CN114409752A - Application of GmUVR8 gene family in improving plant isoflavone content - Google Patents

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

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CN114409752A
CN114409752A CN202210096864.2A CN202210096864A CN114409752A CN 114409752 A CN114409752 A CN 114409752A CN 202210096864 A CN202210096864 A CN 202210096864A CN 114409752 A CN114409752 A CN 114409752A
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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 a GmUVR8 gene family in improving the content of plant isoflavone. The GmUVR8 gene family includes three members: a GmUVR8a gene, a GmUVR8b gene and a GmUVR8c gene. According to the invention, the excessive expression vector construction is carried out on the GmUVR8a, b and c genes, and the stable transformation is carried out in the soybean body, so that the content of secondary metabolites such as daidzein, daidzin, genistein and genistin in the stably transformed soybean obtained by excessive expression of the GmUVR8a, b and c is obviously improved, and the quality of the soybean is further improved. Therefore, overexpression of the GmUVR8 gene family in plants is an effective way to improve plant quality, and has important production and theoretical research significance.

Description

Application of GmUVR8 gene family in improving plant isoflavone content
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a GmUVR8 gene family in improvement of plant isoflavone content.
Background
Sunlight is of great importance in plant growth and development. Light not only can be used as an important source for plant photosynthesis to promote the growth and development of plants, but also realizes plant cell signal transduction through a series of light receptors. Ultraviolet light is an inherent component of sunlight and inevitably affects the growth and development of plants. The ultraviolet light is 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 UV-B due to the presence of the atmosphere on the earth's surface. Although the proportion of the total light energy which is finally reached by the UV-B to the ground is less than 0.1 percent, the UV-B still has vital importance for the growth and development of plants. For example, low intensity UV irradiation inhibits the normal growth of the Arabidopsis hypocotyls. For another example, high intensity ultraviolet radiation damages DNA and proteins, causing the accumulation of plant reactive oxygen species. In response to uv radiation, plants develop a series of self-defense mechanisms, such as the synthesis of the secondary metabolite isoflavone, as a "sunscreen" against damage from uv radiation.
Soy isoflavones are found mainly in leguminous plants, and are present in the highest amounts in soybean seed coats, hypocotyls, and cotyledons. The soybean isoflavone is a natural estrogen, and can make up for the defect of insufficient secretion of female estrogen after long-term administration, and regulate estrogen level in human body, thereby relieving climacteric syndrome. In addition, isoflavones can prevent cardiovascular diseases and osteoporosis, and are natural cancer chemopreventive agents. In soybeans, isoflavones are mainly classified into 3 groups, namely glycitins (Daidzingroups), genistins (genistinroups), and glycitinosides (glycitinroups). Each group exists in 4 forms such as free form, glucoside form, acetyl glucoside form, malonyl glucoside form, etc.
During the growth and development of the plants, the increase of the content of isoflavone can obviously improve the disease resistance of the plants. Research shows that ultraviolet radiation promotes the generation of isoflavone substances and may increase the disease resistance of plants. The UV RESISTANCE LOCUS 8 (UVR8) protein is the only identified ultraviolet light receptor of plants in recent years, so that the over-expression of the GmUVR8 receptor protein can improve the capability of responding to ultraviolet light signals, promote the synthesis of isoflavone, obtain the plant line rich in isoflavone and have certain production and application values.
Disclosure of Invention
The invention aims to provide application of a GmUVR8 gene family in improving the content of plant isoflavone.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of a GmUVR8 gene family in improving the content of plant isoflavone, wherein the GmUVR8 gene family comprises the following three members: a GmUVR8a gene, a GmUVR8b gene and a GmUVR8c gene; the amino acid sequence coded by the GmUVR8a gene is shown as SEQ ID NO. 1; the amino acid sequence coded by the GmUVR8b gene is shown as SEQ ID NO. 2; the amino acid sequence of the GmUVR8c gene code is shown in 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, and genistein.
Further, the application is as follows: increasing the expression level of GmUVR8a gene, GmUVR8b gene or GmUVR8c gene 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: overexpression of exogenous GmUVR8a gene, GmUVR8b gene or GmUVR8c gene in plants.
Furthermore, the above-mentioned exogenous GmUVR8a gene, GmUVR8b gene or GmUVR8c gene which is overexpressed in the plant is a recombinant plant expression vector which is used for transferring the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene which is overexpressed into the plant through a transgenic technology.
Further, the recombinant plant expression vector is specifically constructed by the following method: after the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene fragment is connected to an intermediate vector P221 through a BP reaction, the gene fragment is connected to a final vector PGWB642 through an LR reaction, and thus a recombinant plant expression vector is obtained.
The invention has the following remarkable advantages:
the overexpression of GmUVR8 protein in plants can enhance the capability of responding to ultraviolet light signals and promote the synthesis of isoflavone, so that the plant line rich in isoflavone can be obtained by the method, and the method 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 present invention.
FIG. 2 is a schematic diagram of the determination of the overexpression expression of Gmuvr8a, b, c.
FIG. 3 shows the determination result of the content of daidzein overexpressed in Gmuvr8a, b, c.
FIG. 4 shows the result of determination of the content of over-expressed daidzein in Gmuvr8a, b, c.
FIG. 5 shows the result of determination of the content of the over-expressed genistein in Gmuvr8a, b, c.
FIG. 6 shows the result of determination of the content of GMuvr8a, b, c over-expressed genistin.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. Determination of vector sequencing in the following examples was determined by sequencing by conventional sequencing.
The soybean cultivar Huachun No.6 is a background material of the invention, and is a soybean cultivar bred by agricultural college of southern China agricultural university and by breeding the article Guizao No.1 multiplied by Brazil No. 8. The third meeting of the second national crop variety approval committee passes by 7 and 28 days in 2009, and the approval number is national approval bean 2009012.
Example 1 Soybean Gmuvr8a, Gmuvr8b, Gmuvr8c overexpress seed profiles
03-3 is wild type material; SR1-14 is GmUVR8a gene overexpression material; SR2-1 is GmUVR8b gene overexpression material: SR3-1 is GmUVR8c gene overexpression material. The Gmuvr8a, Gmuvr8b, Gmuvr8c transgenic T3 lines and the wild type line 03-3 seeds are shown in FIG. 1.
EXAMPLE 2 Synthesis of Soybean cDNA
RNA of mature true leaf of No.6 Huachun soybean is extracted, and cDNA is synthesized by using a reverse transcription kit.
EXAMPLE 3 construction of overexpression vectors
With reference to the Gateway kit (available from invitrogen) instructions, the linker-bearing full-length fragment of the gene of interest was amplified by adding the linker to Gmuvr8a by PCR reaction with high fidelity DNA polymerase using primers UVR8a-Forward primer and UVR8a-Reverse primer: UVR8a-Forward primer:
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAATGTTATTTTGATGGACAATACAGGA-3’；
UVR8a-Reverse primer：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAAACACGGAGCCGTTT-3’。
adding a linker to Gmuvr8b by a PCR reaction of high-fidelity DNA polymerase by using primers UVR8b-Forward primer and UVR8b-Reverse primer to amplify a target gene fragment with the linker:
UVR8b-Forward primer：
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAAGATGAAGTGATGAGTGAAGT-3’；
UVR8b-Reverse primer：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATTGACATCACTTTCAGGGAC-3’。
adding a linker to Gmuvr8c by a PCR reaction of high-fidelity DNA polymerase by using primers UVR8c-Forward primer and UVR8c-Reverse primer to amplify a target gene fragment with the linker:
UVR8c-Forward primer：
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTGCACTCTTCAAGTCTAC-3’；
UVR8c-Reverse primer：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAAACTCGGATCCGTTTGACA-3’。
and (3) PCR reaction system: soybean cDNA was used as template 1. mu.L, Primer-F and Primer-R (Primer concentration 10. mu. mol) were each 1. mu.L, dNTP Mix 4. mu.L, Prime STAR GXL 1. mu.L, 5XGXL Buffer 10. mu.L, ddH2O 32. mu.L.
And (3) PCR reaction conditions: 5min at 98 ℃ and 30sec at 98 ℃; annealing at 55 ℃ for 30 sec; extension at 72 ℃ for 1min30sec for a total of 30 cycles.
PCR products are subjected to agarose gel electrophoresis detection, then gel cutting, purification and recovery are carried out, target gene fragments are respectively connected with a pDNOR221 vector (purchased from invitrogen company) for BP reaction and overnight connection at 25 ℃, the connection products are transformed into escherichia coli DH10B competent cells, positive clones are screened for sequencing, correctly read-through clones are extracted to obtain plasmids, and an entry vector is constructed. The entry vector plasmid and the PGWB642 plasmid vector carrying attR sites were subjected to LR reaction, ligated overnight at 25 ℃ and transformed into E.coli DH10B competent cells to verify positive clones. Culturing positive clone, extracting and purifying the plasmid, transferring the transformation plasmid into agrobacterium strain GV3101, and infecting and transforming prepared soybean.
Example 4 Stable transformation of Soybean
Placing the soybeans infected by the agrobacterium into a co-culture medium and culturing for 4 days at 25 ℃; then transferred to a recovery medium for 7 days at 25 ℃ and then transferred to a resistant redifferentiation medium containing Basta for 14 days at 25 ℃, then transferred to a resistant redifferentiation medium containing phytohormone and Basta for 14 days at 25 ℃ and finally transferred to an elongation medium for 14 days at 25 ℃. The plants obtained were transferred to soil for growth and screened for positive seedlings by spraying Basta diluted 1: 1000.
Example 5 Gene expression level analysis
And (3) analyzing the gene expression level of the transgenic line by using a real-time fluorescent quantitative PCR method. Respectively extracting RNAs in leaves of Gmuvr8a, Gmuvr8b and Gmuvr8c transgenic T3 strains and wild strains 03-3, inverting by using a reverse transcription kit to obtain cDNA serving as a template, and performing real-time fluorescence quantitative PCR. GmActin-11 is used as an internal reference gene, the expression level of a wild type strain 03-3 is 1, and analysis is carried out by a-delta C (t) method. 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’。
and (3) amplification procedure: 3min at 94 ℃; 94 ℃ for 5sec, 60 ℃ for 30 sec; for a total of 40 cycles. After the amplification cycle is finished, the temperature is raised to 65 ℃, and then the temperature is raised to 95 ℃ to denature the DNA product.
Amplification conditions: mu.L of cDNA template, 0.3. mu.L each of Primer-F and Primer-R (Primer concentration 10. mu. mol), 7.5. mu.L of 2 XSSYBR Green qPCR Mix, ddH2O 5.9 μL。
The expression levels of the Gmuvr8a, Gmuvr8b and Gmuvr8c genes in the soybean transgenic T3 strain at the transcription level are shown in FIG. 2. SR1-14 and SR1-15 are expression levels of Gmuvr8a, and compared with a wild type, the expression level of SR1-14 is 8.7 times that of the wild type, and the expression level of SR1-15 is 6.8 times that of the wild type; SR2-1 and SR2-10 are expression levels of Gmuvr8b, compared with a wild type, the expression level of SR2-1 is 72 times that of the wild type, and the expression level of SR2-10 is 107 times that of the wild type; SR3-1 and SR3-2 are expression levels of Gmuvr8c, and compared with a wild type, the expression level of SR3-1 is 10.49 times that of the wild type, and the expression level of SR3-2 is 3.7 times that of the wild type.
EXAMPLE 6 isoflavone content determination
The contents of 4 soybean isoflavone compounds, namely daidzin, genistin, daidzein and genistein in seeds of Gmuvr8a, Gmuvr8b, Gmuvr8c transgenic T3 generation strains and wild type strains 03-3 are respectively measured by adopting ultra-high performance liquid chromatography. The method comprises the following steps: a chromatographic column: waters BEH C18100 mm × 2.1mm 1.7 μm, mobile phase: a is 0.1% FA water, B is 0.1% FA ACN4.2, gradient elution; flow rate: 0.3 mL/min; column temperature: at 40 ℃.
The daidzein content in the seeds of the transgenic T3 generation strain is shown in FIG. 3. L14 and L15 are the content measurement of the daidzein of Gmuvr8a, compared with the wild type, the content of the L14 daidzein is 2.19 times of that of the wild type, and the content of the L15 daidzein is 2.21 times of that of the wild type; l10 is the content measurement of daidzein of Gmuvr8b, compared with wild type, the content of L10 daidzein is 1.78 times of that of wild type; l1 and L2 are measured by the content of the daidzein of Gmuvr8c, and compared with the wild type, the content of the daidzein of L1 is 3.67 times that of the wild type, and the content of the daidzein of L2 is 2.77 times that of the wild type.
The content of daidzein in the seeds of the transgenic T3 generation strain of soybean is shown in FIG. 4. L14 and L15 are the content measurement of the soybean glycoside of Gmuvr8a, compared with the wild type, the content of the soybean glycoside of L14 is 1.12 times of that of the wild type, and the content of the soybean glycoside of L15 is 1.08 times of that of the wild type; l10 is the content measurement of the soybean glycoside of Gmuvr8b, compared with the wild type, the content of the soybean glycoside of L10 is 1.1 times of that of the wild type; l1 and L2 are the content measurement of the soybean glycoside of Gmuvr8c, compared with the wild type, the content of the soybean glycoside of L1 is 1.25 times of that of the wild type, and the content of the soybean glycoside of L2 is 1.12 times of that of the wild type.
The content of genistein in the seeds of transgenic T3 generation strain of soybean is shown in FIG. 5. L14 and L15 are the content measurement of Gmuvr8a genistein, compared with wild type, the content of L14 genistein is 1.17 times of that of wild type, and the content of L15 genistein is 1.12 times of that of wild type; l1 is the content measurement of Gmuvr8b genistein, and compared with wild type, the content of L1 genistein is obviously reduced; l1 and L2 are the content measurement of Gmuvr8c genistein, compared with wild type, the content of L1 genistein is 2.42 times that of wild type, and the content of L2 genistein is 1.8 times that of wild type.
The content of genistin in the seeds of the transgenic T3 generation strain of soybean is shown in FIG. 6. L14 and L15 are content measurement of Gmuvr8a genistin, and compared with a wild type, the content of L14 and L15 genistin is not obviously changed; l1 is the content measurement of Gmuvr8b genistin, and compared with the wild type, the content of L1 genistin is obviously reduced; l1 and L2 are the content determination of Gmuvr8c genistin, compared with the wild type, the content of L1 genistin is 1.14 times of that of the wild type, and the content of L2 genistin is 1.12 times of that of the wild type.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
Application of <120> GmUVR8 gene family in improving content of plant isoflavone
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<160> 18
<170> PatentIn version 3.3
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Glu Ser Asp Val Asn
435
<210> 3
<211> 474
<212> PRT
<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 (8)

  1. The application of GmUVR8 gene family in improving the content of plant isoflavone is characterized in that: the GmUVR8 gene family includes three members: a GmUVR8a gene, a GmUVR8b gene and a GmUVR8c gene; the amino acid sequence coded by the GmUVR8a gene is shown as SEQ ID NO. 1; the amino acid sequence coded by the GmUVR8b gene is shown as SEQ ID NO. 2; the amino acid sequence of the GmUVR8c gene code is shown in SEQ ID NO. 3.
  2. 2. Use according to claim 1, characterized in that: 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.
  3. 3. Use according to claim 1, characterized in that: the plant is soybean.
  4. 4. Use according to claim 1, characterized in that: the isoflavone is daidzin, genistin, daidzein and genistein.
  5. 5. Use according to claim 1, characterized in that: increasing the expression level of GmUVR8a gene, GmUVR8b gene or GmUVR8c gene in the plant to increase the isoflavone content of the plant.
  6. 6. Use according to claim 5, characterized in that: the method for improving the expression level of the GmUVR8a gene, the GmUVR8b gene or the GmUVR8c gene in the plant is realized by the following steps: overexpression of exogenous GmUVR8a gene, GmUVR8b gene or GmUVR8c gene in plants.
  7. 7. Use according to claim 6, characterized in that: the recombinant plant expression vector which excessively expresses the exogenous GmUVR8a gene, the exogenous GmUVR8b gene or the exogenous GmUVR8c gene in the plant is used for transferring the recombinant plant expression vector which excessively expresses the GmUVR8a gene, the exogenous GmUVR8b gene or the exogenous GmUVR8c gene into the plant through a transgenic technology.
  8. 8. Use according to claim 7, characterized in that: the recombinant plant expression vector is specifically constructed by the following method: the recombinant overexpression vector is obtained by connecting a GmUVR8a gene, a GmUVR8b gene or a GmUVR8c gene fragment to an intermediate vector P221 through a BP reaction and then connecting to a final vector PGWB642 through an LR reaction.
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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
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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
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