[go: up one dir, main page]

CN113322266B - Application of rice OsRHD1-1 gene in rice male sterile line cultivation - Google Patents

Application of rice OsRHD1-1 gene in rice male sterile line cultivation Download PDF

Info

Publication number
CN113322266B
CN113322266B CN202110720468.8A CN202110720468A CN113322266B CN 113322266 B CN113322266 B CN 113322266B CN 202110720468 A CN202110720468 A CN 202110720468A CN 113322266 B CN113322266 B CN 113322266B
Authority
CN
China
Prior art keywords
rice
gene
osrhd1
male sterile
sterile line
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110720468.8A
Other languages
Chinese (zh)
Other versions
CN113322266A (en
Inventor
甘银波
杨帅淇
吴君宇
黄琳丽
刘一华
刘伯涵
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN202110720468.8A priority Critical patent/CN113322266B/en
Publication of CN113322266A publication Critical patent/CN113322266A/en
Application granted granted Critical
Publication of CN113322266B publication Critical patent/CN113322266B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
    • C12Y501/03002—UDP-glucose 4-epimerase (5.1.3.2), i.e. UDP-galactose 4-epimerase

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

本发明公开了水稻OsRHD1‑1基因在水稻雄性核不育系培育中的应用。本发明通过设计OsRHD1‑1基因的敲除靶点构建基因敲除载体，并在水稻中进行遗传转化，发现OsRHD1‑1基因功能的丧失能够导致花粉不育，有助于新型雄性核不育系的培育，助力杂交育种的发展。

Figure 202110720468

The invention discloses the application of rice OsRHD1-1 gene in the cultivation of rice male sterility lines. The present invention constructs a gene knockout vector by designing a knockout target of the OsRHD1-1 gene, and performs genetic transformation in rice. It is found that the loss of the function of the OsRHD1-1 gene can lead to pollen sterility, which is helpful for a new male male sterile line The development of cross-breeding is facilitated.

Figure 202110720468

Description

Application of rice OsRHD1-1 gene in rice male sterile line cultivation
Technical Field
The invention relates to the technical field of bioengineering, in particular to application of a rice OsRHD1-1 gene in cultivation of a rice male sterile line.
Background
Rice is one of the most important food crops in the world, and how to improve the yield of the rice to meet the increasing food demand is a problem to be urgently solved at present. Because the cultivated land area and the fresh water resource in China are limited, the method for improving the yield per unit of rice is a main way for improving the yield of rice. Factors influencing the yield per unit of rice are many, and intrinsic genetic characteristics influencing the tillering number of each plant, the grain number per ear and the grain weight of the rice are rice yield determining factors; external factors, including abiotic stresses such as illumination, temperature, water and salinity, and biotic stresses of diseases and insect pests have important influence on single birth; in addition, human factors also play a key role in the yield per unit of rice, such as cultivation modes, fertilization modes, pest control levels and the like.
The high yield of rice can not be separated from the excellent variety, and the breeding of the excellent variety can not be separated from the effective breeding method. Currently, rice breeding methods include conventional breeding and molecular breeding. Conventional breeding includes systematic breeding, cross breeding and mutation breeding; the molecular breeding comprises molecular marker-assisted selective breeding, transgenic breeding and molecular design breeding. However, molecular breeding is still greatly limited in application due to the problems of high cost, safety concerns, and incomplete discovery and functional research of major agronomic trait genes of rice. At present, systematic breeding is still the main breeding method, and the application of cross breeding is very wide, thereby greatly improving the yield of rice in China.
The crossbreeding is a breeding method which utilizes two rice subspecies with genetic difference to carry out hybridization, the excellent characters of the two selected subspecies can be complemented, and the progeny can generate heterosis. At present, the cross breeding method comprises a three-line method and a two-line method. The three-line method comprises a sterile line (a nucleoplasm interaction male sterile line and a male genic sterile line), a maintainer line and a restorer line; the two-line method comprises a sterile line (a temperature-sensitive male sterile line and a photosensitive male sterile line) and a restorer line. The three-line sterile lines currently used in China are mainly II-32A, gold 23A, Zhenshan 97A, gang 46A and Longtepu A; the sterile line of the two-line method mainly comprises Y58S and Petai 64S. Therefore, the cultivation of the sterile line plays a key role in the development of the hybrid rice industry. However, the poor quality of the sterile line is an important reason for the poor yield and quality of hybrid rice, so that the cultivation of the sterile line with good rice quality, strong stress resistance and good combining ability is a problem which needs to be solved urgently at present.
In Arabidopsis thaliana, Schiefelbein et al (J.W.Schiefelbein and C.Somerville.1990.genetic Control of Root Hair Development in Arabidopsis thaliana plant Cell,2(3):235-243.) reported that the Arabidopsis thaliana ROOT HAIR DEFECTIVE 1(AtRHD1, At1G64440) gene has an important regulatory role in the Development of Root hairs, and the Atrhd1 mutant shows abnormal Root Hair initiation.
Through sequence alignment analysis, 4 homologous genes with functions similar to that of AtRHD1 exist in rice and code UDP-glucose/galactose epimerase, but no report on the function of the genes for regulating rice pollen development exists.
Disclosure of Invention
The research result of the application shows that the OsRHD1-1 gene (LOC-Os05g51670) plays a key role in pollen development. As OsRHD1-1 is a nuclear gene, and pollen abortion can be caused by knocking out the gene in rice, the OsRHD1-1 can be used as a breeding material for breeding a novel rice male nuclear sterile line, can provide a hybrid combination of excellent varieties for rice hybrid breeding, and further improves the quality and the yield of rice.
Application of rice OsRHD1-1 gene in breeding male sterile line of rice. Preferably, the base sequence of the rice OsRHD1-1 gene is shown as SEQ ID NO. 1.
The invention also provides a cultivation method of the rice male sterile line, which knocks out the OsRHD1-1 gene of rice.
Preferably, the method for cultivating the rice male sterile line comprises the following steps:
(1) constructing a gene knockout vector, wherein the gene knockout vector is a plant expression vector with a sequence for knocking out a rice OsRHD1-1 gene;
(2) and (2) introducing the gene knockout vector obtained in the step (1) into rice cells to knock out the OsRHD1-1 gene of the rice, and culturing to obtain a rice male sterile line.
Preferably, the plant expression vector is pYLCRISPR/Cas9Pubi-H。
When transforming a recipient plant, an agrobacterium-mediated transformation method can be adopted, and the agrobacterium can be specifically agrobacterium EHA 105. And (2) infecting rice after the gene knockout carrier is transferred into agrobacterium.
Preferably, the rice infected by agrobacterium is rice seed-induced callus. In addition to rice seed-induced callus, it may also be callus induced by a tissue sample taken from a rice plant. Or other means of growing plants after transgenesis may be used.
Preferably, the target base sequence targeted by the knockout is 5'-tcctccagcttctccaact-3'. The base sequence of the target site of gene knockout is not limited to the sequence, as long as the function of the gene is lost after the OsRHD1-1 gene of rice is knocked out.
The invention has the following beneficial effects: according to the invention, a gene knockout carrier is constructed by designing a knockout target of an OsRHD1-1 gene, and genetic transformation is carried out in rice, so that pollen sterility can be caused by loss of the function of the OsRHD1-1 gene, and the method is beneficial to cultivation of a novel male sterile line and development of assisted cross breeding.
Drawings
FIG. 1 is a diagram showing the results of the detection of OsRHD1-1 genome sequence editing of OsRHD1-1-1, Osrhd1-1-2 and Osrhd1-1-3 knockout strains.
FIG. 2 is a graph showing the comparison of florets of wild type (NIP) and knockout lines of rice (Osrhd1-1-1, Osrhd1-1-2, Osrhd 1-1-3).
FIG. 3 is a graph showing the comparison results of pollen fertility and anther of wild type (NIP) and rice knockout lines (Osrhd1-1-1, Osrhd1-1-2, Osrhd 1-1-3).
FIG. 4 is a graph showing the comparison results of pistil morphology between wild type (NIP) and rice knockout lines (Osrhd1-1-1, Osrhd1-1-2, Osrhd 1-1-3).
Detailed Description
Example 1: construction of rice OsRHD1-1 gene knockout vector and rice genetic transformation
The base sequence of the rice OsRHD1-1 gene is shown in SEQ ID NO. 1.
(1) According to the website of http:// skl.scau.edu.cn/targettign/, a specific knockout target of the OsRHD1-1 gene is designed, and the sequence is as follows:
knocking out a target: TCCTCCAGCTTCTCCAACT are provided.
And designing an expression sequence for expressing the corresponding sgRNA sequence according to the sequence of the knocked target.
(2) The pYLCRISPR/Cas9Pubi-H vector was double-digested with BsaI endonuclease from NEB (same type of endonuclease at two positions)
The system is as follows:
Figure BDA0003136717220000041
buffer 2. mu.L; 1 mu L of carrier; BsaI endonuclease 1 uL; ddH2O was added to a total volume of 10. mu.L.
The enzyme digestion conditions are as follows: 30 minutes at 37 ℃.
(3) Knock-out target synthesis
The primer target sequences were synthesized as follows:
595100-aFP：5’-GCCGTCCTCCAGCTTCTCCAACT-3’；
595100-aRP：5’-AAACAGTTGGAGAAGCTGGAGGA-3’。
the primer annealing system is as follows:
595100-aFP：9μL；595100-aRP：9μL；ddH2o was added to a total volume of 20. mu.L.
The annealing conditions are as follows: after 5 minutes at 95 ℃, the mixture was taken out and left at room temperature.
(4) Connection of knockout target and enzyme digestion vector
The ligation reaction was carried out using T4 ligase from Taraka as follows:
knocking out a target: 1 mu L of the solution; enzyme digestion vector: 1.5 mu L; t4 ligase buffer (T4 ligase buffer): 0.5 mu L; t4 ligase (T4 ligase): 0.5 mu L; ddH2O was added to a total volume of 5. mu.L.
The ligation reaction conditions were: 3 minutes at 10 ℃, 6 seconds at 6 ℃ (0.2 ℃ per cycle), 3 minutes at 16 ℃, 1 minute at 18 ℃, and 19 cycles from the first step to the fourth step; 15 minutes at 65 ℃; storing at 12 deg.C.
Taking the ligation product to transform escherichia coli DH5 alpha, picking PCR verification positive clone, then carrying out sequencing verification, and extracting plasmid for later use.
(5) EHA105 competent cells were removed from the freezer at-80 ℃ and Agrobacterium transformed by liquid nitrogen freeze-thaw.
And unfreezing the competent cells on ice, adding 5 mu L of target plasmid into the competent cells, uniformly mixing, placing on ice for about 30min, quickly freezing all the mixture in a centrifugal tube in liquid nitrogen for 1 min, then placing in a 37 ℃ water bath kettle for 2 min for thawing, then adding 1mL of non-resistant LB liquid nutrient medium, re-suspending the cells, placing on a shaking table at the temperature of 28 ℃ and 200rpm for resuscitation for 2 h, then coating on YEP plates (Rif (rifampicin) 50mg/L and Kan (kanamycin sulfate) 50mg/L) containing corresponding resistance, and performing inverted culture at the temperature of 28 ℃ for 2 days until single colonies grow out.
(7) Selecting the grown agrobacterium tumefaciens monoclonal, identifying the positive monoclonal by colony PCR,
(8) rice seed (Nipponbare rice) induced callus was transformed by the rapid genetic transformation of rice.
(9) Hygromycin screens resistant callus and induces differentiation to emerge, extracts differentiated seedling leaf DNA, amplifies genome target sequence sequencing to identify target point mutation conditions, and the used primers are as follows:
RHD1-1CAS-F：5’-GAAGCTAGGAAAAAGGGAAATA-3’；
RHD1-1CAS-R：5’-TCGCCCGAATACAGATATAAGT-3’。
example 2: gene editing condition of rice OsRHD1-1 gene knockout strain
In order to detect the development condition of pollen in the small flowers of the OsRHD1-1 gene knockout strain of rice, DNA of a gene editing seedling is extracted, and an amplified fragment is sent to Hangzhou Shanghai company for sequencing.
As shown in FIG. 1, the sequencing results revealed that both DNA single strands of the 3 strains sequenced were mutated.
Example 3: small anther character observation of rice OsRHD1-1 gene knockout strain
The anther of the small flower of the rice OsRHD1-1 gene knockout strain becomes small, the color becomes light and the development is poor.
As shown in FIG. 2, the traits of the florets of the OsRHD1-1 knockout line of rice obtained in example 2 were observed, and the anthers were reduced, the color was lightened, and the development was poor in the florets of the OsRHD1-1 knockout line of rice.
Example 4: pollen observation of rice OsRHD1-1 gene knockout strain
In order to detect the pollen development condition in the small flowers of the OsRHD1-1 gene knockout strain of rice, the pollen is observed by using an iodine staining method.
As shown in FIG. 3, the florets of the OsRHD1-1 knockout line of rice have no pollen and are pollen sterile.
Example 5: observation of pistil of florets of rice OsRHD1-1 gene knockout strain
In order to detect the gynoecium development condition in the small flowers of the OsRHD1-1 gene knockout strain of rice, the gynoecium morphology is observed by using an iodine staining method.
As shown in FIG. 4, the pistils of the florets of the OsRHD1-1 gene knockout line of rice develop normally.
The experimental results of the people prove that the OsRHD1-1 gene of the rice has a key role in the development of pollen with the background of Japanese fine rice, the loss of the function of the gene causes a pollenless phenotype, and the growth of pistil is not influenced.
Sequence listing
<110> Zhejiang university
<120> application of rice OsRHD1-1 gene in rice male sterile line cultivation
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1065
<212> DNA
<213> Rice (Oryza sativa L.)
<400> 1
atggtttcgg ccttgttgcg gacgatcctg gtgacgggcg gcgccggcta catcggcagc 60
cacaccgtcc tccagcttct ccaactcggc ttccgcgttg tcgtcctcga caacctcgac 120
aacgcctccg agctcgccat cctccgcgtc agggaactcg ccggacacaa cgccaacaac 180
ctcgacttcc gcaaggttga cctccgcgac aagcaagcgt tggaccaaat cttctcctct 240
caaaggtttg aggctgtcat ccattttgcc gggctgaaag ctgttggcga gagcgtgcag 300
aagcccctgc tttactacga caacaacctc atcggcacca tcactctcct gcaggtcatg 360
gccgcacatg gctgcaccaa gctggtgttc tcatcatccg caactgtcta cgggtggccc 420
aaggaggtgc cctgcactga agaatcccca ctttgtgcaa tgaaccccta cggcagaaca 480
aagctggtaa tcgaagacat gtgccgggat ctgcatgcct cagacccaaa ctggaagatc 540
atactgctcc gatacttcaa ccctgttgga gctcacccaa gcgggtacat tggtgaggac 600
ccctgcggca tcccaaacaa cctcatgccc ttcgtccagc aggtcgctgt tggcaggagg 660
ccggccctta ccgtctatgg aaccgactac aacaccaagg atggaactgg ggttcgtgac 720
tatatccatg ttgttgatct agcggatggt catatcgccg cgttaaggaa gctctatgaa 780
gattctgata gaataggatg tgaggtgtac aatctgggca ctggaaaggg gacatctgtg 840
ctggaaatgg ttgcagcatt cgagaaagct tctggaaaga aaatcccgct tgtatttgct 900
ggacgaaggc ctggagatgc cgagatcgtt tacgctcaaa ctgccaaagc tgagaaggaa 960
ctgaaatgga aggcaaaata cggggtagag gagatgtgca gggacctgtg gaattgggcg 1020
agcaagaacc cctacgggta tggatcgccg gacagtagca actga 1065
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcctccagct tctccaact 19
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gccgtcctcc agcttctcca act 23
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aaacagttgg agaagctgga gga 23
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaagctagga aaaagggaaa ta 22
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcgcccgaat acagatataa gt 22

Claims (7)

1.水稻OsRHD1-1基因在水稻雄性核不育系培育中的应用，水稻OsRHD1-1基因的碱基序列如SEQ ID NO.1所示。1. The application of the rice OsRHD1-1 gene in the cultivation of rice male sterile lines. The base sequence of the rice OsRHD1-1 gene is shown in SEQ ID NO.1. 2.一种水稻雄性核不育系培育方法，其特征在于，将水稻OsRHD1-1基因敲除，水稻OsRHD1-1基因的碱基序列如SEQ ID NO.1所示。2. A method for cultivating a rice male sterile line, characterized in that the rice OsRHD1-1 gene is knocked out, and the nucleotide sequence of the rice OsRHD1-1 gene is shown in SEQ ID NO.1. 3.如权利要求2所述的水稻雄性核不育系培育方法，其特征在于，包括以下步骤：3. rice male sterile line breeding method as claimed in claim 2, is characterized in that, comprises the following steps: （1）构建基因敲除载体，所述基因敲除载体为带有用于对水稻OsRHD1-1基因进行敲除的序列的植物表达载体；(1) constructing a gene knockout vector, which is a plant expression vector with a sequence for knocking out the rice OsRHD1-1 gene; （2）将步骤（1）基因敲除载体导入水稻的细胞中将水稻OsRHD1-1基因敲除，培养后获得水稻雄性核不育系。(2) Introducing the gene knockout vector of step (1) into rice cells to knock out the rice OsRHD1-1 gene, and after culturing, a rice male sterility line is obtained. 4.如权利要求3所述的水稻雄性核不育系培育方法，其特征在于，所述植物表达载体为pYLCRISPR/Cas9Pubi-H。4. The method for cultivating a rice male sterile line according to claim 3, wherein the plant expression vector is pYLCRISPR/Cas9P ubi -H. 5.如权利要求3所述的水稻雄性核不育系培育方法，其特征在于，步骤（2）将基因敲除载体转入农杆菌后，侵染水稻。5 . The method for cultivating a rice male sterile line according to claim 3 , wherein in step (2), the gene knockout vector is transformed into Agrobacterium to infect rice. 6 . 6.如权利要求5所述的水稻雄性核不育系培育方法，其特征在于，农杆菌侵染的水稻为水稻种子诱导的愈伤组织。6 . The method for cultivating a rice male sterile line according to claim 5 , wherein the rice infected by Agrobacterium is callus induced by rice seeds. 7 . 7.如权利要求3所述的水稻雄性核不育系培育方法，其特征在于，基因敲除针对的靶点碱基序列为5’-tcctccagcttctccaact-3’。7. The method for cultivating a rice male sterile line according to claim 3, wherein the target base sequence for gene knockout is 5'-tcctccagcttctccaact-3'.
CN202110720468.8A 2021-06-28 2021-06-28 Application of rice OsRHD1-1 gene in rice male sterile line cultivation Active CN113322266B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110720468.8A CN113322266B (en) 2021-06-28 2021-06-28 Application of rice OsRHD1-1 gene in rice male sterile line cultivation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110720468.8A CN113322266B (en) 2021-06-28 2021-06-28 Application of rice OsRHD1-1 gene in rice male sterile line cultivation

Publications (2)

Publication Number Publication Date
CN113322266A CN113322266A (en) 2021-08-31
CN113322266B true CN113322266B (en) 2022-03-25

Family

ID=77424998

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110720468.8A Active CN113322266B (en) 2021-06-28 2021-06-28 Application of rice OsRHD1-1 gene in rice male sterile line cultivation

Country Status (1)

Country Link
CN (1) CN113322266B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118185956B (en) * 2024-04-25 2024-11-01 江西省超级水稻研究发展中心(江西省农科院海南水稻育种中心) OsUGE3 gene mutant and application thereof in cultivation of dual-purpose sterile line of rice

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106011167A (en) * 2016-05-27 2016-10-12 上海交通大学 Application of male sterility gene OsDPW2 and rice sterility recovery method
CN110241126A (en) * 2019-07-03 2019-09-17 浙江大学 Application of OsDGD2β Gene in Breeding Male Sterile Rice Varieties

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106011167A (en) * 2016-05-27 2016-10-12 上海交通大学 Application of male sterility gene OsDPW2 and rice sterility recovery method
CN110241126A (en) * 2019-07-03 2019-09-17 浙江大学 Application of OsDGD2β Gene in Breeding Male Sterile Rice Varieties

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GenBank：AB087745.1;佚名;《NCBI》;20080215;参见序列 *
NCBI Reference Sequence: XM_ 015784747.2;佚名;《NCBI》;20180807;参见序列 *
UDP-glucose pyrophosphorylase gene affects mycelia growth and polysaccharide synthesis of Grifola frondosa;Xin-Yi Zan;《International Journal of Biological Macromolecules》;20200607;第1161-1170页 *
水稻OsYAB6和OsUGE1基因克隆与功能分析;刘慧丽;《中国科学院植物研究所博士学位论文》;20110525;全文 *

Also Published As

Publication number Publication date
CN113322266A (en) 2021-08-31

Similar Documents

Publication Publication Date Title
CN107475210B (en) A rice bacterial blight resistance-related gene OsABA2 and its application
CN108503700B (en) Rice grain protein and its encoding gene and application
CN105950651A (en) Application of male-sterility gene OsGEN and method for restoring fertility
CN113151314B (en) Plant ACCase mutant gene and application thereof
CA3211382A1 (en) Method for site-directed mutagenesis of bnhbbd gene of brassica napus l., and use
CN109609527B (en) Application of CDPK18L gene as a negative regulator in improving tomato bacterial leaf spot resistance and high temperature resistance
CN111440804A (en) Application of Maize ZmBES1/BZR1-5 Gene in Breeding Large Grain Plants
CN109234288B (en) Application of rape BnA9-2 gene in improving pod shatter resistance of rape
CN114540374A (en) High herbicide resistance plant gene mutant and corresponding vector and application
CN112522291B (en) Rice OsSH3P2 gene and application thereof
CN113322266B (en) Application of rice OsRHD1-1 gene in rice male sterile line cultivation
CN111153976A (en) A rice defense mechanism regulatory protein and its application in breeding
CN102120763B (en) Rice OsNAC coding sequence and its application
CN114478731A (en) Herbicide-resistant rice mutant protein and application thereof
CN109609516A (en) Application of a disease resistance gene in rice smut resistance improvement
CN112048488B (en) OsPEAMT2 gene for improving heading stage maturing rate of paddy rice under high temperature stress, protein and application thereof
CN113265403A (en) Soybean Dt1 gene editing site and application thereof
CN113462689A (en) Application of soybean gene promoters pEIF1 and pEIF1-I in soybeans, arabidopsis thaliana and tobaccos
CN110407922B (en) Rice cold tolerance gene qSCT11 and its application
CN112322633A (en) Rice root-knot nematode resistance gene OsBetvI and application thereof
CN115896084A (en) Application of Rice OsRHD1-3 Gene in Regulating Rice Pollen Fertility
TWI441591B (en) Method for enhancing thermotolerance of plant and applicatibility of transgenic plant
CN111875689B (en) Method for creating male sterile line by using tomato green stem close linkage marker
CN112080518B (en) Application of maize AL14 gene in drought resistance of plants
CN115141842A (en) Application of GmABI5 gene in regulating plant grain weight

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant