CN102899349A - Method for increasing plant folate content by using synergistic effect of transferred soybean genes of GTPCHI and ADCS - Google Patents
Method for increasing plant folate content by using synergistic effect of transferred soybean genes of GTPCHI and ADCS Download PDFInfo
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Abstract
The invention relates to the field of plant gene engineering, and specifically relates to a method for increasing plant folate content by using a synergistic effect of transferred soybean genes of GTPCHI and ADCS. The gene sequence of GTPCHI synthetic key enzyme of the soybean folate is shown as SEQ ID No. 1; and the gene sequence of ADCS is shown as SEQ ID No. 2. The synergistic effect of the transferred soybean genes of GTPCHI and ADCS can obviously increase the folate content in transgenic plants. Therefore, the method can be used as a method for producing the transgenic plants with high folate content. People can obtain natural folates directly by eating the plants; phenomenon that folate intake is lack can be solved; and adverse side effects brought by long-term taking folate tablets can be overcome.
Description
Technical field
The present invention relates to plant genetic engineering field, particularly, relate to the method that a kind of utilization turns soybean GTPCHI and ADCS gene synergistic function raising leaf acid content.
Background technology
Folic acid (folates) is the general name of tetrahydrofolic acid (THFA) (THF) and derivative thereof, is the water-soluble vitamin B group of a class, is called again FA, VB11, vitamin(e) M, and its chemical structure as shown in figure 12.
Wherein, THF is comprised of three parts: pyridine ring of talking endlessly (2-amino-4-hydroxy-6-methyl talk endlessly pyridine), para-amino benzoic acid pABA is connected poly-Glu with polyglutamic acid) (1-8 Glu λ connects), have another name called FolbaL.The one carbon unit of the different states of oxidation, methyl for example, methylene radical; inferior methylene radical; formyl radical, both form tetrahydrofolic acid (THFA)s (THF), 5-methyl THF, 5-formyl THF, 5, many kinds of folic acid derivatives of 10-methylene radical THF thereby be connected to the N10 of N5, pABA of the pyridine ring of talking endlessly or bridging.THF and derivative thereof are the important factors of mediation one carbon unit shift reaction as donor and the acceptor of one carbon unit.
Thereby plant and microorganism can be satisfied self-demand by de novo synthesis folic acid, and the mankind and animal then can not be synthesized folic acid, can only obtain by diet.Except the content of its folic acid such as spinach, beans and cabbage was relatively high, the folate content of other foods such as food crop, vegetable and fruit etc. was all very low, far can not satisfy human body to the demand of folic acid in the food plant.The requirement of general normal people's folic acid is 200～400 μ g/d, and the suggestion adult of the World Health Organization is at least 200 μ g/d, and pregnant woman and wet nurse are 400 μ g/d.
Folic acid is to keep the necessary type organic matter of the normal vital process of organism, and it namely is confirmed as far back as 1948 to the important trophism of human body.Human (or other animals) can cause megaloblastic anemia and leukopenia as lacking folic acid, and can increase the sickness rate of cardiovascular disorder and cancer; Folic acid is particularly important to the pregnant woman, as lacking folic acid at conceived in 3 months, can cause the fetal neural tube developmental defect, thereby increase the incidence of spina bifida, anencephalia, if folic acid insufficiency of intake within front 6 weeks of pregnancy, the possibility that the baby suffers from anencephaly and Rachischisis can increase by 4 times, secondly, the frequent Supplement of folic acid of pregnant woman can prevent that neonatal weight from kicking the beam, the congenital abnormality such as premature labor and infant cleft palate (harelip); Common erythrocytic generation and the maturation of promoting of folic acid and vitamin B12 is to make the indispensable material of red blood corpuscle; And folic acid participates in the synthetic of DNA, amino acid, pantothenic acid etc. in vivo; Folic acid can provide a large amount of uncombined carbon ions, supplies with the important raw chemical material of making nerve ending and consisting of the transmission impulsion, comes the normal development of underwriter's somatic nervous system.Find also that clinically folic acid has antitumor, as to promote the infant neurocyte and the effects such as brain cell development and Adjunctive Treatment For Schizophrenia.
The phenomenon of folic acid deficiency is at global ubiquity, especially in economic level and lower developing country and the under-developed country of standard of living.Show according to investigations, developing country every year because the baby due defective that folic acid deficiency causes has 200,000 examples at least, cause that because of folic acid deficiency the case of pregnant woman anemia has 10,000,000 at least.The U.S. and some western countries take the policy of interpolation synthetic foliamin in food to satisfy human's demand at present, and this is difficult to unsound in the food-processing industry system, that population distribution is uneven, economic condition is limited developing country's implementation, and whether the folic acid of long-term taking synthetic can bring adverse side effect also very troubling to human body simultaneously.Growing along with biotechnology transformed the folic acid metabolic pathway of synthesizing by engineered means and improves the food Folic Acid and have very large magnetism and extremely important meaning.
After deliberation comparatively clear of the biosynthesizing of folic acid in the plant materials, synthesising part relates to 3 different subcellular structures, and namely plastid, plastosome and tenuigenin are divided into three branches, and its branching diagram is as shown in figure 13.
Wherein, pterin partly is synthetic take GTP as precursor in tenuigenin, and the synthetic of para-amino benzoic acid is synthetic in plastid take chorismic acid as precursor, pterin and amino acid formic acid is transported to plastosome participates in synthetic folic acid.In this process, two key enzymes are arranged, be included in the pterin branch, GTP cyclase I (GTP cyclohydrolase I in the first step reaction, GTPCHI), can form dihydroneopterin triphosphoric acid (DHNTP) by catalysis GTP, and the amino deoxy chorismic acid synthetase (ADCS) in the first step reaction of pABA route of synthesis.The folate content of the coexpression of these two kinds of folic acid key enzymes in plant in can the Effective Raise plant found in research in recent years.(2007) such as Rocio I find that under study for action Arabidopis thaliana GTPCHI crosses in the transgenic Fructus Lycopersici esculenti of expression, and para-amino benzoic acid approach exhaustion in the high fruit of folate content can increase the content of organizing Folic Acid so draw the synthetic of suitable raising pterin.The deficiency of PABA infers that replenishing PABA in the plant that turns the GTPCHI gene then can further improve folate content so that folate content has only improved 2 times.Afterwards, they are the hybridization of the plant of overexpression PABA and overexpression pterin, make these two proterties in conjunction with after, its mature fruit Folic Acid has improved approximately 25 times.The acting in conjunction of this explanation GTPCHI and ADCS in tomato is synthetic very important to folic acid.The content of soybean Folic Acid is higher, and the GTPCHI of soybean and ADCS also do not report the synthetic synergy that whether exists of folic acid.
With GTPCHI and the ADCS coexpression of soybean, for carrying out the conversion of crop vegetables etc., improve the content of food crop vegetables Folic Acid, thereby satisfy human daily demand, reduce neonatal inborn defect, build up resistance, the generation of preventing cardiovascular disease, cancer etc. are significant.
Summary of the invention
The purpose of this invention is to provide the method that a kind of utilization turns soybean GTPCHI and ADCS gene synergistic function raising leaf acid content.
A kind of utilization according to the present invention turns the method for soybean GTPCHI and ADCS gene synergistic function raising leaf acid content, comprise above-mentioned two kinds of soybean genes: the step of GmGTPCHI gene and GmADCS gene transferred plant and coexpression, wherein, the CDS sequence of soybean GmGTPCHI gene is shown in SEQ ID No.1, and the CDS sequence of soybean GmADCS gene is shown in SEQ ID No.2.
Utilization according to the present invention turns the method for soybean GTPCHI and ADCS gene synergistic function raising leaf acid content, and is wherein, that the method for soybean GTPCHI and ADCS gene co-expressing is as follows:
Method (1) makes up respectively the expression vector of soybean GTPCHI and ADCS gene, then imports in the purpose plant coexpression; Or the co-expression carrier of method (2) structure soybean GTPCHI and the series connection of ADCS gene, then import in the purpose plant coexpression.
Particularly, for method (1), the GmGTPCHI of soybean and the CDS of GmADCS gene can be building up to respectively the plant over-express vector, and infect respectively in the plant, wherein, the positive transfer-gen plant of selecting GmGTPCHI to infect is done male parent, and the positive transfer-gen plant that GmADCS infects is done female parent, hybridizes to obtain independently transgenic line.According to embodiments of the invention, with the RT-PCR technology for detection expression level of goal gene in the transgenosis hybridization plant, and measure folate content in these plant, the result shows, the plant of infecting separately than GmGTPCHI and GmADCS all is significantly improved, and has improved 2.5～4.5 times than wild-type.
Perhaps, according to embodiments of the invention, for method (2), soybean GmGTPCHI and GmADCS can be driven by cauliflower mosaic virus (CaMV) 35S promoter respectively, be connected in series on the pCAMBIA3301 carrier, infect plant, the seed of gathering in the crops is screened at the resistance culture base, obtain transfer-gen plant.With the RT-PCR technology for detection expression level of goal gene in the transgenic plant, and measure folate content in these plant, find all to be significantly improved than the plant that GmGTPCHI and GmADCS infect separately, improved 3～4.5 times than wild-type.
Therefore, according to technical scheme of the present invention, with the synthetic key enzyme GTPCHI of the folic acid of soybean and ADCS coexpression in other plant, find that two enzyme actings in conjunction can significantly improve the folate content of Arabidopis thaliana, this is that the conversion of carrying out crop vegetable and fruit etc. is laid a good foundation, the human diet that can pass through directly obtain natural folic acid, solves folic acid and takes in the phenomenon that lacks, and overcome the adverse side effect that long-term taking folic acid tablet brings.
Description of drawings
Fig. 1 is the genotype identification of the positive transfer-gen plant of hybridization.Be depicted as the part electrophorogram of pcr amplification, wherein 4,5,6,7,8,10,11 for changing the plant of GmGTPCHI gene over to, and 1,2,4,5,6,7,8,9,11,12 for changing the plant of GmADCS gene over to.
Fig. 2 detects for GmGTPCHI, GmADCS expression level in the positive transfer-gen plant of hybridization.The negative contrast of wild-type Arabidopis thaliana (WT) all detects the expression of goal gene in the transgenic positive plant, expression amount there are differences in different plants.
Fig. 3 analyzes for hybridization Plant Leaf acid content.The WT wild-type is contrast, and GTPCHI is for changing separately the plant of GmGTPCHI over to, and ADCS is for changing separately the plant of GmADCS over to, and GA1-GA6 is the hybridization strain.
Fig. 4 is the carrier diagram that GmGTPCHI and GmADCS are cloned into pCAMBIA1301 simultaneously.
Fig. 5 infects wild-type Arabidopis thaliana plant genotype identification for the series connection carrier.Wherein C1, C2, C4, C6, the positive transfer-gen plant of C7, C8.
Fig. 6 analyzes for the positive transfer-gen plant gene of series connection carrier GmGTPCHI, GmADCS expression level.The negative contrast of wild-type Arabidopis thaliana (WT) all detect the expression of goal gene in the transgenic positive plant, and expression amount there are differences in different plants.
Fig. 7 analyzes for the positive transfer-gen plant folate content of series connection carrier.
Fig. 8 is for turning GmGTPCHI gene plant genotype identification, and 1-13 is the Arabidopis thaliana plant after GmGTPCHI infects, and 1,3,5-11,13 positive transfer-gen plants.
Fig. 9 is the measurement result that turns GmGTPCHI gene plant folate content, particularly, is the content sum of measuring two kinds of main folic acid existence form 5-methyl THF and 5-formyl THF.
Figure 10 is the genotypic evaluation of transfer-gen plant, wherein the positive transfer-gen plant of 1-4,6,7,10-12.
Figure 11 is the mensuration of pABA content in the transfer-gen plant, finds that transfer-gen plant pABA content increases than wild-type.
Figure 12 is the chemical structure of folic acid.
Figure 13 is the biosynthesizing branching diagram of folic acid in the plant materials.
Embodiment
Make the experimental methods of molecular biology specify in following examples, all carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book, perhaps carry out according to test kit and product description.
1, obtains the est sequence of soybean GTPCHI gene
Because the GTPCHI gene that derives from different plant species should have homology to a certain degree, or there is conservative structural domain., the CDS sequence of the GTPCHI gene of Arabidopis thaliana and the est sequence of soybean are compared at NCBI for this reason, obtain the est sequence of soybean GTPCHI gene.
2, pcr amplification obtains GmGTPCHI gene fragment and sequential analysis thereof
Get soybean leaves 100mg, adopt the Trizol method to extract total RNA, after DNase I processes, detect RNA purity and concentration, get 1 μ g and obtain cDNA with reverse transcription test kit First strand cDNA systhesis (available from Fermentas).Take the est sequence of soybean GTPCHI gene as template design primer, increase take soybean cDNA as template.Obtain approximately 700bp band, checking order is the fragment sequence of soybean GTPCHI, respectively EST and 3 ' the end est sequence of sequence and 5 ' end is assembled splicing in Vector NTI.Carry out respectively pcr amplification (primer sequence sees Table 1) take GTPCHI-3FW, GTPCHI-7RV as primer, obtain the product about 1400bp, these fragments are reclaimed test kit (TIANGEN) with sepharose to be reclaimed, link respectively p-EASY-T1 carrier (available from full formula King Company), transform the bacillus coli DH 5 alpha competent cell, blue hickie screening and bacterium colony PCR obtain positive colony.Through sequencing analysis, obtain 1380bp purpose fragment, called after GmGTPCHI.
3,5 ' and 3 ' RACE obtains the total length of GmGTPCHI gene
Take sequence GmGTPCHI obtained above as template, design respectively 5 ' RACE and 3 ' RACE primer, respectively called after: GmGTPCHI5 ' RACE, GmGTPCHI5 ' RACE nest, GmGTPCHI3 ' RACE, GmGTPCHI3 ' RACE nest (primer sequence sees Table 1).Adopt the Trizol method to extract the total RNA of soybean leaves, obtain the cDNA of 5 ' and 3 ' RACE according to the method on the specification sheets of geneRACE test kit (available from invitrogen), take the cDNA of 5 ' and 3 ' RACE as template, with 5 ' above-mentioned RACE and 3 ' RACE primer, carry out respectively nest-type PRC.PCR is obtained the band recovery and connect the p-EASY-T1 carrier to check order, and sequencing result is consistent with the GmGTPCHI UTR sequence that announce the website.Obtain the complete CDS sequence 1380bp of soybean folic acid key gene GmGTPCHI through sequential analysis.
Table 1 soybean GTPCHI gene clone primer sequence
4, turn the evaluation of GmGTPCHI gene Arabidopis thaliana folate content
The CDS sequence construct of the GmGTPCHI of soybean to plant expression vector pH2GW7, is driven by cauliflower mosaic virus (CaMV) 35S promoter, obtain recombinant vectors pH2GW7-GmGTPCHI.The carrier electric shock is transformed into Agrobacterium LBA4404, and the picking enzyme is cut and is identified correct clone, utilizes the agriculture bacillus mediated flower method of being stained with to infect the wild-type Arabidopis thaliana, and the seed of results is designated as T
0Generation.T
0At first screen at the resistance culture base of 1/2MS+ hygromycin B (25mg/L) for seed, obtain the positive green seedling of 13 strains and (be designated as T
1Generation), and move in the greenhouse and cultivated 20 days, get respectively blade and extract DNA, PCR identifies transfer-gen plant, wherein 1,3,5-11,13 positive transfer-gen plants (as shown in Figure 8).Utilize high performance liquid chromatography to detect folate content in these transgenic positive plant, mainly be to detect 5-methyl THF and these two kinds main folic acid derivatives content of 5-formyl THF, found that the GmGTPCHI transfer-gen plant has improved 1.6～2 times (as shown in Figure 9) than wild-type.
1, obtains the est sequence of soybean ADCS gene
The CDS sequence of the ADCS gene of Arabidopis thaliana and the est sequence of soybean are compared at NCBI, obtain the est sequence of soybean GTPCHI gene.
2, pcr amplification obtains GmADCS gene fragment and sequential analysis thereof
Get soybean leaves 100mg, adopt the Trizol method to extract total RNA, after DNase I processes, detect RNA purity and concentration, get 1 μ g and obtain cDNA with reverse transcription test kit First strand cDNA systhesis (available from Fermentas).Take the est sequence of soybean ADCS gene as template design primer, increase take soybean cDNA as template.Obtain approximately 1300bp band, fragment connects the p-EASY-T1 carrier after reclaiming, and 8 positive colonies of picking check order, and compares with 3 ' EST, finds that wherein 6 clones' sequencing result unanimously and with 3 ' EST mates, and size is 1352bp.With the 1352bp sequence that obtains and the soybean gene group sequence alignment of having announced, find with No. 10 chromosomal one section sequence identity of soybean very high, and the CDS sequence 2304bp of its prediction, according to CDS primers ADCSMF and the ADCSMR (primer sequence sees Table 2) of prediction, pcr amplification obtains most of CDS sequence 2296bp of GmADCS gene.
3,5 ' and 3 ' RACE obtains the total length of GmADCS gene
The Trizol method is extracted soybean leaves and is extracted total RNA, after DNase I processes, gets 5 μ g, according to the method acquisition cDNA of geneRACE test kit (available from invitrogen).According to obtaining sequence 5 ' end 260bp place's design primer ADCS5 ' RACE, 180bp place design primer ADCS5 ' RACE nest (primer sequence sees Table 2).Do afterwards nest-type PRC.Electrophoresis detection obtains the fragment about 800bp, and fragment connects the p-EASY-T1 carrier after reclaiming, and through sequencing analysis, obtains 5 ' terminal sequence and 218bp5 ' the UTR sequence of 483bp on the basis of known array.According to obtaining sequence 3 ' terminal 300bp place design primer ADCS 3 ' RACE, 159bp place design primer ADCS 3 ' RACEnest (primer sequence sees Table 2).Do afterwards nest-type PRC, electrophoresis detection obtains the fragment about 400bp, and fragment connects the p-EASY-T1 carrier after reclaiming, and through sequencing analysis, obtains 3 ' the UTR sequence of 252bp soybean.The sequence that obtains is spliced, finally obtain by analysis complete GmADCS gene order, clone primer ADCS10FLF1 and the ADCS10R1 (primer sequence sees Table 2) of GmADCS gene C DS total length according to the sequences Design that the clone obtains, PCR and to product order-checking obtains CDS sequence 2784bp.
Table 2GmADCS gene clone primer sequence
4, turn the acquisition of GmADCS gene Arabidopis thaliana and the mensuration of the synthetic precursor pABA content of folic acid thereof
The CDS sequence construct of GmADCS is arrived between the restriction enzyme site Nco I and Pml I of plant over-express vector pCAMBIA1302, driven by cauliflower mosaic virus (CaMV) 35S promoter and obtain recombinant expression vector pCAMBIA1302-GmADCS.Above-mentioned recombinant vectors electric shock is transformed Agrobacterium LBA4404, and the picking enzyme is cut and is identified correct clone, utilizes the agriculture bacillus mediated flower method of being stained with to infect the wild-type Arabidopis thaliana, and the seed of results is designated as T
0Generation.T
0At first screen at the resistance culture base of 1/2MS+ hygromycin B (25mg/L) for seed, obtain the positive green seedling of 13 strains and (be designated as T
1Generation), and move in the greenhouse and cultivated 20 days, get respectively blade and extract DNA, PCR identifies transfer-gen plant, 1-4 in the plant of infecting, 6., 7,10-12 is transgenic positive plant (as shown in figure 10).The folic acid that utilizes high performance liquid chromatography (HPLC) to detect in the transgenic positive plant synthesizes precursor pABA content, finds to improve 1.2～2 times (such as Figure 11) than wild-type.
The acquisition of embodiment 3, dual-gene hybridization plant and the mensuration of folate content thereof
The GmGTPCHI of soybean and the CDS sequence of GmADCS are building up to respectively the plant over-express vector, drive by cauliflower mosaic virus (CaMV) 35S promoter.Wherein, the CDS sequence of soybean GmGTPCHI is by the pH2GW7 that recombinates of Gateway system, the CDS sequence construct of the synthetic key gene GmADCS of soybean folic acid obtains recombinant expression vector pCAMBIA1302-GmADCS between the restriction enzyme site Nco I and Pml I of plant over-express vector pCAMBIA1302.Utilize the agriculture bacillus mediated flower method of being stained with to infect the wild-type Arabidopis thaliana.
With the T that changes GmGTPCHI over to that obtains
0In generation,, positive Arabidopis thaliana plant G1-G4 did male parent, and the T0 that changes GmADCS over to that obtains is done female parent for positive Arabidopis thaliana plant A1-5, had carried out following cross combination: G1x A1, G1xA2, G2xA3, G2xA4, G3xA2, G4xA5, the kind pod after the hybridization is gathered in the crops separately respectively (seeing Table 3).
Seed was cultivated in the greenhouse 20 days, got respectively blade and extracted DNA.Design GmGTPCHI and GmADCS gene specific primer (table 4) carry out pcr amplification take DNA as template, and the PCR reaction system is as follows:
The PCR reaction conditions: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of amplifications of Taq enzyme 30s, 35 circulations, PCR product 1% agarose electrophoresis detects, and the fragment that obtains about 400bp is the purpose band.Figure 1 shows that the part electrophorogram of pcr amplification, wherein 4,5,6,7,8,10,11 for changing the plant of GmGTPCHI gene over to, 1,2,4,5,6,7,8,9,11,12 for changing the plant of GmADCS gene over to, therefore, 4,5,6,7,8,11 for changing simultaneously the plant of two goal gene over to.
Further extracted the above-mentioned total RNA that changes simultaneously two goal gene plant over to RNA Trizol method, after DNase I processes, get 1 μ g, method acquisition cDNA according to reverse transcription test kit (Fermentas) makes template, carry out pcr amplification with the gene specific primer shown in the table 4, detect the gene expression dose of GmGTPCHI and GmADCS, find all to can't detect in the wild-type expression of two genes, and can detect the remarkable expression of gene in the transfer-gen plant, and the expression of gene there are differences between different strains, and Fig. 2 is the electrophorogram that part detects.
Get 30 days blade of growth being accredited as the dual-gene plant that changes among the offspring of GA1, GA2, GA3, GA4, GA5, GA6 respectively, extract folic acid, content with high-efficient liquid phase chromatogram technique analysis folic acid, found that with wild-type and compare, the folate content that changes separately GmGTPCHI over to has improved 1.5 times, the Plant Leaf acid content that changes separately GmADCS over to does not change, and hybridization Plant Leaf acid content has improved 2.5～4.5 times (Fig. 3) than wild-type.
Table 3 is hybridized the independent transgenic line of acquisition.
Wherein, G1-G4 is the transgenic line that changes GTPCHI over to, and A1-A5 is the transgenic line that changes GmADCS over to.
Table 4PCR primer sequence
The analysis of embodiment 4, two genes series connection Vector construction and positive transfer-gen plant folate content
Soybean GmADCS gene is cloned into the pCAMBIA1301 carrier with BglII and Pml I, simultaneously the zzzGmGTPCHI gene is switched on the pCAMBIA1301 with Kpn I and Pst I enzyme together with the 35S promoter of its front end and the terminator of rear end, obtain series connection carrier as shown in Figure 4.
Electric shock transforms Agrobacterium LBA4404, and the picking enzyme is cut and identified correct clone, utilizes the agriculture bacillus mediated flower method of being stained with to infect the wild-type Arabidopis thaliana, and the seed of results is designated as T
0Generation.
T
0At first screen at the resistance culture base of 1/2MS+ hygromycin B (25mg/L) for seed, obtain the green seedling of 12 strains and (be designated as T
1Generation), and cultivated 20 days in the immigration greenhouse, getting respectively blade extraction DNA is template.3 ' end design upstream primer GmADCSFw in the GmADCS gene order, at the Fw downstream design vector special primer V-Rv of 400bp place (table 5), carry out PCR, PCR reaction system and condition are with embodiment 3, PCR product 1% agarose electrophoresis detects, and the fragment that obtains about 400bp is the purpose band.As shown in Figure 5 C1, C2, C4, C6, the positive transfer-gen plant of C7, C8.
Further extract total RNA of these positive transfer-gen plants with the Trizol method, after DNase I processes, get 1 μ g, method acquisition cDNA according to reverse transcription test kit (Fermentas) makes template, carry out pcr amplification with the gene specific primer shown in the table 5, detect the gene expression dose of GmGTPCHI and GmADCS, find to can't detect the expression of two genes in the wild-type, and in transfer-gen plant, can detect remarkable expression (Fig. 6).
Get the blade of 30 days transfer-gen plant of growth, extract folic acid, utilize high performance liquid chromatography (HPLC) to analyze folate content in these plant, find to have improved 3～4.5 times than wild-type, the Arabidopis thaliana folate content that transforms separately respectively than two genes increase (Fig. 7).
Table 5PCR primer sequence
Claims (4)
1. a utilization turns the method that soybean GTPCHI and ADCS gene synergistic function improve the leaf acid content, it is characterized in that, described method comprises that the over-express vector that will contain soybean GTPCHI and ADCS gene imports the step that improves the folate content of plant in the purpose plant, by the coexpression of two kinds of genes jointly
Wherein, the nucleotide sequence of described GTPCHI gene is shown in SEQ ID No.1; The nucleotide sequence of described ADCS gene is shown in SEQ ID No.2.
2. utilization according to claim 1 turns the method for soybean GTPCHI and ADCS gene synergistic function raising leaf acid content, it is characterized in that, the method for soybean GTPCHI and ADCS gene co-expressing is as follows:
Method (1) makes up respectively the expression vector of soybean GTPCHI and ADCS gene, then imports in the purpose plant coexpression; Or
Method (2) makes up the co-expression carrier of soybean GTPCHI and the series connection of ADCS gene, then imports in the purpose plant coexpression.
3. utilization according to claim 2 turns the method for soybean GTPCHI and ADCS gene synergistic function raising leaf acid content, it is characterized in that, for method (1), soybean GTPCHI and ADCS gene are made up respectively plant expression vector and be transfected into respectively in the different plants, the positive transfer-gen plant of selecting soybean GTPCHI gene to infect is done male parent, the positive transfer-gen plant of selecting soybean ADCS gene to infect is done female parent, and male parent and female parent are hybridized the dual-gene hybrid plant of acquisition.
4. utilization according to claim 2 turns the method for soybean GTPCHI and ADCS gene synergistic function raising leaf acid content, it is characterized in that, for method (2), soybean GTPCHI and ADCS gene are connected on the expression vector pCAMBIA1301 carrier, carrier is transfected into plant, the seed of results at the enterprising row filter of resistance culture base, is obtained transgenic plant.
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| CN105647942A (en) * | 2016-03-11 | 2016-06-08 | 中国农业科学院生物技术研究所 | Application of maize gene ZmGFT1 in increasing folic acid content in plants |
| CN114703218A (en) * | 2022-05-09 | 2022-07-05 | 兰州大学 | Application of genes MsGCHI and MsADCS in improving plant folic acid content and promoting plant growth |
| CN115997894A (en) * | 2022-12-06 | 2023-04-25 | 苏州金记食品有限公司 | Germinated soybean rich in folic acid, preparation method and protein milk prepared by using germinated soybean |
| CN118480557A (en) * | 2024-07-10 | 2024-08-13 | 海南大学 | Soybean disease-resistant RING-H2 type zinc finger protein gene GmRHF1 and application thereof |
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| CN1792152A (en) * | 2005-12-26 | 2006-06-28 | 中国科学院长春应用化学研究所 | Plant growth accelerating composition contg. soybean vegetable female hormone |
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| CN105647942A (en) * | 2016-03-11 | 2016-06-08 | 中国农业科学院生物技术研究所 | Application of maize gene ZmGFT1 in increasing folic acid content in plants |
| CN105647942B (en) * | 2016-03-11 | 2020-01-14 | 中国农业科学院生物技术研究所 | Application of corn ZmGFT1 gene in improving folic acid content of plants |
| CN114703218A (en) * | 2022-05-09 | 2022-07-05 | 兰州大学 | Application of genes MsGCHI and MsADCS in improving plant folic acid content and promoting plant growth |
| CN115997894A (en) * | 2022-12-06 | 2023-04-25 | 苏州金记食品有限公司 | Germinated soybean rich in folic acid, preparation method and protein milk prepared by using germinated soybean |
| CN118480557A (en) * | 2024-07-10 | 2024-08-13 | 海南大学 | Soybean disease-resistant RING-H2 type zinc finger protein gene GmRHF1 and application thereof |
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