CN102181478B - Method for cultivating transgenic wheat with seeds with improved iron content - Google Patents
Method for cultivating transgenic wheat with seeds with improved iron content Download PDFInfo
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- CN102181478B CN102181478B CN2011101127684A CN201110112768A CN102181478B CN 102181478 B CN102181478 B CN 102181478B CN 2011101127684 A CN2011101127684 A CN 2011101127684A CN 201110112768 A CN201110112768 A CN 201110112768A CN 102181478 B CN102181478 B CN 102181478B
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Abstract
The invention discloses a method for cultivating transgenic wheat with seeds with improved iron content. The method for cultivating a transgenic plant with improved iron content comprises a step of importing an expression cassette consisting of an endosperm-specific promoter, an encoding gene of soybean ferritin and a terminator into a starting plant to obtain a target transgenic plant with the iron content higher than that of the starting plant. The experiment proves that the iron content in the seeds of the transgenic wheat is remarkably improved and the transgenic wheat with high iron content is obtained. The method has significance for improving the nutritional quality of wheat and the daily diet structure of people.
Description
Technical field
The present invention relates to a kind of method of cultivating the transgenic wheat that iron level improves in the seed.
Background technology
Iron is indispensable important element in the many normal physiological processes of human body.The shortage of human iron nutrition has become one of the most serious nutritive deficiency in the world today.The portion report that provides according to United Nations system nutrition problem standing committee in 2004 shows that the world population more than 1/2 receives the bad influence of micro-nutrients (WHO, 1992).According to estimates, the whole world has 2,000,000,000 people to suffer from iron deficiency, wherein with women and children (Long Xinxian, 1998) in the majority.The Chinese government also pays special attention to the mineral deficiency problem of disadvantaged group.
Adult human body contains the iron of about 3～5g, is the highest trace element of people's in-vivo content.Wherein 70% iron is present in oxyphorase and the myohaemoglobin, contains 25% iron that exists with the ferritin form in liver, kidney, the marrow, and small portion iron is oxidasic cofactor (Wang Jie, 2005).Iron in the oxyphorase is the conveying person of oxygen in the body, and it is transported to each tissue with the oxygen that lung absorbs, and supplies cellular oxidation, and the carbonic acid gas that simultaneously cellular oxidation is produced is transported to lung's exhalation and goes.Iron still is the staple of cytopigment enzyme and other several kinds of coenzyme.Have every day the iron of 30mg in tissue, to be transformed into serum levels of iron approximately and advance people's marrow, be used for hematopoiesis, good circulation transport way between formative tissue iron, serum levels of iron and the marrow iron is keeping running balance (Zheng Hua, 2009).
Iron deficiency is very big to the influence of human body, when the body too little iron, will develop into iron-deficiency anaemia gradually, and muscle cell utilizes the energy-producing function of oxygen to reduce the source of having reduced heat energy.Can influence human organ during slight iron deficiency, show that the neural system aspect may make children mental retardation and cause the unusual of behavior aspect.Show that the immunologic function aspect is that the children immune function of iron deficiency is very poor, suffer from multiple disease easily, all relevant like Wilson's disease, Parkinson's disease etc. with iron nutritive deficiency.Digestion and absorption function also can suffer damage during children's iron deficiency in addition, and appetite is poor, diarrhoea easily, and anthropometic is also just very poor.And the muscular movement function also is poor.Anaemia also can take place during except above-mentioned symptom in the people of too little iron, a series of as expiratory dyspnea, heart speed speed, the sleep symptom (weighing apparatus moral virtue, 2001) of anaemia aspect such as bad occurs.Thus, visible iron deficiency is the disease very big to human health damage.
According to estimates, in developing country, there are 50% pregnant woman and 40% non-pregnant women to suffer from iron-deficiency anaemia.There are some researches show that pregnant woman's different pregnancy phases, sideropenia in various degree are influential to newborn infant's bleeding of the umbilicus iron level, the slight sideropenia of pregnant woman can influence newborn infant's iron stocks, and pregnant woman's iron deficiency is early bigger more to the influence that newborn infant's iron is stocked.And the neonatal iron level of iron-deficiency anaemia carrier mothers is starkly lower than the neonatal iron level of normal carrier mothers, and the neonatal probability of iron-deficiency anaemia is also apparently higher than the neonatal probability of normal carrier mothers iron-deficiency anaemia (Jiang Yuehua, 2005).
Iron mainly is present in the animal food, and content is low in plant food, and the general specific absorption of the iron of plant food is very low, how below 10%, is 1% like rice, and corn and black soya bean are 3%, and lettuce is 4%, and wheat is 5%.Major cause is the inhibition factor that plant food exists more iron to absorb; Form insoluble molysite like phytate, oxalate, carbonate and iron; And the promoting factor that iron absorbs is less like amino acid and vitamins C content in plant that protein, promotion iron absorb.In order to overcome iron nutritive deficiency, can take a series of measure, as improve the diet structure, perhaps utilize reinforced iron food and additional chalybeate.But both expensive, and need investment year after year.This is a large economy burden for the weak developing country of economic base.
Summary of the invention
An object of the present invention is to provide a kind of method of cultivating the transgenic plant of iron level raising.
The method of the transgenic plant that cultivation iron level provided by the present invention improves comprises the steps: in the plant that sets out, to import the encoding sox of soybean ferritin, obtains the purpose transgenic plant that iron level is higher than the said plant that sets out.
In the aforesaid method, said iron level is the iron level in the seed.
In above-mentioned arbitrary said method, the said plant that sets out is farm crop.
In above-mentioned arbitrary said method, said farm crop are wheat.
In above-mentioned arbitrary said method, the aminoacid sequence of said soybean ferritin is shown in SEQ ID NO:1; And/or the nucleotide sequence of the encoding sox of said soybean ferritin is shown in SEQ ID No:2.
In above-mentioned arbitrary said method, said encoding sox imports through following expression cassette.
In above-mentioned arbitrary said method, said expression cassette is made up of the encoding sox and the terminator of endosperm specificity promoter, soybean ferritin.
Wherein, said endosperm specificity promoter is the endosperm tissue specific expressing promoter 1Dx5 of high molecular weight glutenin subunit of wheat through gene.
Wherein, the aminoacid sequence of said soybean ferritin is shown in SEQ ID NO:1; And/or the nucleotide sequence of the encoding sox of said soybean ferritin is shown in SEQ ID No:2; And/or the nucleotide sequence of said expression cassette is shown in SEQ ID NO:3.
The said method that in the plant that sets out, imports the encoding sox of soybean ferritin comprises the steps:
1) wheat immature embryo is connected in the rataria inducing culture, cultivated 3 days;
2) change over to jointly in the rataria that step 1) obtains with said expression cassette with as the carrier of resistance screening mark; Rataria after transforming is cultivated at the enterprising row filter of the rataria inducing culture that contains Bialaphos, obtained resistant calli;
3) said resistant calli is transferred to division culture medium, carries out differentiation culture, obtain breaking up seedling;
4) will break up seedling and move to the strong plantlets and rootage substratum, carry out root culture.
The composition of rataria inducing culture: solvent is the MS substratum, and solute and concentration thereof are following: 2,4-D 2mg/L, sucrose 30g/L, agar 7g/L powder; The pH value of rataria inducing culture is 5.8.
The composition that contains the rataria inducing culture of Bialaphos: solvent is said rataria inducing culture, and solute and concentration thereof are Bialaphos 0.5mM;
The composition of division culture medium: solvent is the MS substratum, and solute and concentration thereof are following: 10mg/L zein, 1mg/LIAA, 0.5mM Bialaphos, 30g/L sucrose, 7g/L agar powder; The pH5.8 of division culture medium;
The composition of strong plantlets and rootage substratum: solvent is the MS substratum, and solute and concentration thereof are following: 10mg/L IAA, 80g/L sucrose, 7g/L agar powder; The pH of strong plantlets and rootage substratum is 5.8.
Said carrier as the resistance screening mark is pBAC35SIH3.
Another object of the present invention provides a kind of expression cassette.
Expression cassette provided by the present invention is made up of the encoding sox and the terminator of endosperm specificity promoter, soybean ferritin.
In the above-mentioned expression cassette, said endosperm specificity promoter is the endosperm tissue specific expressing promoter 1Dx5 of high molecular weight glutenin subunit of wheat through gene.
In above-mentioned arbitrary said expression cassette, the aminoacid sequence of said soybean ferritin is shown in SEQ ID NO:1; And/or the nucleotide sequence of the encoding sox of said soybean ferritin is shown in SEQ ID No:2; And/or the nucleotide sequence of said expression cassette is shown in SEQ ID NO:3.
Another object of the present invention provides a kind of encoding sox of soybean ferritin.
The encoding sox of soybean ferritin provided by the present invention, its nucleotide sequence is shown in SEQ ID NO:2.
The application in the iron level in improving wheat grain of the encoding sox of soybean ferritin shown in soybean ferritin shown in the SEQ ID NO:1 or the SEQ ID No:2 also belongs to protection scope of the present invention.
The present invention has made up by wheat endosperm specific promoter 1Dx5 and has driven the expression vector pBAC47P-fer that the soybean ferritin is expressed, and obtains to comprise the linear expression cassette of ferritin of the carrier free framework of endosperm specific promotor 1Dx5 and NOS terminator with the method that column purification was cut, crossed to enzyme.Expression cassette is imported in the wheat, make soybean ferritin specifically expressing in the wheat grain endosperm.Experiment showed, that iron level is improved significantly in the seed of transgenic wheat, obtained transgenic high ferro wheat.The present invention is for the improvement nutritional quality of wheat, and it is significant to improve the daily diet formula of people.
Description of drawings
Fig. 1 is the structure iron of carrier pBAC47P-fer.
Fig. 2 is the cleavage map of expression cassette.
Fig. 3 is the PCR qualification result of transfer-gen plant.
Fig. 4 is the PCR-southern qualification result of transfer-gen plant.
Fig. 5 is a RT-PCR qualification result in the seed of transfer-gen plant.
Fig. 6 is the content of iron in the transgenic wheat seed.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
One, experiment material
1, the structure of pBAC47P carrier:
PSP72 carrier with Promega company is the carrier that sets out, and between HindIII and Sal I, inserts the promotor of wheat high-molecular-weight glutelin subunit 1Dx5, between Kpn I and EcoR I, inserts the NOS terminator.The NOS terminator comes from the pBI121 carrier of U.S. Clontech company.The 1Dx5 promotor derives from plant expression vector pBPC30.
The nucleotide sequence of the promotor of wheat high-molecular-weight glutelin subunit 1Dx5 is shown in the 1-857 position Nucleotide of 5 of SEQ ID NO:3 ' end.The nucleotide sequence of NOS terminator is shown in the 1625-1887 position Nucleotide of 5 of SEQ ID NO:3 ' end.
2, to spend be for No. 1 that wheat commercial variety is examined in the capital that the Beijing City Agriculture and Forestry Institute crop is cultivated in the capital, at document " Hu Daofen, Yuan Zhendong, Tang Yunlian, Liu Jianping.Plant cell engineering---No. 1 breeding spent in winter wheat training new variety capital.Chinese science (B collects), in March, 1986,285-292 " in disclosed, the public can obtain from China Agricultural University.
3, the structure of pBAC35SIH3 carrier:
PSP72 carrier with Promega company is the carrier that sets out, and between HindIII and Sal I, inserts the CaMV 35S promoter, between Kpn I and EcoR I, inserts the NOS terminator, inserts the bar gene at BamH I and Sac I restriction enzyme site.CaMV 35S promoter and NOS terminator come from the pBI121 carrier of U.S. Clontech company.The bar gene source is in plant expression vector pBPC30.Plant expression vector pBPC30 is at document " Zhang Xiaodong, Liang Rongqi, Chen Xuqing, Yang Fengping, Zhang Liquan.The acquisition of high-quality HMW gluten subunit transgenic wheat and genetic stability thereof and quality trait analysis.Science Bulletin, 2003,48 (5): 474-479 " disclosed in, the public can obtain from China Agricultural University.
4, soybean " is closed rich 42 " (close hand over 9526-3) is the precocity bred of Hejiang, the Heilongjiang Province institute of agricultural sciences, high oily, of short stem, soybean varieties with super high-yielding potentiality, and promoted by Heilongjiang Province's crop varietal approval committee in March, 2002.Available from the Hejiang institute of agricultural sciences of Heilongjiang Institute of Agricultural Sciences (rich kind Ltd is already closed by the Heilongjiang Province), catalog number is " the black beans 2002007 of examining ".Soybean " closes rich 42 " at document " Hu Xiping.Close the research of rich No. 42 soybean high-qualitys, high-yield culture technique.The soybean science, 2005,24 (1): 48-51 " disclosed in, the public can obtain from China Agricultural University.
Two, experimental technique and result
1, the encoding sox of soybean ferritin
Can obtain encoding sox shown in the SEQ ID NO:2 by synthetic, also can prepare the encoding sox of soybean ferritin according to following method.
The young root that soybean " is closed rich 42 " with the Fe-EDTA solution of 100mM (with FeSO47H
2O and Na
2EDTA2H
2O places 450mL zero(ppm) water respectively, and heating and continuous the stirring make it dissolving.Then, slowly pour the former into the latter while hot, the limit bevelling stirs, and after mixing, regulates pH value to 5.5, and the adding distil water constant volume is to 1L.) handle 24h, get the plant young root after the processing, adopt Trizol RNA to extract test kit (day root company) and extract its total RNA.With primer SoybeanFer-F and SoybeanFer-R are carried out the RT-PCR amplification, amplified production is the encoding sox of purpose soybean ferritin.
The wherein little write sequence of SoybeanFer-F:5 '-ggtaccATGGCTCTTGCTCCATCCAAAGTT-3 ' is the Kpn1 restriction enzyme site;
The wherein little write sequence of SoybeanFer-R:5 '-gagctcCTAATCAAGAAGTCTTTGATCAA-3 ' is the Sac1 restriction enzyme site.
2, recombinant expression vector:
Two ends being had dna fragmentation shown in the SEQ ID NO:2 of synthetic of Kpn1 restriction enzyme site and Sac1 restriction enzyme site or above-mentioned pcr amplification product links on pGEM-T Easy Vector System (Promega company) carrier and uses; Utilize the alkaline lysis method of extracting plasmid, utilize the sp6/T7 primer to carry out the plasmid order-checking.Choosing the correct plasmid of order-checking then cuts with Sac I and Kpn I enzyme; Use sepharose DNA to reclaim test kit (centrifugal column type; It root company) reclaims target gene fragment; With target gene fragment be connected with pBAC47P carrier after Kpn I enzyme is cut through Sac I, obtain recombinant vectors, note is made pBAC47P-fer; With carrier pBAC47P-fer heat shock transformed into escherichia coli, carry out the amicillin resistance screening, the picking mono-clonal carries out enlarged culturing and sequence verification with positive monoclonal.The structure of carrier pBAC47P-fer is as shown in Figure 1.
Sequencing result shows:
1), between the Kpn I of this recombinant vectors pBAC47P-fer and Sac I site along from the gene order of the direction of Kpn I to Sac I shown in SEQ ID NO:2.The proteic aminoacid sequence of this genes encoding is shown in SEQ ID NO:1.
2), between the Xho I and EcoRI site of this recombinant vectors pBAC47P-fer the gene order of (along direction) from Xho I to EcoR I shown in SEQ ID NO:3.Wherein, The 1-857 position Nucleotide terminal from 5 of SEQ ID NO:3 ' is the endosperm tissue specific expressing promoter 1Dx5 of high molecular weight glutenin subunit of wheat through (HMW-GS) gene; 864-1616 position Nucleotide is the encoding sox of soybean ferritin, and 1623-1887 position Nucleotide is the Nos terminator.
3, transform
Spending No. 1 with the capital is experiment material.
With Hind III and EcoRI digested plasmid pBAC47P-fer, reclaim the fragment of about 1.8Kb, i.e. acquisition comprises the linear expression cassette (Fe-box) (Fig. 2 arrow indication, about 1.8Kb) of ferritin of the frameless construction of endosperm specific promotor 1Dx5 and NOS terminator.Among Fig. 2, M:1kb ladder Marker 1: recombinant plasmid 2: enzyme is cut product.
Get the back 12 days prematurity seed of pollination; With 70% alcohol rinsing 2 minutes, with 0.1% mercury chloride sterilization 15 minutes, sterilized water washing 3 times; Under aseptic condition, choose rataria and be inoculated in rataria inducing culture (interpolation 2mg/L 2,4-D in the MS minimum medium; 30g/L sucrose, 7g/L agar powder, pH5.8) last 3 day.With particle bombardment with in ferritin expression cassette (Fe-box) and 3 days the rataria of selection markers genophore pBAC35SIH3 cotransformation inducing culture.With the rataria that does not change any gene and carrier over to is the wild-type contrast, contrasts as empty carrier with the rataria that changes pBAC35SIH3 over to.
Rataria after the conversion screens on the rataria inducing culture that contains Bialaphos (0.5mM).Resistant calli is transferred to division culture medium, and (the MS minimum medium adds 10mg/L zein, 1mg/L IAA, 0.5mM Bialaphos, 30g/L sucrose, 7g/L agar powder; PH5.8) on, the differentiation seedling grows and it is moved to the strong plantlets and rootage substratum behind the stem (the MS substratum adds 10mg/L IAA, 80g/L sucrose, 7g/L agar powder; PH5.8) on, treat that seedling grows 2 young leaves, behind the 3-4 bar main root, clean substratum, be transplanted in the little furrow of covered with plastic film of solarium.
Screening culture medium, division culture medium contain weedicide glufosinates (Bialaphos), and the bar gene can detoxify the glufosinates acetylize.Thereby the callus that has only changed the bar gene over to could be survived and emerge.Because no bar gene in the expression cassette is so transform with pBAC35SIH3.Can replace to other carrier, as contain the carrier of epsp gene, screening culture medium, division culture medium are with the interpolation herbicide glyphosate at this moment.
Obtain T according to the method described above
0For transfer-gen plant 276 strains.
4, transfer-gen plant is identified
(1) PCR identifies:
To T
0Identify as follows for plant.Work as T
0When growing to 3-4 sheet blade, choose the wheat spire, extract the wheat leaf blade genomic dna in a small amount with the CTAB method for wheat.With primer SoybeanFer-F/SoybeanFer-R is identified.
Primer: SoybeanFer-F 5 '-GGTACCATGGCTCTTGCTCCATCCAAAGTT-3 '
SoybeanFer-R?5’-GAGCTCTAATCAAGAAGTCTTTGATCAA-3’
Spend No. 1 the negative contrast of plant, the positive contrast of plasmid pBAC47P-fer with unconverted wheat capital.
The result detects through PCR.Detect positive transfer-gen plant 65 strains (Fig. 3) altogether.Among Fig. 3, M:DL2000 Marker; +: positive control; 0: water;-: the wild-type plant; All the other are transfer-gen plant.
With the positive contrast of plasmid pBPC47P-fer, the not negative contrast of transfer-gen plant, water is blank.Detect the fer gene in the transfer-gen plant, the identical positive plant of plant that is 750bp of amplified fragments with positive control.The transfer-gen plant that is positive is spent in the capital No. 1: 12,18,23,124,125,128, and 133-139,141,143,144,146,147; 148,154,158,161,163,169-186,196,219,214,217,223-229; 231,233,235,237,245,249,250,253,256,263,265.
(2) PCR-Southern identifies:
To T
0Identify as follows for plant.Through PCR-Southern transfer-gen plant is further identified.The PCR product is got 3 μ L samples be splined on sepharose, change film hybridization behind the electrophoresis, can see, hybridization signal can appear in corresponding positive plant and plasmid bands of a spectrum place, and negative control does not have hybrid belt.Swimming lane 1 is a plasmid pBPC47P-fer positive control among Fig. 4,2 negative contrasts, the positive plant of 3-10.Detected result is identical with the PCR detected result.
PCR and southern result thereof prove that expression cassette has been integrated into the genome of wheat.
(3) T
0For T that transfer-gen plant is tied
1The RT-PCR of seed detects
65 strain T of difference extraction step (1)
0For the filling stage seed RNA of transfer-gen plant, SoybeanFer-F/SoybeanFer-R is carried out the RT-PCR amplification with primer.
SoybeanFer-F：5’-ggtaccATGGCTCTTGCTCCATCCAAAGTT-3’；
SoybeanFer-R：5’-gagctcCTAATCAAGAAGTCTTTGATCAA-3’。
The result is as shown in Figure 5, M:DL2000Marker; +: positive control (pBPC47P-fer); 0: water;-: the non-transgenic plant; All the other: transfer-gen plant.65 strain T with step (1)
0For 29 strains that have of the seed of transfer-gen plant, be respectively 12,23,128,133,134,137,141,143,144,146 in the rna level tests positive; 148,161,171,172,173,174,175,178,182,184; 185,209,217,223,224,227,231,235,250,253.
RT-PCR result proves that the ferritin expression cassette expresses in the transgenic wheat seed.
5, T
1Mensuration for the iron level of transgenic seed
The ripe seed of the transfer-gen plant of RT-PCR tests positive is carried out the mensuration of iron level.
Adopt atomic absorption spectrometry; A kind of relative measurement method that this method absorbs based on the atom pairs characteristic light; When its ultimate principle is steam through sample of the characteristic spectrum of element to be measured that radiation of light source is gone out, absorbed by the ground state atom of element to be measured, under certain condition; Incident light be absorbed and in the degree that weakens and the sample content of element to be measured be proportionate the content of element to be measured in the derived sample thus.
Experimental procedure: get 6 100mL volumetric flasks; Add successively 1.00,2.00,3.00,4.00,5.00 and the 100 μ g/mL ironworkers of 6.00mL make standardized solution (ferrous ion concentration is the ferrous sulfate aqueous solution of 100 μ g/mL), be diluted to scale with deionized water, shake up; (the lamp long duration current 2.9mA at wavelength 248.3nm place; PM voltage 360V, acetylene flow 1.2L/min), measure absorbance value with the production standard curve.Earlier sample (being wheat grain) is cleaned with tap water, used the zero(ppm) water rinsing again 2 times, placed 80 ℃ of thermostatic drying chambers dry 1 day, smash to pieces, take by weighing the 4g whole wheat flour in the 100mL Erlenmeyer flask, add 4: 1 HNO
3: HClO
4Nitration mixture 30mL, hot digestion is handled on hot plate.Reaction 30～40min treats that white cigarette emits to the greatest extent, takes off cooling, adds 1: 1 hydrochloric acid 10mL again, boils dissolving, suitable dilution to be cooled, and after fixed dissolving, (lamp long duration current 2.9mA, PM voltage 360V, acetylene flow 1.2L/min) measures at wavelength 248.3nm place.Replication 3 times is got its MV.
T
1Mensuration result for the iron level of transgenic seed shows (table 1), and the iron level comparison of the transgenic line seed of surveying is according to improving, and some strain system reaches significantly and utmost point level of signification.Explain that this ferritin expression cassette can improve the iron level of transgenic wheat seed.In the table 1, CK is the not transgenic wheat kind " spend No. 1 in the capital " of wild-type.The T1 of 26 transfer-gen plants of the positive that SXY001---SXY119 representes to identify is for seed, and MV is the mg number of iron in every gram seed, and S is a standard error of the mean.The result who changes the empty carrier control group is consistent with the wild-type result.
Iron level (unit: mg/g) in table 1, the wheat grain
Claims (5)
1. method of cultivating the transgenic plant that iron level improves comprises the steps: in the plant that sets out, to import the encoding sox of soybean ferritin, obtains the purpose transgenic plant that iron level is higher than the said plant that sets out;
Said iron level is the iron level in the seed;
The said plant that sets out is farm crop, and said farm crop are wheat;
The aminoacid sequence of said soybean ferritin is shown in SEQ ID NO:1; And/or the nucleotide sequence of the encoding sox of said soybean ferritin is shown in SEQ ID No:2.
2. method according to claim 1 is characterized in that: said encoding sox imports through the said expression cassette of claim 3.
3. an expression cassette is made up of the encoding sox and the terminator of endosperm specificity promoter, soybean ferritin;
Said endosperm specificity promoter is the endosperm tissue specific expressing promoter 1Dx5 of high molecular weight glutenin subunit of wheat through gene;
The aminoacid sequence of said soybean ferritin is shown in SEQ ID NO:1; And/or the nucleotide sequence of the encoding sox of said soybean ferritin is shown in SEQ ID No:2; And/or the nucleotide sequence of said expression cassette is shown in SEQID NO:3.
4.SEQ soybean ferritin shown in the ID NO:1 is the application in the iron level in improving wheat grain.
5.SEQ the encoding sox of soybean ferritin shown in the ID No:2 is the application in the iron level in improving wheat grain.
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|---|---|---|---|---|
| CN1339067A (en) * | 1999-09-30 | 2002-03-06 | 默里斯坦治疗公司 | Synthetic and chimeric promoters, expresion cassettes, plasmids, vectors, transgenic plants and seeds containing them, and method for producing them |
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| CN1339067A (en) * | 1999-09-30 | 2002-03-06 | 默里斯坦治疗公司 | Synthetic and chimeric promoters, expresion cassettes, plasmids, vectors, transgenic plants and seeds containing them, and method for producing them |
Non-Patent Citations (4)
| Title |
|---|
| Anderson O.D.Triticum aestivum Glu-1D-1d gene for high molecular weight flutenin subunit.《Genbank》.2010, * |
| Fumiyuki Goto.Iron fortification of rice seed by the soybean ferritin gene..《Nature Biotechnology》.1999,第17卷 * |
| Fumiyuki Goto.Iron fortification of rice seed by the soybean ferritin gene.《Nature Biotechnology》.1999,第17卷 * |
| Wang, Y.B.chloroplast ferritin.《Genbank》.2007, * |
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