WO2020211747A1 - 一种固定植物杂种优势的种子分选方法 - Google Patents

一种固定植物杂种优势的种子分选方法 Download PDF

Info

Publication number
WO2020211747A1
WO2020211747A1 PCT/CN2020/084695 CN2020084695W WO2020211747A1 WO 2020211747 A1 WO2020211747 A1 WO 2020211747A1 CN 2020084695 W CN2020084695 W CN 2020084695W WO 2020211747 A1 WO2020211747 A1 WO 2020211747A1
Authority
WO
WIPO (PCT)
Prior art keywords
expression
gene
seeds
promoter
expression cassette
Prior art date
Application number
PCT/CN2020/084695
Other languages
English (en)
French (fr)
Inventor
曹孟良
夏玉梅
詹祎捷
唐宁
卜小兰
余木兰
袁隆平
Original Assignee
湖南杂交水稻研究中心
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 湖南杂交水稻研究中心 filed Critical 湖南杂交水稻研究中心
Publication of WO2020211747A1 publication Critical patent/WO2020211747A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen

Definitions

  • the invention belongs to the field of plant molecular biology and agricultural biotechnology, and in particular relates to a pollen-specific gene switch system to regulate screening marker genes, and then sort cloned seeds to obtain cloned seeds that meet the purity requirements, thereby realizing a hybrid hybrid-free system Method of application.
  • a complete plant contains sporophyte generations (2n), which has a specific reproductive structure (floral organs), in which pistil and stamen undergo meiosis and genetic recombination to produce megaspores (1n) and microspores (1n) respectively;
  • the large and microspores form female and male gametophytes through several mitosis.
  • the female gametophyte embryonic sac
  • the male gametophyte polyonic sac
  • contains 7 cells (1 egg cell, 2 helper cells, 1 central cell with 2 nuclei, 3 antipodal cells
  • the male gametophyte (pollen) contains 1 vegetative cell and 2 sperm; after pollination, the pollen tube enters the embryo sac and double fertilization occurs.
  • sperm male gamete
  • egg cell female gamete
  • Apomixis is a reproductive method in which plants produce embryos and seeds without fusion of sperm and egg cells during the process of producing sporophytes from gametophytes.
  • haploid apomixis Including two basic types of haploid apomixis and diploid apomixis. Since diploid apomixis can produce offspring with the same genotype as the mother plant and can fix any excellent genotype, it is especially valued by breeders.
  • the apomictic breeding we usually refer to is also mainly specialized Refers to this type, which includes: Adventive embryo (Adventive Embryony) reproduction, Apospory (Apospory) and Diploid Spore reproduction (Diplospory).
  • the regenerated plants had obvious variations in floral morphology, structure and reproductive characteristics, and multiple anthers appeared. , Multiple ovules and multiple embryo sac structures and other specific reproductive phenomena. However, through molecular breeding technology and protoplast fusion technology, rice apomictic germplasm with breeding value has not yet been created.
  • the ratio of cloned seeds in the apomictic reproduction system is only 29%, which is lower than the purity requirement of 96% in production, and the cloned seeds and the seeds produced by normal pollination and fertilization cannot be sorted. So that it is impossible to realize the application of hybrid apomixis.
  • the purpose of the present invention is to provide a method for using a pollen-specific gene switch system to regulate selection marker genes, and then to sort cloned seeds, thereby realizing the application of a hybrid system without fusion.
  • the present invention also provides nucleic acids encoding the autonomous embryo generation, the expression of selection markers, and the expression of pollen-specific regulation and selection genes, and genetic engineering intermediates (such as expression cassettes, recombinant vectors, transgenic cell lines or recombinant bacteria) to determine whether plants are The identification method obtained by the method of the present invention.
  • a seed sorting method for fixing plant heterosis including the following steps:
  • the acceptor material is a monocot or a dicot.
  • the preferred monocot is rice, more preferably hybrid rice.
  • the MiMe mutant can promote mitosis to replace meiosis.
  • the pathway of autonomous embryo development in step A includes but is not limited to the expression of BBM1 driven by an egg cell-specific expression promoter or mutants of OsPLA, MATRILINEAL (MTL), NOT LIKE DAD (NLD);
  • the egg cell of E1 Specific expression promoters include but are not limited to AtDD45, Os03g0296600 pro, ECA1-like1 pro, DCL2, AT1G74480.1, ZmEAlpromoter
  • the embryo autonomous genes of E1 include but are not limited to BBM1, WUS, LEC, CLAVATA, MYB115.
  • the E2 selection marker gene includes but is not limited to fluorescent gene.
  • the E2 promoter is connected with a terminator, so that the selection marker gene driven by the promoter cannot be expressed, the terminator flanks the recognition sequence of E3, and the E2 promoter is an embryo-specific expression promoter , Including but not limited to Lec1, OsESP1, EL5, pBnaA09g21960D.
  • the pollen-specific gene switch expression of E3 can cut off the terminator connected to the promoter in E2, so that the selection marker gene can be expressed.
  • the regulation system of E3 includes but not limited to double-strand break inducing enzyme system, genome editing system, and recombination Enzyme system, transposon system, pollen-specific expression promoter in E3 includes but not limited to PG47.
  • An expression cassette comprising the polynucleotide of claims 1-3 operably linked to a heterologous polynucleotide of interest.
  • a vector that knocks out MiMe and inserts three linkage expression cassettes the expression cassette of the autonomous embryogenic gene BBM1, the expression cassette of the fluorescent selection marker gene RFP and the expression cassette of the Cre recombinase switch regulation; the autonomous embryogenic gene BBM1
  • the nucleotide sequence of BBM1 in the expression cassette is shown in SEQ ID NO. 6
  • the nucleotide sequence of RFP in the fluorescent screening marker gene REP expression cassette is shown in SEQ ID NO. 8
  • Cre in the Cre recombinase switch regulating expression cassette The nucleotide sequence of the recombinase is shown in SEQ ID NO.11.
  • the knockout of MiMe refers to the knockout of PAIR1, REC8, and OSD1 genes
  • the embryo autonomous gene BBM1 carries the egg cell-specific expression promoter AtDD45 and the Nos terminator, the REP carries the embryo-specific expression promoter OsESP1, loxP-Nos-loxP and the Nos terminator, and the Cre recombinase is initiated by pollen-specific expression.
  • Sub PG47 carries and is connected to the Nos terminator.
  • nucleic acid which can be DNA or RNA, preferably DNA.
  • the nucleic acid of the second aspect of the present invention can be constructed such that the expression cassette is inserted into any vector transformed into a cell.
  • the primer pair that amplifies the full length or any fragment of the aforementioned nucleic acid fragment is also within the protection scope of the present invention.
  • Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing nucleic acid fragments of the second aspect are also within the protection scope of the present invention.
  • the preferred cell is a plant cell, preferably a rice cell, more preferably a hybrid rice cell, and the nucleic acid of the second aspect of the present invention contained in the plant cell may be transgenic technology (as described in the specific embodiment of the present invention)
  • the introduction into plant cells includes introduction into the nucleus, chloroplasts, mitochondria and/or plastids of plant cells.
  • the nucleic acid of the second aspect of the present invention may also be present in plant cells by other techniques.
  • the present invention provides the application of the aforementioned expression cassettes in fixing plant heterosis.
  • the embryogenic gene expression cassette E1 driven by the egg cell-specific expression promoter can make the parthenogenesis of the oocyte-containing cells produce asexual embryos with fixed heterosis;
  • the selection marker gene expression cassette E2 regulated by E3 and the pollen-specific expression cassette E2 E3 can sort the asexual embryo seeds produced by parthenogenesis and the zygotic embryo seeds formed by pollination.
  • the cloned seeds retain the heterosis and can be used for production, and the remaining seeds can be used for commercial use.
  • the present invention provides a method for identifying the plant of the fourth aspect of the present invention, that is, determining whether the plant contains the nucleic acid of the second aspect. By this method, it can be judged whether the plant belongs to the plant of the present invention, that is, whether it belongs to the plant of the fourth aspect of the present invention.
  • the steps of determination can be through conventional nucleic acid detection and sequencing. Exemplary methods include nucleic acid sequencing, polymerase chain reaction (PCR) detection, screening marker observation, fluorescence quantitative sorting, probe hybridization detection, sequence determination, and flow cytometry. Analysis etc.
  • the beneficial effect obtained by the present invention is that the pollen-specific gene switch system is used to regulate the selection marker gene, and then the cloned seeds are sorted, thereby realizing the application of the hybrid non-fusion system.
  • the present invention uses the egg cell-specific expression promoter to drive the autonomous embryogenic expression cassette E1 to make the egg cell parthenogenetic to form an embryo, or it can be obtained by mutants such as haploid inducing genes OsPLA, MATRILINEAL (MTL), NOT LIKE DAD (NLD), etc.
  • Asexual embryos, the selection marker gene expression cassette E2 and the expression cassette E3 driven by the pollen-specific expression promoter constitute a conditional gene knockout system, which can control the specific expression of the selection marker gene.
  • This method of apomictic reproduction can produce and sort out cloned seeds with fixed heterosis without affecting seed yield, reduce the complicated seed production procedures of hybrid seeds, reduce labor intensity, reduce the production cost of hybrid seeds, and To achieve the industrialization requirements of the one-line method for the utilization of heterosis, and to improve the efficiency of the hybrid seed industry as a whole.
  • the invention will have broad application and market prospects in the agricultural field.
  • FIG 1 shows the fluorescence of Ep30TLK and Ep30TL plates
  • FIG. 2 shows the fluorescence of Ep30TLK and Ep30TLEP tubes
  • Figure 3 is the green fluorescence result of p26LK1 and its red fluorescence control
  • Figure 4 is the green fluorescence result of p26LK2 and its red fluorescence control
  • Figure 5 is a schematic diagram of the structure of the apomictic reproduction vector p24DB of the present invention
  • Ep30TLK Ep30TLK
  • Ep30TL After the electric shock is completed and resuscitated, take an appropriate amount (usually 100-200 ⁇ L) of the transformed product and spread it on the ampicillin + kana resistant LB solid medium; take Ep30TL and spread it on the ampicillin resistant LB solid medium to verify The prepared Ep30TL is competent. Invert the petri dish at 37°C and incubate overnight. Observe the growth of the colony after 12-16h.
  • Ep30TLK contains both p30TL and p30TK vectors.
  • the primer sequences are as follows. The other part is 1mL and placed in a 1.5mL EP tube. After centrifugation at 500rpm/min for 1min, the supernatant is discarded. The precipitate is used to observe the green fluorescence and Ep30TL bacterial solution is used as a control. The results are shown in Figure 2. Under natural light conditions, the green of Ep30TLK is visible to the naked eye; under the observation of hand-held fluorescent glasses, Ep30TLK emits green fluorescence, while the control Ep30TL has no green fluorescence.
  • Reverse sequencing primer (Ep30TL): 5’AGATCTCGATCCCGCGAAAT 3’
  • Reverse sequencing primer 5’GTATATGTGCCTACTAACGC 3’
  • the sequence seq26LK1 is connected to the vector pLJ02 through HindIII/HindIII, the target fragment is in the same direction as the Ubi-promoter, named p26LK1.
  • the sequence seq26LK2 was linked to the vector pLJ02 through HindIII/HindIII, the target fragment was in the same direction as the Ubi-promoter, named p26LK2.
  • the vectors p26LK1 and p26LK2 are scratched, and 1L of a single colony is selected, and the plasmid is extracted for use;
  • the petri dish was cultured in a dark room at 27°C.
  • the transient expression of fluorescence was observed and photographed under a fluorescence microscope. See Figure 3 and Figure 4 for the fluorescence expression results.
  • Example 2 Construction of a plant expression vector carrying three linked gene expression cassettes of autonomous embryogenic genes, fluorescent selection marker genes and regulatory selection marker gene expression cassettes
  • I determined an egg cell-specific expression promoter AtDD45Pro its nucleotide sequence is shown in SEQ ID NO.5; I determined an embryogenesis gene BBM1, and its nucleotide sequence is shown in SEQ ID NO.6; OK 1 embryo-specific expression promoter OsESP1, its nucleotide sequence is shown in SEQ ID NO.7; the red fluorescent gene RFP is determined to be a selection marker gene, and its nucleotide sequence is shown in SEQ ID NO.8; the pollen-specific expression promoter is determined See SEQ ID NO.9 for its nucleotide sequence.
  • Design 1 targets in PAIR1, REC8, OSD1,
  • the embryo autonomous gene BBM1 carries the egg cell-specific expression promoter AtDD45 and the Nos terminator, and its nucleotide sequence is shown in SEQ ID NO.10;
  • the RP coding sequence on the vector pLJ02 carries the embryo-specific expression promoter OsESP1, loxP-Nos-loxP and Nos terminator;
  • pollen-specific expression promoter PG47 carries Cre recombinase, and its nucleotide sequence is shown in SEQ ID NO.11 and Nos terminator. See Figure 5 for the complete vector construction map.
  • the correctly constructed plant expression vector was used to transform Agrobacterium EHA105 using electric shock.
  • the introduction method adopts the electric shock conversion method, mainly referring to the instruction manual of the electric shock meter of bio-rad company. The specific steps are as follows:
  • a single colony of the plant expression vector p24DB Agrobacterium was inoculated on LB medium containing 50mg/L kanamycin and cultured in the dark at 26°C for 2 days.
  • the Agrobacterium cells were washed with NB-AS liquid medium and cultured in liquid shaking at 28°C at 180rpm for 90- 120min. Adjust the colony concentration to OD600 0.8-1.0, transform hybrid rice and hybrid wheat, sterilize hybrid rice seeds, pick full-grained seeds, soak them in 75% alcohol for 30s, pour off the alcohol, rinse with sterile water, and disinfect with HgCl2 for 8 minutes , Wash with sterile water twice, soak for 1 min each time, and soak in sterile water for 1 hour. The sterilized seeds were inoculated on the induction medium and grown for 7 days under light.
  • Collect the aseptic calluses together Put it into the Agrobacterium suspension, soak for 5-10min, take it out, and dry it with filter paper. Inoculate the co-cultivation medium, co-cultivation for 2 days, wash the co-cultured callus 6 times, dry with filter paper, and then inoculate the hygromycin-resistant selection medium for 45 days.
  • the resistant callus was transferred to the differentiation medium. After 2 weeks of culture, the callus began to turn green, and after 3 weeks, young shoots and roots would grow. Transfer the seedlings to small triangular flasks containing rooting medium, one plant per bottle, continue to cultivate under light, when the plant grows to about 7-10cm, carry out indoor refining, and transplant it into the soil after 3-4 days. .
  • the positive plants were screened out by observation of fluorescence and PCR.
  • DNA extraction Take 1.0g leaves, grind them into powder with liquid nitrogen, transfer to 2ml EP tube, add 700 ⁇ l of preheated CTAB solution. In a water bath at 65°C for 30-60 minutes, mix gently during the period. After cooling, add an equal volume of chloroform: isoamyl alcohol (24:1), mix well, and centrifuge at 12000 rpm for 10 minutes. Propanol, let stand at -20°C for 30-60min. The precipitate was collected at 4°C, 12000 rpm, 10 min, and the supernatant was discarded. Wash the precipitate twice with 70% ethanol, blow dry the residue of alcohol, and dissolve the precipitate with 50-100 ⁇ l ddH 2 O for use.
  • PCR analysis Design primers with RP gene as a template, and the amplified product fragment is 431bp.
  • PCR reaction system DNA 30-90ng, 10 ⁇ Buffer 2.0 ⁇ l, 1mM dNTP 1.8 ⁇ l, 25mM MgCl 2 1.5 ⁇ l, 10uM primer 0.5 ⁇ l each, Tag enzyme 1.5U, add ddH 2 O to 20 ⁇ l reaction volume.
  • the PCR cycle conditions were 94°C, 3min; 94°C, 1min, 64°C, 1.5min, 72°C 30s, 40 cycles; 72°C, extension 5min. Use 1.4% agarose gel electrophoresis to detect, photograph and record the electrophoresis results.
  • Fluorescence quantitative analysis Use the plant total RNA extraction kit (Tiangen DP432) to extract RNA from the T0 generation transgenic hybrid rice and wild-type hybrid rice control.
  • the method refers to the kit instructions. Reverse transcription synthesis of corresponding cDNA using total RNA as template. With Actin as the internal reference gene and wild-type plants as the control, the RP gene was detected by RT-PCR by quantitative PCR.
  • Southern blot analysis Take total DNA from rice and wheat for EcoR I digestion, electrophoresis, and transfer to nitrocellulose membrane Hybond-N. Use random primer labeling kit (Promega) and [ ⁇ -32P]dATP (Beijing Yahui Company) to prepare ⁇ -32P-labeled RP gene fragments by random primer method, as molecular probes, and transform according to molecular cloning methods such as Sambrook Southern hybridization analysis of plants.
  • the pollen fertility and seed setting of transgenic plants that have been molecularly identified were investigated. Take the anthers at flowering and dye them with conventional I2-KI to observe the ratio of sterile pollen and fertile pollen. Compared with the wild-type control, observe the agronomic characteristics of the transgenic plants such as plant height, tiller number, flag leaf length, seed setting rate, and 1000-seed weight.
  • the hybrid rice and hybrid wheat carrying the apomictic vector are selfed.
  • Self-bred seeds can be screened out with red fluorescence by photoelectric sorting, and the seeds with red fluorescence can be obtained as zygotic embryo seeds for commercial use; seeds without red fluorescence are cloned seeds and continue to be used for hybridization Propagation of rice and hybrid wheat.

Landscapes

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

Abstract

提供了一种利用花粉特异性基因开关系统分选克隆种子的方法。将3个紧密连锁的基因表达盒和敲除了3个关键减数分裂基因REC8,PAIR1及OSD1的MiMe敲除载体同时转入杂交植物，3个基因表达盒包括：1)胚自主发生基因表达盒E1；2)受E3调控的筛选标记基因表达盒E2；3)花粉特异性调控E2的表达盒E3。通过花粉特异性基因开关调控的筛选标记在杂合转基因植物的自交结实种子中分选无性胚种子和合子胚种子，克隆种子保留了杂种优势可用于生产，其余种子可以商用。

Description

一种固定植物杂种优势的种子分选方法 技术领域
本发明属于植物分子生物学和农业生物技术领域，尤其涉及一种利用花粉特异性基因开关系统调控筛选标记基因，进而分选克隆种子，获得达到纯度要求的克隆种子，从而实现杂交种无融合体系应用的方法。
背景技术
一个完整的植株包含孢子体世代(2n)，它具有特定的生殖结构(花器官)，其中的雌蕊、雄蕊经过减数分裂和遗传重组，分别产生大孢子(1n)和小孢子(1n)；大、小孢子经过几次有丝分裂形成雌、雄配子体，雌配子体(胚囊)含有7个细胞(1个卵细胞、2个助细胞、1个具2核的中央细胞、3个反足细胞)并深埋于母体的胚珠组织中，雄配子体(花粉)含有1个营养细胞和2个精子；传粉后，花粉管进入胚囊，发生双受精作用，其中一个精子(雄配子)和卵细胞(雌配子)结合形成合子胚(2n)，另一个精子和中央细胞结合形成胚乳(3n)，由此完成一个生活周期，并开始新一轮孢子体世代。无融合生殖是植物在由配子体产生孢子体的过程中不经过精卵细胞融合而产生胚和种子的生殖方式。包括单倍体无融合生殖和二倍体无融合生殖两种基本类型。二倍体无融合生殖由于能产生与母体植株基因型完全相同的后代，能固定任何优良的基因型，所以，特别受育种家们的重视，我们通常所指的无融合生殖育种也主要是专指这一类型，它包括：不定胚(Adventive embryony)生殖，无孢子生殖(Apospory)和二倍体孢子生殖(Diplospory)。
我国水稻无融合生殖研究从1979年开始，在“七五”期间，我国水稻无融合生殖研究的热点就是筛选和鉴定水稻多胚苗材料，进而确定其胚胎学特性及育种价值。然而，通过对多胚苗水稻的胚胎发育进行深入研究后发现，部分多胚苗水稻具有单倍体无融合生殖特性，其多胚来源于卵细胞的受精作用和助细胞无配子生殖。要利用现有的多胚苗水稻来固定水稻的杂种优势难度相当大，甚至是不可能的。尽管有人报道在水稻中已经发现了具有不定胚生殖特性的材料，甚至已经成功地培育出无融合生殖水稻，但这类消息似乎还缺乏使人信服的胚胎学证据。我国水稻无融合生殖研究中的另一项重要内容就是利用现代生物技术，将具有无融合生殖特性的远缘物种的遗传物质导入水稻中，进而筛选水稻 无融合生殖种质的探索性试验。通过穗茎注射法将大黍(Panicam max imum)总DNA导入籼型水稻之后获得了一些变异个体。利用粳稻广亲和品系02428与无融合生殖大黍品系OK85进行不对称体细胞杂交后成功地获得了再生植株，再生植株在花器形态、结构和生殖特性上发生了明显的变异，出现了多花药、多胚珠和多胚囊结构等特异的生殖现象。然而，通过分子育种技术和原生质体融合技术仍然没创造出具育种价值的水稻无融合生殖种质。
近年来植物生物技术飞速发展，通过分子生物学以及基因工程的手段去研究和改良作物在农业生产中取得了很好的应用前景。近期通过分子生物学以及基因工程的手段在无融合生殖研究领域取得了重要进展。2018年底，美国加州大学戴维斯分校Venkatesan Sundaresan教授团队发表的水稻无融合生殖体系；通过编辑PAIR1、REC8、OSD1(MiMe)三个基因，结合卵细胞中异位表达BBM1(Synthetic-Apomictic)，实现了水稻的无融合生殖。但是该无融合生殖体系中克隆种子的比率只达到29％，元低于生产上96％的纯度要求，且克隆种子和正常授粉受精而成的种子无法进行分选。以至于还无法实现杂交种无融合生殖体系的应用。
发明内容
针对现有技术的不足，本发明目的在于提供了一种利用花粉特异性基因开关系统调控筛选标记基因，进而分选克隆种子，从而实现杂交种无融合体系应用的方法。另外，本发明还提供了编码胚自主发生、筛选标记表达和花粉特异性调控筛选基因表达的核酸以及基因工程中间体(如，表达盒、重组载体、转基因细胞系或重组菌)，判断植物是否采用本发明方法获得的鉴定方法。
为了解决上述技术问题，本发明的技术方案如下：
一种固定植物杂种优势的种子分选方法，包括如下步骤：
A、构建胚自主发生基因表达盒E1；
B、构建受E3调控的筛选标记基因表达盒E2；
C、构建花粉特异性调控E2的表达盒E3；
D、创制MiMe突变体(PAIR1、REC8、OSD1)；
E、创制转基因，其转入有上述A、B、C3个连锁表达盒和携带MiMe突变位点。
所述E3和E2构成一个基因开关系统，E3的花粉特异性表达可以切除E2中与启动子相连的终止子，使其所驱动的筛选标记基因得以表达，孤雌生殖产生的固定了杂种优势的无性胚种子和授粉受精形成的合子胚种子可以通过筛选标记基因分选，从而获得达到种子纯度要求的孤雌生殖种子，使一系法杂种优势固定达到生产应用的要求。
在本发明的具体实施方式中，所述受体材料为单子叶植物或双子叶植物，优选的单子叶植物是水稻，更优选是杂交水稻。
优选的，所述MiMe突变体除了3个关键减数分裂基因REC8,PAIR1及OSD1，可以促使有丝分裂替代减数分裂。
优选的，所述步骤A中胚自主发生的途径包括但不限于卵细胞特异表达启动子驱动的BBM1的表达或OsPLA、MATRILINEAL(MTL)、NOT LIKE DAD(NLD)的突变体；所述E1的卵细胞特异表达启动子包括但不限于AtDD45、Os03g0296600 pro、ECA1-like1 pro、DCL2、AT1G74480.1、ZmEAlpromoter，所述E1的胚自主发生基因包括但不限于BBM1、WUS、LEC、CLAVATA、MYB115。
优选的，所述E2的筛选标记基因包括但不限于荧光基因。
优选的，所述E2的启动子连有一个终止子，致使该启动子所驱动的筛选标记基因不能表达，终止子侧翼携带E3的识别序列，所述E2的启动子为胚特异性表达启动子，包括但不限于Lec1、OsESP1、EL5、pBnaA09g21960D。
优选的，E3的花粉特异性基因开关表达可以切除E2中与启动子相连的终止子，使筛选标记基因得以表达，E3的调控系统包括但不限于双链断裂诱导酶系统、基因组编辑系统、重组酶系统、转座子系统，E3中所述花粉特异表达启动子包括但不限于PG47。
一种表达盒，所述表达盒包含可操作连接到目的异源多核苷酸的权利要求1-3所述的多核苷酸。
一种载体，所述载体敲除MiMe，并插入3个连锁表达盒：胚自主发生基因BBM1表达盒、荧光筛选标记基因RFP表达盒和Cre重组酶开关调控表达盒；所述胚自主发生基因BBM1表达盒中BBM1的核苷酸序列见SEQ ID NO.6；所述荧光筛选标记基因REP表达盒中RFP的核苷酸序列见SEQ ID NO.8；所述Cre重组酶开关调控表达盒中Cre重组酶的核苷酸序列见SEQ ID NO.11。
所述敲除MiMe是指对PAIR1、REC8、OSD1基因的敲除；
所述胚自主发生基因BBM1携带卵细胞特异表达启动子AtDD45和Nos终止子，所述REP携带胚特异表达启动子OsESP1、loxP-Nos-loxP和Nos终止子，所述Cre重组酶由花粉特异表达启动子PG47携带并连接Nos终止子。
在第二方面，本发明提供了核酸，所述核酸可以是DNA，也可以是RNA，优选是DNA。
在第三方面，本发明第二方面的核酸可被构建成表达盒插入转化进细胞中的任何载体中。扩增上述核酸片段的全长或任意片段的引物对也是本发明保护的范围。含有第二方面核酸片段的重组载体、表达盒、转基因细胞系或重组菌也是本发明的保护范围。优选的细胞是植物细胞，优选是水稻细胞，更优选是杂交水稻细胞，所述植物细胞中包含的本发明第二方面的核酸可以是通过转基因技术(如本发明具体实施方式所述的技术)导入植物细胞的，包括导入到植物细胞的核、叶绿体、线粒体和/或质体中，也可以是通过其它技术使得本发明第二方面的核酸存在于植物细胞中的。
在第四方面，本发明提供了前述各表达盒在固定植物杂种优势中的应用。卵细胞特异表达启动子驱动的胚自主发生基因表达盒E1，可以使含卵细胞孤雌生殖产生固定了杂种优势的无性胚；受E3调控的筛选标记基因表达盒E2和花粉特异性调控E2的表达盒E3，可以使经过孤雌生殖产生的无性胚种子和授粉授精形成的合子胚种子得以分选。克隆种子保留了杂种优势可用于生产，其余种子可用于商用。
在第五方面，本发明提供了鉴定本发明第四方面的植物的方法，即测定所述植物是否含有第二方面所述的核酸。通过该方法，可以判断植物是否属于本发明的植物，即是否属于本发明第四方面的植物。测定的步骤可以通过常规的核酸检测和测序，示例性的方法包括核酸测序、聚合酶链式反应(PCR)检测、筛选标记观察、荧光定量分选、探针杂交检测、序列测定和流式细胞分析等。
与现有技术相比，本发明的有益效果如下：
本发明取得的有益效果在于利用花粉特异性基因开关系统调控筛选标记基因，进而分选克隆种子，从而实现杂交种无融合体系的应用。本发明将卵细胞特异表达启动子驱动的胚自主发生表达盒E1可以使卵细胞孤雌生殖形成胚，或通过单倍体诱导基因OsPLA、MATRILINEAL(MTL)、NOT LIKE DAD(NLD)等的突变体获得无性胚，筛选标记基因表达盒E2和受花粉特异表达启动子驱动的表达盒E3构成条件性基因敲除系统，可以控制筛选标记基因的特定表达。将这3个表达盒转入MiMe突变体，转 基因后代中除了卵细胞经过孤雌生殖产生的克隆种子外，还有经过正常授粉受精产生的合子胚种子。通过条件性基因敲除系统使通授粉受精产生的合子胚中含有红色荧光，通过光电分选，可高效分选出不含红色荧光的克隆种子，用于繁殖，含有红色荧光的合子胚种子可以用于商用。这种无融合生殖方法可以在不影响种子产量的前提下产生并分选出固定了杂种优势的克隆种子，减除杂交种子的复杂制种程序，减轻劳动强度，降低杂交种子的生产成本，并达到杂种优势利用一系法的产业化要求，整体提高杂交种业的效益。本发明在农业领域将具有广阔的应用和市场前景。
附图说明
图1是Ep30TLK和Ep30TL平板荧光情况
图2是Ep30TLK和Ep30TLEP管中荧光情况
图3是p26LK1绿色荧光结果及其红色荧光对照
图4是p26LK2绿色荧光结果及其红色荧光对照
图5是本发明的无融合生殖载体p24DB结构示意图
具体实施方式
以下将结合实施例来详细说明本发明。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
实施例1：花粉特异基因开关系统的建立
1.基因开关系统原核表达载体的构建和表达检测
1.1基因开关系统原核表达载体的构建
(1)基因锁Ep30TL的构建
合成序列：EcoRI+LoxP+spacer+FRT+Nos+LoxP+spacer+FRT+eGFP+SacI，将该序列通过EcoRI和SacI构建在载体pET-21a(+)(amp抗性)，命名为p30TL，再转化大肠杆菌BL21，命名为Ep30TL。p30TL序列见SEQ ID NO.1。
(2)基因钥匙p30TK的构建
合成序列:EcoRI+FLP+Nos+SacI，将该序列通过EcoRI和SacI构建于载体pRSFDuet-1(kana抗性)，命名为p30TK。p30TK序列见SEQ ID NO.2。
(3)Ep30TLK的构建
将p30TK转化到Ep30TL后，命名为Ep30TLK。
(4)制备Ep30TL电击感受态
(5)将p30TK电击转化到Ep30TL感受态中(Ep30TLK)
电击完成并复苏后，取适量(一般为100-200μL)的转化产物涂布在含有氨苄+卡纳抗性的LB固体培养基上；取Ep30TL涂布在氨苄抗性LB固体培养基上，验证所制备的Ep30TL感受态活性。将培养皿倒置在37℃培养内，过夜培养。12-16h后观察菌落生长情况。
1.2基因开关系统原核表达检测
从37℃烘箱中取出转化后的LB固体平板，在荧光体式显微镜观察菌落荧光情况。只涂布了Ep30TL感受态的氨苄抗性LB平板若长满了白色单菌落，证明Ep30TL感受态制备成功；在涂布有转化产物的氨苄+卡纳抗性的LB平板上，若看到大量绿色荧光单菌落，证明FLP重组酶系统作为基因开关在原核中的表达成功，结果附图1所示。
挑取绿色荧光单菌落于LB+Kana+Amp液体培养基摇菌过夜，将扩繁完毕的菌液提取质粒，一部分送公司进行序列检测，检测Ep30TLK中同时含有p30TL和p30TK两个载体。在p30TL和p30TK上分别设置一对引物进行检测，引物序列如下。另一部分取1mL放置在1.5mL EP管中，在500rpm/min离心1min后，弃去上清液，将沉淀用于观察绿色荧光情况，并用Ep30TL菌液作为对照，结果如附图2所示，在自然光条件下，Ep30TLK的绿色肉眼可见；在手持荧光眼镜的观察下，Ep30TLK发出绿色荧光，对照Ep30TL无绿色荧光。
正向测序引物(Ep30TL)：5’CCGGATATAGTTCCTCCTTTC 3’
反向测序引物(Ep30TL)：5’AGATCTCGATCCCGCGAAAT 3’
正向测序引物(p30TK)：5’CTTTCATCAATTGTGGAAGA 3’
反向测序引物(p30TK)：5’GTATATGTGCCTACTAACGC 3’
2.1基因开关系统真核表达载体的构建
合成序列seq26LK1，长度6090bp。其序列见SEQ ID NO.3。将序列seq26LK1通过HindIII/HindIII连接到载体pLJ02，目的片段与Ubi promoter的方向一致，命名p26LK1。
合成序列seq26LK2，长度6945bp。其序列见SEQ ID NO.4。将序列seq26LK2通过HindIII/HindIII连接到载体pLJ02，目的片段与Ubi promoter的方向一致，命名p26LK2。
2.2基因开关系统真核表达载体的表达检测
2.2.1.质粒抽提
载体p26LK1、p26LK2划板，挑单菌落摇菌1L，大提质粒备用；
2.2.2花粉的收集和处理
早上8～9点选取刚扬花或即将扬花的单株水稻，用套袋的方式收集散粉的花药，将收集的花粉抖落在花粉固体培养基上(100g/L蔗糖+20mg/L硼酸+40mg/L氯化钙)；
2.2.3基因枪转化
将制备好的花粉材料放入基因枪进行轰击；
2.2.4观察瞬时表达情况
轰击完成后，将培养皿置于27℃暗室培养，第2天开始在荧光显微镜下观察荧光的瞬时表达并拍照。荧光表达结果详见附图3和附图4。
实施例2：携带胚自主发生基因、荧光筛选标记基因和调控筛选标记基因表达盒3个连锁基因表达盒的植物表达载体构建
1.特异启动子和目标基因的选择
通过查阅文献和网页搜索，确定1个卵细胞特异表达启动子AtDD45Pro，其核苷酸序列见SEQ ID NO.5；确定1个胚胎发生基因BBM1，其核苷酸序列见SEQ ID NO.6；确定1个胚特异表达启动子OsESP1，其核苷酸序列见SEQ ID NO.7；确定红色荧光基因RFP为筛选标记基因，其核苷酸序列见SEQ ID NO.8；确定1个花粉特异表达启动子，其核苷酸序列见SEQ ID NO.9。
2.无融合生殖载体p24DB的构建
分别在PAIR1、REC8、OSD1设计1个靶标，
5′-GCGCTCGCCGACCCCTCGGG-3′；
5′-GGTGAG GAGGTTGTCGTCGA-3′；
5′-GTGTGGCGATCGTGTACGAG-3′。
构建MiMe敲除载体。在敲除载体上插入3个连锁表达盒：胚自主发生基因BBM1表达盒、荧光筛选标记基因RP表达盒和Cre酶开关调控表达盒。胚自主发生基因BBM1携带卵细胞特异表达启动子AtDD45和Nos终止子，其核苷酸序列见SEQ ID NO.10；载体pLJ02上的RP编码序列携带胚特异表达启动子OsESP1、loxP-Nos-loxP和Nos终止子；花粉特异表达启动子PG47携带Cre重组酶，其核苷酸序列见SEQ ID NO.11和Nos终止子。完整的载体构建图谱见附图5。
3.重组质粒转化
(1)取一管200μL大肠杆菌感受态细胞DH5a与5μL连接产物混合，冰浴30min；
(2)迅速置于42℃恒温水浴锅中，热激90s，冰浴2min；
(3)加入500μL LB液体培养基，混匀；
(4)37℃、200rpm,培养45min，使细胞恢复正常生长状态；
(5)将菌液均匀涂布于LB固体培养基平板上；
(6)30min后，置37℃恒温培养箱，过夜培养。
(7)挑取正确的单克隆接菌，抽提质粒，酶切验证。
4.重组菌的获得
将构建正确的植物表达载体使用电激法转化农杆菌EHA105。导入方法采用电激转化方法，主要参考bio-rad公司的电击仪使用说明书进行，具体步骤如下：
将储存于-80℃的EHA105感受态细胞取出放在冰上冻融，1mm电击杯放在冰上预冷，把冻存的SOC放置在37℃解冻。将干净的EP离心管插在冰上预冷，一般EP离心管的数目比要转化的样品多两个，一个用来做阴性对照(没有加入DNA)、另一个做阳性对照(加入1μl的10ng/μl pUC19)。分别吸取1μl要转化的DNA样品放入预冷的EP离心管中，然后将融化的EHA105感受态细胞20μl轻轻取出放入预冷的离心管底部，轻轻的将两者混匀，尽量不要产生气泡，离心管底部不要用手接触，避免温度变化对感受态转化效率造成影响，操作过程尽量的快。设置转化参数，电阻200Ω，电容25μF，电压1800V，电激杯规格1mm，BioRad电激仪一般有推荐使用的参数。轻轻吸取感受态和DNA 混合物，放入电激杯中，轻敲使混合物均匀的分布于杯底。盖上盖子放入电激槽中，关上安全盖，按下电激红色按钮，待电激完成后，将在37℃预热好的SOC放入电激杯中，将混合物旋起，用吸头将混合物转入摇菌管中，于28℃恒温摇床，180rpm,2h。取50μl菌液涂布在含卡那霉素(50μg/mL)和利福平(25μg/mL)的LB固体培养基上，28℃暗培养2d，挑取农杆菌转化子单菌落，接种到添加了相同抗生素的LB液体培养基中，28℃条件下，振荡培养2d。取适量菌液加入等体积的50％浓度的无菌甘油混合，于-80℃条件下保存、备用。
实施例3：转基因植株的获得
挑选植物表达载体p24DB农杆菌单菌落接种于含50mg/L kanamycin的LB培养基上26℃暗培养2天后，用NB-AS液体培养基洗下农杆菌菌体，28℃180rpm液体振荡培养90-120min。调整菌落浓度至OD600为0.8-1.0，转化杂交稻和杂交小麦，杂交水稻种子消毒，挑选子粒饱满的种子，用75％的酒精浸泡30s，倒掉酒精，无菌水冲洗一遍，用HgCl2消毒8min，无菌水清洗2遍，每次浸泡1min，用无菌水浸泡1h。消毒后的种子接种到诱导培养基上，光照下生长7d。
将无菌愈伤集中在一起。放入农杆菌悬浮液中，浸泡5-10min，取出，用滤纸晾干。接种到共培养培养基，共培养2天，将共培养的愈伤，清洗6遍，并用滤纸晾干后接种到具有潮霉素抗性的筛选培养基上45天。
将抗性愈伤转移到分化培养基上，培养2周后愈伤开始转绿，3周后即可长出幼芽，随之根也长出。将幼苗转移到含生根培养基的小三角瓶内，每瓶一株，继续光照培养，待小植株长至7-10cm左右时进行室内炼苗，3～4d后将其移栽到土壤中生长。通过观察荧光和PCR检测筛选出阳性植株。
实施例4：转基因植株的分子检测
DNA抽提：取1.0g叶片，液氮研磨至粉末状，转入2mlEP管中，加入700μl预热的CTAB溶液。于65℃水浴30-60min，期间轻轻混匀，冷却后加入等体积氯仿：异戊醇(24:1)，混匀，12000rpm离心10min,取上清至新的离心管中，加入500μl异丙醇，-20℃静置30-60min。4℃，12000rpm,10min收集沉淀，弃上清。70％乙醇清洗沉淀2次，吹干酒精残留，50-100μl ddH 2O溶解沉淀，备用。
PCR分析：以RP基因作为模板设计引物，扩增产物片段为431bp。
正向引物：
F:5'CCCAGTTCCAGTACGGCTCCAAG 3'；
反向引物：
R:5'CTCGTTGTGGGAGGTGATGTCCAG 3'。
PCR反应体系：DNA 30-90ng,10×Buffer 2.0μl,1mM dNTP 1.8μl,25mM MgCl 2 1.5μl,10uM primer两种各0.5μl,Tag酶1.5U，加ddH 2O至20μl反应体积。PCR循环条件94℃，3min；94℃,1min，64℃,1.5min，72℃30s，40个循环；72℃，延伸5min。用1.4％琼脂糖凝胶电泳检测，照相记录电泳结果。
荧光定量分析：使用植物总RNA提取试剂盒(天根DP432)对T0代转基因杂交稻和野生型杂交稻对照进行RNA抽提，方法参考试剂盒说明书。以总RNA为模板逆转录合成相应的cDNA。以Actin为内参基因，野生型植株为对照，用RT-PCR的方法对RP基因进行定量PCR检测。
Southern印迹分析：取水稻和小麦总DNA进行EcoR Ⅰ酶切、电泳、转移至硝酸纤维素膜Hybond-N。利用随机引物标记试剂盒(Promega公司)和[α-32P]dATP(北京亚辉公司)，随机引物法制备α-32P标记的RP基因片段，作为分子探针，按照Sambrook等分子克隆方法进行转化植株的Southern杂交分析。
实施例5：转基因植株的农艺性状考察
对经过分子鉴定的转基因植株考察花粉育性和结实情况。取开花时的花药，经常规的I2-KI染色后，观察花粉不育花粉和可育花粉比率。与野生型对照比较，观察转基因植株的株高、分蘖数、剑叶长、结实率、千粒重等农艺性。
实施例6：杂交植物的无融合生殖繁殖
将携带无融合生殖载体的杂交水稻和杂交小麦自交。自交种子通过对红色荧光的光电分选，可以筛选出具有红色荧光的种子，获得具有红色荧光的种子为合子胚种子用于商用；而不具有红色荧光的种子为克隆种子，继续用于杂交水稻和杂交小麦的繁殖。
上述实施例阐明的内容应当理解为这些实施例仅用于更清楚地说明本发明，而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落入本申请所附权利要求所限定的范围。
Figure PCTCN2020084695-appb-000001
Figure PCTCN2020084695-appb-000002
Figure PCTCN2020084695-appb-000003
Figure PCTCN2020084695-appb-000004
Figure PCTCN2020084695-appb-000005
Figure PCTCN2020084695-appb-000006
Figure PCTCN2020084695-appb-000007
Figure PCTCN2020084695-appb-000008
Figure PCTCN2020084695-appb-000009

Claims (4)

  1. 一种固定植物杂种优势的种子分选方法，其特征在于，其包括如下步骤：
    A、构建胚自主发生基因表达盒E1；
    B、构建受E3调控的筛选标记基因表达盒E2；
    C、构建花粉特异性调控E2的表达盒E3；
    D、创制MiMe突变体；
    E、创制转基因植物，转入上述E1、E2、E3 3个连锁表达盒和携带MiMe突变位点；
    所述E3和E2构成一个基因开关系统，E3的花粉特异性表达可以切除E2中与启动子相连的终止子，使其所驱动的筛选标记基因得以表达，孤雌生殖产生的固定了杂种优势的无性胚种子和授粉受精形成的合子胚种子可以通过筛选标记基因分选，从而获得达到种子纯度要求的孤雌生殖种子，使一系法杂种优势固定达到生产应用的要求；
    所述植物为单子叶植物或双子叶植物；
    所述步骤B中E2的启动子连有一个终止子，致使该启动子所驱动的筛选标记基因不能表达，终止子侧翼携带E3的识别序列；所述E2的启动子为胚特异性表达启动子，包括但不限于Lec1、OsESP1、ESD4Like5、pBnaA09g21960D；所述E2的筛选标记基因包括但不限于荧光基因；
    所述步骤C中E3的花粉特异性基因开关表达可以切除E2中与启动子相连的终止子，使筛选标记基因得以表达；所述E3的调控系统包括但不限于双链断裂诱导酶系统、基因组编辑系统、重组酶系统、转座子系统，也包括不同调控系统的组合和联用；所述E3中花粉特异表达启动子包括但不限于PG47；
    所述步骤D中MiMe突变体包括但不限于PAIR1、REC8、OSD1的突变。
  2. 如权利要求1所述的方法，其特征在于，所述步骤A中胚自主发生的途径包括但不限于卵细胞特异表达启动子驱动的胚自主发生基因的表达或OsPLA、MATRILINEAL、NOT LIKE DAD的突变体，所述E1的胚自主发生基因包括但不限于BBM1、WUS、LEC、CLAVATA、MYB115，所述E1的卵细胞特异表达启动子包括但不限于AtDD45、Os03g0296600 pro、ECA1-like1 pro、DCL2、AT1G74480.1、ZmEAlpromoter。
  3. 一种载体，所述载体敲除MiMe，并插入3个连锁表达盒：胚自主发生基因BBM1表达盒、荧光筛选标记基因RFP表达盒和Cre重组酶开关调控表达盒；所述胚自主发生基因BBM1表达盒中BBM1 的核苷酸序列见SEQ ID NO.6；所述荧光筛选标记基因RFP表达盒中RFP的核苷酸序列见SEQ ID NO.8；所述Cre重组酶开关调控表达盒中Cre重组酶的核苷酸序列见SEQ ID NO.11。
    所述敲除MiMe是指对PAIR1、REC8、OSD1基因的敲除；
    所述胚自主发生基因BBM1携带卵细胞特异表达启动子AtDD45和Nos终止子，所述REP携带胚特异表达启动子OsESP1、loxP-Nos-loxP和Nos终止子，所述Cre重组酶由花粉特异表达启动子PG47携带并连接Nos终止子。
  4. 如权利要求1-2任一项所述的方法、权利要求3所述的载体在固定杂种优势、克隆种子方面的应用。
PCT/CN2020/084695 2019-04-17 2020-04-14 一种固定植物杂种优势的种子分选方法 WO2020211747A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910307717.3A CN110093354B (zh) 2019-04-17 2019-04-17 一种固定植物杂种优势的种子分选方法
CN201910307717.3 2019-04-17

Publications (1)

Publication Number Publication Date
WO2020211747A1 true WO2020211747A1 (zh) 2020-10-22

Family

ID=67445055

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/084695 WO2020211747A1 (zh) 2019-04-17 2020-04-14 一种固定植物杂种优势的种子分选方法

Country Status (2)

Country Link
CN (1) CN110093354B (zh)
WO (1) WO2020211747A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110093354B (zh) * 2019-04-17 2020-06-05 湖南杂交水稻研究中心 一种固定植物杂种优势的种子分选方法
CN111118056B (zh) * 2020-01-07 2023-05-09 湖南杂交水稻研究中心 一种实现水稻杂种优势固定的方法
CN114181965B (zh) * 2020-09-14 2024-07-09 湖南杂交水稻研究中心 核酸分子、载体、细胞和引物及其应用以及基于双重调控的植物高纯度克隆种子分选方法
CN114507671B (zh) * 2020-11-16 2024-08-09 湖南杂交水稻研究中心 核酸分子、载体和细胞及其应用及通过无性胚和无胚种子高效筛选植物无融合生殖克隆种子
CN112779283B (zh) * 2021-01-22 2025-01-03 湖南杂交水稻研究中心 核酸分子、载体和细胞及其应用及基于父系印记基因的植物无融合生殖克隆种子的筛选方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1301300A (zh) * 1998-02-20 2001-06-27 曾尼卡有限公司 花粉特异性启动子
CN101194018A (zh) * 2005-02-02 2008-06-04 英特拉克森公司 位点特异性丝氨酸重组酶和它们的使用方法
CN102905517A (zh) * 2010-04-23 2013-01-30 先锋国际良种公司 基因开关组合物及使用方法
CN102972285A (zh) * 2012-12-05 2013-03-20 湖南杂交水稻研究中心 一种杂交作物的育种方法
CN104082128A (zh) * 2014-07-25 2014-10-08 湖南杂交水稻研究中心 一种水稻智能不育系的繁殖方法
WO2016048909A1 (en) * 2014-09-22 2016-03-31 Pioneer Hi-Bred International, Inc. Methods for reproducing plants asexually and compositions thereof
CN109072178A (zh) * 2015-11-18 2018-12-21 先正达参股股份有限公司 单倍体诱导组合物及其使用方法
CN110093354A (zh) * 2019-04-17 2019-08-06 湖南杂交水稻研究中心 一种固定植物杂种优势的种子分选方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2014008243A (es) * 2012-01-06 2015-02-20 Pioner Hi Bred International Inc Un metodo para seleccionar plantas por elementos geneticos que inducen la partenogenesis en plantas.
WO2014154141A1 (zh) * 2013-03-29 2014-10-02 湖南杂交水稻研究中心 一种利用雌性不育的杂交稻机械化制种方法
CN108220333B (zh) * 2018-03-28 2021-01-05 中国农业科学院作物科学研究所 一种高效植物受体孤雌生殖单倍体筛选方法
CN109486853A (zh) * 2018-11-21 2019-03-19 湖南杂交水稻研究中心 一种利用基因组编辑技术快速创制适合机械化制种的工程雌性不育系的方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1301300A (zh) * 1998-02-20 2001-06-27 曾尼卡有限公司 花粉特异性启动子
CN101194018A (zh) * 2005-02-02 2008-06-04 英特拉克森公司 位点特异性丝氨酸重组酶和它们的使用方法
CN102905517A (zh) * 2010-04-23 2013-01-30 先锋国际良种公司 基因开关组合物及使用方法
CN102972285A (zh) * 2012-12-05 2013-03-20 湖南杂交水稻研究中心 一种杂交作物的育种方法
CN104082128A (zh) * 2014-07-25 2014-10-08 湖南杂交水稻研究中心 一种水稻智能不育系的繁殖方法
WO2016048909A1 (en) * 2014-09-22 2016-03-31 Pioneer Hi-Bred International, Inc. Methods for reproducing plants asexually and compositions thereof
CN109072178A (zh) * 2015-11-18 2018-12-21 先正达参股股份有限公司 单倍体诱导组合物及其使用方法
CN110093354A (zh) * 2019-04-17 2019-08-06 湖南杂交水稻研究中心 一种固定植物杂种优势的种子分选方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN H ET AL.: "Application of Cre-lox Gene Switch to Limit the Cry Expression in Rice Green Tissues", SCIENTIFIC REPORTS, vol. 7, no. 1,, 6 November 2017 (2017-11-06), XP055746162, ISSN: 2045-2322, DOI: 20200703101523 *
KHANDAY I ET AL.: "A Male-Expressed Rice Embryogenic Trigger Redirected for Asexual Propagation Through Seeds", NATURE, vol. 565, no. 7737, 1 February 1999 (1999-02-01), ISSN: 1476-4687, DOI: 20200706094126 *
WANG C ET AL.: "Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes", NATURE BIOTECHNOLOGY, vol. 37, no. 3, 31 March 2019 (2019-03-31), XP036900604, ISSN: 1546-1696, DOI: 20200706083816 *
WANG Y ET AL.: "Engineering stable heterosis", JOURNAL OF GENETICS AND GENOMICS, vol. 46, no. 1,, 20 January 2019 (2019-01-20), XP085616542, ISSN: 1673-8527, DOI: 20200703102152 *
XIE E ET AL.: "A Strategy for Generating Rice Apomixis by Gene Editing", JOURNAL OF INTEGRATIVE PLANT BIOLOGY, vol. (61), no. 8, 30 January 2019 (2019-01-30), XP055746158, ISSN: 1744-7909, DOI: 20200703101827 *

Also Published As

Publication number Publication date
CN110093354A (zh) 2019-08-06
CN110093354B (zh) 2020-06-05

Similar Documents

Publication Publication Date Title
US20220411810A1 (en) Method for conducting site-specific modification on entire plant via gene transient expression
WO2020211747A1 (zh) 一种固定植物杂种优势的种子分选方法
US20200216853A1 (en) Method for rapidly and efficiently creating directed gene mutated non-transgenic plants and its applications
Chumakov et al. Agrobacterium-mediated in planta transformation of maize via pistil filaments
CN110467658B (zh) 黄瓜CsGL2-LIKE基因及其参与调控雄花部分败育中的应用
CN117737078A (zh) MADS-box基因RhAGL6及其在调节月季花器官发育中的应用
CN108588120A (zh) 一种玉米农杆菌转化受体的制备方法及农杆菌介导的玉米转化方法
CN107858370B (zh) 一种制备育性减低植物的方法
CN110029126B (zh) 一种固定植物杂种优势的方法
CN112094862B (zh) 一种带有标记的棉花雄性不育系的创制方法
CN111690679A (zh) 一种用于培育黄瓜雄性不育系的重组表达载体及其构建方法和应用
CN114946639B (zh) 两种小麦诱导系在小麦单倍体幼胚及籽粒鉴别中的应用
CN110204600A (zh) BnSPL14基因、蛋白及其在控制甘蓝型油菜株型中的应用
CN114763555B (zh) 利用基因编辑实现高产优质育种的方法及试剂
CN114181965B (zh) 核酸分子、载体、细胞和引物及其应用以及基于双重调控的植物高纯度克隆种子分选方法
CN109879945B (zh) 甘蓝型油菜抗裂角基因BnIND的功能及应用
CN115011607A (zh) 芝麻育性调控基因及其表达载体和应用
CN112779283A (zh) 核酸分子、载体和细胞及其应用及基于父系印记基因的植物无融合生殖克隆种子的筛选方法
CN110904120B (zh) 一种胚胎发育调控基因drr1及其编码的蛋白质和应用
CN117305326B (zh) 青花菜BoCENH3基因及其在单倍体诱导中的应用
US20240140998A1 (en) Method for inducing embryogenesis of seed plant without fertilization, protein used therein, nucleic acid, vector, and recombinant seed plant in which embryo can be generated without fertilization
CN113604485B (zh) 拟南芥AtGSNOR基因、蛋白及应用
Junhao et al. High-frequency of twin-embryo seeds with clonal and sexually produced embryos drives apomixis in hybrid ric
CN117887756A (zh) 一种提高植物无融合生殖诱导率的方法
CN107099534A (zh) 一种在植物生长的特定时期内表达的水稻种子特异性启动子

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20791155

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20791155

Country of ref document: EP

Kind code of ref document: A1