CN114409752B - Application of GmUVR8 gene family in improving isoflavone content of plants - Google Patents

Application of GmUVR8 gene family in improving isoflavone content of plants Download PDF

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CN114409752B
CN114409752B CN202210096864.2A CN202210096864A CN114409752B CN 114409752 B CN114409752 B CN 114409752B CN 202210096864 A CN202210096864 A CN 202210096864A CN 114409752 B CN114409752 B CN 114409752B
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陈栩
徐慧芳
刘秋林
高震
陈娇梅
黄来妹
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Fujian Agriculture and Forestry University
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Abstract

The invention discloses application of GmUVR8 gene family in improving isoflavone content of plants. The GmUVR8 gene family includes the following three members: the GmUVR8a gene, the GmUVR8b gene and the GmUVR8c gene. According to the invention, through constructing an over-expression vector for genes GmUVR8a, b and c and performing stable transformation in soybean bodies, the content of secondary metabolites in the stable transformed soybean obtained by over-expression of the GmUVR8a, b and c is obviously improved, such as daidzein, genistein and genistin, so that the quality of the soybean is improved. Therefore, the overexpression of the GmUVR8 gene family in plants is an effective way for improving plant quality, and has important production and theoretical research significance.

Description

Application of GmUVR8 gene family in improving isoflavone content of plants
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a GmUVR8 gene family in improving the content of plant isoflavone.
Background
Sunlight is of vital importance in plant growth and development. Light not only can serve as an important source of plant photosynthesis to promote plant growth and development, but also can achieve plant cell signaling through a range of photoreceptors. Ultraviolet light is an inherent component of sunlight and inevitably affects the growth and development of plants. Ultraviolet light can be classified into UV-A (320-400 nm), UV-B (280-320 nm) and UV-C (100-280 nm) according to wavelength. The ozone layer in the atmosphere absorbs UV-C and most of the UV-B due to the presence of the atmosphere at the earth's surface. Although the proportion of UV-B which finally reaches the ground is less than 0.1% of the total light energy, the UV-B is still critical to the growth and development of plants. For example, low intensity ultraviolet radiation inhibits normal growth of the hypocotyl of Arabidopsis thaliana. For another example, high intensity ultraviolet radiation damages DNA and proteins, causing the accumulation of plant active oxygen. In order to combat ultraviolet radiation, plants produce a range of self-defense mechanisms, such as the anabolic secondary metabolite isoflavones, as "sunscreens" to combat damage by ultraviolet radiation.
Soy isoflavones are mainly found in leguminous plants, with the highest levels in the soybean hulls, hypocotyls, and cotyledons. Soybean isoflavone is a natural estrogen, and can be taken for a long time to compensate for the defect of insufficient secretion of female estrogen, and regulate the estrogen level in human body, thereby relieving climacteric syndrome. In addition, isoflavone can prevent cardiovascular diseases and osteoporosis, and is a natural cancer chemopreventive agent. Among soybeans, isoflavones are largely classified into 3 classes, namely, daidzein (Daidzingroups), genistin (Genistingroups), glycitin (glycitin roups). Each group exists in 4 forms of free form, glucosyl form, acetylglucosyl form, malonyl glucosyl form and the like.
During the growth and development of plants, the increase of isoflavone content can obviously improve the disease resistance of plants. Research shows that ultraviolet radiation promotes the generation of isoflavone substances and can increase the disease resistance of plants. UV RESISTANCE LOCUS 8 (UVR 8) protein is the only ultraviolet light receptor identified in recent years, so that over-expression of GmUVR8 receptor protein may improve the ability to respond to ultraviolet light signals, promote the synthesis of isoflavone, obtain isoflavone-rich plant strain, and have certain production and application values.
Disclosure of Invention
The invention aims to provide application of GmUVR8 gene family in improving isoflavone content of plants.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
use of the GmUVR8 gene family for increasing the isoflavone content of a plant, the GmUVR8 gene family comprising three members: the GmUVR8a gene, the GmUVR8b gene, and the GmUVR8c gene; the amino acid sequence of the GmUVR8a gene is shown as SEQ ID NO. 1; the amino acid sequence of the GmUVR8b gene is shown as SEQ ID NO. 2; the amino acid sequence of the GmUVR8c gene is shown as SEQ ID NO. 3.
Further, the CDS sequence of the GmUVR8a gene is shown as SEQ ID NO. 4; the CDS sequence of the GmUVR8B gene is shown as SEQ ID NO. 5; the CDS sequence of the GmUVR8c gene is shown as SEQ ID NO. 6.
Further, the plant is soybean.
Further, the isoflavone is daidzin, genistin, daidzein, genistein.
Further, the above application is: the expression level of the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene is increased in the plant to increase the isoflavone content of the plant.
Further, the above-mentioned improvement of the expression level of the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene in plants is achieved by the following method: the exogenous GmUVR8a gene, gmUVR8b gene or GmUVR8c gene is overexpressed in plants.
Furthermore, the above-mentioned recombinant plant expression vector which overexpresses exogenous GmUVR8a gene, gmUVR8b gene or GmUVR8c gene in plants is transformed into plants by transgenic technology.
Furthermore, the recombinant plant expression vector is specifically constructed and obtained by the following method: after the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene fragment is linked to the intermediate vector P221 by BP reaction, it is linked to the final vector PGWB642 by LR reaction, thereby obtaining a recombinant plant expression vector.
The invention has the remarkable advantages that:
the over-expression of GmUVR8 protein in plants may enhance the ability to respond to ultraviolet light signals and promote the synthesis of isoflavone, so that the plant strain rich in high isoflavone can be obtained by the invention, and has certain production and application values.
Drawings
FIG. 1 is a schematic representation of wild-type and Gmuvr8a, b, c overexpressing seeds obtained according to the invention.
FIG. 2 is a schematic representation of the measurement of the overexpression level of Gmuvr8a, b, c.
FIG. 3 shows the results of the measurement of the content of the overexpressed daidzein in Gmuvr8a, b, c.
FIG. 4 shows the results of the measurement of the content of the overexpressed soybean glycoside of Gmuvr8a, b, c.
FIG. 5 shows the results of the measurement of the content of the over-expressed genistein of Gmuvr8a, b, c.
FIG. 6 shows the results of measurement of the content of the overexpressed genistin in Gmuvr8a, b, c.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The sequencing of the vectors in the examples below was determined by sequencing by conventional sequencing companies.
The soybean cultivar Huachun No.6 is a background material of the invention, and is a soybean cultivar obtained by breeding by agricultural college of agricultural university in south China, and breeding by using product seeds Guizao No.1 and Brazil No. 8. The third conference of the second crop variety approval committee of 7 months 28 days 2009 is approved, and the approval number is national audit bean 2009012.
Example 1 soybean Gmuvr8a, gmuvr8b, gmuvr8c overexpressing seed profile
03-3 is a wild type material; SR1-14 is GmUVR8a gene over-expression material; SR2-1 is GmUVR8b gene over-expression material: SR3-1 is GmUVR8c gene over-expression material. Gmuvr8a, gmuvr8b, gmuvr8c transgenic T3 generation strain and wild type strain 03-3 seed are shown in FIG. 1.
EXAMPLE 2 Synthesis of Soybean cDNA
RNA of mature leaves of Huachun No.6 soybean is extracted, and cDNA is synthesized by using a reverse transcription kit.
EXAMPLE 3 construction of the overexpression vector
Referring to the Gateway kit (purchased from the company invitrogen) instruction, the primers UVR8a-Forward primer and UVR8a-Reverse primer are used for adding a linker to Gmuvr8a through a PCR reaction of high-fidelity DNA polymerase, so that the full-length fragment of the target gene with the linker is amplified: UVR8a-Forward primer:
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAATGTTATTTTGATGGACAATACAGGA-3’；
UVR8a-Reverse primer：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAAACACGGAGCCGTTT-3’。
the primers UVR8b-Forward primer and UVR8b-Reverse primer are used for adding a linker to Gmuvr8b through a PCR reaction of high-fidelity DNA polymerase, and a target gene fragment with the linker is amplified:
UVR8b-Forward primer：
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAAGATGAAGTGATGAGTGAAGT-3’；
UVR8b-Reverse primer：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATTGACATCACTTTCAGGGAC-3’。
the primers UVR8c-Forward primer and UVR8c-Reverse primer are used for adding a linker to Gmuvr8c through a PCR reaction of high-fidelity DNA polymerase, and a target gene fragment with the linker is amplified:
UVR8c-Forward primer：
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTGCACTCTTCAAGTCTAC-3’；
UVR8c-Reverse primer：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAAACTCGGATCCGTTTGACA-3’。
PCR reaction system: soybean cDNA was used as a template of 1. Mu.L, each of Primer-F and Primer-R (Primer concentration: 10. Mu. Mol) of 1. Mu.L, dNTP Mix of 4. Mu.L, prime STAR GXL of 1. Mu.L, 5xGXL Buffer of 10. Mu.L, and ddH2O of 32. Mu.L.
PCR reaction conditions: 98℃for 5min at 98℃for 30sec; annealing at 55 ℃ for 30sec; the extension is carried out at 72℃for 1min for 30sec for a total of 30 cycles.
And (3) performing gel cutting, purifying and recycling after agarose gel electrophoresis detection of the PCR product, respectively connecting target gene fragments with a pDOR 221 vector (purchased from the company of invitrogen) to perform BP reaction, connecting the target gene fragments overnight at 25 ℃, converting the connecting products into competent cells of escherichia coli DH10B, screening positive clones, sequencing, and correctly reading clone to extract plasmids, so as to construct an entry vector. The entry vector plasmid and PGWB642 plasmid vector carrying attR site were subjected to LR reaction, ligated overnight at 25℃to transform E.coli DH10B competent cells, and positive clones were verified. Positive clones are cultivated, plasmids are extracted and purified, and finally transformed plasmids are transferred into agrobacterium strain GV3101, and infection transformation is carried out on prepared soybeans.
EXAMPLE 4 Stable transformation of soybeans
Placing soybeans infected by agrobacterium tumefaciens on a co-culture medium, and culturing for 4 days at 25 ℃; then transferred to recovery medium at 25℃for 7 days, then to Basta-containing resistant redifferentiation medium at 25℃for 14 days, then to phytohormone and Basta-containing resistant redifferentiation medium at 25℃for 14 days, and finally to elongation medium at 25℃for 14 days. The obtained plants were transferred to soil for growth and screened for positive seedlings by spraying with Basta diluted 1:1000.
Example 5 analysis of Gene expression levels
The gene expression level analysis of the transgenic lines was performed by a real-time fluorescent quantitative PCR method. RNA in the T3 generation strain and the wild type strain 03-3 leaves of Gmuvr8a, gmuvr8b and Gmuvr8c transgenes are respectively extracted, and cDNA is obtained by reversing with a reverse transcription kit as a template, so that real-time fluorescence quantitative PCR is performed. GmActin-11 was used as a reference gene and analyzed by the method of-DeltaC (t) with the expression level of the wild-type strain 03-3 being 1. The primers used were as follows:
UVR8a-Forward primer：
5’-GATTCTCTTGTGCCTCAGA-3’，
UVR8a-Reverse primer：
5’-AGATTGTCACCGACTCCA-3’；
UVR8b-Forward primer：
5’-GGACAGATTGGAGTTGGTAA-3’，
UVR8b-Reverse primer：
5’-TTGGAACATTGCGGTCTAT-3’；
UVR8c-Forward primer：
5’-TGATCGTTGCTCTCCTGT-3’，
UVR8c-Reverse primer：
5’-CCACACTCAATGCCTCAA-3’。
amplification procedure: 94 ℃ for 3min;94℃for 5sec and 60℃for 30sec; a total of 40 cycles. After the amplification cycle is completed, the temperature is raised to 65℃and then the DNA product is denatured by heating to 95 ℃.
Amplification conditions: cDNA template 1. Mu.L, primer-F and Primer-R (Primer concentration 10. Mu. Mol) each 0.3. Mu.L, 2 x SYBR Green qPCR Mix 7.5. Mu.L, ddH 2 O 5.9 μL。
The expression levels of the Gmuvr8a, gmuvr8b and Gmuvr8c genes in the soybean transgenic T3 generation lines are shown in FIG. 2. SR1-14 and SR1-15 are the expression level of Gmuvr8a, compared with the wild type, SR1-14 expression level is 8.7 times of the wild type, SR1-15 expression level is 6.8 times of the wild type; SR2-1 and SR2-10 are the expression level of Gmuvr8b, compared with the wild type, the expression level of SR2-1 is 72 times of the wild type, and the expression level of SR2-10 is 107 times of the wild type; SR3-1 and SR3-2 are expressed in Gmuvr8c, the SR3-1 is expressed in 10.49 times of wild type and the SR3-2 is expressed in 3.7 times of wild type.
EXAMPLE 6 isoflavone content determination
And (3) respectively measuring the contents of 4 soybean isoflavone compounds, namely soybean glycoside, genistin, daidzein and genistein in the T3 generation strain of the Gmuvr8a, the Gmuvr8b and the Gmuvr8c transgenosis and the wild strain 03-3 seeds by adopting an ultra-high performance liquid chromatography. The method comprises the following steps: chromatographic column: waters BEH C18 100mm x 2.1mm 1.7 μm, mobile phase: a is 0.1% FA water, B is 0.1% FA ACN4.2, and gradient elution is carried out; flow rate: 0.3 mL/min; column temperature: 40 ℃.
The daidzein content of the soybean transgenic T3-generation strain seed is shown in FIG. 3. L14 and L15 are measured as the content of Gmuvr8a daidzein, and compared with the wild type, the content of L14 daidzein is 2.19 times that of the wild type, and the content of L15 daidzein is 2.21 times that of the wild type; l10 is the content measurement of Gmuvr8b daidzein, and compared with a wild type, the content of the L10 daidzein is 1.78 times that of the wild type; l1 and L2 are measured as the content of Gmuvr8c daidzein, and compared with the wild type, the content of L1 daidzein is 3.67 times that of the wild type, and the content of L2 daidzein is 2.77 times that of the wild type.
The soybean glycoside content in the soybean transgenic T3 generation strain seed is shown in FIG. 4. L14 and L15 are content measurement of Gmuvr8a soyabean glycoside, compared with the wild type, the content of L14 soyabean glycoside is 1.12 times of that of the wild type, and the content of L15 soyabean glycoside is 1.08 times of that of the wild type; l10 is the content measurement of Gmuvr8b soyabean glycoside, and compared with a wild type, the content of the L10 soyabean glycoside is 1.1 times of that of the wild type; l1 and L2 are content measurement of Gmuvr8c soyaside, compared with the wild type, the content of L1 soyaside is 1.25 times of that of the wild type, and the content of L2 soyaside is 1.12 times of that of the wild type.
The genistein content in the soybean transgenic T3-generation seed is shown in fig. 5. L14 and L15 are content measurement of Gmuvr8a genistein, compared with a wild type, the content of the L14 genistein is 1.17 times of that of the wild type, and the content of the L15 genistein is 1.12 times of that of the wild type; the content of the L1 is measured by the content of the Gmuvr8b genistein, and compared with a wild type, the content of the L1 genistein is obviously reduced; l1 and L2 are content measurement of Gmuvr8c genistein, and compared with a wild type, the content of L1 genistein is 2.42 times of that of the wild type, and the content of L2 genistein is 1.8 times of that of the wild type.
The genistin content of the soybean transgenic T3-generation strain seed is shown in FIG. 6. L14 and L15 are the content measurement of Gmuvr8a genistin, and compared with the wild type, the content of the L14 and L15 genistin is not changed obviously; the content of L1 is measured by the content of Gmuvr8b genistin, and compared with a wild type, the content of L1 genistin is obviously reduced; l1 and L2 are content measurement of Gmuvr8c genistin, compared with a wild type, the content of the L1 genistin is 1.14 times that of the wild type, and the content of the L2 genistin is 1.12 times that of the wild type.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> Fujian university of agriculture and forestry
Application of <120> GmUVR8 gene family in improving isoflavone content of plants
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<213> soybean (Glycine max (l.) merr.)
<400> 3
Met Leu His Ser Ser Ser Leu Leu Phe Leu Ser Pro Ser Thr Gly Pro
1 5 10 15
Ile Asn Val Ile Asn Pro Cys Pro Ser Phe Ser Ser Leu Phe Pro Pro
20 25 30
Ile Thr Met Glu Leu Glu Val Thr Ala Ala Thr Ser Pro Pro Ser Arg
35 40 45
Val Leu Leu Ile Ser Ala Gly Ala Ser His Thr Val Ala Leu Leu Ser
50 55 60
Gly Asn Val Val Cys Ser Trp Gly Arg Gly Glu Asp Gly Gln Leu Gly
65 70 75 80
His Gly Asp Thr Asp Asp Arg Leu Leu Pro Thr His Leu Ser Ala Leu
85 90 95
Asp Ala Gln Gln Ile Asp Ser Ile Ala Cys Gly Ala Asp His Thr Leu
100 105 110
Ala Tyr Ser Glu Ser Arg Asn Glu Leu Tyr Ser Trp Gly Trp Gly Asp
115 120 125
Phe Gly Arg Leu Gly His Gly Asn Ser Ser Asp Leu Leu Ile Pro Gln
130 135 140
Pro Ile Ile Ala Leu Gln Gly Leu Arg Ile Lys Gln Ile Ala Cys Gly
145 150 155 160
Asp Ser His Cys Leu Ala Val Thr Met Glu Gly Glu Val Gln Ser Trp
165 170 175
Gly Arg Asn Gln Asn Gly Gln Leu Gly Leu Gly Thr Ser Glu Asp Ser
180 185 190
Leu Val Pro Gln Lys Ile Gln Thr Phe Gln Gly Val Pro Ile Lys Met
195 200 205
Val Ala Ala Gly Ala Glu His Ser Val Ala Ile Thr Glu Asn Gly Glu
210 215 220
Leu Tyr Gly Trp Gly Trp Gly Arg Tyr Gly Asn Leu Gly Leu Gly Asp
225 230 235 240
Arg Asn Asp Arg Trp Ile Pro Glu Lys Val Ser Ser Val Asp Cys Asp
245 250 255
Lys Met Val Met Val Ala Cys Gly Trp Arg His Thr Ile Ser Val Ser
260 265 270
Ser Leu Gly Gly Leu Tyr Thr Tyr Gly Trp Ser Lys Tyr Gly Gln Leu
275 280 285
Gly His Gly Asn Phe Glu Asp Ser Leu Val Pro Gln Lys Leu Gln Ala
290 295 300
Leu Ser Asp Lys Leu Ile Ser Gln Val Ser Gly Gly Trp Arg His Ser
305 310 315 320
Met Ala Leu Thr Ser Thr Gly Leu Leu Tyr Gly Trp Gly Trp Asn Lys
325 330 335
Phe Gly Gln Val Gly Val Gly Asp Asn Val Asp Arg Cys Ser Pro Val
340 345 350
Gln Val Lys Phe Pro His Asp Gln Lys Val Val Gln Ile Ser Cys Gly
355 360 365
Trp Arg His Thr Ile Ala Val Thr Glu Lys Glu Asn Val Phe Ser Trp
370 375 380
Gly Arg Gly Thr Asn Gly Gln Leu Gly His Gly Asp Thr Val Asp Arg
385 390 395 400
Asn Ser Pro Lys Ile Ile Glu Ala Leu Ser Val Asp Gly Ser Ser Gly
405 410 415
Pro His Ile Glu Ser Ser Asn Thr Asp Leu Leu Ser Gly Lys Ser Gly
420 425 430
Ala Ser Leu Ser Glu Arg Tyr Ala Val Val Pro Asp Glu Thr Val Ser
435 440 445
Gly Gln Thr Ala Ser Ser Ser Ser Gly Asp Arg Leu Asp Ile Ser Val
450 455 460
Pro Glu Ser Asp Val Lys Arg Ile Arg Val
465 470
<210> 4
<211> 1449
<212> DNA
<213> soybean (Glycine max (l.) merr.)
<400> 4
atgaaatgtt attttgatgg acaatacagg ataagatgcg aaaaaacaac agagaaatca 60
aattctttca tcatcaatga tataaactcc ttgctcctct tgactcattc attcactccc 120
aatatgatgg aattggaagc caccgccgct acttccccac cttctcgcgt tctcctcatc 180
tccgcaggtg ccagccacac cgttgccctt ctctctggga atgttgtgtg ttcgtggggt 240
cgcggagagg acggacagtt aggccatggt gacaccgatg ataggccctt acccacgcag 300
ctcagtgcat tggatgccca acacattgat tctattgcct gtggagctga tcataccctt 360
gcgtattccg aatcacgcaa tgaactctat agttggggat ggggtgattt tggaaggttg 420
ggtcatggca attctagtga tttgctcatt cctcagccta ttatagcatt gcaaggtcta 480
aggataaagc aaattgcctg tggggatagc cactgtttgg cagttaccat ggaaggcgag 540
gttcagagtt gggggaggaa tcaaaatggt caacttggac ttggcaacac agaggattct 600
cttgtgccac aaaagattca agcgtttaag ggagttccta tcaaaatggt tgctgcaggt 660
gcagaacaca gtgtagctat tactgaaaat ggagaactgt atggatgggg ttggggccga 720
tatggaaatt tggggttggg agatagaaat gatcgctgga atcctgagaa agtttcttct 780
gttgattgtg acaagatggt catggttgct tgtggttggc ggcatacaat ttctgtttca 840
tcttctggtg gcatatacac atacgggtgg agcaaatatg gccagctagg acatggaaat 900
tttgaggatt ctcttgtgcc tcagaagctt caagccttga gtgataagtt aatctctcag 960
gtatcaggtg gttggaggca tagtatggca ctcacgtcta ctggactact atttggatgg 1020
ggttggaata agtttggaca ggttggagtc ggtgacaatc ttgatcactg ctctcctgtg 1080
caagtgaagt tcccccaaga tcagagagta gttcagatct catgtgggtg gaggcacaca 1140
attgctgtaa ctgaaaagga aaatgtattt tcttggggaa gaggcacaaa tgggcaactt 1200
gggcatgggg ataccattga ccggaattct ccaaagatta ttgaggcatt gagtgtggat 1260
ggatctgctg ggccgcacat agaatcctca aacactgatc tattgtcagg gaaaagtggc 1320
gtctccttat ctgagagata tgcagttgtg ccggacgaaa ctgtctcagg acaaactgct 1380
agttcaagca gtggagatag gcttgaaatt agtgtcccag aaagtgatgt caaacggctc 1440
cgtgtttga 1449
<210> 5
<211> 1314
<212> DNA
<213> soybean (Glycine max (l.) merr.)
<400> 5
atggaagatg aagtgatgag tgaagtggct gctccacctc gtcgtgttct tcttgtatcg 60
gctggtgcca gccacagtgt ggcacttctg agtggaaatg ttgtttgttc gtggggacga 120
ggagaggatg gacagttagg ccatggtgac actgatgatc gactatttcc tacaaaactg 180
agtgcattgg atggccaaga cataatatgt gttacttgtg gagctgatca tactatggca 240
cgttctgagt ctggcaggga tgtatatagt tggggatggg gtgactttgg aaggttgggt 300
catggtgatc atagtgactt gctcattcct catcccataa aagcattaca gggtctaatg 360
atacaacaaa ttgcctgtgg ggacagtcat tgtttggcag ttaccatgga cagccaggtg 420
ctgagttggg gacgcaatca aaatggtgaa cttggacttg gaaccgcaga ggactctctt 480
ctgccacaaa aaattcaaat ttttgaggaa atacctatca aaatggttgc tgctggtgct 540
gaacatagtg tagcaatcac taaagatggg aatttgtatg gatggggctg gggccgatat 600
ggaaacttgg gattgggtga cagaaacgat cgattgctgc ctgagaaagt gactgttgat 660
ggtgacaaga tggccatggt tgcttgtggc tggcggcaca caatatgtgt ttcatcttct 720
ggtggattat acacaaatgg atggggcaaa tacggccagc taggacatgg ggattttgag 780
gatcatcttg tgcctcgcaa ggttcaagcc ttgagtgata agttcatttc gcaggtatca 840
ggtgggtgga ggcatagtat ggcactcacg tctagtggac aacttttggg ctggggatgg 900
aataagttcg gacagattgg agttggtaac aattttgatt gttgctctcc catgcaagtg 960
aactttcccc atgatcagaa agtacaaatg atctcatgcg ggtggagaca cacgattgct 1020
gttactgaac gtgagaatgt atattcttgg ggaagaggag caaatggaca acttgggaat 1080
ggagaaacca tagaccgcaa tgttccaaca attattgagg ccttcagtgt tgatggatct 1140
tccggacagc acatagaatc ctcaaaacct tatccatcat cagggaaatc ctcgtcctcc 1200
atatcagaga gatatgcaat tgttccagat gaaactgcct cgggatcaca acctacaact 1260
tcagaagagg gaaataggca tgataccagt gtccctgaaa gtgatgtcaa ttga 1314
<210> 6
<211> 1425
<212> DNA
<213> soybean (Glycine max (l.) merr.)
<400> 6
atgctgcact cttcaagtct actctttctc tctccttcca ctggtcccat caatgttata 60
aacccttgcc cctctttctc ttctctcttc cctcccatta ccatggaatt ggaagtcacc 120
gccgctactt ctccaccttc tcgcgttctc ctcatctccg ccggtgccag ccacaccgtt 180
gcccttctct ctgggaatgt tgtgtgctcg tggggtcgcg gagaggatgg acagttaggc 240
catggtgaca ccgatgatag actcttaccc actcatctca gtgcattgga tgctcaacaa 300
attgattcta ttgcatgtgg agctgatcat acccttgcgt attccgaatc acgcaatgaa 360
ctctatagtt ggggatgggg tgattttgga aggttgggtc atggcaattc tagtgatttg 420
ctcattcctc agcctattat agcattgcaa ggtctaagga taaagcaaat tgcctgtggg 480
gatagccact gtttggcagt taccatggaa ggcgaggttc agagttgggg gaggaatcaa 540
aatggtcaac ttggacttgg cacctcggag gattctcttg tgccacaaaa gattcaaaca 600
tttcagggag tacctatcaa aatggttgct gcaggtgcag aacacagtgt agctattact 660
gaaaatggag aactgtatgg atggggttgg ggccgatatg gaaatttggg gttgggggat 720
agaaatgatc gctggattcc tgagaaagtt tcttctgttg attgtgacaa gatggtcatg 780
gttgcttgtg gttggcggca tacaatttct gtttcatctt tgggtggctt atacacatat 840
gggtggagca aatatggcca gttaggacat ggaaattttg aggattctct tgtgcctcaa 900
aagcttcaag ccttgagtga taagttaatc agtcaggtat cgggtggttg gaggcatagt 960
atggcactga cgtctactgg actactatat ggatggggtt ggaataagtt tggacaggtt 1020
ggagttggtg acaatgttga tcgttgctct cctgtgcaag tgaagttccc ccatgatcag 1080
aaagtagttc agatctcatg tgggtggaga cacacaattg ctgtaactga aaaggaaaat 1140
gtattttctt ggggaagagg cacaaatggg caacttgggc atggggatac cgttgaccgg 1200
aattctccaa agattattga ggcattgagt gtggatggat cttccgggcc gcacatagaa 1260
tcctcaaaca ctgatctatt gtcagggaaa agtggtgcct ccttatctga gagatatgca 1320
gttgttccgg atgaaactgt ctcaggacaa actgctagtt caagcagtgg agataggctt 1380
gatatcagtg tcccagaaag tgatgtcaaa cggatccgag tttga 1425
<210> 7
<211> 62
<212> DNA
<213> artificial sequence
<400> 7
ggggacaagt ttgtacaaaa aagcaggctt aatgaaatgt tattttgatg gacaatacag 60
ga 62
<210> 8
<211> 48
<212> DNA
<213> artificial sequence
<400> 8
ggggaccact ttgtacaaga aagctgggtt tcaaacacgg agccgttt 48
<210> 9
<211> 57
<212> DNA
<213> artificial sequence
<400> 9
ggggacaagt ttgtacaaaa aagcaggctt aatggaagat gaagtgatga gtgaagt 57
<210> 10
<211> 54
<212> DNA
<213> artificial sequence
<400> 10
ggggaccact ttgtacaaga aagctgggtt tcaattgaca tcactttcag ggac 54
<210> 11
<211> 53
<212> DNA
<213> artificial sequence
<400> 11
ggggacaagt ttgtacaaaa aagcaggctt aatgctgcac tcttcaagtc tac 53
<210> 12
<211> 52
<212> DNA
<213> artificial sequence
<400> 12
ggggaccact ttgtacaaga aagctgggtt tcaaactcgg atccgtttga ca 52
<210> 13
<211> 19
<212> DNA
<213> artificial sequence
<400> 13
gattctcttg tgcctcaga 19
<210> 14
<211> 18
<212> DNA
<213> artificial sequence
<400> 14
agattgtcac cgactcca 18
<210> 15
<211> 20
<212> DNA
<213> artificial sequence
<400> 15
ggacagattg gagttggtaa 20
<210> 16
<211> 19
<212> DNA
<213> artificial sequence
<400> 16
ttggaacatt gcggtctat 19
<210> 17
<211> 18
<212> DNA
<213> artificial sequence
<400> 17
tgatcgttgc tctcctgt 18
<210> 18
<211> 18
<212> DNA
<213> artificial sequence
<400> 18
ccacactcaa tgcctcaa 18

Claims (4)

  1. The application of GmUVR8 gene family in improving the isoflavone content of plants is characterized in that: the GmUVR8 gene family includes the following three members: the GmUVR8a gene, the GmUVR8b gene, and the GmUVR8c gene; the amino acid sequence of the GmUVR8a gene is shown as SEQ ID NO. 1; the amino acid sequence of the GmUVR8b gene is shown as SEQ ID NO. 2; the amino acid sequence coded by the GmUVR8c gene is shown as SEQ ID NO. 3;
    the CDS sequence of the GmUVR8a gene is shown in SEQ ID NO. 4; the CDS sequence of the GmUVR8B gene is shown in SEQ ID NO. 5; the CDS sequence of the GmUVR8c gene is shown in SEQ ID NO. 6;
    the plant is soybean;
    the isoflavone is daidzin, genistin, daidzein and genistein;
    the application is specifically as follows: the expression level of the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene is increased in the plant to increase the isoflavone content of the plant.
  2. 2. The use according to claim 1, characterized in that: the expression level of the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene in the plant is improved by the following method: the exogenous GmUVR8a gene, gmUVR8b gene or GmUVR8c gene is overexpressed in plants.
  3. 3. The use according to claim 2, characterized in that: the expression vector of the recombinant plant, which is used for over-expressing exogenous GmUVR8a genes, gmUVR8b genes or GmUVR8c genes in plants, is transferred into the plants through a transgenic technology.
  4. 4. A use according to claim 3, characterized in that: the recombinant plant expression vector is specifically constructed and obtained by the following method: the GmUVR8a gene, gmUVR8b gene or GmUVR8c gene fragment is connected to the intermediate vector P221 through BP reaction, and then connected to the final vector PGWB642 through LR reaction, so that the recombinant super-expression vector is obtained.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054636A (en) * 1997-09-17 2000-04-25 E. I. Du Pont De Nemours And Company Isoflavone biosynthetic enzymes
CN108728451A (en) * 2018-06-14 2018-11-02 福建农林大学 A kind of transcription factor GmPHR of the special responding low-phosphor of soybeanLPAnd application
CN109486850A (en) * 2018-12-04 2019-03-19 厦门大学 The light genetic tool of UV-B light down regulation chromatin interaction over long distances
CN110878313A (en) * 2019-12-21 2020-03-13 吉林大学 Soybean Gm-SEIPIN2 family gene and application thereof in increasing oil content of plant seeds
CN111303256A (en) * 2018-12-10 2020-06-19 中国科学院上海生命科学研究院 MYB and UVR8 are combined with each other in a UV-B dependent mode to regulate and control growth and development of plant roots
CN111978388A (en) * 2020-09-02 2020-11-24 厦门大学 Rice UV-B light receptor gene OsUVR8a and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054636A (en) * 1997-09-17 2000-04-25 E. I. Du Pont De Nemours And Company Isoflavone biosynthetic enzymes
CN108728451A (en) * 2018-06-14 2018-11-02 福建农林大学 A kind of transcription factor GmPHR of the special responding low-phosphor of soybeanLPAnd application
CN109486850A (en) * 2018-12-04 2019-03-19 厦门大学 The light genetic tool of UV-B light down regulation chromatin interaction over long distances
CN111303256A (en) * 2018-12-10 2020-06-19 中国科学院上海生命科学研究院 MYB and UVR8 are combined with each other in a UV-B dependent mode to regulate and control growth and development of plant roots
CN110878313A (en) * 2019-12-21 2020-03-13 吉林大学 Soybean Gm-SEIPIN2 family gene and application thereof in increasing oil content of plant seeds
CN111978388A (en) * 2020-09-02 2020-11-24 厦门大学 Rice UV-B light receptor gene OsUVR8a and application thereof

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Effects of UV-B radiation on the isoflavone accumulation and physiologicalbiochemical changes of soybean during germination Physiological-biochemical change of germinated soybean induced by UV-B;Meng Ma等;Food Chemistry;第250卷;第259-267页 *
NCBI GenPept XP_003523212.2，ultraviolet-B receptor UVR8 isoform X2[Glycine max];无;NCBI GenPept;全文 *
NCBI GenPept XP_003525133.1，ultraviolet-B receptor UVR8[Glycine max];无;NCBI GenPept;全文 *
NCBI GenPept XP_003526878.4，ultraviolet-B receptor UVR8 isoform X2[Glycine max];无;NCBI GenPept;全文 *
Ultraviolet-B radiation: a potent regulator of flavonoids biosynthesis, accumulation and functions in plants;Avantika Pandey等;CURRENT SCIENCE;第119卷(第2期);第176-186页 *
紫外辐射对大豆异黄酮含量变化的影响;李先佳等;大豆科学;第28卷(第2期);第357-359页 *

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