CN111850000A - Application of recombinant expression vector containing rice gene OsNAR2.1 and promoter thereof - Google Patents
Application of recombinant expression vector containing rice gene OsNAR2.1 and promoter thereof Download PDFInfo
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Abstract
The invention discloses an application of a recombinant expression vector containing a rice gene OsNAR2.1 and a promoter thereof. A recombinant expression vector is characterized by containing a promoter of a rice gene OsNAR2.1 and the rice gene OsNAR2.1; the nucleotide sequence of the promoter of the rice gene OsNAR2.1 is SEQ ID NO.1, and the nucleotide sequence of the rice gene OsNAR2.1 is SEQ ID NO. 2. The recombinant expression vector disclosed by the invention is applied to reducing the methane emission of the paddy field. The invention reports for the first time that the promoter gene OsNAR2.1 for expressing the rice gene OsNAR2.1 can reduce the content of methanogens in the soil of the rice rhizosphere, increase the content of methane-oxidizing bacteria and reduce the application of the discharge of methane in the rice field.
Description
Description of the cases
The invention relates to a divisional application of a Chinese patent application with the application date of 2018, 3 and 2, the application number of 2018101756349 and the name of 'a recombinant expression vector containing a rice gene OsNAR2.1 and a promoter thereof and application thereof'.
Technical Field
The invention belongs to the field of genetic engineering, and relates to an application of a recombinant expression vector containing a rice gene OsNAR2.1 and a promoter thereof.
Background
Methane (CH)4) Is one of the main global greenhouse gases, and has 25 times higher capability than carbon dioxide in global warming greenhouse effect. In addition, CH4It also affects the chemical and oxidizing capacity of the atmosphere (IPCC. changes in atmospheric chemistry and in radial compliance. Pp.2.9.2-2.9.3 in S. Solomon, D.Qin, and M.Manning, et al. eds. simulation change 2007: the physical science basis. control of Working Group I to the following description of the innovative implementation change. 2007. Cambri. Univ.Press, Cambridge, U.K.).
The rice field is atmosphere CH4One of the main emission sources of (2) accounts for 5 to 19 percent of the total emission. China 'S rice planting area accounts for 23% of the global rice planting area, and in order to guarantee the agricultural production output, China' S rice field uses nitrogen fertilizer in large quantity, which causes the emission of greenhouse gas to increase, and affects a series of environmental ecological effects (Frolding S, Qiu J, bones S, animal. combining fresh and ground center data todevelop new maps of the distribution of rice aggregation in China. Global biogeochemical cycles.2002.16(4): 38-1-38-10.). Research shows that the application of urea in rice field can increase CH 4Emission of (Hu RG, Hatano R, Kusa K, et al. Effect of nitrogen deferrizationn on methane flux in astructured clay soil cultivated with onion in Central Hokkaido,Japan.SoilScience&Plant Nutrition,2002,48(6): 797-. Application of urea alters the abundance of rice root microorganisms (X Fan, H Yu, Q Wu, J Ma, H xu. et al. effects of fertilization on microbiological association and emission of greenhouse gases (CH)4and N2O) in rice paddyfields, ecology and Evolution,2016,6(4):1054) the only biogenic source of methane production in paddy soil is methanogens. The last step of CH4 production is controlled by the mcrA gene of methanogens, and the pmoA gene of methanogens encodes the use of CH4The first key enzyme of (1), will be CH4Oxidized to methanol.
The promoter for expressing the rice gene OsNAR2.1 and starting the rice gene OsNAR2.1 can obviously increase the expression abundance of the soil methane-oxidizing bacteria pmoA gene of the paddy field and reduce the expression abundance of the methanogen mcrA gene, thereby reducing the discharge of the methane in the paddy field.
Disclosure of Invention
The invention aims to provide a recombinant expression vector containing a rice gene OsNAR2.1 and a promoter thereof.
Another object of the present invention is to provide the use of the recombinant expression vector.
The purpose of the invention can be realized by the following technical scheme:
A recombinant expression vector is characterized by containing a promoter of a rice gene OsNAR2.1 and the rice gene OsNAR2.1; the nucleotide sequence of the promoter of the rice gene OsNAR2.1 is SEQ ID NO.1, and the nucleotide sequence of the rice gene OsNAR2.1 is SEQ ID NO. 2.
In a preferred embodiment of the present invention, the starting vector of the recombinant expression vector is pTCK303 vector.
As a preferred embodiment of the invention, the promoter of the rice gene OsNAR2.1 in the recombinant expression vector is connected with the rice gene OsNAR2.1 through corresponding enzyme cutting sites to form a pOsNAR.1: OsNAR2.1 recombinant fragment, and the recombinant fragment is introduced into an issuing vector.
The recombinant expression vector is applied to reducing the methane emission of the paddy field; preferably, the method is applied to the aspects of reducing the content of methanogenic bacteria in rice rhizosphere soil, increasing the content of methane-oxidizing bacteria and reducing the discharge of methane in a rice field.
Has the advantages that:
1. through systematic research, the biological function of the rice gene OsNAR2.1 started by the rice gene OsNAR2.1 promoter is provided for the first time, the expression abundance of the soil methane-oxidizing bacteria pmoA gene of the rice field can be increased, and the expression abundance of the methanogen mcrA gene is reduced (figure 1).
2. The inventor provides a transgenic plant of a rice gene OsNAR2.1 promoter for starting the rice gene OsNAR2.1 for the first time, and the transgenic plant shows that the discharge yield of methane in a rice field is remarkably reduced by 33-90 percent in the rice field (figure 3-5).
Drawings
FIG. 1: schematic diagram of rice gene OsNAR2.1 promoter promoting OsNAR2.1 vector.
FIG. 2: and (4) analyzing the copy number of the soil methane bacteria at the rhizosphere of the rice.
a: methanogen (mcrA) content. b: methane oxidizing bacteria (pmoA) content.
FIG. 3: analysis of methane emission in rice field at different times
FIG. 4 analysis of discharge amount of methane from rice field at different times
FIG. 5 analysis of discharge amount of methane from rice field at different times
Detailed description of the preferred embodiments
Cloning of rice gene OsNAR2.1 promoter sequence
1) Extraction of genomic DNA of Rice
When the rice (Nipponbare) seedlings grow to 3 leaves, about 0.1g of leaves are weighed, ground by liquid nitrogen, fully ground and added into a 2ml centrifuge tube, 1ml of DNA extracting solution (purchased from TIANGEN, China) is rapidly added, and after fully shaking and shaking, the rice genome DNA is extracted.
2) Cloning of OsNAR2.1 Gene full Length
The promoter of the NAR2.1(AP004023.2) gene of rice was retrieved from the gene database of NCBI website (http:// www.ncbi.nlm.nih.gov /), and the promoter sequence of the NAR2.1 gene was amplified from the genomic DNA of rice by designing a Primer sequence (see below) using software Primer 5.0.
P1:5’-TGCTGACAAACCAAACCGACT-3’(SEQ ID NO.3)
P2:5’-CCCCACCTCTCCCACCTCAC-3’(SEQ ID NO.4)
Using the rice genomic DNA obtained in step 1) as a template, performing PCR amplification using high fidelity enzyme (Prime Star HS DNApolymerase available from Takara), the PCR procedure was as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 30s, 30 cycles, and constant temperature at 72 deg.C for 5min and 4 deg.C. The DNA fragment was subjected to agarose electrophoresis separation, excised, recovered, cloned into a pMD-19 vector (purchased from Takara Co., Ltd.), and sequenced correctly to obtain the promoter sequence (SEQ ID NO.1) of the rice gene OsNAR2.1 having the entire coding region.
Secondly, cloning of rice gene OsNAR2.1 sequence
1) Extraction of Total RNA
Rice (Nipponbare) seedlings grow to 3-leaf stage, after treatment with 0.2mM nitrate nitrogen for 6 hours, roots are taken out immediately and placed in liquid nitrogen for freezing and preservation, about 0.1g of roots are weighed, ground by liquid nitrogen, ground and fully added into a 1.5ml centrifuge tube, 1ml of Trizol reagent (purchased from Invitrogen, USA) is rapidly added, and after fully shaking and shaking, total RNA is extracted.
2) Cloning of OsNAR2.1 Gene full Length
The NAR2.1 gene series OsNAR2.1(AP004023.2) of rice was searched from a gene database of NCBI website (http:// www.ncbi.nlm.nih.gov /). Primers (below) were designed to hook the full-length sequence of OsNAR2.1 from tissue cDNA library.
P3:5’-CAATGGCGAGGCTAGCCGGCGTT-3’(seq id no.5)
p4:5’-CGATCTACTTGTCCTTCTTGCGCTTCT-3’(SEQ ID NO.6)
Using the total RNA obtained in step 1) as a template, synthesizing a first cDNA strand by reverse transcription, and performing PCR amplification by using high fidelity enzyme (PrimeStar HS DNA polymerase from Takara), wherein the PCR program is as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 30s, 30 cycles, and constant temperature at 72 deg.C for 5min and 4 deg.C. Agarose electrophoresis separation, cutting gel, recovering, cloning to pMD-18 vector (purchased from Takara company), and obtaining the full-length sequence (SEQ ID NO.2) of the rice high-affinity nitrate transport protein gene OsNAR2.1 with complete coding region after correct sequencing.
Thirdly, obtaining a rice gene expression OsNAR2.1 promoter promoting OsNAR2.1 transgenic strain
1) Construction of rice gene OsNAR2.1 promoter promoting OsNAR2.1 vector
Designing a Primer sequence (as follows) by using software Primer 5.0 according to the promoter sequence of the rice gene OsNAR2.1 obtained in the step one and a sequence shown as SEQ ID NO. 1:
P5:5’-GAGGCGCGCCTGCTGACAAACCAAACCGACT-3’(ASC I)(SEQ ID NO.7)
P6:5’-CATTAATTAACCCCACCTCTCCCACCTCAC-3’(Pac I)(SEQ ID NO.8)
PCR amplification was performed using the pMD-18 vector for cloning the OsNAR2.1 promoter obtained in step one as a template, using high fidelity enzyme (PrimeStar HS DNA polymerase available from Takara Inc.) according to the following PCR protocol: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 30s, 30 cycles, and constant temperature at 72 deg.C for 5min and 4 deg.C. And (3) carrying out agarose electrophoresis separation and gel cutting to obtain PCR products of OsNAR2.1 promoters with enzyme cutting sites Asc I and Pac I added to the F end and the R end respectively. And carrying out double enzyme digestion on the recovered product by using restriction enzymes Asc I and Pac I, simultaneously carrying out double enzyme digestion on the plant overexpression vector pTCK303 plasmid by using Asc I and Pac I, and then respectively recovering the PCR fragment and the vector after enzyme digestion. After recovery, the linearized vector was ligated with the digested PCR fragment by T4 ligase overnight at 4 ℃ and transformed into E.coli DH5a competent cells, which were plated in 50. mu.gmL cells containing kanamycin -1After 12h of growth on LB solid culture medium, positive colonies are picked, plasmids are extracted, and after the size of fragments is verified to be correct by Asc I and Pac I double enzyme digestion, the bacterial liquid is subjected to DNA sequencing, and the clone containing the correct sequencing is named as pOsNAR2.1-pTCK 303.
According to the full-length sequence of the rice gene OsNAR2.1 obtained in the step two, see the sequence of SEQ ID NO.2, a software Primer 5.0 is used for designing a Primer sequence (as follows):
P7:5’-GAGAATTCCAATGGCGAGGCTAGCCGGCGTT-3’(ECOR I)(SEQ ID NO.9)
P8:5’-GAGGCGCGCCCGATCTACTTGTCCTTCTTGCGCTTCT-3’(Asc I)(SEQ ID NO.10)
and (3) performing PCR amplification by using the pMD-18 vector with the full length of the cloned OsNAR2.1 gene obtained in the step two as a template and using high fidelity enzyme (Prime Star HS DNA polymerase from Takara company), wherein the PCR program is as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 30s, 30 cycles, and constant temperature at 72 deg.C for 5min and 4 deg.C. And carrying out agarose electrophoresis separation, cutting gel and recovering to obtain the PCR products of OsNAR2.1 gene full length with enzyme cutting sites EcoR I and Asc I added to the F end and the R end respectively. And carrying out double digestion on the recovered product by using restriction enzymes EcoR I and Asc I, simultaneously carrying out double digestion on the pOsNAR2.1-pTCK303 plasmid by using EcoR I and Asc I, and then respectively recovering the digested PCR fragment and the vector. After recovery, the linearized vector was ligated with the digested PCR fragment by T4 ligase overnight at 4 ℃ and transformed into E.coli DH5a competent cells, which were plated in 50. mu.gmL cells containing kanamycin -1After 12h of growth on LB solid culture medium, positive colonies are picked, plasmids are extracted, and after the sizes of fragments are verified to be correct by EcoR I and Asc I double enzyme digestion, DNA sequencing is carried out on the bacterial liquid, and the clone containing the correct sequencing is named as pOsNAR2.1-OsNAR2.1-pTCK 303. Namely, the expression vector, the vector is schematically shown in FIG. 1).
Obtaining transgenic plants
The obtained agrobacterium transformed with the vector infects rice callus, co-cultures the rice callus for 60 hours, and obtains T through selective culture, differentiation, rooting and seedling hardening0Transgenic plants are generated. The propagation is carried out twice on all the transgenic materials, and the T1 generation and T2 generation materials which are stably inherited are obtained.
Through extracting DNA of rice rhizosphere soil to carry out methane copy number identification, the fact that methanogens (mcrA) in the rhizosphere soil of a transgenic line of expressing a rice gene OsNAR2.1 promoter and starting OsNAR2.1 is reduced by 18.5% and methane-oxidizing bacteria (pmoA) is increased by 125.4% compared with WT (figure 2) is found, and finally, the methane emission is reduced by 30% -90% (figure 3-5).
In conclusion, the invention discovers that the promoter for expressing the rice gene OsNAR2.1 can obviously reduce the content of methanogen (mcrA) in rice rhizosphere soil, increase the content of methane-oxidizing bacteria (pmoA) and reduce the emission of methane in a rice field by starting the OsNAR2.1; the OsNAR2.1 promoter of the present invention can be used to drive an OsNAR2.1 expression vector, which may include an enhancer, whether a transcription enhancer or a translation enhancer, if necessary. To simplify the identification of transformed cells, selectable markers including enzymes resistant to antibiotics, enzymes which utilize compounds recognized by a color change (e.g., B-glucuronidase GUS) or luminescence (e.g., luciferase) may be used, and marker-free selection may also be used. As the expression vector, a Ti plasmid, Ri plasmid, plant virus vector or the like can be used. Transformation methods plants can be transformed by Agrobacterium-mediated transformation, particle gun, pollen tube channel or other methods. The expression of the vector can reduce the content of methanogens in the rhizosphere soil of the rice, increase the content of methane-oxidizing bacteria and reduce the emission of methane in the rice field.
Sequence listing
<110> Nanjing university of agriculture
<120> application of recombinant expression vector containing rice gene OsNAR2.1 and promoter thereof
<160>10
<170>SIPOSequenceListing 1.0
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<213> Rice (Oryza sativa)
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gattccccac ctctcccacc tcactcctac ctcactccta gtcctctgcc gaaagtactt 60
cctccgtttc acaatgtaag tcattctaat atttttcaca ttcatattga tgtttgaatc 120
tagattgata tatatgttta gattcgttag catcgatatg aatatgggaa atgctagaat 180
gacttatatt gtgaaacaga gtgagtatca tgtaaaagtt agaaggaaaa aaatagagct 240
gtttgtgatg atatgggtgt ggttgtgttg tgtgagccga tgtccattgt actgtactca 300
ttttaaatgt acgtaccgtt aacttatata gttatatgcg tttgatcatt tgtcaaaatt 360
tagtgaaact ttaaaattta ttatacttaa agtatattta atgataaatt taaaataaaa 420
taaactttca gctgttatgt tcaaaatcaa catcgtcaga tattttaaat taaaggtagt 480
acttttaaaa aaaggatttt tgcggtgtgt cgtggcgaaa ctgctaccaa gtttcaatga 540
tcatatgcca tttcatagga taattactct catcgtggta agtaagaatc gattgcctat 600
tttcggcagg ctgttgtttc aaagcatcga tctgcttgga caacttgagc aaagctagct 660
agaactgggt cgatataatt gcagcactag gcaatcaaga gacggagctg ccaccagcta 720
gctgagctga gctgatatga tcaacacagt gcagacttgg tcgtgttcga gttcgatcga 780
cggatggctg tcctgctctt gcgctcatgc atgtcatctc ttcggaagta ggagtacagc 840
agtacttgag gaatattatt agagagtaag ttgaactgtt ttcaatagtt cagggtgtaa 900
actaagctga ggaattgtta ggaggttaaa tgctgtggca aaatagtttg gaggagcgaa 960
atgatttttt tttcatatga aaaacatcta aatttatttt ttgccaaaac actagtatat 1020
catcaaattt tcatccatta agaacgcctt ctcaatatta ataattccaa tgtgatatct 1080
taatgctcaa tgaacctaaa atagtttgga tgagtgaaat ggactctttt tgagtttttt 1140
tccatatgaa aacatctaaa tttatttttt tttgccaaaa cactggtata tcatcaaatt 1200
ttcctccatt aagaacgcct tctcaacgtt aataactcca atgttattat cttaatgcca 1260
aatgaaccta ccatgaacgt catgctcaca atttaattaa caacaaccgg gcactcaaga 1320
tcattcgcgg ttgccgcttc tcaccggttg cctgaaccct tgggacccct ccaaaagctt 1380
aattaccccc aaaaccgcat gatctctctc ttctcttctc ttctcacacg tcgtcaaagc 1440
ctctgacttt ggatatcccc gaccccacta aacttaatca acttgatcat tacaacaatt 1500
aagttgcctc ttgaatccaa cgaagtagct ggtcaactct ccgagctcta gcctcgctct 1560
cccgcctata aattcaccga tcgatcgatc gatcgatctc agcatcagca gcagcagcag 1620
attcatttct tggtcttcgt ctccgtctcc gtccttgggt tgatatccag aatcagtcgg 1680
tttggtttgt cagcaatg 1698
<210>2
<211>621
<212>DNA
<213> Rice (Oryza sativa)
<400>2
atggcgaggc tagccggcgt tgctgctctc tcgttggtgc tcgtcttgct cggcgccggc 60
gtgccccggc cggcggccgc cgccgcggcg aagacgcagg tgttcctctc caagctgccc 120
aaagcgctcg tcgtcggcgt ctcgcccaag cacggtgaag tcgtgcacgc cggcgagaac 180
acggtgacgg tgacgtggtc gctgaacacg tcggagccgg cgggcgccga cgcggcgttc 240
aagagcgtga aggtgaagct gtgctacgcg ccggcgagcc ggacggaccg cgggtggcgc 300
aaggcctccg acgacctgca caaggacaag gcgtgccagt tcaaggtcac cgtgcagccg 360
tacgccgccg gcgccggcag gttcgactac gtggtggcgc gcgacatccc gacggcgtcc 420
tacttcgtgc gcgcctacgc ggtggacgcg tccggcacgg aggtggccta cgggcagagc 480
tcgccggacg ccgccttcga cgtcgccggg atcaccggca tccacgcctc cctcaaggtc 540
gccgccggcg tcttctccac cttctccatc gccgcgctcg ccttcttctt cgtcgtcgag 600
aagcgcaaga aggacaagta g 621
<210>3
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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tgctgacaaa ccaaaccgac t 21
<210>4
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<213> Artificial Sequence (Artificial Sequence)
<400>4
<210>5
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
caatggcgag gctagccggc gtt 23
<210>6
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
cgatctactt gtccttcttg cgcttct 27
<210>7
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<213> Artificial Sequence (Artificial Sequence)
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gaggcgcgcc tgctgacaaa ccaaaccgac t 31
<210>8
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
cattaattaa ccccacctct cccacctcac 30
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<213> Artificial Sequence (Artificial Sequence)
<400>9
gagaattcca atggcgaggc tagccggcgt t 31
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<213> Artificial Sequence (Artificial Sequence)
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gaggcgcgcc cgatctactt gtccttcttg cgcttct 37
Claims (4)
1. The application of a recombinant expression vector in reducing the methane emission of a paddy field; the recombinant expression vector contains a promoter of a rice gene OsNAR2.1 and the rice gene OsNAR2.1; the nucleotide sequence of the promoter of the rice gene OsNAR2.1 is SEQ ID NO.1, and the nucleotide sequence of the rice gene OsNAR2.1 is SEQ ID NO. 2.
2. The use according to claim 1, wherein the starting vector of said recombinant expression vector is pTCK303 vector.
3. The use of claim 2, wherein the promoter of the rice gene OsNAR2.1 and the rice gene OsNAR2.1 in the recombinant expression vector are connected by corresponding restriction enzyme sites to form a recombinant pOsNAR.1: OsNAR2.1 fragment, which is introduced into the starting vector.
4. The application of any one of claims 1-3, wherein the recombinant expression vector is applied to the aspects of reducing the content of methanogens in the rhizosphere soil of rice, increasing the content of methane-oxidizing bacteria and reducing the emission of methane in rice fields.
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| CN108315346A (en) * | 2018-03-02 | 2018-07-24 | 南京农业大学 | Recombinant expression carrier and its application of a kind of OsNAR2.1 containing paddy gene and its promoter |
| CN114941007A (en) * | 2022-06-02 | 2022-08-26 | 河南农业大学 | Recombinant expression vector and application thereof |
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| CN108341858B (en) * | 2018-03-02 | 2021-02-26 | 南京农业大学 | Application of rice gene OsNAR2.1 in drought resistance |
| CN108977460B (en) * | 2018-08-15 | 2021-11-16 | 中国农业科学院深圳农业基因组研究所 | Recombinant expression vector and application thereof in increasing rice yield and reducing cadmium concentration |
| CN110923241B (en) * | 2019-11-05 | 2022-10-14 | 南京农业大学 | Application of key gene OsLSD1.1 for controlling formation of aeration tissue of rice in reduction of methane emission |
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| CN108341858B (en) * | 2018-03-02 | 2021-02-26 | 南京农业大学 | Application of rice gene OsNAR2.1 in drought resistance |
| CN108315346B (en) * | 2018-03-02 | 2021-03-09 | 南京农业大学 | Recombinant expression vector containing rice gene OsNAR2.1 and promoter thereof and application thereof |
| CN108977460B (en) * | 2018-08-15 | 2021-11-16 | 中国农业科学院深圳农业基因组研究所 | Recombinant expression vector and application thereof in increasing rice yield and reducing cadmium concentration |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108315346A (en) * | 2018-03-02 | 2018-07-24 | 南京农业大学 | Recombinant expression carrier and its application of a kind of OsNAR2.1 containing paddy gene and its promoter |
| CN108315346B (en) * | 2018-03-02 | 2021-03-09 | 南京农业大学 | Recombinant expression vector containing rice gene OsNAR2.1 and promoter thereof and application thereof |
| CN114941007A (en) * | 2022-06-02 | 2022-08-26 | 河南农业大学 | Recombinant expression vector and application thereof |
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| CN108315346A (en) | 2018-07-24 |
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