Soybean isoflavone synthesis regulatory gene IC15 and application thereof

Abstract

The invention relates to a soybean isoflavone synthesis regulation gene IC15 and application thereof, and aims to improve the total content and yield of soybean isoflavone. The IC15 gene is cloned from a high isoflavone soybean variety through a PCR (Polymerase Chain Reaction) method, and the gene has two genotypes: IC15-ZD and IC15-HF. By means of genetic transformation, the content of soybean isoflavone, especially daidzein and genistein, can be remarkably increased by overexpressing the IC15-ZD genotype. Besides, the invention also develops a molecular marker closely linked with the isoflavone content, and the molecular marker is used for directionally improving soybean varieties so as to cultivate new soybean varieties which are more beneficial to human health.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and crop genetic breeding, and in particular to a soybean isoflavone synthesis regulating gene IC15 and its application in improving the total content and yield of soybean isoflavones. Background Art

[0002] Soy isoflavones have extensive and outstanding benefits for human health. They can alleviate menopausal symptoms, reduce the risk of cardiovascular disease and osteoporosis, and inhibit the spread of certain cancers. Soybean seeds are the main source of isoflavones for the human body. However, the isoflavone content of the main soybean varieties on the market is generally not high (1.2-2.5 mg / g); there are only more than 10 high-isoflavone soybean varieties bred in China (>4.0 mg / g) and the agronomic traits are average. As people pay more attention to their health, the demand for isoflavones will increase, making it difficult for existing varieties to meet the isoflavone demand in the medical and health care fields. Therefore, creating new high-value-added, high-isoflavone, high-quality and high-yield soybean varieties is crucial to meeting people's health needs and maintaining the safety of my country's soybean industry.

[0003] However, the determination method of isoflavones is complex, time-consuming and labor-intensive. It is difficult to track the isoflavone content in time in conventional breeding, which makes the breeding of high-isoflavone soybean varieties more difficult. Using genes associated with high-isoflavone traits to directly screen breeding materials will greatly improve breeding efficiency. However, as secondary metabolites, soybean isoflavones are easily affected by the environment, and their regulatory network is complex and their mechanism of action is still unclear. At present, there is still no effective isoflavone regulatory gene for breeding application. Therefore, the key genes for the synthesis and regulation of soybean isoflavones are cloned by genetic means to quickly and accurately cultivate new varieties of high-isoflavone soybeans to meet human health and medical care needs; at the same time, the soybean yield can be improved from the secondary metabolism level, which will provide important new clues and practical significance for the selection of new high-yield soybean varieties.

[0004] It should be noted that the information disclosed in the above background technology section is only used for understanding the background of the present application, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the invention

[0005] The main purpose of the present invention is to overcome the defects existing in the above-mentioned background technology and provide soybean isoflavone synthesis regulatory gene IC15 and its application.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, the soybean isoflavone synthesis regulatory gene IC15 is represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] In the second aspect of the present invention, the protein encoded by the soybean isoflavone synthesis regulatory gene IC15 is encoded by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] In the third aspect of the present invention, the soybean isoflavone synthesis regulatory gene IC15 having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is used to increase the total content and yield of soybean isoflavones.

[0010] In a fourth aspect of the present invention, a method for cloning a soybean isoflavone synthesis regulatory gene IC15 comprises the following steps:

[0011] RNA is extracted from soybean variety ZD27 to obtain the IC15-ZD gene shown in SEQ ID NO.1; or RNA is extracted from soybean variety HF55 to obtain the IC15-HF gene shown in SEQ ID NO.2;

[0012] The extracted RNA was treated with DNase I;

[0013] Synthesize cDNA;

[0014] IC15 gene was amplified using forward primer F1 and reverse primer R1;

[0015] The PCR product was purified and sequenced to obtain the IC15 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0016] In a fifth aspect of the present invention, a method for obtaining IC15 gene-transgenic soybean hairy roots comprises the following steps:

[0017] Construct a vector, and connect the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.2 into the plant expression vector pG2RNAi;

[0018] Transform the target vector into Agrobacterium K599 competent cells;

[0019] Infect soybean seedlings to obtain soybean hairy roots;

[0020] The T0 generation transgenic seedlings were identified to confirm transgenic positive seedlings containing the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.2.

[0021] In a sixth aspect of the present invention, a method for developing molecular markers in soybean breeding applications comprises the following steps:

[0022] Based on the reference genome of soybean 'Williams 82', dCAPS primers for the IC15 gene shown in SEQ ID NO.1 and SEQ ID NO.2 were designed;

[0023] Using the recombinant inbred line population, the near-isogenic line of IC15 gene was obtained through PAGE gel electrophoresis identification;

[0024] Develop a dCAPS molecular marker that is tightly linked to isoflavone content and use it to select and directionally improve soybean varieties containing the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.2.

[0025] In the seventh aspect of the present invention, the molecular markers are used in the directional and rapid breeding of new soybean varieties.

[0026] The present invention has the following beneficial effects:

[0027] The present invention clones the total isoflavone content regulating gene IC15 from high isoflavone soybean varieties by PCR method, and there are two genotypes, IC15-ZD and IC15-HF. The present invention overexpresses different genotypes of IC15 gene in soybean hair roots by genetic transformation. It is found that compared with the control, the content of isoflavone aglycone components in the hair roots of soybeans with IC15-ZD gene is significantly increased by 88.26 mg / kg (P<0.05) than that of the hair roots with IC15-HF gene, an increase of 12.51%. Figure 4 As shown, especially daidzein and genistein increased by 3.03 times and 1.18 times respectively. Further, the present invention obtains the near-isogenic line of IC15 gene, and the daidzein, genistein and total isoflavone contents from IC15-ZD genotype seeds are significantly higher than those in IC15-HF genotype seeds, and the results are consistent with the results in transgenic hairy roots. It shows that the IC15-ZD genotype mainly increases the total isoflavone content of soybeans by promoting the synthesis of daidzein and genistein in daidzein, and plays a key role in the soybean isoflavone synthesis pathway. These results will also enhance the understanding of the molecular mechanism of soybean isoflavone synthesis.

[0028] The isoflavone content of soybean seeds is closely related to human health. The present invention further develops a molecular marker that closely links the soybean isoflavone regulatory gene IC15 with the isoflavone content, which can be used to select and improve cultivars to obtain new soybean varieties that are more beneficial to human health.

[0029] Other beneficial effects of the embodiments of the present invention will be further described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1This is the electrophoresis diagram of the PCR amplification of the IC15 gene according to the embodiment of the present invention.

[0031] Figure 2 This is a molecular identification diagram of IC15 gene-transformed soybean hairy roots according to an embodiment of the present invention.

[0032] Figure 3 This is a diagram showing the determination of isoflavone components in the hairy roots of soybeans transgenic for IC15 according to an embodiment of the present invention.

[0033] Figure 4 This is a graph showing the determination of isoflavone components in the seeds of the IC15 near-isogenic line according to an embodiment of the present invention.

[0034] Figure 5 This is a development diagram of the IC15 gene molecular marker according to an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention.

[0036] In general, the present invention provides a soybean isoflavone synthesis regulatory gene IC15 and its encoded protein and application. The soybean isoflavone synthesis regulatory gene IC15 of the present invention has a non-synonymous mutation in the nucleotide of the gene, and there are two genotypes, IC15-ZD and IC15-HF, and the nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table, respectively. The present invention develops a molecular marker for the soybean isoflavone synthesis regulatory gene IC15. Application of the soybean isoflavone synthesis regulatory gene IC15 of the present invention in increasing the total content of isoflavones.

[0037] In some embodiments, the present invention provides a soybean isoflavone synthesis regulatory gene IC15 having a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0038] In other embodiments, the present invention provides a protein encoded by the soybean isoflavone synthesis regulatory gene IC15 encoded by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0039] In other embodiments, the present invention provides a use of a soybean isoflavone synthesis regulatory gene IC15 having a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 in increasing the total content and yield of soybean isoflavones.

[0040] In some other embodiments, the present invention provides a method for cloning a soybean isoflavone synthesis regulatory gene IC15, comprising the following steps:

[0041] RNA is extracted from soybean variety ZD27 to obtain the IC15-ZD gene shown in SEQ ID NO.1; or RNA is extracted from soybean variety HF55 to obtain the IC15-HF gene shown in SEQ ID NO.2; the extracted RNA is treated with DNase I; cDNA is synthesized; the IC15 gene is amplified with forward primer F1 and reverse primer R1; the PCR product is purified and sequenced to obtain the IC15 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0042] In some other embodiments, the present invention provides a method for obtaining soybean hairy roots transgenic for IC15 gene, comprising the following steps: constructing a vector, linking the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.2 into the plant expression vector pG2RNAi; transforming the target vector into Agrobacterium K599 competent cells; infecting soybean seedlings to obtain soybean hairy roots; and identifying T0 generation transgenic seedlings to confirm transgenic positive seedlings containing the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.2.

[0043] In some other embodiments, the present invention provides a method for developing molecular markers in soybean breeding applications, comprising the following steps: designing dCAPS primers for the IC15 gene shown in SEQ ID NO.1 and SEQ ID NO.2 based on the reference genome of soybean 'Williams 82'; using a recombinant inbred line population to obtain near-isogenic lines of the IC15 gene through PAGE gel electrophoresis identification; developing dCAPS molecular markers tightly linked to isoflavone content for selection and directed improvement of soybean varieties containing the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.2.

[0044] In some further embodiments, the present invention also provides the use of the molecular markers in the directional and rapid breeding of new soybean varieties.

[0045] The present invention successfully cloned the gene IC15 regulating the total content of soybean isoflavones, and revealed the key role of its two genotypes IC15-ZD and IC15-HF in regulating the synthesis of soybean isoflavones. Through genetic transformation, the present invention found that the IC15-ZD genotype can significantly increase the content of isoflavone aglycones in soybean hair roots, especially the content of daidzein and genistein, compared with the IC15-HF genotype, thereby increasing the total isoflavone content of soybeans. This discovery not only promotes the understanding of the molecular mechanism of soybean isoflavone synthesis, but also provides important gene resources for cultivating new soybean varieties with high isoflavone content. In addition, the present invention also develops molecular markers closely linked to isoflavone content, which provides a new strategy for rapid and directional improvement of soybean varieties to improve their nutritional value and yield, meets the growing demand for healthy food, and helps to maintain the safety of the soybean industry. Through these technical advantages, the present invention not only improves the nutritional value of soybeans, but also enhances the market competitiveness of soybean crops, which is of great significance to promoting sustainable agricultural development and improving human health.

[0046] The specific embodiments of the present invention are further described below.

[0047] Example 1: Cloning of the soybean isoflavone total content regulatory gene IC15

[0048] First, using the soybean variety 'Zhongdou 27' (abbreviated as: ZD27) with high isoflavone content and the soybean variety 'Hefeng 55' (abbreviated as: HF55) with low isoflavone content as experimental materials, the total RNA of immature seeds at different stages was extracted according to the operation manual of the TRIzol kit of Invitrogen Company;

[0049] Second, the total RNA extracted in step 1 was treated with DNase I;

[0050] 3. Take 1 μg of the total RNA treated in step 2 for cDNA synthesis. The cDNA synthesis operation is purchased from BD SMART T The RACE cDNA Amplification Kit was used according to the user manual to obtain cDNA;

[0051] 4. Using the cDNA obtained in step 3 as a template, refer to TaKaRa's HS DNA Polymerase operating instructions, use forward primer F1 and reverse primer R1 to amplify IC15 gene. PCR reaction conditions are as follows: pre-denaturation 95℃, 3min; follow the program (denaturation 95℃, 15s; annealing 59℃, 20s; extension 72℃, 1min 45s) for 35 cycles; final extension 72℃, 5min; 4℃, ∞.

[0052] Forward primer F1: 5'-TCAATGACGGAGTCAGAAGAAGC-3'

[0053] Reverse primer R1: 5'-GCCAGCTTTTATTTCTCTAGCAGTT-3'

[0054] Figure 1 The electrophoresis diagram of PCR amplification of IC15 gene is shown. M: DNAMarker DL 5000; 1: IC15-ZD gene PCR amplification band; 2: empty lane; 3: IC15-HF gene PCR amplification band.

[0055] Finally, the PCR product (such as Figure 1 ) was purified and sequenced on an ABI3130 sequencer (ABI). The sequencing results showed that the IC15 gene consisted of 3237 bases, and its nucleotide sequence was different in soybean varieties with significant differences in isoflavone content, with a single nucleotide variation. In the soybean variety ZD27 with high isoflavone content, the sequence is shown in SEQ ID NO: 1, recorded as IC15-ZD; in the soybean variety HF55 with low isoflavone content, the sequence is shown in SEQ ID NO: 2, recorded as IC15-HF.

[0056] Example 2: Obtaining IC15 Gene Transgenic Soybean Hairy Roots

[0057] 1. Vector construction: The cDNA amplified fragments in Example 1 were respectively connected into the plant expression vector pG2RNAi (provided by the Soybean Molecular Breeding Laboratory, Northeast Center of Geography and Agriculture, Chinese Academy of Sciences) to form CaMV 35S promoter-driven IC15-ZD and IC15-HF fusion plasmids, denoted as pG2RNAi-IC15-ZD and pG2RNAi-IC15-HF, respectively.

[0058] 2. The target vectors were transformed into Agrobacterium K599 competent cells, and the steps are as follows:

[0059] (1) Thaw the competent cells of Agrobacterium rhizogenes K599 on ice. Pipette 1 μL of the empty plasmid and the recombinant plasmid constructed in the above step, add 50 μL of the competent cells, gently stir the bottom of the tube to mix, and place on ice for 5 min.

[0060] (2) Immediately perform the following operations in sequence: place in liquid nitrogen for 5 min, bath in 37°C water for 5 min, and bath in ice for 5 min.

[0061] (3) Add 700 μL of antibiotic-free YEP liquid culture medium and resuscitate at 180 rpm at 28°C for 2-3 h.

[0062] (4) Centrifuge at 5000 r / min for 1 min and discard 600 μL of supernatant. After resuspending the cells, take 100 μL of the bacterial solution and spread it on a YEP plate containing 50 μg / mL kanamycin and 50 μg / mL streptomycin, and culture it upside down at 28°C for 2-3 days.

[0063] (5) Pick a single colony for PCR identification. Place the correct plaque corresponding to the electrophoresis band in YEP liquid culture medium containing 50 μg / mL kanamycin and 50 μg / mL streptomycin for propagation.

[0064] (6) Add an equal volume of 30% (V / V) glycerol to preserve the bacteria and store at -80°C.

[0065] 3. Obtaining soybean hairy roots

[0066] The following steps were used to infect soybean seedlings with Agrobacterium K599 bacterial solution to obtain soybean hairy roots.

[0067] (1) 5 μL of Agrobacterium rhizogenes K599 culture medium containing pG2RNAi-IC15-ZD and pG2RNAi-IC15-HF and blank control culture medium (Agrobacterium rhizogenes K599 containing the original plasmid vector pG2RNAi) were placed in 25 mL of YEP medium (with kanamycin and streptomycin contents of 50 μg / mL) and cultured at 28°C and 180 r / min for 12 h.

[0068] (2) Take 5 mL of activated bacterial solution and place it in 50 mL of YEP medium (the content of kanamycin and streptomycin is 50 μg / mL each). Cultivate at 28°C and 180 r / min until OD600 = 0.6-0.8, and then resuspend in double distilled water.

[0069] (3) Sowing 'Dongnong 50' soybeans in vermiculite. After 4 days, take the cotyledons and hypocotyls of healthy seedlings, cut them off 1.5 cm from the cotyledon node, and pierce three holes under each node with a syringe needle.

[0070] (4) The treated seedlings were immersed in K599 bacterial suspension containing an empty vector, K599 bacterial suspension containing pG2RNAi-IC15-ZD, and K599 bacterial suspension containing pG2RNAi-IC15-HF for 30 min.

[0071] (5) Add moist vermiculite to the pot and cover it with a layer of filter paper. Place the infected seedlings on the filter paper, seal with plastic wrap to keep it moist, and culture in an artificial climate chamber (16 h light / 8 h dark, 28°C / 26°C).

[0072] (6) When new roots 2 cm long grow from the bottom of the hypocotyl, transfer the seedlings to vermiculite for further cultivation.

[0073] 4. Identification of Transgenic Soybean Hairy Roots

[0074] To T 0 The transgenic seedlings were amplified using the crude extracted DNA as a template according to the EasyTaq DNA Polymerase instruction manual of Quanshijin Company with CaMV 35S promoter-Bar gene primers (F3 and R3) to obtain transgenic positive seedlings.

[0075] Forward primer F3: 5'-CCTGTGCCTCCAGGGAC-3'

[0076] Reverse primer R3: 5'-GCGGTCTGCACCATCGTC-3'

[0077] The molecular identification results of IC15 transgenic soybeans are as follows Figure 2 Compared with the control 'Dongnong 50', IC15-ZD or IC15-HF gene was significantly overexpressed in transgenic soybean. The above results prove that the transgenic materials obtained in this experiment are indeed overexpressed in the target gene, expressed as IC15-ZD-OE and IC15-HF-OE.

[0078] Example 3: Identification of the content of isoflavone components in transgenic soybean hairy roots

[0079] (1) Sample preparation: Three groups of 'Dongnong 50' soybean hairy roots cultured for 45 days were quickly ground in liquid nitrogen, 0.50 g of the sample was weighed into a 15 mL centrifuge tube, and 9 mL of 90% methanol solution was quickly added.

[0080] (2) Sample treatment: Place the centrifuge tube in an ultrasonic cleaner at 60°C for 30 minutes, shaking it upside down every 10 minutes. After the extract has cooled to room temperature, centrifuge it at 7000r / min for 10 minutes. Transfer the supernatant to a 25mL volumetric flask, add 6mL of 90% methanol solution to the remaining residue and repeat the extraction twice. After combining the supernatants, transfer them to a 25mL volumetric flask, dilute to the mark with 10% methanol solution, and shake well. Pipette 1mL of the extract, pass it through a 0.45μm organic filter membrane into a 2mL brown injection bottle, store it at 4°C away from light, and wait for HPLC determination.

[0081] (3) Chromatographic analysis: According to the method in the national standard (GB / T26625-2011) "High Performance Liquid Chromatography for Determination of Soybean Isoflavone Content in Grain and Oil Inspection", the contents of six isoflavone components in the seeds of the ZH-BIL population were detected. The water bath-ultrasound-assisted extraction method was adopted to extract soybean isoflavones with 90% methanol aqueous solution as solvent. Then, the HPLC method was used with RP-C18 stainless steel column as chromatographic column, 0.1% acetic acid aqueous solution and 0.1% acetic acid acetonitrile solution as mobile phase, and gradient elution was used to determine the contents of six isoflavone components including daidzein, glycitein, genistin, soyizin, glycitein and genistein. The gradient elution program is shown in Table 1. The chromatographic conditions are shown in Table 2.

[0082] Table 1 HPLC mobile phase gradient elution program

[0083]

[0084] Table 2 HPLC column conditions

[0085]

[0086] (4) Data processing: Using CLASS-VPV6.1 software and the peak area method, the contents of the six soy isoflavone components, namely daidzein, glycitein, genistin, daidzein, glycitein and genistein, the glucoside content (the sum of the contents of the three glucosides, namely daidzein, glycitein and genistin), the aglycone content (the sum of the contents of the three aglycones, namely daidzein, glycitein and genistein), and the total isoflavone content (the sum of the contents of the three glucosides and the three aglycones) in the sample were calculated using the standard samples of the six soy isoflavone components.

[0087] After the "Dongnong 50" soybean hairy roots were formed by Agrobacterium rhizogenes K599, the isoflavone content in the hairy roots of IC15-ZD and IC15-HF gene overexpressing plants and blank control (CK) plants was determined to clarify the difference between the two genes in isoflavone biosynthesis. After 45 days of growth, the hairy roots of IC15-ZD gene-transfected, IC15-HF gene-transfected and blank control "Dongnong 50" soybeans all showed green fluorescence in the fluorescence field, further confirming that the K599 strain carrying the pG2RNAi-IC15-ZD and pG2RNAi-IC15-HF recombinant plasmid vectors had been successfully transferred into soybean plants.

[0088] After determining the isoflavone content in the hairy roots of soybeans with IC15-ZD and IC15-HF genes, Figure 3As shown in the results, the total isoflavone content in hairy roots containing IC15-ZD gene was significantly increased by 88.26mg / kg (P<0.05) compared with that containing IC15-HF gene, an increase of 12.51%. Among them, the content of daidzein in the former was also extremely significantly higher than that in the latter by 15.04mg / kg (P<0.01), an increase of 3.03 times; the content of genistein in the former was also extremely significantly higher than that in the latter by 62.43mg / kg (P<0.01), an increase of 1.18 times. This shows that the IC15-ZD genotype mainly increases the content of total isoflavones in soybeans by promoting the synthesis of daidzein and genistein in daidzein, and plays a key role in the synthesis pathway of soybean isoflavones.

[0089] Example 4: Development of molecular markers closely related to soybean IC15 gene and isoflavone content

[0090] First, dCAPS primers for the IC15 gene were designed based on the reference genome of soybean 'Williams 82'.

[0091] Second, using the recombinant inbred line population that had been constructed in the early stage, with ZD27 as the female parent and HF55 as the male parent, they hybridized to produce the F1 population; then the F1 population was backcrossed with the male parent ("Hefeng 55") to obtain the F2 population; and then using the single seed inheritance method, an F7 backcross recombinant inbred line containing 212 offspring was obtained, referred to as the ZH-BIL population.

[0092] 3. PAGE gel electrophoresis was performed on the ZH-BIL population to obtain the typing results of 212 plants in the F7 generation, and a near isogenic line (NIL) of the IC15 gene, #40, was obtained. Among them, #40-1 was IC15-ZD type, recorded as NIL-IC15 ZD ; 40-2 is IC15-HF type, recorded as NIL-IC15 HF .

[0093] The content of each component of soybean isoflavones in seeds of each genotype of IC15 near-isogenic line was determined. The determination method and data processing method were the same as those in Example 3. The determination results are shown in FIG. Figure 4 As shown. NIL-IC15 ZD The contents of daidzein, genistein and aglycone in seeds were higher than those of NIL-IC15 HF seeds significantly increased by 123.33, 88.67 and 214.75 mg / kg (P<0.001); although, NIL-IC15 ZD The genistin content of seeds was higher than that of NIL-IC15 HF Seeds are low, but total isoflavone content is NIL-IC15 ZDHigher than NIL-IC15 in seeds HF 278.57 mg / kg (P<0.05), an increase of 7.11%. The above results show that the IC15-ZD genotype can significantly promote the synthesis of aglycone isoflavones. This analysis result is consistent with the results of transgenic hairy roots in the example, indicating that the IC15 gene is a key gene for regulating the synthesis of soybean isoflavones.

[0094] Furthermore, based on different IC15 genotypes, the present invention developed dCAPS molecular markers that are closely linked to isoflavone content in the IC15 gene, such as Figure 5 Overall, the IC15-ZD genotype can be used to select and improve cultivars to create new soybean varieties that are more beneficial to human health.

[0095] Soybean isoflavone synthesis regulatory gene IC15-ZD gene sequence (SEQ ID NO: 1):

[0096] >SEQ ID NO: 1

[0097]

[0098] Soybean isoflavone synthesis regulatory gene IC15-HF gene sequence (SEQ ID NO: 2):

[0099] >SEQ ID NO: 2

[0100]

[0101] The above contents are further detailed descriptions of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can also make several substitutions or modifications to these described embodiments without departing from the concept of the present invention, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention.

Claims

1. A soybean isoflavone synthesis regulatory gene IC15 having a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The protein encoded by the soybean isoflavone synthesis regulatory gene IC15 encoded by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. Application of the soybean isoflavone synthesis regulatory gene IC15 having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 in increasing the total content and yield of soybean isoflavones.

4. A method for cloning the soybean isoflavone synthesis regulatory gene IC15, characterized in that: The following steps are involved: RNA was extracted from soybean variety ZD27 to obtain the IC15-ZD gene shown in SEQ ID NO.1; Alternatively, RNA is extracted from soybean variety HF55 to obtain the IC15-HF gene shown in SEQ ID NO.2; The extracted RNA was treated with DNase I; Synthesize cDNA; IC15 gene was amplified using forward primer F1 and reverse primer R1; The PCR product was purified and sequenced to obtain the IC15 gene sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

5. A method for obtaining IC15 gene-transgenic soybean hairy roots, characterized in that: The following steps are involved: Construct a vector, and connect the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.2 into the plant expression vector pG2RNAi; Transform the target vector into Agrobacterium K599 competent cells; Infect soybean seedlings to obtain soybean hairy roots; The T0 generation transgenic seedlings were identified to confirm transgenic positive seedlings containing the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.

2.

6. A method for developing molecular markers in soybean breeding applications, characterized in that: The following steps are involved: Based on the reference genome of soybean 'Williams 82', dCAPS primers for the IC15 gene shown in SEQ ID NO.1 and SEQ ID NO.2 were designed; Using the recombinant inbred line population, the near-isogenic line of IC15 gene was obtained through PAGE gel electrophoresis identification; Develop a dCAPS molecular marker that is tightly linked to isoflavone content and use it to select and directionally improve soybean varieties containing the IC15 gene shown in SEQ ID NO.1 or SEQ ID NO.

2.

7. Application of the molecular marker developed by the method as claimed in claim 5 in the directional rapid breeding of new soybean varieties.

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