CN112553217B - Application of GmVIT1 gene of soybean and recombinant vector containing GmVIT1 gene - Google Patents
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Abstract
The invention discloses application of a soybean GmVIT1 gene and a recombinant vector containing the GmVIT1 gene, belongs to the technical field of genetic engineering, and aims to solve the problem of how to promote the plant to improve the seed setting rate.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to application of a soybean GmVIT1 gene and a recombinant vector containing the GmVIT1 gene.
Background
Soybean (glycine max l.) is a very important commercial crop, and the soybean gene bank contains abundant genetic resources. It is an available approach to explore beneficial genetic resources and to improve crop yield. The planted crop variety mainly takes the characteristics of large seed setting amount, wide adaptability, strong growth potential and the like as indexes. The seed setting amount is an important component of crop yield and is also a main technical index for fine variety breeding. The low seed yield seriously affects the stability of the yield and the quality of crops and is not beneficial to the fine variety breeding of the crops. The method has great economic benefit for improving the seed setting rate of the plants.
Disclosure of Invention
The invention aims to solve the problem of improving the seed setting rate of plants, and provides application of a GmVIT1 gene of soybean in promoting seed setting of the plants.
Further limited, the sequence of the GmVIT1 gene is shown as SEQ ID NO. 3.
Further defined, the plant is tobacco.
The invention also provides a recombinant vector containing the GmVIT1 gene, wherein the vector is a recombinant vector pBI121-GmVIT1.
Further limiting, the method for promoting the plant seed setting of the GmVIT1 gene of the soybean comprises the following specific steps:
(1) Constructing the recombinant vector pBI121-GmVIT1;
(2) Preparation of Agrobacterium carrying the GmVIT1 Gene: transferring the recombinant vector pBI121-GmVIT1 obtained in the step (1) into an agrobacterium infected state for culture to obtain agrobacterium carrying a GmVIT1 gene;
(3) Preparation of plants carrying the GmVIT1 gene: culturing seeds of the plants to reach a seedling stage, then performing pre-culture treatment on the plants, then infecting the plants subjected to the pre-culture treatment with the agrobacterium obtained in the step (2), and then performing bud induction, rooting induction and transplantation domestication to obtain plants carrying GmVIT1 genes;
(4) Culturing a plant carrying the GmVIT1 gene:
further limiting, the construction method of the recombinant vector in step (1) is to utilize restriction enzymes to respectively enzyme-cut the vectors pMD18T-GmVIT1 and pBI121 plasmids to obtain the enzyme-cut products of the sequence segment of the GmVIT1 gene and the pBI121 plasmid, and then to perform a ligation reaction of the sequence segment of the GmVIT1 gene and the enzyme-cut products of the pBI121 plasmid, wherein the ligation reaction system is as follows: 5 mu.L of pBI121 plasmid restriction enzyme products, 3 mu.L of GmVIT1 gene sequence fragments, 1 mu.L of 10 XT 4 DNA ligation buffer solution and 1 mu.L of T4 DNA ligase, wherein the reaction temperature is 16 ℃, and the reaction time is 12h.
Further defined, the restriction enzymes are Xba I and Sma I.
Further defined, the seed of the plant in step (3) is reached.
Further defined, the step of infecting the plant with Agrobacterium in step (3) is as follows:
1) Coating agrobacterium carrying a recombinant vector pBI121-GmVIT1 on a YEB solid culture medium containing kanamycin, streptomycin and rifampicin, and culturing for 48h at 28 ℃;
2) Resuspending the bacterial colony of the agrobacterium obtained in the step 1) by using a 1/2MS liquid culture medium to obtain a bacterial liquid of the agrobacterium, and then diluting the bacterial liquid by using 1/2MS until the OD600 value is 0.4-0.6;
3) And (3) taking plant leaves, infecting the plant leaves in the bacterial liquid of the agrobacterium obtained in the step 2) for 6-7min, taking out the plant leaves, placing the plant leaves on sterile filter paper, sucking off the attached bacterial liquid, placing the plant leaves on an MS1 culture medium, and culturing for 2 days under a dark condition.
Further, in the pre-culture treatment in the step (3), the plant leaves are cut into a square shape of 1cm × 1cm and pre-cultured for 48 hours.
Has the advantages that: compared with wild type, the seed setting quantity of the transgenic tobacco obtained by the agrobacterium-mediated leaf disc method is obviously improved, which shows that the GmVIT1 gene can improve the seed setting rate of plants.
Drawings
FIG. 1 is the vector diagram of pBI 121;
FIG. 2 is a graph showing the results of PCR for positive transgenic tobacco, wherein M is DL200,0 is blank control, -is negative control, + is positive control, and 1-17 are transgenic tobacco tested;
FIG. 3 is a picture of positive transgenic tobacco tissue, wherein A is callus, B is Km resistant shoot, C is rooted culture of resistant tobacco shoot, and D is rooted tobacco.
FIG. 4 is a photograph of transgenic tobacco seed setting, wherein A, B is transgenic tobacco and C is control.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Experimental materials: wild type tobacco seeds (Nicotiana tabacum L.cv.SR-1) were stored in the laboratory.
Test reagents: gel recovery kit (TIANGEN), RNA extraction kit (TIANGEN), PCR kit (TAKARA), vector ligation kit (TAKARA), and plasmid extraction kit were purchased from AXYGEN, inc.
Example 1 method for constructing GmVIT 1-containing Gene
The specific steps for constructing the pBI121-GmVIT1 recombinant vector are as follows:
respectively carrying out enzyme digestion on the vectors pMD18T-GmVIT1 and pBI121 plasmids by using Xba I and SmaI restriction endonucleases to obtain an enzyme digestion product of a sequence fragment of a GmVIT1 gene and a pBI121 plasmid (a vector diagram is shown in figure 1), and then carrying out a ligation reaction on the sequence fragment of the GmVIT1 gene and the enzyme digestion product of the pBI121 plasmid, wherein the ligation reaction system is as follows: 5 mu L of pBI121 plasmid restriction enzyme products, 3 mu L of GmVIT1 gene sequence fragments, 1 mu L of 10 XT 4 DNA Ligase Buffer and 1 mu L of T4 DNA Ligase, wherein the reaction temperature is 16 ℃, and the reaction time is 12h, thus obtaining the recombinant vector pBI121-GmVIT1.
Example 2 application of GmVIT1 gene of alfalfa in promoting flowering of plants
A recombinant vector pBI121-GmVIT1 containing soybean GmVIT1 genes is constructed, and the recombinant vector is transferred into agrobacterium tumefaciens to be cultured together with plantlets to obtain transgenic plantlets, and the method specifically comprises the following steps:
(1) Constructing a recombinant vector pBI121-GmVIT1: the recombinant vector pBI121-GmVIT1 was obtained by the method of reference example 1;
(2) Preparation of Agrobacterium carrying the GmVIT1 Gene: transferring the pBI121-GmVIT1 vector obtained in the step (1) into an agrobacterium infected state for culture to obtain agrobacterium carrying a GmVIT1 gene;
(3) Preparation of plants carrying the GmVIT1 gene: culturing the seeds of the plants to a seedling stage, then carrying out pre-culture treatment on the plants, then infecting the agrobacterium carrying the GmVIT1 gene obtained in the step (2) on the plants after the pre-culture treatment, and then carrying out bud induction, rooting induction and transplantation domestication to obtain the plants carrying the GmVIT1 gene;
(4) Culturing plants carrying the GmVIT1 gene:
the experiment effect is verified by using the experiment:
1. the method for extracting the RNA of the soybean comprises the following steps:
(1) 0.1g of soybeans was put in a mortar precooled with liquid nitrogen and ground into a finely divided powder.
(2) The powder was transferred to a 1.5mL sterile centrifuge tube and 450. Mu.L RL (confirming the addition of β -mercaptoethanol) was added.
(3) All the liquid was transferred to a filtration column, centrifuged at 12000rpm for 5min and the supernatant carefully pipetted into a new RNase-Free centrifuge tube.
(4) Slowly adding 0.5 times of the volume of the supernatant of absolute ethyl alcohol, mixing uniformly, transferring the obtained solution and the precipitate into an adsorption column, centrifuging at 12000rpm for 60s, pouring off the waste liquid in the collection tube, and putting the adsorption column back into the collection tube.
(5) Add 350. Mu.L deproteinized liquid to the adsorption column, centrifuge at 12000rpm for 60s, discard the filtrate, and place the adsorption column back into the collection tube.
(6) Preparing DNase working solution: add 70 μ LRDD solution into 10 μ L DNase I reaction solution, mix gently.
(7) Adding 80 μ LDNase working solution onto the filter membrane of the adsorption column, and standing for 15min.
(8) Add 350. Mu.L deproteinized solution to the centrifugation and adsorption column, centrifuge at 12000rpm for 60s, discard the filtrate, and place the adsorption column back into the collection tube.
(9) Adding 300 μ L of rinsing solution into centrifugal adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 60s, discarding filtrate, and placing adsorption column back into collecting tube.
(10) And (5) repeating the step (9).
(11) Centrifuging at 12000rpm for 2min, discarding the filtrate, and standing the adsorption column at room temperature for 5min to completely air-dry the residual rinse solution in the adsorption column.
(12) Transferring the centrifugal adsorption column into RNase-free collection tube, adding 30 μ L RNA eluent, standing for 1-2min, centrifuging at 12000rpm for 2min to obtain RNA sample, and storing at-80 deg.C.
2. Method for obtaining cDNA by RNA reverse transcription of soybean
Using the extracted RNA as a template, obtaining cDNA by a reverse transcription PCR method, wherein the reaction process comprises the following steps:
(1) Preparing a reaction solution I:
(2) And mixing the reaction solution I, placing the mixture in a water bath at 65 ℃ for 5min, and quickly placing the mixture in ice after the water bath.
(3) Preparing a reaction solution II:
reaction procedure:
30℃ 10min
42℃ 20min
99℃ 5min
the primer sequence is as follows: the sequence of GmVIT 1F is shown as SEQ ID NO.1, namely 5'-ATGGCATCAGATCACTCG-3'
The sequence of GmVIT 1R is shown as SEQ ID NO.2, namely 5'-CTAAAGACCACTTGACCCA-3'
PCR amplification procedure: 94 ℃ for 5min,30 cycles of 94 ℃ for 30s,59 ℃ for 30s,72 ℃ for 1min, and finally 72 ℃ for 10min; detecting by 1% agarose gel electrophoresis;
gel recovery of target gene bands: and recovering a target gene fragment GmVIT1 (soybean VIT 1) obtained by PCR amplification by adopting a DNA gel recovery kit, wherein the sequence is shown as SEQ ID NO. 3.
3. Method for constructing vector pMD18T-GmVIT1
(1) Reaction system: mu.L of pMD18-T vector, 3. Mu.L of target gene fragment solution, and 1. Mu.L of ddH 2 O, 5 mu L Solution I. The reaction temperature is 16 ℃, and the reaction time is more than 12h (overnight).
(2) Connecting a GmVIT1 target gene fragment to a cloning vector pMD18T, transferring a connection product into an escherichia coli competence by using a heat shock method, wherein the specific contents are as follows:
taking out the Escherichia coli competence from a refrigerator at minus 80 ℃, quickly putting the Escherichia coli competence on ice, adding 20 mu L of the ligation product after the Escherichia coli competence is dissolved, and carrying out ice bath for 30min;
taking out, rapidly placing in 42 deg.C water bath for 1min15 s, rapidly inserting into ice, and ice-cooling for 2min. Adding 800 μ L LB liquid culture medium, placing on 200rpm shaking table, 37 deg.C, and shaking for 1h;
taking out the centrifuge tube, centrifuging at 4000rpm for 5min, taking out 800 μ L of supernatant, re-suspending the residual thallus, and smearing the residual 200 μ L of bacterial liquid on two LB solid culture media containing ampicillin resistance according to the volume ratio of 3:1; carrying out inverted culture in an incubator at 37 ℃ overnight; and selecting a single colony on a culture medium for identification, selecting a positive colony for amplification culture, and extracting a pMD18T-GmVIT1 plasmid by using a plasmid extraction kit.
4. Method for constructing recombinant vector pBI121-GmVIT1
(1) Plasmids for pMD18T-GmVIT1 and pBI121 were extracted: extracting plasmids of pMD18T-GmVIT1 and pBI121 by a plasmid extraction kit;
(2) The plasmid pMD18T-GmVIT1 and pBI121 is digested:
plasmid DNA of pBI121 is subjected to double digestion by Xba I and Sma I, and the reaction system of double digestion is as follows: mu.L of pBI121 plasmid, 10. Mu.L of TAKARA10 XM Buffer, 3.5. Mu.L of Xba I, 3.5. Mu.L of Sma I and 10. Mu.L of BSA gave a digested product of pBI121 plasmid.
Xba I and Sma I are adopted to carry out double enzyme digestion on plasmid DNA of pMD18T-GmVIT1, and the reaction system of the double enzyme digestion is as follows: 50 μ L of the PCR product, 10 μ L of 10 XM Buffer, 2.5 μ L of Xba I, 2.5 μ L of Sma I and 5 μ L of BSA and 30 μ L of ddH 2 O, obtaining a sequence fragment of GmVIT1.
(3) Recovering the sequence fragment of the GmVIT1 and the enzyme digestion product of the pBI121 plasmid by adopting a DNA gel recovery kit; the expression vector pBI121 is connected with the sequence segment of GmVIT1, and the reaction system during connection is as follows: 5 μ L of plasmid DNA cleavage product of pBI121, 3 μ L of sequence fragment of GmVIT1, 1 μ L of 10 XT 4 DNA ligation buffer and 1 μ L of T4 DNA ligase; the reaction conditions are as follows: the reaction temperature is 16 ℃, and the reaction time is 12-18 h to obtain the connecting product.
(4) Identification of recombinant plasmids
Colony PCR identification is carried out, gmVIT1 gene primers containing Xba I and Sma I enzyme cutting sites are used for carrying out PCR identification, and the reaction system is as follows: 7.7 μ L of ddH 2 O, 1. Mu.L of 10 XPCR Buffer (TAKARA),0.8. Mu.L of dNTP Mix (2.5 mmol/L), 0.4. Mu.L of GmVIT1-Xba I (10. Mu. Mol/L), 0.4. Mu.L of GmVIT1-Sma I (10. Mu. Mol/L), 0.1. Mu.L of rTaq DNApolymerase (5. Mu. Mol/L), and 1. Mu.L of the colony samples to be detected.
Single colony reaction procedure: (30 cycles)
Extracting recombinant plasmid DNA of pBI121-GmVIT1 for enzyme digestion verification, wherein the verified reaction system is as follows: 6.6. Mu.L of the pBI121-GmVIT1 recombinant plasmid, 1. Mu.L of 10 XT Buffer, 1. Mu.L of BSA, 0.3. Mu.L of Xba I, 0.3. Mu.L of Sma I, 0.8. Mu.L of ddH 2 And O. The desired band of about 660bp was obtained.
5. Method for preparing agrobacterium carrying GmVIT1 gene
(1) The recombinant plasmid pBI121-GmVIT1 is transformed by a freeze-thawing method, and the method comprises the following steps:
(1) the competent cells, which were stored at-80 ℃, were thawed on ice.
(2) 10 mu L of the recombinant plasmid of pBI121-GmVIT1 is added into 100 mu L of the agrobacterium-infected cell GV3101, and the mixture is stirred by hand to the bottom of the tube and mixed evenly.
(3) Standing on ice for 5min, liquid nitrogen for 5min, water bath at 37 deg.C for 5min, and ice bath for 5min.
(4) Adding 700 μ L of YEB liquid culture medium without antibiotics, and culturing at 28 deg.C for 2-3h under shaking.
(5) The cells were harvested by centrifugation at 6000rmp for 1min, 100. Mu.L of the supernatant was retained and the pellet was resuspended, spread on YEB solid medium containing kanamycin (50 mg/mL) + (Str) streptomycin (50 mg/mL) + (Rif) rifampicin (50 mg/mL), and placed in a 28 ℃ incubator for 2-3 days in the dark while inverted.
Identifying the transformed agrobacterium; the identification method comprises the following steps: selecting single colonies with relatively uniform growth, and performing colony PCR amplification by using gene specific primers; the reaction system identified was: template DNA colony, 1. Mu.L of 10 XBuffer, 0.8. Mu.L of dNTP Mix, 0.2. Mu.L of GmVIT1-Xba I, 0.2. Mu.L of GmVIT1-Sma I, 0.1. Mu.L of rTaq, and 7.7. Mu.L of ddH 2 O; the reaction conditions are as follows: (1) 94 ℃ for 5min; (2) 94 ℃ and 30s; (3) 59 ℃ for 30s; (4) 72 ℃ for 1min; (5) 72 ℃ for 10min; (6) 4 ℃ and 1h; (1) circulating for 30 times;
6. method for preparing plant carrying GmVIT1 gene
Pre-culturing and treating plant seedlings:
(1) Placing wild type tobacco seed in sterilized centrifuge tube, sterilizing with 75% alcohol for 3min, and washing with sterilized water for 6-8 times.
(2) Then treated with 1mL of 10% sodium hypochlorite for 5min, and washed with sterilized water for 6-8 times.
(3) And uniformly spreading the treated seeds on an MS solid culture medium, and culturing to grow seedlings.
(4) And (4) transferring the plantlets into a bottle filled with an MS solid culture medium, continuously culturing until the plantlets grow to a seedling stage, and using the leaves for infection experiments.
(5) The tobacco leaves were cut into a square of about 1cm × 1cm and pre-cultured for 48 hours.
Activating the positive agrobacterium obtained in the step 5: coating pBI121-GmVIT1 positive bacteria on YEB solid culture medium containing Km, str and Rif in a superclean bench, culturing at 28 deg.C for 48 hr in an inverted manner, suspending with 1/2MS liquid culture medium to obtain bacterial liquid, and diluting with 1/2MS to OD 600 About 0.4 to about 0.6. The bacterial solution used above was transferred to a 50mL Erlenmeyer flask for infestation of tobacco.
Taking out the leaves pre-cultured in the above steps, infecting in the bacterial liquid for 6min, continuously reversing to ensure that the leaves are fully contacted with the bacterial liquid, taking out the leaves, placing on sterile filter paper, sucking off the attached bacterial liquid, placing the back of the leaves upwards on a culture medium of MS1, and culturing in dark for 2d.
And (3) bud induction: the leaves were transferred to MS2 medium for bud induction, with medium changes every 3 days, and after 3 consecutive times, every 2 weeks.
Rooting induction: when the differentiated buds grow to about 2cm, the differentiated buds are cut off and transferred to an MS3 culture medium for rooting culture.
Transplanting and domesticating: and transferring the rooted tobacco seedlings to soil for culture.
Method for identifying positive plants: PCR identification is carried out on Km resistant plants, pBI121-GmVIT1 plasmid is used as a positive control, a fragment of about 660bp can be amplified, the amplified fragment is consistent with the positive control fragment, and the PCR identification result of part of the PCR identification result is shown in figure 2 after GmVIT1 is transferred into tobacco.
As shown in FIG. 3, the GmVIT1 gene is genetically transformed into tobacco by the agrobacterium-mediated leaf disc method, and resistant plants of the GmVIT1 gene can be obtained.
7. Culturing a plant carrying the GmVIT1 gene, and transplanting the grown resistant plant to a soil-containing: vermiculite is 1:1, counting the seed setting condition of the plants.
The statistical analysis results are shown in table 1 and fig. 4, and the method for transferring the GmVIT1 gene can improve the seed setting rate of the plant by about 2 times. The weight of the seeds of the transgenic line averagely reaches more than 2 times of that of the control, and the statistical analysis shows that the weight of the seeds of the transgenic line has significant difference (P is less than 0.05).
TABLE 1 seed set statistics
Note: the seed number is counted by weight in g.
SEQUENCE LISTING
<110> university of Harbin
Application of GmVIT1 gene of soybean and recombinant vector containing GmVIT1 gene
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> GmVIT1 F
<400> 1
<210> 2
<211> 19
<212> DNA
<213> GmVIT1 R
<400> 2
ctaaagacca cttgaccca 19
<210> 3
<211> 660
<212> DNA
<213> GmVIT1 Gene
<400> 3
atggcatcag atcactcgtc cgtcaaccaa cccaaatttg tctatccaaa caatgaactt 60
gagcaacaag aagccctaga ggtcgaaacc aaggactttg actactcaaa gagatctcaa 120
tggctacgtg ctgctgtttt aggtgccaat gacgggttgg tctcaacagc atcactaatg 180
atgggtgtgg gagcagtgaa gcaagacatc aaagtcatga tcctaacagg ttttgcaggg 240
ctagttgctg gggcatgcag catggccatt ggggagtttg tgtcggttta ctcgcagctg 300
gacatagaag ttgctcaaat gaaaagagag aaagaaaggg acaacataga tcaagaggaa 360
gatggttatg agaaagagaa attgccaaac cctatacatg ctgcagcagc atcagctctt 420
gcattttcag ttggtgcatt ggtgcctctc cttgcagcct cttttataag ggactataag 480
gtgaggcttg gtgtgatttt gggagcagtg agtttagctc ttgtggtgtt tggttggttg 540
ggggcagtgt tgggtaaggc tccaacattt aggtcttgtg tgagagtctt gcttgggggg 600
tggctagcca tggctattac ttttgggcta accaagttga ttgggtcaag tggtctttag 660
Claims (7)
1. Of soybeanGmVIT1The application of the gene in promoting plant seed setting is characterized in that the plant is tobacco; saidGmVIT1The sequence of the gene is shown in SEQ ID NO. 3.
2. Use according to claim 1, characterized in that of soyaGmVIT1The method for promoting the plant seed setting by the gene comprises the following specific steps:
(1) Construction of the recombinant vector pBI121-GmVIT1；
(2) Preparation carrierGmVIT1Genetic agrobacterium: the recombinant vector pBI121-GmVIT1Transferring into Agrobacterium to be cultured in an infected state to obtain carrierGmVIT1An agrobacterium of a gene;
(3) Preparation carrierGmVIT1A plant of the gene: culturing the seeds of the plant to the seedling stage, then carrying out pre-culture treatment on the plant, then infecting the plant subjected to the pre-culture treatment by the agrobacterium obtained in the step (2), and then carrying out bud induction, rooting induction and transplantation domestication to obtain the carrierGmVIT1A plant of the gene;
(4) Culture carrierGmVIT1A plant of the gene.
3. The use according to claim 2, wherein the recombinant vector in step (1) is constructed by using restriction endonuclease to cleave the vector pMD18T-GmVIT1And pBI121 plasmid to obtainGmVIT1The product of the enzyme digestion of the sequence fragment of the gene and the pBI121 plasmid is obtained, and thenGmVIT1The sequence fragment of the gene and the restriction enzyme product of the pBI121 plasmid are subjected to a ligation reaction, and the ligation reaction system is as follows: 5. mu.L of pBI121 plasmid cleavage product, 3. Mu.L ofGmVIT1The sequence fragment of the gene, 1. Mu.L of 10 XT 4 DNA ligation buffer and 1. Mu.L of T4 DNA ligase, the reaction temperature was 16 ℃ and the reaction time was 12h.
4. The use according to claim 3, wherein the restriction enzyme isXba IAndSma I。
5. use according to claim 2, wherein the seeds of the plants in step (3) reach the seedling stage.
6. The use according to claim 2, wherein the step of infecting the plant with Agrobacterium in step (3) is as follows:
1) Will carry the recombinant vector pBI121-GmVIT1Is plated on YEB solid medium containing kanamycin, streptomycin and rifampicin at 28Culturing at 48h deg.C;
2) Resuspending the bacterial colony of the agrobacterium obtained in the step 1) by using a 1/2MS liquid culture medium to obtain a bacterial liquid of the agrobacterium, and then diluting the bacterial liquid to OD by using 1/2MS 600 The value is 0.4-0.6;
3) And (3) taking plant leaves, infecting the plant leaves for 6 min-7 min in the bacterial liquid of the agrobacterium obtained in the step 2), taking the plant leaves out, placing the plant leaves on sterile filter paper, sucking off the attached bacterial liquid, placing the plant leaves on an MS1 culture medium, and culturing for 2 days under a dark condition.
7. The use according to claim 2, wherein the preculture treatment in step (3) is to cut the plant leaves into 1cm x 1cm squares, preculture 48h.
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| Title |
|---|
| GenBank：MN547956.1;Guo，D;《GenBank》;20200523;全文 * |
| Molecular Evolution of the Vacuolar Iron Transporter ( VIT) Family Genes in 14 Plant Species;Jun Cao;《Genes (Basel)》;20190214;第10卷(第2期);第1-16页 * |
| 大豆GmVIT1克隆及耐盐功能分析;李永光等;《东北农业大学学报》;20200225;第51卷(第2期);第1.3-1.4节 * |
| 高等植物体内铁运输机制研究进展;贾凯等;《基因组学与应用生物学》;20101228;第29卷(第6期);第1152-1158页 * |
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