CN110592114A - Application of oryza sativa auxin glycosyl transferase gene - Google Patents
Application of oryza sativa auxin glycosyl transferase gene Download PDFInfo
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- CN110592114A CN110592114A CN201910954271.3A CN201910954271A CN110592114A CN 110592114 A CN110592114 A CN 110592114A CN 201910954271 A CN201910954271 A CN 201910954271A CN 110592114 A CN110592114 A CN 110592114A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8267—Seed dormancy, germination or sprouting
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/1048—Glycosyltransferases (2.4)
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Abstract
The invention discloses an application of an auxin glycosyl transferase gene. The nucleotide sequence of the gene OsIAGLU is shown as SEQ ID NO.1, and the amino acid sequence of the coded corresponding protein is shown as the sequence table SEQ ID NO. 2. The OsIAGLU gene is reported to regulate the activity of rice seeds for the first time in rice, and experiments show that the mutation of the gene influences the seed germination and seedling establishment capability under normal conditions. The OsIAGLU gene of the invention is proved to regulate and control the rice seed vigor, and the gene is beneficial to screening and cultivating rice varieties with high seed vigor and is beneficial to direct seeding rice production.
Description
Technical Field
The invention belongs to the technical field of seed biology, and relates to an application of a rice auxin glycosyl transferase gene.
Background
Rice (Oryza sativa L.) is one of the longest food crops in the cultivation history in China. In recent years, with the development of economy, rural labor is increasingly in short supply, and direct-seeded rice production becomes more and more common. The direct seeding rice production has high requirements on seed sowing quality, and poor seed quality can cause slow seed germination speed, low field seedling rate, uneven seedling growth vigor and even seriously affect crop yield. Auxin is an important factor for regulating and controlling the growth and development of plants, few reports are made on the research on regulating and controlling the germination of rice seeds and the growth of seedlings by using auxin glycosyltransferase genes, and no report is made on the application of screening and cultivating high-activity rice varieties by using the auxin glycosyltransferase genes. Therefore, the auxin glycosyl transferase gene OsIAGLU participating in the regulation and control of rice seed germination and seedling growth is utilized to provide help for screening and cultivating high-activity rice varieties, and has important significance for direct-seeded rice production.
Disclosure of Invention
The invention aims to provide separation, cloning, functional verification and application of an auxin glycosyltransferase gene OsIAGLU for controlling rice seed germination and seedling growth.
The purpose of the invention can be realized by the following technical scheme:
a gene OsIAGLU from the oryza sativa auxin glycosyltransferase, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The amino acid sequence of the protein coded by the oryza sativa auxin glycosyltransferase gene OsIAGLU is shown in SEQ ID NO. 2.
The rice OsIAGLU gene or protein disclosed by the invention is applied to screening or breeding of rice varieties with high seed vigor.
The rice OsIAGLU gene mutant obtaining and gene function verification method comprises the following steps:
(1) obtaining the nucleotide sequence and the amino acid sequence of the OsIAGLU gene of the rice;
(2) designing a target site and a primer thereof, carrying out PCR amplification by taking pCBC-MT1T2 as a template, purifying and recovering a PCR product, and obtaining an MT1T2-PCR vector;
(3) constructing the MT1T2-PCR glue recovery product with the target fragment of the OsIAGLU gene obtained in the step (2) onto a pHUE411 vector to obtain a pHUE411+ MT1T2-PCR vector;
(4) transforming the plasmid with the OsIAGLU gene target fragment obtained in the step (3) into agrobacterium; transforming the agrobacterium with the transformation plasmid into rice;
(5) screening and identifying rice mutants, and identifying seed vitality under normal conditions.
Further, in the step (1), a primer sequence of the gene is cloned by using rice cDNA as a template through PCR, wherein an upstream primer sequence of the PCR is shown as SEQ ID NO.3, and a downstream primer sequence is shown as SEQ ID NO. 4.
Further, the rice OsIAGLUCRISPR/Cas9 mutant gRNA target sequence (target fragment of 19bp in OsIAGLU gene) constructed in the step (2) is shown as a sequence table SEQ ID NO.5 and SEQ ID NO. 6; the primer sequence for PCR amplification by taking pCBC-MT1T2 as a template is shown in a sequence table SEQ ID NO.7/SEQ ID NO.8 and SEQ ID NO.9/SEQ ID NO. 10;
in step (3), the pHUE411 vector is digested with BsaI, and the pHUE411+ MT1T2-PCR vector is obtained by the homologous recombination method.
In the step (5), the upstream primer sequence used for screening the homozygous mutant is shown as SEQ ID NO.11, and the downstream primer sequence is shown as SEQ ID NO. 12. The rice seed vitality identification comprises seed germination and seedling growth under normal conditions.
The method for detecting the OsIAGLU gene expression level in the seed germination stage is characterized by comprising the following steps of:
(1) taking rice seeds in different germination stages;
(2) RNA was extracted from each sample using a TransZol Plant kit (Transgen, www.transgen.com);
(3) by usingII Reverse transcription of cDNA formed by Vazyme Biotech Co., Ltd kit, using it as template;
(4) and (3) analyzing by using fluorescent quantitative PCR, and detecting a primer sequence by using the fluorescent quantitative PCR, wherein the upstream primer sequence is shown as SEQ ID NO.13 of the sequence table, and the downstream primer sequence is shown as SEQ ID NO.14 of the sequence table.
Further, in the step (1), the rice seeds are cultured in a 10mL distilled water culture dish at 25 ℃, and are sampled after 0, 4, 12, 24, 36, 48, 60 and 72 hours of imbibition; the seeds were rapidly ground into powder after freezing treatment with liquid nitrogen and the samples were stored at-80 ℃ and sampled in triplicate at each time point.
In the step (4), a rice reference gene OsActin primer is adopted, the sequence of the upstream primer is shown as a sequence table SEQ ID NO.15, and the sequence of the downstream primer is shown as a sequence table SEQ ID NO. 16.
Has the advantages that: the OsIAGLU gene is separated and cloned from rice, and the function of the gene in the aspect of seed vigor regulation is identified, so that the OsIAGLU gene has important significance for screening or breeding high-vigor rice varieties.
The invention has the following advantages:
(1) the OsIAGLU gene is obtained by separating and cloning from rice, and the gene is proved to participate in the regulation and control of rice seed vigor for the first time by constructing a CRISPR/Cas9 mutant.
(2) The invention provides a foundation for screening rice varieties with high seed vigor, provides important gene resources for improving the rice seed vigor, and has important significance for direct seeding rice production.
Drawings
FIG. 1: seed vigor expression of rice OsIAGLU mutant under normal conditions
FIG. 2: expression condition of rice OsIAGLU gene in seed germination process
Detailed Description
The invention is further explained by combining the attached drawings and specific embodiments, the methods used in the embodiments are all conventional methods without special description, and the primers and sequencing are completed by Guangzhou Tianyihui Gene technology, Inc.; various restriction enzymes, ligase, DNA Marker, Tag DNA polymerase, dNTPs and the like used in the experiment are purchased from Guangzhou Shuicheng Biotech limited; the reverse transcription kit is purchased from Novozan Biotechnology Co., Ltd; the plasmid extraction kit, the gel recovery kit and the genome extraction kit are purchased from Meiji Biotechnology Ltd, and the methods are carried out according to the specifications.
Example 1: cloning of genes
The sequence of the OsIAGLU gene is cloned by PCR by using a Nipponbare cDNA of a japonica rice variety as a template, wherein the sequence of an upstream primer of the PCR is shown as SEQ ID NO.3, and the sequence of a downstream primer is shown as SEQ ID NO. 4. Obtaining the nucleotide sequence and the amino acid sequence of the OsIAGLU gene of the rice, wherein the nucleotide sequence is shown as a sequence table SEQ ID NO.1, and the amino acid sequence is shown as an SEQ ID NO. 2.
Example 2: mutant construction
Logging into a websitehttp://www.genome.arizona.edu/crispr/CRISPRsearch.htmlAnd screening target spots. The target sequences are shown as SEQ ID NO.5 and SEQ ID NO.6, and the primers are designed according to the target sequences and are shown as SEQ ID NO.7/SEQ ID NO.8 and SEQ ID NO.9/SEQ ID NO. 10. And (3) carrying out four-primer PCR amplification by taking pCBC-MT1T2 as a template, purifying and recovering a PCR product to obtain an MT1T2-PCR vector. The pHUE411 vector was digested with BsaI, and the pHUE411+ MT1T2-PCR vector was obtained by homologous recombination.
The obtained pHUE411+ MT1T2-PCR vector is used for transforming agrobacterium; agrobacterium with transformation plasmid is transformed into japonica rice of wild type Nipponbare; sequencing the PCR amplification product, comparing with wild type, screening homozygous mutant, the upstream primer is shown as SEQ ID NO.11, and the downstream primer is shown as SEQ ID NO. 12.
Example 3: phenotypic analysis of Gene mutants
Seed germination tests were performed using the OsIAGLU CRISPR/Cas9 mutants, osiglu-1, osiglu-2, and osiglu-3 seeds, which were successfully constructed in example 2, and wild-type Nipponbare (WT) rice varieties. The specific method comprises the following steps: selecting 50 healthy and plump seeds repeatedly each time, sterilizing the surfaces of the seeds with 0.1% mercuric chloride solution for 5min, washing the seeds with distilled water for 3 times, wiping the surfaces of the seeds dry, placing the seeds in a culture dish (with the diameter of 9cm) padded with two layers of filter paper, adding 10mL of distilled water, placing the seeds in a 15 ℃ condition, culturing the seeds for 12h in light/dark for 7d respectively, and finally counting the seedling rate. The experiment was repeated 3 times. The results show that the germination speed of the mutant seeds is slower and the seedling growth is significantly weaker compared to the control seeds (fig. 1). Therefore, the gene plays an important role in improving the seed germination speed and seedling growth.
Example 4: analysis of Gene expression during seed Germination
Using Ningpo, repeatedly selecting 50 healthy and plump seeds each time, sterilizing the surface of the seeds for 5min by using 0.1% mercuric chloride solution, washing the seeds for 3 times by using distilled water, wiping the surfaces of the seeds dry, placing the seeds into a culture dish (the diameter is 9cm) padded with two layers of filter paper, adding 10mL of distilled water, placing the seeds in a dark incubator at 25 ℃ for imbibition for 0, 4, 12, 24, 36, 48, 60 and 72 hours respectively, and then sampling respectively. The seeds were frozen in liquid nitrogen and then rapidly ground into powder and the samples stored at-80 ℃. The experiment was repeated 3 times.
RNA was extracted from each sample using a TransZol Plant kit (Transgen, www.transgen.com); by usingII Reverse transcription of cDNA formed by Vazyme Biotech Co., Ltd kit, using it as template; and (3) analyzing by using fluorescent quantitative PCR (polymerase chain reaction), and detecting a primer sequence of OsIAGLU by using the fluorescent quantitative PCR, wherein the upstream primer sequence is shown as SEQ ID No.13, and the downstream primer sequence is shown as SEQ ID No. 14. A rice internal reference gene OsActin primer is adopted, the sequence of the upstream primer is shown as SEQ ID NO.15, and the sequence of the downstream primer is shown as SEQ ID NO. 16. The results show that the expression level of the OsIAGLU gene has a trend of increasing, then decreasing and then increasing in the seed germination process (figure 2). OsIAGLU gene expression is highest when seeds are imbibed for 48 hours. Therefore, the gene is induced and expressed in the seed germination process, and the gene expression plays an important role in seed germination.
Sequence listing
<110> southern China university of agriculture
<120> application of oryza sativa auxin glycosyltransferase gene
<160> 16
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Rice (Oryza sativa)
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atgcatttct tgatcgtgtc gggggcggcg caggggcaga tcacgccggc gaggcggctg 60
gcgcgcgcgc tggtggcggc ggcggagcct ggggtgatca tcagggccac gctggccgtg 120
ccgctgtcgg cgctgaggag gatgttcccc gggaaggcgg ccggcgcagc cgccggtgaa 180
ggggccgtgg tgctgtcgga cggggccggc gtcgactacg ccgcgttcac cgacgggttc 240
gacgacgggt tccagcccga gcggtgcgac ggcgcggcgt tcgtcgggag gctccagctc 300
gtcggcccgg cgtcgctggc ccggctggcg gcggcgctgc gcgcgcgggg gaggcccgtg 360
acgtgcgtcg tgtacaccct gctccttccg ttcgccgccg ccgtcgccag ggacctcgac 420
gtgccggcgt acttcttctg gaccatgccg gcggccgtgc tttcagtcta ctaccattac 480
ttccacggcc gccatggcct cgtcgacgcc gccgccggag tccgggacga ccccaaccgc 540
cgcgtccaag tccccggcct cgagttcctc cgcgcccgcg acctcccgtc gctgctcacc 600
gggtcaagcc cctacctccc ggccttccgg gagatgttcc acgtcgtcga ggccaccgcc 660
gccgcgtcgt gccatgccca tggccagagt ggcgcgaagc cgtgggtgct tgtgaacacc 720
ttcgacgcgc tcgagccgaa ggcgctcgcg tccgtccccg gcattgacct catcccggtt 780
ggacccatgg tcaccgacac ggaggccgac ggcggcggcg acctcttcga gcaagacgac 840
gacgcaggct acatgcaatg gctcgacaag cagcgggacg cctccgtcgt gtacgtcgcg 900
ttcggcagcc tcgccgtgct ctcgccgagg cagctggagg agatacgcca ctgcctcgag 960
gtgaccggac ggcccttcct ctgggtggtc cggcgcgaca gccgcgacgg cggcggcggc 1020
ggcggcgcgg ccaccggatt attgccgccg gcaggcggga tggtggtgga gtggtgcagc 1080
caggcgcgcg tgctggcgca ccgggcggtg gggtgcttcg tcacccactg cgggtggaac 1140
tcgacgctgg agaccgtcgc gtgcggcgtg ccagcggtaa tggcgccgca gtggtccgac 1200
caggcgacga acgcgcgcat ggccgaggcg cggtggggcg tcggcgtgcg cgcggagacc 1260
gcggccgacg gcaccgtgct ctcgtcggag ctctcacgcg gcatcgacgc cgtcatgggg 1320
gacagcgacg gcgcccgcgc gatacgccgg cgcgcaagaa catggaaggc gcgcgccgcc 1380
atggcgctgg acgccgccgc cgacgacgcc gagtttgacg gggacgccac ggcggcgcgc 1440
aacctgagac gatttgtgca gggcgtacgt agcagagaac gggagcgcga gcaaaagcaa 1500
gcagggcaga gttaa 1515
<210> 2
<211> 504
<212> PRT
<213> Rice (Oryza sativa)
<400> 2
Met His Phe Leu Ile Val Ser Gly Ala Ala Gln Gly Gln Ile Thr Pro
1 5 10 15
Ala Arg Arg Leu Ala Arg Ala Leu Val Ala Ala Ala Glu Pro Gly Val
20 25 30
Ile Ile Arg Ala Thr Leu Ala Val Pro Leu Ser Ala Leu Arg Arg Met
35 40 45
Phe Pro Gly Lys Ala Ala Gly Ala Ala Ala Gly Glu Gly Ala Val Val
50 55 60
Leu Ser Asp Gly Ala Gly Val Asp Tyr Ala Ala Phe Thr Asp Gly Phe
65 70 75 80
Asp Asp Gly Phe Gln Pro Glu Arg Cys Asp Gly Ala Ala Phe Val Gly
85 90 95
Arg Leu Gln Leu Val Gly Pro Ala Ser Leu Ala Arg Leu Ala Ala Ala
100 105 110
Leu Arg Ala Arg Gly Arg Pro Val Thr Cys Val Val Tyr Thr Leu Leu
115 120 125
Leu Pro Phe Ala Ala Ala Val Ala Arg Asp Leu Asp Val Pro Ala Tyr
130 135 140
Phe Phe Trp Thr Met Pro Ala Ala Val Leu Ser Val Tyr Tyr His Tyr
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Phe His Gly Arg His Gly Leu Val Asp Ala Ala Ala Gly Val Arg Asp
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Asp Pro Asn Arg Arg Val Gln Val Pro Gly Leu Glu Phe Leu Arg Ala
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Arg Asp Leu Pro Ser Leu Leu Thr Gly Ser Ser Pro Tyr Leu Pro Ala
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Phe Arg Glu Met Phe His Val Val Glu Ala Thr Ala Ala Ala Ser Cys
210 215 220
His Ala His Gly Gln Ser Gly Ala Lys Pro Trp Val Leu Val Asn Thr
225 230 235 240
Phe Asp Ala Leu Glu Pro Lys Ala Leu Ala Ser Val Pro Gly Ile Asp
245 250 255
Leu Ile Pro Val Gly Pro Met Val Thr Asp Thr Glu Ala Asp Gly Gly
260 265 270
Gly Asp Leu Phe Glu Gln Asp Asp Asp Ala Gly Tyr Met Gln Trp Leu
275 280 285
Asp Lys Gln Arg Asp Ala Ser Val Val Tyr Val Ala Phe Gly Ser Leu
290 295 300
Ala Val Leu Ser Pro Arg Gln Leu Glu Glu Ile Arg His Cys Leu Glu
305 310 315 320
Val Thr Gly Arg Pro Phe Leu Trp Val Val Arg Arg Asp Ser Arg Asp
325 330 335
Gly Gly Gly Gly Gly Gly Ala Ala Thr Gly Leu Leu Pro Pro Ala Gly
340 345 350
Gly Met Val Val Glu Trp Cys Ser Gln Ala Arg Val Leu Ala His Arg
355 360 365
Ala Val Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser Thr Leu Glu
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Thr Val Ala Cys Gly Val Pro Ala Val Met Ala Pro Gln Trp Ser Asp
385 390 395 400
Gln Ala Thr Asn Ala Arg Met Ala Glu Ala Arg Trp Gly Val Gly Val
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Arg Ala Glu Thr Ala Ala Asp Gly Thr Val Leu Ser Ser Glu Leu Ser
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Arg Gly Ile Asp Ala Val Met Gly Asp Ser Asp Gly Ala Arg Ala Ile
435 440 445
Arg Arg Arg Ala Arg Thr Trp Lys Ala Arg Ala Ala Met Ala Leu Asp
450 455 460
Ala Ala Ala Asp Asp Ala Glu Phe Asp Gly Asp Ala Thr Ala Ala Arg
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Asn Leu Arg Arg Phe Val Gln Gly Val Arg Ser Arg Glu Arg Glu Arg
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Glu Gln Lys Gln Ala Gly Gln Ser
500
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgcatttct tgatcgtgtc gg 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttaactctgc cctgcttgct tt 22
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gccgccttcc cggggaaca 19
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gatgatcacc ccaggctcc 19
<210> 7
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aataatggtc tcaggcggcc gccttcccgg ggaaca 36
<210> 8
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggccgccttc ccggggaaca gttttagagc tagaaatagc 40
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gatgatcacc ccaggctccc gcttcttggt gcc 33
<210> 10
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
attattggtc tctaaacgat gatcacccca ggctcc 36
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<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cggcgcaggg gcagatcac 19
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gcaccgctcg ggctggaac 19
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<213> Artificial Sequence (Artificial Sequence)
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gcgtacttct tctggaccat gc 22
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gccgtggaag taatggtagt agac 24
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aggaaggctg gaagaggacc 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cgggaaattg tgagggacat 20
Claims (2)
1. Application of oryza sativa auxin glycosyltransferase gene OsIAGLU in screening or cultivating paddy rice varieties with high seed vigor.
2. The use of the oryza sativa auxin glycosyltransferase gene OsIAGLU of claim 1 in genetic engineering for improving germination viability of rice seeds and promoting growth of seedlings.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112501188A (en) * | 2020-12-02 | 2021-03-16 | 华南农业大学 | Application of oryza sativa auxin glycosyl transferase gene in cultivation of flooding-resistant rice variety |
CN113862279A (en) * | 2021-08-25 | 2021-12-31 | 上海师范大学 | Gene OsACO for inhibiting growth of rice seedlings and application thereof |
CN114891811A (en) * | 2022-05-12 | 2022-08-12 | 华南农业大学 | Application of rice phosphoinositide kinase gene |
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CN108715903A (en) * | 2018-05-03 | 2018-10-30 | 南京农业大学 | The application of rice α-isopropylmalate synthase gene |
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CN108715903A (en) * | 2018-05-03 | 2018-10-30 | 南京农业大学 | The application of rice α-isopropylmalate synthase gene |
Non-Patent Citations (1)
Title |
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XIAO‑LU YU ET AL.: "Overexpression of OsIAAGLU reveals a role for IAA–glucose", 《PLANT CELL REPORTS》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112501188A (en) * | 2020-12-02 | 2021-03-16 | 华南农业大学 | Application of oryza sativa auxin glycosyl transferase gene in cultivation of flooding-resistant rice variety |
CN113862279A (en) * | 2021-08-25 | 2021-12-31 | 上海师范大学 | Gene OsACO for inhibiting growth of rice seedlings and application thereof |
CN113862279B (en) * | 2021-08-25 | 2024-03-12 | 上海师范大学 | Gene OsACO for inhibiting growth of rice seedlings and application thereof |
CN114891811A (en) * | 2022-05-12 | 2022-08-12 | 华南农业大学 | Application of rice phosphoinositide kinase gene |
CN114891811B (en) * | 2022-05-12 | 2023-05-26 | 华南农业大学 | Application of rice inositol triphosphate kinase gene |
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