CN104761627A - Application of GmbHLH transcription factor in promotion of synthesizing soy isoflavone - Google Patents

Abstract

The invention relates to an application of GmbHLH transcription factor in promotion of synthesizing soy isoflavone, belonging to the field of plant genetic engineering. The amino acid sequence of the GmbHLH transcription factor is shown as SEQ ID No.2, the nucleotide sequence of the encoding gene is shown as SEQ ID No.1. The selected GmbHLH transcription factor is only expressed in soy cell nucleus, and the content of the soy isoflavone can be increased by regulating the expression quantity of gene through combination with DNA sequence. No metabolites are generated during the whole process of GmbHLH as a transcription factor, so that the hidden potential hazard of the transgenic metabolism can be reduced to the greatest extent.

Description

GmbHLH transcription factor is promoting the application in soybean isoflavones synthesis

Technical field

The present invention relates to plant genetic engineering field, be specifically related to the application that a kind of soybean bHLH transcription factor and encoding gene thereof improve isoflavone content level in genetically engineered soybean tissue.

Background technology

Isoflavones, as a kind of secondary metabolite distinctive in leguminous plants, plays the effect of plant protecting chemical in plant materials, particularly in the invasion of Genes For Plant Tolerance external fungi, plays important provide protection.Meanwhile, isoflavones also serves vital role as the analogue of human estrogen in promotion human health.Of common occurrence for the article of description isoflavones in biology and medical science in recent years, mainly set forth isoflavones in the effect promoted health and in preventing disease.These contents concentrate on mostly: isoflavones balance women estrogen level, prevention and therapy Menopause complication; Regulate cardiovascular systems, improve myocardial ischemia, reduce blood steroid substance content; Anti-cancer and anticancer effect, particularly reduce the function of mammary cancer; In addition, isoflavones also has curative effect such as prevention senile dementia and osteopathy etc.

Along with the generally improvement of our people's standard of living, the input of people in improving the quality of living is more, particularly adds for family's healthy aspect spending.On the other hand, China proportion of aged population is also progressively increasing, and this ratio making healthy aspect expenditure account for total financial aspects expenditure increases further.Along with people are to science and technology and the more deep and comprehensive understanding of knowledge, similarly be that isoflavone formulation can be accepted as healthcare products.Isoflavones can some multiple stubborn diseases of prevention and therapy, and have good curative effect, therefore also increase its consumers demand thereupon.Soybean, as one of main cash crop of our times, is also the main separation and Extraction source of soybean isoflavones (soybeanisoflavone).So the isoflavone content improving soybean plant strain has very high economy and social value.

The transcription factor of plant is generally positioned in the nucleus of plant, by conjunction with specific DNA sequence dna, regulates the expression of downstream gene, plays the effect of same metabolic pathway sometimes, thus the downstream product of this metabolic pathway is accumulated in a large number.The metabolism of plant phenylalanine is mainly divided into metabolic process and later stage metabolic process in earlier stage, in early days in metabolic process, namely under the effect of looking into youngster's ketone isomerase CHI, 4 ' is generated, 5 ', 7 '-trihydroxy-flavanone, this material will become multiple zymolyte, under flavanone-3 hydroxylase (F3H) and dihydro reductase enzyme (DFR) effect, generate anthocyanidin on the one hand, under isoflavone synthase (IFS) and 2-hydroxyl isoflavanone dehydratase (HID) effect, synthesize isoflavones on the other hand.So, suppress the expression of anthocyanidin synthesis related gene, substrate can be impelled to flow to isoflavones compound direction.The research of Xie (2013) is pointed out, overexpression AtbHLH3 transcription factor can reduce the anabolism of Arabidopsis anthocyanin.

At present, transgenic method is the important means improving crop quality and output.Because traditional breeding method is for the raising limitation of crop yield, and new variety the cultivation time very long, hinder the development and progression of agricultural industry, cause a large amount of waste dropped into simultaneously.Transgenic method well solves this problem, shortens the breeding time limit, and significantly improves the object proterties of crop.But genetically modified safety problem also receives extensive concern.In order to solve Transgene-safty problem, being on the one hand step up to improve censorship, is step up to study the impact for genetically modified crops inner metabolism product of the foreign gene that imports on the other hand, and the toxicity research of oneself protein.At present, the process LAN of native gene can solve the safety problem that genetically modified crops run into a certain extent.Native gene comes from crop itself, improves corresponding plant trait by native gene overexpression, thus reduction foreign gene imports the hidden danger that meta-bolites is failed to understand in the generation caused greatly, is also convenient to carrying out of gene self research work.

Summary of the invention

The invention provides a kind of GmbHLH transcription factor promoting the application in soybean isoflavones synthesis, to solve the security hidden trouble of gene meta-bolites.

GmbHLH transcription factor is promoting the application in soybean isoflavones synthesis.

Described GmbHLH transcription factor, is characterized in that its aminoacid sequence is as described in SEQ ID No.2.

The gene of the soybean bHLH transcription factor described in coding, is characterized in that its nucleotide sequence is as described in SEQ ID No.1.

A kind of recombinant vectors, utilize plant expression vector pCAMBIA3301H, the part of cloning the GmbHLH3a transcription factor open reading frame obtained is configured to recombinant vectors pCAMBIA3301H-GmbHLH3a, containing 35s promotor in described pCAMBIA3301H carrier.

The bHLH transcription factor of raising isoflavone content provided by the present invention derives from Jilin 32 soybean varieties, bHLH transcription factor gene called after GmbHLH3a.

SEQ ID No.1 is by 1515 based compositions; SEQ ID No.2 is made up of 504 amino-acid residues, contain a HLH structural domain from N-terminal 51 to 227 amino acids residue containing a bHLH-MYC_N structural domain and 358 to 407 amino acids residues, learn that these two structural domains belong to MYC class transcription factor family member by comparison and prediction; Containing a nuclear localization signal: from N-terminal 342 to 343 amino acids residue.

Utilize plant expression vector pCAMBIA3301H, the part of cloning the GmbHLH3a transcription factor open reading frame obtained is configured to recombinant vectors pCAMBIA3301H-GmbHLH3a.Containing 35s promotor in pCAMBIA3301H carrier, thus make GmbHLH3a can high expression in plant; PCAMBIA3301H carrier also comprises glyphosate-tolerance Bar gene, has verified the function with antiweed in multiple genetically modified crops, does not find that it has the toxicity of harmful to human so far simultaneously.PCAMBIA3301H-GmbHLH3a recombinant plant expression vector can be directed in various soybean varieties by particle gun and Agrobacterium tumefaciens mediated mode, also comprises in various dicotyledons.Finally, the soybean varieties of homoisoflavone content can be obtained.

The invention has the beneficial effects as follows: the present invention has cloned a soybean GmbHLH3a transcription factor gene relevant to soybean phenylalanine metabolic pathway, the homologous gene of this transcription factor is obtained by evolutionary analysis, learn that it belongs to bHLH gene family, especially itself and Arabidopis thaliana AtbHLH3 homology, therefore probably has close function; Prove that its basic structure is identical with MYC transcription factor by experiment further; The pCAMBIA3301H-GmbHLH3a recombinant plant expression vector soybean transformation Agrobacterium rhizogenes will built further, detects transgenosis root of hair isoflavone content, proves to which raises each component concentration of soybean isoflavones.These results of study are all that pCAMBIA3301H-GmbHLH3a recombinant vectors importing soybean raising isoflavone content provides effective foundation in basic theory and actual effect.

The GmbHLH3a transcription factor that the present invention selects, only expresses in soya cells core, by conjunction with having the expression amount of gene under DNA sequence dna regulation and control thus improve the content of soybean isoflavones.Whole process is transcription factor protein due to GmbHLH3a, does not produce meta-bolites, thus reduces the potential safety hazard of transgenosis meta-bolites to greatest extent.

Accompanying drawing explanation

Fig. 1 is the pcr amplification result figure of GmbHLH3a gene, M:2000bp maker.1-3:GmbHLH3 amplification;

Fig. 2 is MYC class transcription factor evolution tree graph;

Fig. 3 is the expression pattern figure of soybean MYC class transcription factor;

Fig. 4 is the expression pattern figure of GmbHLH3a recombinant protein, M:Protein Marker; 1:pET28a empty carrier induces bacterium without IPTG; 2:pET28a empty carrier IPTG induces bacterium; 3:pET28a-MYC2a does not induce bacterium; 4-8: be respectively pET28a-GmbHLH3a IPTG and induce 4h, 6h, 8h, 10h and 12h thalline;

Fig. 5 is GmbHLH3a gene Grain Development expression pattern figure;

Fig. 6 is GmbHLH3a gene Subcellular Localization test chart;

Fig. 7 is reporter plasmid, effect plasmid vector construct schematic diagram;

Fig. 8 is the screening schematic diagram of transgenosis root of hair positive findings, in figure:

A: transform K599pCAMBIA1300h – GmbHLH3 Soybean Root,

B: transform K599 (CK) pCAMBIA1300h Soybean Root,

C: obtain positive findings,

D: obtain negative findings;

Fig. 9 is the qRT-PCR qualification result of GmbHLH3a gene in transgenosis root of hair, wherein

1-3:pCAMBIA1300h–GmbHLH3；

4-6:pCAMBIA1300H；

Figure 10 is the measurement result figure of transgenosis root of hair isoflavone content high-efficient liquid phase chromatogram HPLC, wherein:

1. total isoflavone 2. Genistoside 3. glycitin 4. daidzin.

Embodiment

The Cloning and sequence analysis of embodiment 1:GmbHLH3a gene

The extraction of soybean RNA and the synthesis of cDNA: extract the soybean Jilin 32 Grain Development total serum IgE of 30 days with RNAiso Plus (purchased from TaKaRa company), with M-MLV reverse transcriptase (RNase H ^-) (purchased from TaKaRa) carry out reverse transcription, synthesis Article 1 cDNA.

The Design and synthesis of primer: carry out Blast comparison by 3 of soybean varieties Jilin 32 period (20,30 and 50 days) immature embryo express spectras order-checking gained sequence (Hua Da gene completes) input phytozome10.1, obtains the global cDNA sequence of a bHLH3-like higher with bHLH transcription factor protein sequence homology in plant.According to acquired cDNA sequence, its nucleotide sequence as described in Sequence N0.1, design and synthesis primer, GmbHLH3a-RT-F:5 '-CGCCAAATCACAATACCC-3 ', GmbHLH3a-RT-R:5 '-GCCACTTAACCTACAAGACAGA-3 '.With above-mentioned cDNA for template, pcr amplification is carried out: altogether 25: total system according to following reaction system and condition, include 10, include and carry out μ 0 according to following reaction system and condition, the primer GmbHLH3a-RT-F of 2.5mM dNTP mix 2 μ dNTP μ d and each 1 μm of primer GmbHLH3a-RT-R, cDNA 2 μ D, Ex Taq (purchased from TaKaRa) 0.5 μ a, mends deionized water to 25 μ 5.Reaction conditions: denaturation 94 DEG C of 8min; 94 DEG C of 40s, 58 DEG C of 90s, 72 DEG C of 1min, 30 circulations; 72 DEG C extend 8min.Be connected with cloning vector pMD18-T (purchased from TaKaRa) after being reclaimed by above-mentioned amplified fragments, send Hua Da gene sequencing after qualification, authentication sequence correctly retains for subsequent use afterwards.

Obtain through pcr amplification the sequence including GmbHLH3a full length gene ORF, the protein be made up of 502 amino-acid residues of encoding, comprise a conservative HLH binding domain, and the distinctive bHLH-MYC_N structural domain of MYC transcription factor, a nuclear localization signal.

The phylogenetic analysis of embodiment 2:GmbHLH3a gene

The soybean bHLH transcription factor announced according to plantTFDB online database and Arabidopis thaliana bHLH transcription factor data, use ClustalW2 software to produce unrooted evolutionary tree to two groups of Data Synthesis, the grouping into sub-families mark (classification schemes is with reference to the categorised content of calendar year 2001 Ralf Stracke article) using MEGA 6.0 to carry out evolutionary tree therefrom draws soybean and Arabidopis thaliana MYC class transcription factor family.The MYC class transcription factor obtained is carried out blast in ncbi database, detects relevant research information.Arabidopis thaliana or soybean transcription factor related names are changed to the research name of having announced, GmMYC genetic homology and the functional analysis of corresponding A tMYC transcription factor better can be understood.

Finally obtain 19 soybean MYC classes transcription factor (as Fig. 2) by analysis, because soybean belongs to ancient source tetraploid, so generally all multiple copied can be there is with the soybean GmMYC of AtMYC homology.GmbHLH3a belongs to the very high homology gene of AtbHLH3, and illustrate that GmbHLH3a and AtbHLH3 is consistent on protein structure, functional similarity degree also should be high.It is reported that AtbHLH3 has suppresses the metabolism of Arabidopis thaliana DFR gene to be expressed, thus suppresses the function of anthocyanidin synthesis.

Embodiment 3: soybean bHLH-MYC class transcription factor gene Grain Development expression pattern

By 3 of soybean varieties Jilin 32 period (20,30 and 50 days) immature embryo express spectra order-checking gained sequences, perl language is used relevant MYC transcription factor to be screened.The expression pattern cluster analysis using MeV to carry out gene finds 9 soybean MYC class transcription factor expression, and the MYC2 transcription factor expression amount activating DFR genetic expression declines, and suppresses the transcription factors such as GmbHLH3, GmbHLH13 of DFR genetic expression to significantly improve (as Fig. 3) at Grain Development expression amount.

The result height that this result and the budding isoflavones of soybean kernel significantly promote to accumulation rate during 50 days after 30 days conforms to.

The expression of embodiment 4:GmbHLH3a gene in prokaryotic system

According to GmbHLH3a gene order design and synthesis primer, and thereon, introduce EcoR I and Xho I restriction enzyme site respectively, GmbHLH3a-CP-F:5 '-GGAATTCATGGTGGGTGAGAAGTTTT-3 ' in downstream primer; GmbHLH3a-CP-R:5 '-CCGCTCGAGTCAACTTTTGGATAGGGAT-3 '.With pMD18-T-GmbHLH3a plasmid for template, carry out pcr amplification.Amplified production is reclaimed after test kit (purchased from Shanghai Pu Luomaige) purifying through sepharose DNA purifying, with Sac I and Hind III (various restriction enzyme is all purchased from TaKaRa) double digestion, after reclaiming purifying, connect with the same pET28a carrier with EcoR I and Xho I double digestion.Connect product conversion intestinal bacteria (E.coli) DH5 α, after empirical tests, proceed in Host Strains Rosetta (DE3) and express.

The prokaryotic expression of recombinant protein: respectively by positive Rosetta (DE3) bacterial strain with transform pET28a (+) empty carrier Rosetta (DE3) inoculation in 50ul/ml Kan solid LB media, 37 DEG C of overnight incubation.Picking list colony inoculation in 50ul/ml Kan LB liquid medium, 200rpm, 37 DEG C of shaking culture 12h.Arranging by the ratio of 1:100 is seeded in 10ml LB liquid nutrient medium with pipettor absorption bacterium liquid, 200rpm, 37 DEG C of shaking culture, and 3 ~ 4h detects OD600=0.4 ~ 0.6; Adding in IPTG (100mM) 101ul bacterium liquid to final concentration is 1mM, prepares one group of bacterium liquid simultaneously and does not add IPTG induction as a control group, 30 DEG C of shaking culture; Shaking culture was got 1ml bacterium liquid in 1.5ml centrifuge tube, the centrifugal 1min of room temperature 13000rpm, remove portion supernatant liquor, is left the resuspended thalline of 50ul LB nutrient solution after cultivating 4h every 2 hours; In thalline, add 50ul 2xloading Buffer, vortex mixes; Heating in water bath to 100 DEG C, boils 10min, the centrifugal 1min of 13000rpm, Aspirate supernatant, is transferred in new 1.5ml centrifuge tube, 4 DEG C of preservations;

The SDS-PAGE of expression product analyzes: carry out according to SDS-PAGE electrophoresis ordinary method.

After IPTG induction, GmbHLH3a recombinant protein (molecular size range is about 60kDa) can stablize expression (Fig. 4) effectively in Rosetta (DE3) bacterial strain, and GmbHLH3a albumen does not have toxicity in prokaryotic system.

Embodiment 5:GmbHLH3a gene Grain Development expression pattern

Respectively extract soybean Jilin 32 20 days, 30 days, 50 days embryos total serum IgE and reverse transcription becomes cDNA, method is with embodiment 1.With above-mentioned cDNA for template carries out real-time fluorescence quantitative PCR, according to the cDNA sequence of GmbHLH3a design real-time fluorescence quantitative PCR primer (OL3879:5 '-AAATTGGCTGGTCTTCACACTG-3 '; OL3880:5 '-CATTTCCACAACACCCTGCTC-3 ').With soybean constitutive expression gene β-actin with soybean composing type for internal reference (OL1016:CGGTGGTTCTATCTTGGCATC-3 '; OL1017:GTCTTTCGCTTCAATAACCCTA-3 ').Real-time fluorescence quantitative PCR instrument (Agilent AgilentMX3001) is utilized to carry out real-time RT-PCR analysis.Reaction system is containing 2 × SYBR Premix Ex Taq (purchased from TaKaRa) 10 μ L, cDNA 2 μ L, Primer F 0.4 μ L, Primer R 0.4 μ L, and moisturizing is to cumulative volume 20 μ L.Response procedures is 95 DEG C of 30s; 95 DEG C of 5s, 58 DEG C of 34s, 72 DEG C of 30s, 40 circulations.Adopt 2 ^{-Δ Δ CT}method analytical data, determines the relative expression quantity of gene.Each sampling spot establishes 3 technology to repeat, and test establishes 3 secondary pollutants to repeat (as Fig. 5) altogether.

Embodiment 6:GmbHLH3a gene Subcellular Localization is tested

Build GmbHLH3::eGFP and GmMYC2a::eGFP fusion gene, use BamH I, Kpn I and XbaI, Kpn I double digestion pMD18-T-GmbHLH3::eGFP, pMD18-T-GmMYC2a::eGFP and object carrier pCAMBIA3301 respectively.Final structure pCAMBIA3301H-GmbHLH3::eGFP and pCAMBIA3301H-GmMYC2a::eGFP Subcellular Localization carrier.Electric shocking method transform Agrobacterium tumefaciens EHA105.The single bacterium colony of picking agrobacterium tumefaciens, is inoculated in (50ul/ml Kan, 50mg/L Rifampin) YEP liquid nutrient medium, 28 DEG C, lucifuge shaking culture 16 ~ 20h; Draw 500ul agrobacterium tumefaciens bacterium liquid, be inoculated in 50ml (50ul/ml Kan, 50mg/L Rifampin) YEP liquid nutrient medium, 28 DEG C, lucifuge shaking culture 14 ~ 16h, survey OD600=0.5 ~ 0.7; 5000rpm, the centrifugal 10min of room temperature, outwells top waste liquid, the MS liquid nutrient medium resuspended bacterium liquid of preparation containing (10mM MgCl2,100mM AS), and room temperature leaves standstill 30min; Agrobacterium tumefaciens-mediated transformation transforms onion epidermis cell; After infecting end, epidermic cell inside onion is placed on aseptic filter paper and blots bacterium liquid, be transferred to by epidermic cell inside green onion subsequently and be covered with in the MS solid medium of aseptic filter paper containing (100uM AS), 25 DEG C, lucifuge cultivates 48 ~ 72h; By the 90% glycerine mounting of epidermic cell inside onion, laser copolymerization is used to show burnt micro mirror observation of cell; When after discovery green fluorescence, epidermic cell adds Hoechst inside onion and soak 5min, again observe the same area cell, to take pictures preservation with laser confocal microscope.

Result shows, GmbHLH3a and GmMYC2a schedules plant nucleolus (Fig. 6), this conclusion shows the only enforcement effect in nucleus of GmbHLH3a gene, do not have discovery to move to high expression in other plant cell structures, further demonstrate the security of the genetic material as genetically modified crops.

Embodiment 7: turn the preparation of GmbHLH3a transgenic soybean root of hair and the screening of assaypositive tissue

Structure pCAMBIA3301H-GmbHLH3 carrier and pCAMBIA3301H empty carrier proceed in Agrobacterium rhyzogenesK599.The Agrobacterium rhyzogenesK599 of picking positive colony, is inoculated in (100ul/ml Kan) YEP solid medium, and 28 DEG C of lucifuges cultivate 48h; The single bacterium colony of picking agrobacterium tumefaciens, is inoculated in (100ul/ml Kan) YEP liquid nutrient medium, 28 DEG C, lucifuge shaking culture 16 ~ 20h; Draw 500ul agrobacterium tumefaciens bacterium liquid, be inoculated in 50ml (100ul/ml Kan) YEP liquid nutrient medium, 28 DEG C, lucifuge shaking culture 14 ~ 16h, survey OD600=0.5 ~ 0.7.Infect soybean Hairy root and root growth is sent out in induction strong: the seedling of sprouting is removed seed coat, cotyledon is divided into two (method transforms similar to cotyledonary node) together with plumular axis along axis, remove terminal bud and axillalry bud part, laterally 3 ~ 5 cuttves are drawn at cotyledonary node and plumular axis junction, put into Agrobacterium and infect liquid, infect 6h, wherein change once when 3h and new infect liquid; Explant adaxial and its surface being inoculated in down is covered with in the Dual culture substratum of an aseptic filter paper, and limit mouth seals, and in 16h illumination, 8h lucifuge replaces lower cultivation 5 days; Explant is put into the sterilizing 1/2MS substratum being added with 250mg/L cephamycin, 250mg/L Pyocianil, cleaning and dipping 30min, removes surperficial bacterium liquid, subsequently by cotyledonary node adaxial and its surface upward, inserted by hypocotyl in induction of hairy roots substratum, induction of hairy roots produces after 20 days; Obtain having the positive root of hair tissue of soybean of fluorescence with laser confocal microscope screening, then RNA is organized in extraction further, reverse transcription forms Article 1 cDNA, utilizes qRT-PCR to detect the expression amount (method reference, embodiment 5) of GmbHLH3a.At least obtain transgenosis root of hair and the empty carrier transgenosis root of hair each three groups (Fig. 8 ~ 9) of process LAN GmbHLH3a.

Embodiment 8:HPLC method measures the isoflavone content of transgenosis root of hair

Accurately take Jilin 32 soybean positive transgenic root of hair fresh weight and be about 0.5g, put into mortar, add the methyl alcohol of 80% in 400g/100mg fresh weight ratio, fully grind, lapping liquid is transferred in 10ml centrifuge tube; Seal the centrifugal mouth of pipe, be placed in 80 DEG C of water-bath water-bath 14h; 12000g, centrifugal 15min, by the further removal of impurities of supernatant liquor 0.45um organic phase membrane filtration; Isoflavones extracting solution after removal of impurities is transferred to 10ml volumetric flask, adds the methanol constant volume of 80% to 10ml, lucifuge 4 DEG C preservation; HPLC method measures Immature Embryo Culture of Soybean isoflavone content (Figure 10).

Detected result shows that turning GmbHLH3a gene can improve isoflavone content to a certain extent.

Claims (4)

1.GmbHLH transcription factor is promoting the application in soybean isoflavones synthesis.

2. GmbHLH transcription factor as claimed in claim 1, is characterized in that its aminoacid sequence is as described in SEQ IDNo.2.

3. the gene of soybean bHLH transcription factor as claimed in claim 2 of encoding, is characterized in that its nucleotide sequence is as described in SEQ ID No.1.

4. a recombinant vectors, it is characterized in that: utilize plant expression vector pCAMBIA3301H, the part of cloning the GmbHLH3a transcription factor open reading frame obtained is configured to recombinant vectors pCAMBIA3301H-GmbHLH3a, containing 35s promotor in described pCAMBIA3301H carrier.