in example 3.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

The following examples are for better understanding of the present invention, but are not intended to limit the present invention. The experimental procedures in the following examples, unless otherwise specified, were selected from conventional procedures or according to commercial instructions. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Soybean plant:

dongnong 50(DN50), hereinafter WT (wild type), was used as the wild type soybean, and this variety is a trial variety of Heilongjiang province (also called "black trial bean 2007022") and is commercially available (for example, Niilongjiang dragon family group Co., Ltd.). DN50 is a small bean variety (black bean 2007022) with a sub-limiting podding habit. The height of the plant is about 106 cm, and the plant has branches, white flowers, sharp leaves, grey fuzz and normal density of the fuzz.

PI 547739 is Glycine max L, available from the national department of agriculture, and has a website http:// www.ars-grin. gov/npgs/acc/acc _ queries. html, and PI 547739 plants are not hairy.

Gene:

the GmP1 gene was cloned from PI 547739.

Carrier:

the pTF101.1 vector was purchased from the China plasmid vector strains cell Gene Collection (Biovector Science Lab, Inc).

Agrobacterium strain GV3101 was purchased from the China plasmid vector Strain cell Gene Collection (Biovector Science Lab, Inc).

The kit comprises:

consumables such as enzyme digestion recovery kit were purchased from New England Biolabs and Tiangen Biotech (Beijing) Ltd.

Sequencing company: beijing Liu He Hua Dagen science and technology Co.

Definition of no fuzz: the overground part of the plant is visually observed, and the surface is smooth without hairy hair.

Example 1 discovery of GmP1 protein and Gene encoding the same

Based on a large amount of sequence analysis and functional verification, the inventor discovers a protein from a soybean variety PI 547739, names the protein as GmP1 protein (P1 protein for short), the amino acid of the protein is shown as SEQ ID NO:1 in a sequence table, names a gene for coding the GmP1 protein as GmP1 gene, the genome sequence of the gene is shown as SEQ ID NO:7 (including 2 exons) in the sequence table, and the cDNA sequence of the gene is shown as SEQ ID NO:8 in the sequence table.

Example 2 functional verification of GmP1 protein

Construction of recombinant plasmid

1. The apical meristem in soybean variety PI 547739 was isolated from the plants and DNA was extracted to obtain the apical meristem DNA of soybean variety PI 547739.

2. And (3) performing PCR amplification by using the DNA obtained in the step (1) as a template and using a primer pair consisting of F1 and R1 to obtain a PCR amplification product 5693 bp.

F1：(SEQ ID NO:9)；

R1：(SEQ ID NO:10)。

3. PTF101.1 vector was double digested with restriction enzymes EcoR I and Hind III, recovering a vector backbone of about 9138 bp.

4. And (3) carrying out homologous recombination and connection on the PCR product in the step (2) and the vector framework in the step (3) by utilizing a homologous recombinase (product number TRANSGEN, CU101) to obtain a recombinant plasmid GmP1-PTF 101.1. According to the sequencing result, the structure of the recombinant plasmid GmP1-PTF101.1 is described as follows: the construction of the expression cassette is completed by inserting a GmP1 promoter (shown as SEQ ID NO: 15), a double-stranded DNA molecule shown as SEQ ID NO:7 in a sequence table and a terminator (shown as SEQ ID NO: 16) between EcoR I and Hind III enzyme cutting sites of a PTF101.1 vector.

II, obtaining of GmP1 complementary transgenic plant

1. The recombinant plasmid GmP1-PTF101.1 is introduced into an agrobacterium strain GV3101 by a common method to obtain recombinant agrobacterium, and the recombinant agrobacterium is frozen at the temperature of minus 80 ℃ and preserved by glycerol.

2. The recombinant Agrobacterium obtained in the step 1 is transformed into a receptor plant DN50 by a cotyledonary node transformation method (a mosaic M.P. et al 2004 Assessment of conditioning and manipulating Agrobacterium-mediated soybean transformation. Euphytoica 136: 167-179), and T1 generation seeds are harvested. The specific operation steps are as follows:

(1) seed sterilization and germination

Round and plump DN50 soybean seeds with smooth surface and no disease spots are selected and placed in a culture dish of 120 mm. Putting the culture dish into a dryer, putting a 250ml beaker into the dryer, adding 100ml sodium hypochlorite solution, slowly adding 4ml concentrated hydrochloric acid along the beaker, immediately covering the cover of the dryer, sterilizing the soybean seeds for 18h by using chlorine gas, and covering the soybean seeds in a super clean bench to blow off residual chlorine gas after sterilization. The sterilized soybean seeds with umbilicus facing downwards are evenly placed in a germination culture medium, and 30-35 seeds are placed in each dish. Then, the seeds are wrapped by a fresh-keeping bag, a ventilation opening is cut, and the seeds are placed in a dark incubator, wherein the germination condition is 22 ℃, and the germination time is more than 16 hours.

(2) Infection with Agrobacterium and explant co-culture

Taking germinated seeds, cutting off a part of cotyledons, and longitudinally cutting the seeds into two symmetrical parts along hypocotylsAnd lightly scraping a pair of true leaves at the cotyledonary node under a microscope, and finally lightly pricking the cotyledonary node with a scalpel for several times to obtain the explant for transformation. Thawing frozen-stored recombinant Agrobacterium with-80 deg.C glycerol, dipping in small amount of bacterial liquid in a sterilized gun head, culturing on YEP solid culture medium containing Kan (kanamycin) and Gen (gentamicin) at 28 deg.C for 2 days, coating on YEP solid culture medium containing Kan and Gen, culturing overnight, and resuspending cultured overnight Agrobacterium with liquid coculture medium to OD₆₀₀The value is 0.6. Putting the prepared explant into the resuspended agrobacterium tumefaciens bacterial liquid, placing the explant into a dark incubator at 22 ℃ for infection overnight, then sucking the redundant bacterial liquid on the surface by using sterile filter paper, flatly paving the cotyledon node on a solid co-culture medium paved with the sterile filter paper, and carrying out dark infection at 22 ℃ for 5 days.

The composition of the solid co-culture medium was B5 salt, B5 vitamins, 30g/L sucrose, 0.6g/L MES (2-morpholinoethanesulfonic acid), 1.6 mg/L6-BA (benzyladenine), 100mg/L L-Cys,0.1M DDT (bis-P-chlorophenyltrichloroethane), 0.5mg/L GA3 (gibberellin), 0.2% (w/v) phytogel (Sigma-P8169), pH 5.4)

(3) Transgenic seedling acquisition

Obliquely inserting the cotyledon node after 5 days of co-culture into a bud induction culture medium I (SI-I), culturing for 7 days under 16h of light for 8h of darkness and 5000-6000Lux of light intensity, shearing off overlong hypocotyl, transferring into a bud induction culture medium II (SI-II) containing 8mg/ml of PPT (glufosinate ammonium), and continuously culturing for 14-20 days.

The cluster buds were excised from the hypocotyl, transferred to a bud elongation medium (SEM) containing 4mg/ml PPT, subcultured every 10 days at 25 ℃ for 16h under light intensity of 5000-6000Lux for 8h in the dark until the buds elongated to about 5 cm. Cutting off the bud which is extended to about 5cm, directly inserting the bud into a rooting culture medium, irradiating for 16h at 25 ℃ for 8h in darkness and under the illumination intensity of 5000-6000Lux until the root is extended to 3-4cm, and preparing for transplanting.

In this step, the composition of each medium was as follows:

the composition of the bud induction medium I is B5 salt, B5 vitamin, 30g/L sucrose, 0.6g/L MES, 1.6 mg/L6-BA, 50mg/L cefamycin, 150mg/L timentin (Tim, Phototech-T869-10 g), 4g/L glufosinate, 0.2% (w/v) plant gel, and the pH value is 5.7;

the composition of the bud induction medium II is B5 salt, B5 vitamin, 30g/L sucrose, 0.6g/L MES, 1.6 mg/L6-BA, 50mg/L Cef, 150mg/L Tim, 8g/L glufosinate, 0.2% (w/v) plant gel, pH 5.7;

the composition of the shoot elongation medium was MS salt, B5 vitamin, 30g/L sucrose, 0.6g/L MES, 0.5mg/L GA3, 1mg/L ZR (zeatin riboside (trans)), 50mg/L L-Glu, 50mg/L Asp, 0.1mg/L IAA (indoleacetic acid), 50mg/L Cef, 100mg/L Tim, 4g/L glufosinate, 0.2% (w/v) phytogel, pH 5.8;

the rooting medium consists of MS salt, B5 vitamin, 20g/L sucrose, 0.6g/L MES, 50mg/L L-Glu, 50mg/L Asp, 1.5mg/L IBA (indolebutyric acid), 25mg/L Tim, 0.2% (w/v) plant gel and pH 5.8.

(4) Exercising, transplanting and screening

Removing a sealing film from the tissue culture seedling to be transplanted, adding a small amount of sterile water, irradiating for 16h for 8h in darkness at 25 ℃ and under the illumination intensity of 5000-6000Lux, transplanting the seedling after culturing for two days, uniformly mixing vermiculite and turfy soil in equal amount, putting the mixture into a tray with water, then pulling out the tissue culture seedling from a rooting culture medium, washing out the residual culture medium at the root, and transferring the tissue culture seedling into nutrient soil fully absorbing water. Soybean leaves are smeared with herbicide containing 0.1 percent Basta, and the plants without yellowing reaction after 3 days are judged as transgenic positive plants. Two transgenic lines, namely a CE-1 line and a CE-2 line (an expression vector constructed by a self promoter, namely CE, Complementary expression) are randomly selected from the plants judged to be positive by the transgenes for subsequent identification.

Example 3 changes in the traits of trichome in transgenic plants overexpressing GmP1

1. Identification of expression level of Gene

The recipient strain DN50, the complementary transgenic strain CE-1 strain GmP1 and the CE-2 strain are respectively identified as follows:

(1) the apical meristem of the seedling line was taken, total RNA was extracted and reverse transcribed into cDNA.

(2) And (2) identifying the expression quantity of the GmP1 gene by using the cDNA extracted in the step (1) as a template and a primer pair consisting of F3 and R3, and identifying an internal reference gene (expression quantity of an Actin gene) by using F2 and R2.

Primer sequences used in this example

F2：(SEQ ID NO:11)；R2：(SEQ ID NO:12)。

F3：(SEQ ID NO:13)；R3：SEQ ID NO:14)。

The expression level of GmP1 gene in the obtained different materials was shown in FIG. 1 by quantitative PCR amplification using the cDNA as a template and each specific primer.

The results show that, as can be seen from FIG. 1, the gene expression level of GmP1 is significantly higher than that of the control DN50 by about 2 times in the two complementary expression lines of GmP1, CE-1 and CE-2. It was confirmed that there was overexpression of GmP1 in lines CE-1 and CE-2.

2. Comparison of the number of fuzz of receptor strains DN50 and GmP1 complementary expression transgenic strains

The whole soybean plants in the three groups were observed by naked eyes, and it could be seen that the receptor strain DN50 was normal density of fuzz, and in the CE-1 and CE-2 groups, no fuzz was produced by the plants.

We carried out comparison of the experiments on the number of hairs of the recipient lines DN50 and GmP1 after planting the transgenic lines CE-1 and CE-2, photographing the main stem of the plant by using a camera (the picture is shown in figure 3), counting the number of hairs of 0.5cm long petioles with the same node, counting ten plants in each line, taking off one root by using tweezers, and counting the result as shown in figure 2, wherein the scale bar in the figure represents 1 cm.

As can be seen from fig. 2 and fig. 3, the GmP1 complementation expression transgenic line showed no fuzz phenotype compared with DN50, which indicates that GmP1 gene controls the generation and development of the fuzz of soybean in a presence or absence mode, and the gene mutation thereof can make the soybean show no fuzz phenotype. The method is suggested to be used as a means for researching the stress resistance and increasing the yield stability of soybean plants.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Industrial applicability

The invention provides a gene related to the property of trichome, and the use of the protein related to the property of trichome-free property and the coding gene thereof can research and improve the physical biological stress resistance of soybean plants, the biological stress resistance of glandular hairs, the property of coping with abiotic stress and the like. It is expected that the editing, e.g., introduction or deletion, of genes related to epidermal hair, e.g., genes related to density of dense trichome, transformation techniques, etc., can be applied to the selection and utilization of soybean varieties.

Soybean is one of the important food crops, providing a source of oil and protein for humans and animals. At present, the shortage of soybean yield in China is filled mainly by imported soybeans. In the genetic breeding of soybean, how to improve the stress resistance and increase the stable yield is always a difficult problem which troubles the soybean production. The results of the above examples show that the GmP1 gene in soybean is involved in regulating the development of soybean fuzz, and complementary expression of the gene results in the no-fuzz phenotype of soybean.

Therefore, in production practice, on one hand, a molecular marker can be designed according to the gene and used for screening of the antler hair varieties in breeding, and on the other hand, the soybean antler hair characters can be properly changed according to the target point of gene editing designed by the gene, so that soybean materials with different antler hair characters can be created.

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Claims

1. a protein which is:

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO; or

2. A gene encoding the protein of claim 1.

3. The gene according to claim 2, comprising the following nucleotide sequence:

(1) the nucleotide sequence is shown as SEQ ID NO. 7; or

(2) The nucleotide sequence is shown as SEQ ID NO. 8; or

4. An expression cassette comprising the gene of claim 2 or 3, preferably further comprising a promoter, preferably a Gmp1 promoter having the nucleotide sequence shown in SEQ ID NO. 15, and a terminator, independently, preferably having the nucleotide sequence shown in SEQ ID NO. 16.

5. A recombinant vector comprising the gene of claim 2 or 3 or the expression cassette of claim 4.

6. A recombinant expression transformant comprising the recombinant vector of claim 5, preferably a plasmid, cosmid, phage or viral vector.

7. A method of genetically engineering a transgenic soybean plant comprising: modulating the expression of the protein of claim 1, or the gene of claim 2 or 3, in a soybean plant, thereby modulating the development process of soybean trichome, e.g. by transferring to the original soybean plant an expression cassette or recombinant vector modulating the expression of said protein or gene, e.g. by transferring to the expression cassette of claim 4, or the recombinant vector of claim 5;

8. Use of the protein of claim 1 or the gene of claim 2 or 3 in soybean genetic engineering.

9. Use of a molecular marker of the gene according to claim 2 or 3 for selection of an antler-hair variety in breeding.

10. Use of the gene of claim 2 or 3 as a target for gene editing for altering the density of soybean fuzz and/or creating soybean material of varying fuzz density.