CN114316002B - Soy fuzz-free related protein, and encoding gene and application thereof - Google Patents

Abstract

The invention provides a protein which is separated from soybean and is related to fuzz-free character, a coding gene and application thereof, and also provides an expression cassette containing the gene, a recombinant vector and a recombinant expression transformant thereof, and a method for obtaining a corresponding genetically engineered soybean plant.

Description

Soy fuzz-free related protein, and encoding gene and application thereof

Technical Field

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

Background

The plant epidermal hair (Trichome) develops from epidermal cells and is a specialized structure that is present in the epidermal tissue of the aerial parts of most terrestrial plants. The morphology, location and nature of the coat can be classified as single or multicellular, branched or unbranched, and glandular or glandular.

The plant surface fur forms a natural physical barrier between the surface layer and the environment, can increase the reflectivity of leaves, reduce the plant transpiration rate, enhance the cold injury or ultraviolet radiation resistance of plants, protect plant tissues from insect damage and the like (Kang et al, 2010;Schilmiller et al, 2009;Tingey,1991;Wagner et al, 2004; wang et al, 2008;Wester et al, 2009), and plays an extremely important biological function in the growth and development process of plants. The plant epidermal hair is used as a model system for studying plant cell fate decisions due to its simple structure and easy observation (Schellmann and Hulskamp, 2005).

Plants are subjected to various stresses from living or non-living sources throughout the life cycle, and the plant surface coat, which is the outermost structure of the plant, has the effect of protecting against external environmental injury (Johnson, 1975;Loughner et al, 2008;Traw and Bergelson,2003). The protection of plant epidermis hair against biotic stress mainly acts through physical barriers, reducing the damage of organisms to plant bodies. It was found in arabidopsis that the non-epidermal hair is more vulnerable to phytophagous insects (Loe et al, 2007).

In addition, the glandular hairs of plants can be repelled, trapped or chemically poisoned by secreting large amounts of secondary metabolites such as terpenes, phenols, alkaloids and sterols etc. to protect the plants themselves from attack (Valverde et al, 2001). In upland cotton, researchers found that the number of glandular hairs increased with increasing age of the plant, as well as the amount of n-tridecan-2-one secreted, and thus the insect resistance increased (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 capsicum, the material with the coat hair has a better resistance to pepper spotting than the fuzz-free material (Kim et al, 2011). In tomato, secondary metabolites secreted in type IV and type VI glandular hairs (gingerol and acyl sugars) can be very resistant to phytophagous insects such as bemisia tabaci, myzus persicae, red spiders, asparagus caterpillar, spider mites and the like (Liedl et al, 1995; maluf et al, 2001). In addition, reduced coat hair and altered morphology in tomato mutants significantly lead to reduced levels of secondary metabolites, such as terpenes, flavonoids and polyphenols, and the like, which ultimately manifest as reduced insect resistance (Kang et al, 2016; kang et al, 2010).

In addition to having a role in protecting against attack by foreign organisms, plant coat hair also has a very important role in enhancing plant body response to abiotic stress. For example, dense skin tomato materials have higher drought and cold resistance under the same conditions (Johnson, 1975), and olive leaf skin can protect plants from adverse conditions such as ultraviolet light and low temperatures (Karabourniotis et al, 1992).

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

Various studies have been made on methods for imparting disease resistance and insect resistance to soybean. 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, etc. Meanwhile, there are researches on anti-mosaic virus transgenesis aiming at virus resistance, anti-soybean cyst nematode, and introduction of other exogenous DNA such as chickpea, gleditsia sinensis lam and the like, and artificially synthesized insecticidal gene expression vectors. However, the current transgenic technology research mainly includes Bt, chitinase, proteinase inhibitor and plant lectin genes, and the available genes are limited in number and need to be further screened and identified. Moreover, there are disadvantages in that the conventional screening markers, antibiotic genes and herbicide resistance genes may affect human health and destroy ecological environment.

Compared with the method, the soybean self genes from various soybean variety resources are utilized for carrying out transgene, other exogenous genes are not introduced into the obtained plants, the gene functions are easier to realize, and the safety is greatly improved. Further, if natural traits such as root density, epidermal hair, etc., which are easily observed 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 and corn, the research of soybean is started later, only genes for individually regulating important agronomic traits including pod bearing habit, flowering time, pod bearing and the like are reported at present, and few genes related to coat hair are reported in the soybean. For example, the use of the fuzz-associated gene soybean flavonoid 3' -hydroxylase has been reported to distinguish gray-fuzz-soybean from brown-fuzz-soybean (2014), but there is less current knowledge of the regulatory genes associated with fuzz density.

Disclosure of Invention

The invention aims to provide a soybean fuzz-free related protein, and a coding gene and application thereof.

The inventor discovers a protein related to the fuzz property from 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 of the invention is over-expressed by using a plant expression vector such as agrobacterium, the fuzz character of the obtained plant is obviously changed from that of the source germ line. Such effects are unexpected based on the prior art, and thus the inventors have found an isolated protein which is a fuzz-free soybean-related protein, and have disclosed for the first time its natural coding gene sequence in soybean.

Phenotypic analysis of transgenic plants and wild-type plants shows that overexpression of the soybean fuzz-free related protein GmP1 of the invention (in the examples, specifically complementary expression using the self promoter) is capable of rendering the soybean transgenic plants into a fuzz-free phenotype.

The invention provides an isolated protein GmP1, which is a protein related to controlling soybean fuzz-free traits, comprising:

(a) A protein consisting of the amino acid sequence set forth in SEQ ID NO. 1; or (b)

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

(c) A protein expressing a purification tag is attached to the amino-terminus or carboxyl-terminus of the protein (a) or (b).

Wherein, the expression purification tag can be various protein expression purification tags conventionally described in the art, 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, and the specific reference is shown in Table 1.

TABLE 1 expression of the purification tag sequences

Label name	Number of residues	Preferred sequences
			Poly-Arg	5-6 (usually 5)	SEQ ID NO:2
Poly-His	2-10 (usually 6)	SEQ ID NO:3
			FLAG	8	SEQ ID NO:4
Strep-tag II	8	SEQ ID NO:5
			c-myc	10	SEQ ID NO:6

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 following nucleotide sequence:

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

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

(3) A nucleotide sequence which hybridizes under stringent conditions to the DNA sequence defined in (1) or (2); or (b)

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

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

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 comprising the nucleotide sequence shown as SEQ ID NO. 15, and independently the terminator preferably comprises the nucleotide sequence shown as SEQ ID NO. 16.

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) A protein consisting of the amino acid sequence set forth in SEQ ID NO. 1; or (b)

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

(c) A protein expressing a purification tag is connected to the amino terminal or carboxyl terminal of the protein (a) or the protein (b), and the amino acid sequence of the expression purification tag is preferably shown in any one of SEQ ID NO. 2-SEQ ID NO. 6.

2. A gene encoding the protein according to item 1.

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

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

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

(3) A nucleotide sequence which hybridizes under stringent conditions to the DNA sequence defined in (1) or (2); or (b)

(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 of item 2 or 3, preferably further comprising a promoter, preferably the Gmp1 promoter of the nucleotide sequence shown as SEQ ID NO. 15, and a terminator, independently, preferably comprising the nucleotide sequence shown as SEQ ID NO. 16.

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

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

7. A method of genetic engineering of a transgenic soybean plant comprising: modulating the expression of the protein of item 1, or the gene of item 2 or 3, in a soybean plant, thereby modulating the soybean fuzz development process, e.g., by transferring into the original soybean plant an expression cassette or recombinant vector that modulates the expression of the protein or gene, e.g., by transferring into 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 modulated in the development of soybean fuzz by confirming expression of the protein or the gene,

optionally, the detection is performed using PCR, preferably using primer pairs of the sequences shown as SEQ ID NO. 9, SEQ ID NO. 10,

optionally, the transfer is preferably performed using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electric conduction, agrobacterium-mediated.

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

9. Use of the molecular marker of the gene according to claim 2 or 3 for screening of a fuzz breed in breeding.

10. The use of the gene of claim 2 or 3 as a target for gene editing for altering the fuzz density of soybean and/or creating soybean material of different fuzz densities.

Wherein the recombinant vector can be obtained by conventional methods in the art, such as: the gene or the expression cassette is constructed by connecting various expression vectors. The expression vectors are conventional in the art and preferably comprise: various plasmids, cosmids, phages or viral vectors, etc., the pTF101.1 vector is preferred in the present invention.

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

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

When the gene is used for constructing a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter can be added before the transcription initiation gene, and the enhanced promoter or the constitutive promoter can be used alone or in combination with other plant promoters; in addition, when constructing a plant expression vector using the gene of the present invention, enhancers, including translational enhancers 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 sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection 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 or chemical resistance marker genes which are expressed in the plants, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

To achieve the above object, the present invention also provides a recombinant expression transformant comprising the aforementioned recombinant vector.

Wherein the recombinant expression transformant can be obtained by a conventional method in the art, such as: the 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 capable of stably autonomously replicating the above recombinant vector and allowing the above gene or the above 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 by introducing the aforementioned gene or the aforementioned expression cassette into a soybean of interest to obtain a transgenic soybean plant exhibiting a fuzz-free phenotype as compared with the soybean of interest.

Among them, the method of introducing the soybean of interest may be by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and culturing the transformed soybean cells or tissues into plants.

In order to achieve the above purpose, the invention also provides an application of the protein or the gene in soybean genetic engineering.

Among them, the soybean genetic engineering is preferably one aimed at regulating the fuzz development process of soybean.

Drawings

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

FIG. 2 shows the experimental results of the number of hairs of wild-type and transgenic plants in example 3, wherein the ordinate shows the number of hairs and the abscissa shows the line number.

FIG. 3 shows corresponding photographs of wild type and transgenic plant fuzz numbers in example 3.

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 experimental methods in the following examples, unless otherwise specified, were either conventional or selected according to the commercial specifications. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Soybean plants:

as the wild type soybean, dongnong 50 (DN 50), hereinafter referred to as WT (wild type), which is a black longjiang province variety (also called black bean 2007022), is used and commercially available (for example, black longjiang family group ltd). DN50 is (black bean 2007022) small grain variety, sub-limited pod habit. The plant height is about 106 cm, and branches, white flowers, pointed leaves and gray fuzz are present, so that the fuzz density is normal.

PI 547739 is Glycine max L. From the United states department of agriculture and its website is http:// www.ars-grin. Gov/npgs/acc/acc_queries. Html, PI 547739 plants have no fuzz.

Gene:

the GmP1 gene was cloned from PI 547739.

And (3) a carrier:

pTF101.1 vector was purchased from China plasmid vector strain cell Gene collection (Biovector Science Lab, inc).

Agrobacterium strain GV3101 was purchased from China plasmid vector strain cell gene collection (Biovector Science Lab, inc).

The kit comprises:

consumables such as the enzyme digestion recovery kit are purchased from New England Biolabs and Tiangen Biochemical technologies (Beijing) limited.

Sequencing company: beijing Liuhua Dada Gene technology Co.

Definition of no fuzz: the overground tissues of the plants were visually inspected for fuzz-free growth and smooth surfaces.

Example 1 discovery of GmP1 protein and its coding Gene

Based on a large number of sequence analyses and functional verification, the inventor discovers a protein from soybean variety PI 547739, and names the protein as GmP1 protein (P1 protein for short), wherein the amino acid is shown as SEQ ID NO:1 in a sequence table, the gene for encoding the GmP1 protein is named as GmP1 gene, the genome sequence of the gene is shown as SEQ ID NO:7 (comprising 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

1. Construction of recombinant plasmids

1. The top meristem in soybean variety PI 547739 is separated from the plant, DNA is extracted, and the top meristem DNA of soybean variety PI 547739 is obtained.

2. And (3) carrying out 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 5693bp.

F1：(SEQ ID NO:9)；

R1：(SEQ ID NO:10)。

3. The PTF101.1 vector was digested with the restriction enzymes EcoR I and Hind III, and the vector backbone of about 9138bp was recovered.

4. And (3) carrying out homologous recombination connection on the PCR product of the step (2) and the vector skeleton of the step (3) by using (homologous recombinase, product number TRANSGEN, CU 101) to obtain a recombinant plasmid GmP1-PTF101.1. Based on the sequencing results, the recombinant plasmid GmP1-PTF101.1 was structurally described as follows: the construction of the expression cassette is completed by inserting the GmP1 promoter (shown as SEQ ID NO: 15), the double-stranded DNA molecule shown as SEQ ID NO:7 in the sequence Listing, and the terminator (shown as SEQ ID NO: 16) between EcoR I and Hind III cleavage sites of the PTF101.1 vector.

2. Acquisition of GmP1 complementation transgenic plants

1. Recombinant plasmid GmP1-PTF101.1 is introduced into agrobacterium strain GV3101 by a common method to obtain recombinant agrobacterium, and the recombinant agrobacterium is frozen at-80 ℃ and stored in glycerol.

2. And (3) transforming the recombinant agrobacterium obtained in the step (1) into a receptor plant DN50 by adopting a cotyledonary node transformation method (Margie M.P.et al2004Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node display. Euphytica 136:167-179), and harvesting the T1 generation seeds. The specific operation steps are as follows:

(1) Seed sterilization and germination

DN50 soybean seeds with full round seeds and smooth surface and no disease spots are selected in a 120mm culture dish. Placing the culture dish into a dryer, placing 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 soybean seeds by using chlorine for 18h, and after sterilization, uncovering the cover in an ultra-clean bench to blow off residual chlorine. Uniformly placing the sterilized soybean seeds in germination culture medium with the umbilicus facing downwards, and 30-35 seeds per dish. Then wrapping with fresh-keeping bag, cutting air vent, placing into dark incubator, germinating at 22deg.C for more than 16 hr.

(2) Infection of Agrobacterium and co-cultivation of explants

Taking germinated seeds, firstly cutting off a part of cotyledons, longitudinally cutting the seeds into two symmetrical parts along the hypocotyl, gently scraping off a pair of true leaves at cotyledonary nodes under a microscope, and finally gently pricking the cotyledon nodes with a surgical knife to obtain the explant for transformation. Thawing recombinant Agrobacterium frozen at-80deg.C on ice, drawing lines with a sterilizing gun head, culturing on YEP solid medium containing 100ug/ml Kan (kanamycin) and 100ug/ml Gen (gentamicin), activating at 28deg.C for 2 days, spreading on new YEP solid medium containing Kan and Gen, culturing overnight, and re-suspending the cultured Agrobacterium with liquid co-culture medium to OD ₆₀₀ The value is 0.6. Placing the prepared explant into resuspended agrobacterium tumefaciens bacteria solution, placing the agrobacterium tumefaciens bacteria solution into a dark incubator at 22 ℃ for infection overnight, then sucking the superfluous bacteria solution on the surface by using sterile filter paper, spreading cotyledonary nodes on a solid co-culture medium paved with the sterile filter paper, and carrying out dark infection at 22 ℃ for 5 days.

The solid co-culture medium consists of B5 salt, B5 vitamin, 30g/L sucrose, 0.6g/L MES (2-morpholinoethanesulfonic acid), 1.6 mg/L6-BA (benzyladenine), 100mg/L L-Cys,0.1MDDT (bis-P-chlorophenyl trichloroethane), 0.5mg/L GA3 (gibberellin), 0.2% (w/v) plant gel (Sigma-P8169), pH 5.4

(3) Transgenic seedling acquisition

The cotyledonary node after co-culture for 5 days is obliquely inserted into a bud induction culture medium I (SI-I), the cotyledonary node is dark under the condition of 25 ℃ and 16h illumination for 8h, the illumination intensity is 5000-6000Lux, the culture is resumed for 7 days, the cotyledonary node is cut off too long and then is transferred into a bud induction culture medium II (SI-II) containing 8mg/ml PPT (glufosinate), and the culture is continued for 14-20 days.

The cluster buds are excised from the hypocotyl and transferred into bud elongation culture medium (SEM) containing 4mg/ml PPT, at 25deg.C under illumination for 8h in the dark, with illumination intensity of 5000-6000Lux, and subcultured every 10 days until the buds are elongated to about 5 cm. Cutting off the buds extending to about 5cm, inserting into rooting culture medium, irradiating for 8h at 25deg.C for darkness, and irradiating with 5000-6000Lux until the roots extend to 3-4cm, and preparing for transplanting.

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

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

the composition of the bud induction culture medium II comprises 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 and pH 5.7;

the composition of the bud elongation culture medium comprises MS salt, B5 vitamin, 30g/L sucrose, 0.6g/L MES,0.5mg/L GA3,1mg/L ZR (zeatin nucleoside (trans)), 50mg/L L-Glu,50mg/LAsp,0.1mg/L IAA (indoleacetic acid), 50mg/L Cef,100mg/L Tim,4g/L glufosinate, 0.2% (w/v) plant gel and pH 5.8;

the rooting culture 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) Seedling hardening, transplanting and screening

Removing a sealing film from tissue culture seedlings to be transplanted, adding a small amount of sterile water, darkening at 25 ℃ for 16h under the illumination of 8h, and culturing for two days, transplanting the seedlings, uniformly mixing vermiculite and turfy soil in equal quantity, placing the mixture into a tray with water, extracting the tissue culture seedlings from a rooting culture medium, flushing the residual culture medium at the root, and transferring the culture medium into nutrient soil fully absorbing water. Soybean leaves were coated with 0.1% basta herbicide and no yellowing response after 3 days was judged as a transgenic positive plant. Two transgenic lines, namely a CE-1 line and a CE-2 line (an expression vector constructed by a self promoter, namely Complementary Expressed and complementation expression), are randomly selected from plants judged to be transgenic positive, and subsequent identification is carried out.

Example 3 variation of fuzz trait in GmP1 overexpressing transgenic plants

1. Identification of Gene expression level

The receptor lines DN50, gmP1 complementation transgene lines CE-1 and CE-2 were identified as follows:

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

(2) Using the cDNA extracted in step (1) as a template, the expression level of the GmP1 gene was identified using a primer pair consisting of F3 and R3, and the reference gene (expression level of an action gene) was identified 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 levels of the GmP1 gene in the different materials obtained by quantitative PCR amplification using the cDNA as a template and each specific primer are shown in FIG. 1.

As can be seen from FIG. 1, the gene expression level of GmP1 was significantly higher than that of the control DN50 by about 2-fold in the two GmP1 complementation expression lines CE-1 and CE-2. The presence of overexpression of GmP1 in lines CE-1 and CE-2 was confirmed.

2. Comparison of the number of fuzz of the recipient Strain DN50, gmP1 complementarily expressed transgenic Strain

The whole soybean plants in the three groups were visually observed to see that the recipient line DN50 was normal fuzz density, and in the CE-1 and CE-2 groups, the plants were not visually fuzz producing.

Comparing the experiment of fuzz number after the complementation expression transgenic lines CE-1 and CE-2 of the receptor lines DN50 and GmP1 are planted, taking a picture of the main stem of the plant by using a camera (the picture is shown in figure 3), counting the fuzz number of the petioles with the length of 0.5cm of the same node, counting ten plants by using tweezers, taking one root by using tweezers, and counting the results as shown in figure 2, wherein the scale in the figure represents 1cm.

As can be seen from fig. 2 and 3, the GmP1 complementarily expressed transgenic lines exhibited a fuzz-free phenotype compared to DN50, indicating that the GmP1 gene controls soybean fuzz generation, development in a selective or non-selective manner, and that overexpression thereof can cause soybeans to exhibit a fuzz-free phenotype. It is suggested that it can be used as a means for researching the stress resistance and increasing the stable yield of soybean plants.

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.

Industrial applicability

The invention provides a fuzz-associated gene, which can be used for researching and improving physical anti-biotic stress property of soybean plants, anti-biotic stress property of glandular hairs, and abiotic stress response property and the like. It is expected that editing of coat-associated genes, such as dense fuzz density-associated genes, for example, introduction or deletion, transformation techniques, and the like will be applied to variety selection and utilization of soybean.

Soybeans are one of the important food crops, providing an oil and protein source for humans and animals. At present, the soybean yield in China is in demand, and the blank is filled mainly by imported soybeans. In genetic breeding of soybeans, how to improve stress resistance and stable yield is always a great difficulty in 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 complementation of expression of the gene results in a fuzz-free phenotype of soybean.

Therefore, in production practice, the molecular marker can be designed according to the gene to be used for screening fuzz varieties in breeding, and on the other hand, the fuzz character of soybean can be moderately changed according to the target point edited by the gene design gene, so that soybean materials with different fuzz characters are created.

Reference is made to:

Kang,J.,Shi,F.,Jones,A.D.,Marks,M.D.,and Howe,G.A.(2010).Distortion of trichome morphology by the hairless mutation of tomato affects leaf surface chemistry.J.Exp.Bot.61,1053-1064.

Schilmiller,A.L.,Schauvinhold,I.,Larson,M.,Xu,R.,Charbonneau,A.L.,Schmidt,A.,Wilkerson,C.,Last,R.L.,and Pichersky,E.(2009).Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.Proc.Natl.Acad.Sci.USA 106,10865-10870.

Tingey,W.M.(1991).Potato glandular trichomes-defensive activity against insect attack.ACS Symp.Ser.449,126-135.

Wagner,G.J.,Wang,E.,and Shepherd,R.W.(2004).New approaches for studying and exploiting an old protuberance,the plant trichome.Ann.Bot.93,3-11.

Wang,G.,Tian,L.,Aziz,N.,Broun,P.,Dai,X.,He,J.,King,A.,Zhao,P.,and Dixon,R.A.(2008).Terpene biosynthesis in glandular trichomes of hop.Plant Physiol.148,1254-1266.

Wester,K.,Digiuni,S.,Geier,F.,Timmer,J.,Fleck,C.,and Hulskamp,M.(2009).Functional diversity of R3 single-repeat genes in trichome development.Development 136,1487-1496.

Schellmann,S.,and Hulskamp,M.(2005).Epidermal differentiation:trichomes in Arabidopsis as a model system.Int.J.Dev.Biol.49,579-584.

Johnson,H.B.(1975).Plant pubescence:an ecological perspective.Bot.Rev.41,233-258.

Loughner,R.,Goldman,K.,Loeb,G.,and Nyrop,J.(2008).Influence of leaf trichomes on predatory mite(Typhlodromus pyri)abundance in grape varieties.Exp.Appl.Acarol.45,111-122.

Traw,M.B.,and Bergelson,J.(2003).Interactive effects of jasmonic acid,salicylic acid,and gibberellin on induction of trichomes in Arabidopsis.Plant Physiol.133,1367-1375.

Loe,G.,Torang,P.,Gaudeul,M.,and Agren,J.(2007).Trichome production and spatiotemporal variation in herbivory in the perennial herb Arabidopsis lyrata.Oikos 116,134-142.

Valverde,P.L.,Fornoni,J.,and Nunez-Farfan,J.(2001).Defensive role of leaf trichomes in resistance to herbivorous insects in Datura stramonium.J.Evol.Biol.14,424-432.

Leite,G.L.D.,Picanco,M.,Guedes,R.N.C.,and Zanuncio,J.C.(2001).Role of plant age in the resistance of Lycopersicon hirsutum f.glabratum to the tomato leafminer Tuta absoluta(Lepidoptera:Gelechiidae).Sci.Hortic.(Amst.)89,103-113.

Laue,G.,Preston,C.A.,and Baldwin,I.T.(2000).Fast track to the trichome:induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda.Planta 210,510-514.

Kim,H.J.,Han,J.H.,Kim,S.,Lee,H.R.,Shin,J.S.,Kim,J.H.,Cho,J.,Kim,Y.H.,Lee,H.J.,Kim,B.D.,et al.(2011).Trichome density of main stem is tightly linked to PepMoVresistance in chili pepper(Capsicum annuum L.).Theor.Appl.Genet.122,1051-1058.

Liedl,B.E.,Lawson,D.M.,White,K.K.,Shapiro,J.A.,Cohen,D.E.,Carson,W.G.,Trumble,J.T.,and Mutschler,M.A.(1995).Acylsugars of wild tomato LycopersiconPennellii alters settling and reduces oviposition of bemisia argentifolii(Homoptera:Aleyrodidae).J.Econ.Entomol.88,742-748.

Maluf,W.R.,Campos,G.A.,and Cardoso,M.D.(2001).Relationships betweentrichome types and spider mite(Tetranychus evansi)repellence in tomatoes with respect tofoliar zingiberene contents.Euphytica 121,73-80.

Kang,J.,Campos,M.L.,Zemelis-Durfee,S.,Al-Haddad,J.,Jones,A.D.,Telewski,F.W.,Brandizzi,F.,and Howe,G.A.(2016).Molecular cloning of the tomato Hairless geneimplicates actin dynamics in trichome-mediated defense and mechanical properties of stemtissue.J.Exp.Bot.67,5313-5324.

Karabourniotis,G.,Papadopoulos,K.,Papamarkou,M.,and Manetas,Y.(1992).Ultraviolet-B Radiation Absorbing Capacity of Leaf Hairs.Physiol.Plant.86,414-418.

Hill,C.B.,Li,Y.,and Hartman,G.L.(2006).Soybean aphid resistance in soybeanJackson is controlled by a single dominant gene.Crop Sci.46,1606-1608.

Claims (7)

1. a method of genetic engineering of a transgenic soybean plant comprising: over-expressing the protein shown as SEQ ID NO. 1, or the gene shown as SEQ ID NO. 7 or SEQ ID NO. 8 in a soybean plant gives a transgenic soybean with a fuzz-free phenotype compared to the original soybean plant.

2. The method of claim 1, wherein the over-expression is performed by transferring into the original soybean plant an expression cassette or recombinant vector that regulates the expression of the protein or gene, the expression cassette comprising the amino acid sequence as set forth in SEQ ID NO:7 or as set forth in SEQ ID NO:8, and the recombinant vector comprises a gene as shown in SEQ ID NO:7 or as set forth in SEQ ID NO:8 or said expression cassette.

3. The method of claim 1, comprising detecting expression of the protein or the gene.

4. The method of claim 1, comprising selecting a transgenic soybean having a fuzz-free phenotype by confirming expression of the protein or the gene.

5. The method according to claim 3, wherein the detection is performed using PCR using a primer pair having the sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10.

6. The method of claim 2, wherein said transferring is performed using Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium-mediated.

7. Use of a protein as defined in claim 1 or a gene as defined in claim 2 or 3 for the overexpression in soybean genetic engineering of a fuzz-free phenotype.