CN115851755B - Mongolian hybrid agro-grass tillering angle regulating factor and application thereof - Google Patents
Mongolian hybrid agro-grass tillering angle regulating factor and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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Abstract
The invention discloses application of a Mongolian hybrid agro-weed tillering angle regulating factor, and belongs to the technical field of plant genetic engineering. The invention uses Mongolian hybrid agro-hybrid tillering node RNA as a material, and adopts an ORF full-length cloning method to obtain the full-length sequence of Meng Nong hybrid agro-hybrid tillering angle regulatory factor LAZY1 or WOX gene. Constructing a plant over-expression vector pCAMBIA1300-UBI, transferring the pCAMBIA1300-UBI into receptor material rice by using an agrobacterium-mediated method, and enabling transgenic rice plants over-expressing LAZY1 to show the phenomenon of large tillering angles. Therefore, the regulatory gene provided by the invention can be involved in the regulation of the tillering angle of plants, can improve the plant type of the plants in the plant genetic engineering and accelerate the breeding process of high-yield close-planted varieties.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a Mongolian hybrid agropyron tillering angle regulatory factor WOX11 gene and LAZY1 gene.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The control of the tillering angle of the plant is a complex biological process, is commonly controlled by environmental factors such as temperature, illumination, nutrients and non-environmental factors such as hormone, genetic genes and the like, and has important significance in constructing an ideal plant type of the plant. In gramineous plants, such as cereal crops like rice, the number of tillers and the size of the tillering angle have a direct relationship with the yield of the crop.
Transcription factors (Transcription factor, TF) are a class of trans-acting factors that can regulate gene expression. A single transcription factor often can regulate the expression of multiple functional genes simultaneously relative to the functional genes. Therefore, it is very interesting to analyze and identify the gene functions of transcription factors. Through the transgenic technology, the transcription factors capable of simultaneously regulating and controlling a plurality of functional genes are transformed into plants, so that the rapid cultivation of new varieties with excellent comprehensive properties is hopeful to be realized.
Disclosure of Invention
Aiming at the problems existing in the prior art, the technical problem to be solved by the invention is to provide the application of the Mongolian hybrid agronomic crop tillering angle regulating factor.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
A Mongolian hybrid agronomic crop tillering angle regulating factor is a WOX11 gene or a LAZY1 gene, and the nucleotide sequences of the Meng Nong hybrid agronomic crop tillering angle regulating factor are shown as SEQ_NO.1 and SEQ_NO. 2.
A plant over-expression vector containing the Meng Nong hybrid agropyron cristatum tillering angle regulatory factor.
Furthermore, the plant over-expression vector of the Meng Nong hybrid agropyron cristatum tillering angle regulating factor is pCAMBIA1300-UBI.
An application of the Meng Nong hybrid agropyron cristatum tillering angle regulating factor in plant breeding.
Further, the application comprises the following steps:
1) Constructing Meng Nong plant overexpression vectors of hybrid agrocybe aegerita tillering angle regulating factors;
2) Transforming the plant over-expression vector of the Meng Nong hybrid agrocybe aegerita tillering angle regulating factor into a plant or a plant cell;
3) And cultivating and screening to obtain transgenic plants with different tillering angles.
Further, hygromycin is used as a screening marker of transgenic plants in the vector pCAMBIA1300-UBI, or the screening of transgenic plants is carried out by using the calicheamicin.
Further, the plant is a gramineous plant, and further, the plant is rice.
Further, the application comprises the improvement of plant type of plants, and further, the regulation of plant tillering angle.
Compared with the prior art, the invention has the beneficial effects that:
The invention discloses application of a Mongolian hybrid agro-weed tillering angle regulating factor, and belongs to the technical field of plant genetic engineering. The invention takes Mongolian hybrid agro-hybrid tillering node RNA as a material, adopts an ORF full-length cloning method to obtain full-length sequences of Meng Nong hybrid agro-hybrid tillering angle regulatory factors WOX11 gene and LAZY1 gene, and is shown as SEQ_WOX11 and SEQ_LAZY1. Constructing a plant over-expression vector pCAMBIA1300-UBI, transferring the pCAMBIA1300-UBI into receptor material rice by using an agrobacterium-mediated method, and enabling transgenic rice plants over-expressing WOX11 or LAZY1 to show the phenomenon of increased tillering angles. Therefore, the regulatory gene provided by the invention can be involved in the regulation of the tillering angle of plants, can improve the plant type of the plants in the plant genetic engineering and accelerate the breeding process of high-yield close-planted varieties.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a diagram showing the construction of a plant expression vector pCAMBIA 1300-UBI;
FIG. 2 is a map of the tiller angle phenotype of transgenic rice overexpressing LAZY1 gene;
FIG. 3 is a chart showing the tillering angle phenotype of transgenic rice overexpressing WOX11 gene.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The material used in the application is Mongolian hybrid agro-grass tillering section, which is taken from the greenhouse of the university of inner Mongolia agriculture in 12 months of 2019.
Example 1: cloning of full-length ORF of WOX11 Gene
Based on Meng Nong hybrid agropyron cristatum tillering node transcriptome sequencing results, a specific primer was designed to amplify the full-length Open Reading Frame (ORF) sequence of the WOX11 gene.
(1) ORF sequence amplification: based on Meng Nong hybrid agropyron cristatum tiller transcriptome sequencing results, designing a specific primer to amplify an Open Reading Frame (ORF) full-length sequence of the WOX11 gene, wherein the ORF specific primer comprises: WOX11 forward primer: 5'-TAGCTCGTCTGCTAGCTCG-3' (SEQ. NO. 3) and WOX11 reverse primer: 5'-CATTAATTTGACCTCGCG-3' (SEQ. RTM. NO. 4). The Trizol method is adopted to extract the RNA of the tillering node of Mongolian hybrid agropyron, and the first strand of cDNA is synthesized by REVERTAID FIRST STRAND CDNA SYNTHESIS KIT of Fermentas company. The ORF sequence of WOX11 gene was amplified using KOD FX Neo (1U/ul) enzyme, PCR amplification procedure: 98 ℃ for 5min;98 ℃ for 10s; 30s at 55 ℃;72℃for 2min (35 cycles); and at 72℃for 10min. The PCR products are subjected to electrophoresis separation by using 1% agarose gel, a gel imaging system is used for photographing and gel cutting, a DNA agarose gel recovery kit is used for recovering amplified fragments, then fresh recovered products are subjected to T4 cloning vector connection and transformation of competent cells of escherichia coli, and finally positive bacterial liquid detected by bacterial liquid PCR is sent to a biological engineering company for sequencing.
(2) According to the sequencing peak diagram, sequence splicing is carried out, after the carrier sequence is removed, alignment is carried out on NCBI, and the final result is further determined, wherein the final result is shown as SEQ_NO. 1.
Example 2: construction and genetic transformation of WOX11 gene plant expression vector
And constructing an over-expression vector of the WOX11 gene. PCR amplification was performed using specific PCR primers and cDNA as a template, and the WOX11 gene open reading frame sequence was constructed into the vector pCAMBIA1300-UBI. Positive clones are selected from a screening culture plate for PCR detection and sequencing verification, and after the PCR detection and the sequencing verification, the construction success of the excessive expression vector is confirmed, the excessive expression vector is named as pCAMBIA1300-UBI (figure 1), the gene is positioned behind a promoter UBI, and the pCAMBIA1300 can be efficiently expressed in a receptor plant under the drive of the promoter UBI.
The specific method for constructing the vector comprises the following steps:
① Amplification of the Gene fragment of interest
Design of specific primers based on the target Gene sequence
E.coli DH5 alpha (pGEM-T-easy-target gene) is used as a template, and the following PCR reaction system is established to amplify the pre-expressed target gene fragment;
Pre mixtaq 25uL
1uL of upstream primer
Downstream primer 1uL
PGEM-T-easy-target gene 1uL
Water 22uL
Total volume of 50uL
The PCR reaction conditions are as follows: 94 ℃ 5 min;94℃for 30 s,58℃for 30 s,72℃for 60 s, for a total of 35 cycles; the extension is 10min at 72 ℃.
The PCR product was electrophoresed on a 1% agarose gel, the target fragment was recovered using E.N.Z.A DNA gel recovery kit, and double cleavage was performed with KpnI/BamHI, the reaction system was as follows:
10×Buffer 5uL
PCR product 35uL
Water 5uL
KpnI 2.5uL
BamHI 2.5uL
Total volume of 50uL
After incubation at 37℃for 3 h%, 1% agarose gel electrophoresis was performed, and the digested fragment of interest was recovered using a DNA gel recovery kit.
② Enzyme digestion preparation of over-expression vector
Picking a DH5a (pCAMBIA 1300-UBI) freshly cultured colony, inoculating the colony into 10 mL LB liquid culture medium (containing 50 mug/mL of kanamin), and carrying out shaking culture at 37 ℃ for 12 h; the plasmid pCAMBIA1300-UBI was extracted with E.Z.N.A.miniprep kit; double cleavage with KpnI/BamHI was performed and the reaction system was as shown in tables 4-3: after incubation at 37℃for 3h, the samples were subjected to 1% agarose gel electrophoresis, 100V electrophoresis, 1 h. The digested plasmid was recovered using the E.Z.N.A.DNA gel recovery kit.
10×Buffer 5uL
PCAMBIA1300-UBI vector 35uL
Water 5uL
KpnI 2.5uL
BamHI 2.5uL
Total volume of 50uL
③ Ligation of vector after cleavage with target Gene after cleavage
The following ligation reactions were established and ligation was performed overnight at 16 ℃.
10 XDNA ligation buffer 1uL
PCAMBIA1300-UBI vector 2uL after cleavage
6UL of target gene fragment after enzyme digestion
T4 DNA ligase 1uL
Total volume of 10uL
④ Transformation of ligation products into E.coli DH 5. Alpha. By CaCl2 method
⑤ PCR identification of transformed clones
Some colonies were picked and inoculated into 10 mL LB medium (containing 50. Mu.g/mL of carbapenem) and cultured overnight at 37℃with shaking, and the cultures were subjected to the following PCR reaction to screen positive clones.
Pre mixtaq 25uL
1UL of upstream primer
Downstream primer 1uL
Template 1uL
Water 22uL
Total volume of 50uL
PCR procedure: the procedure was followed as ①.
10. Mu.L of the PCR reaction product was subjected to 1% agarose gel electrophoresis, and the result was observed under an ultraviolet detector.
Cultures of clones identified as positive by the PCR reaction were sent to sequencing company for sequencing of the recombinant plasmid, and the recombinant plasmid was analyzed for changes in the reading frame of the foreign gene. Sequencing primer is
UBI (forward universal primer) 5'-ACACCCTCTTTCCCCAAC-3' as shown in SEQ ID No. 5.
E9 TER (reverse Universal primer) 5'-GCAATGAAACTGATGCATTG-3' as shown in SEQ ID No. 6.
The constructed pCAMBIA1300-UBI over-expression vector is transferred into an agrobacterium EHA105 strain by a liquid nitrogen freeze thawing method, and genes are transferred into rice by an agrobacterium-mediated genetic transformation method. Transgenic rice plants overexpressing WOX11 gene exhibited a phenomenon in which tillering angle became large (fig. 3). The WOX11 gene is a key regulator of the rice tillering angle, and has important application value in the research of the gramineous plant genetic engineering.
Example 3: cloning of full-Length ORF of LAZY1 Gene
Based on Meng Nong hybrid agropyron cristatum tillering node transcriptome sequencing results, a specific primer was designed to amplify the full-length Open Reading Frame (ORF) sequence of the LAZY1 gene.
(1) ORF sequence amplification: based on Meng Nong hybrid agropyron cristatum tiller transcriptome sequencing results, designing a specific primer to amplify an Open Reading Frame (ORF) full-length sequence of the LAZY1 gene, wherein the ORF specific primer comprises: LAZY1 forward primer: 5'-GCCTCTTCAAGATGACGC-3' (SEQ. NO. 7) and LAZY1 reverse primer: 5'-CTTCCAGTTCATCCGTAC-3' (SEQ. RTM. NO. 8). The Trizol method is adopted to extract the RNA of the tillering node of Mongolian hybrid agropyron, and the first strand of cDNA is synthesized by REVERTAID FIRST STRAND CDNA SYNTHESIS KIT of Fermentas company. ORF sequence of LAZY1 gene was amplified using KOD FX Neo (1U/ul) enzyme, PCR amplification procedure: 98 ℃ for 5min;98 ℃ for 10s; 30s at 55 ℃;72℃for 2min (35 cycles); and at 72℃for 10min. The PCR products are subjected to electrophoresis separation by using 1% agarose gel, a gel imaging system is used for photographing and gel cutting, a DNA agarose gel recovery kit is used for recovering amplified fragments, then fresh recovered products are subjected to T4 cloning vector connection and transformation of competent cells of escherichia coli, and finally positive bacterial liquid detected by bacterial liquid PCR is sent to a biological engineering company for sequencing.
(2) According to the sequencing peak diagram, sequence splicing is carried out, after the carrier sequence is removed, alignment is carried out on NCBI, and the final result is further determined, wherein the final result is shown as SEQ_NO. 2.
Example 4: construction and genetic transformation of LAZY1 Gene plant expression vector
And constructing an over-expression vector of the LAZY1 gene. PCR amplification was performed using specific PCR primers and cDNA as a template, and the LAZY1 gene open reading frame sequence was constructed into the vector pCAMBIA1300-UBI. Positive clones are selected from a screening culture plate for PCR detection and sequencing verification, and the PCR detection and sequencing verification prove that the construction of the excessive expression vector is successful, and the excessive expression vector is named as pCAMBIA1300-UBI (figure 1), and the gene is positioned behind a promoter UBI and can be efficiently expressed in a receptor plant under the drive of the promoter UBI.
The specific method for constructing the vector comprises the following steps:
① Amplification of the Gene fragment of interest
Design of specific primers based on the target Gene sequence
E.coli DH5 alpha (pGEM-T-easy-target gene) is used as a template, and the following PCR reaction system is established to amplify the pre-expressed target gene fragment;
Pre mixtaq 25uL
1uL of upstream primer
Downstream primer 1uL
PGEM-T-easy-target gene 1uL
Water 22uL
Total volume of 50uL
The PCR reaction conditions are as follows: 94 ℃ 5 min;94℃for 30 s,58℃for 30 s,72℃for 60 s, for a total of 35 cycles; the extension is 10min at 72 ℃.
The PCR product was electrophoresed on a 1% agarose gel, the target fragment was recovered using E.N.Z.A DNA gel recovery kit, and double cleavage was performed with KpnI/BamHI, the reaction system was as follows:
10×Buffer 5uL
PCR product 35uL
Water 5uL
KpnI 2.5uL
BamHI 2.5uL
Total volume of 50uL
After incubation at 37℃for 3 h%, 1% agarose gel electrophoresis was performed, and the digested fragment of interest was recovered using a DNA gel recovery kit.
② Enzyme digestion preparation of over-expression vector
Picking a DH5a (pCAMBIA 1300-UBI) freshly cultured colony, inoculating the colony into 10 mL LB liquid culture medium (containing 50 mug/mL of kanamin), and carrying out shaking culture at 37 ℃ for 12 h; the plasmid pCAMBIA1300-UBI was extracted with E.Z.N.A.miniprep kit; double cleavage with KpnI/BamHI was performed and the reaction system was as shown in tables 4-3: after incubation at 37℃for 3h, the samples were subjected to 1% agarose gel electrophoresis, 100V electrophoresis, 1 h. The digested plasmid was recovered using the E.Z.N.A.DNA gel recovery kit.
10×Buffer 5uL
PCAMBIA1300-UBI vector 35uL
Water 5uL
KpnI 2.5uL
BamHI 2.5uL
Total volume of 50uL
③ Ligation of vector after cleavage with target Gene after cleavage
The following ligation reactions were established and ligation was performed overnight at 16 ℃.
10 XDNA ligation buffer 1uL
PCAMBIA1300-UBI vector 2uL after cleavage
6UL of target gene fragment after enzyme digestion
T4 DNA ligase 1uL
Total volume of 10uL
④ Transformation of ligation products into E.coli DH 5. Alpha. By CaCl2 method
⑤ PCR identification of transformed clones
Some colonies were picked and inoculated into 10 mL LB medium (containing 50. Mu.g/mL of carbapenem) and cultured overnight at 37℃with shaking, and the cultures were subjected to the following PCR reaction to screen positive clones.
Pre mixtaq 25uL
1UL of upstream primer
Downstream primer 1uL
Template 1uL
Water 22uL
Total volume of 50uL
PCR procedure: the procedure was followed as ①.
10. Mu.L of the PCR reaction product was subjected to 1% agarose gel electrophoresis, and the result was observed under an ultraviolet detector.
Cultures of clones identified as positive by the PCR reaction were sent to sequencing company for sequencing of the recombinant plasmid, and the recombinant plasmid was analyzed for changes in the reading frame of the foreign gene. Sequencing primer is
UBI (forward universal primer): 5'-ACACCCTCTTTCCCCAAC-3'
E9 TER (reverse Universal primer): 5'-GCAATGAAACTGATGCATTG-3'
The constructed pCAMBIA1300-UBI over-expression vector is transferred into an agrobacterium EHA105 strain by a liquid nitrogen freeze thawing method, and genes are transferred into rice by an agrobacterium-mediated genetic transformation method. Transgenic rice plants overexpressing the LAZY1 gene exhibited a phenomenon in which the tillering angle became large (fig. 2). The LAZY1 gene is a key regulator of the rice tillering angle, and has important application value in the research of the gramineous plant genetic engineering.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing describes the embodiments of the present invention, it should be understood that the present invention is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.
Claims (2)
1. The application of the Mongolian hybrid agronomic crop tillering angle regulating factor in plant breeding is characterized in that the Meng Nong hybrid agronomic crop tillering angle regulating factor is a WOX11 gene or a LAZY1 gene, and the nucleotide sequences of the Mongolian hybrid agronomic crop tillering angle regulating factor are shown as SEQ_NO.1 and SEQ_NO. 2;
The plant is rice;
The application is specifically to control the tillering angle of plants.
2. The use according to claim 1, characterized by the steps of:
1) Constructing Meng Nong plant overexpression vectors of hybrid agrocybe aegerita tillering angle regulating factors;
2) Transforming the plant over-expression vector of the Meng Nong hybrid agrocybe aegerita tillering angle regulating factor into a plant or a plant cell;
3) And cultivating and screening to obtain transgenic plants with different tillering angles.
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| CN115851755B true CN115851755B (en) | 2024-05-24 |
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| CN202210809312.1A Active CN115851755B (en) | 2022-07-11 | 2022-07-11 | Mongolian hybrid agro-grass tillering angle regulating factor and application thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796760A (en) * | 2012-08-10 | 2012-11-28 | 福建农林大学 | Application of zinc finger protein gene in adjusting paddy rice root growth |
| CN104177481A (en) * | 2013-05-21 | 2014-12-03 | 中国农业大学 | Plant lodging and flower organ development regulation protein ZmLA1, and coding gene and application thereof |
| CN104450711A (en) * | 2014-12-31 | 2015-03-25 | 湖南农业大学 | Application of OsmiR156f gene in rice effective tillering increasing |
| CN106676128A (en) * | 2015-11-09 | 2017-05-17 | 中国科学院上海生命科学研究院 | Paddy rice OsWOX11 protein and application of paddy rice OsWOX11 protein for coding gene |
| CN108085319A (en) * | 2017-12-25 | 2018-05-29 | 中国科学院遗传与发育生物学研究所 | Plant tillering angle GAP-associated protein GAP and its encoding gene and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101144624B1 (en) * | 2010-01-22 | 2012-05-11 | 경상대학교산학협력단 | OsMPT gene modifying plant architecture and uses theoreof |
| US20170088846A1 (en) * | 2015-09-25 | 2017-03-30 | The United States Of America, As Represented By The Secretary Of Agriculture | Functional Analysis of LAZY1 in Arabidopsis thaliana and Prunus Trees |
-
2022
- 2022-07-11 CN CN202210809312.1A patent/CN115851755B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796760A (en) * | 2012-08-10 | 2012-11-28 | 福建农林大学 | Application of zinc finger protein gene in adjusting paddy rice root growth |
| CN104177481A (en) * | 2013-05-21 | 2014-12-03 | 中国农业大学 | Plant lodging and flower organ development regulation protein ZmLA1, and coding gene and application thereof |
| CN104450711A (en) * | 2014-12-31 | 2015-03-25 | 湖南农业大学 | Application of OsmiR156f gene in rice effective tillering increasing |
| CN106676128A (en) * | 2015-11-09 | 2017-05-17 | 中国科学院上海生命科学研究院 | Paddy rice OsWOX11 protein and application of paddy rice OsWOX11 protein for coding gene |
| CN108085319A (en) * | 2017-12-25 | 2018-05-29 | 中国科学院遗传与发育生物学研究所 | Plant tillering angle GAP-associated protein GAP and its encoding gene and application |
Non-Patent Citations (8)
| Title |
|---|
| A Core Regulatory Pathway Controlling Rice Tiller Angle Mediated by the LAZY1-Dependent Asymmetric Distribution of Auxin;Ning Zhang等;The Plant Cell;20180618;第30卷(第7期);摘要，第1466页左栏最后1段-右栏第3段 * |
| LAZY1 controls rice shoot gravitropism through regulating polar auxin transport;Peijin Li等;Cell Research;20070430;第17卷(第5期);摘要，第404页右栏第2段 * |
| NCBI.PREDICTED: Triticum dicoccoides WUSCHEL-related homeobox 11-like (LOC119358936), mRNA.GenBank Database.2020,Accession No.XM_037625316.1. * |
| NCBI.PREDICTED:Triticum urartu protein LAZY 1 (LOC125515610), mRNA.GenBank Database.2022,Accession No.XM_048681112.1. * |
| PREDICTED: Triticum dicoccoides WUSCHEL-related homeobox 11-like (LOC119358936), mRNA;NCBI;GenBank Database;20201113;Accession No.XM_037625316.1 * |
| PREDICTED:Triticum urartu protein LAZY 1 (LOC125515610), mRNA;NCBI;GenBank Database;20220616;Accession No.XM_048681112.1 * |
| 中国水稻功能基因组研究进展;肖景华;吴昌银;韩斌;薛勇彪;邓兴旺;张启发;;中国科学(C辑:生命科学);20091015(10);第909-924页 * |
| 基因枪法获得转CBF4基因蒙农杂种冰草的研究;徐春波;王勇;赵来喜;赵海霞;米福贵;;中国草地学报;20130925(05);第24-28页 * |
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