WO2022042619A1 - 一种植物花粉管生长基因及应用 - Google Patents

一种植物花粉管生长基因及应用 Download PDF

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WO2022042619A1
WO2022042619A1 PCT/CN2021/114625 CN2021114625W WO2022042619A1 WO 2022042619 A1 WO2022042619 A1 WO 2022042619A1 CN 2021114625 W CN2021114625 W CN 2021114625W WO 2022042619 A1 WO2022042619 A1 WO 2022042619A1
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osptd1
gene
seq
pollen tube
tube growth
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French (fr)
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张毅
陈云
杜双林
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云南大学
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    • 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
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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)
    • C12N15/8216—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

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  • the invention belongs to the field of genetic engineering, and relates to a rice pollen tube growth-related gene OsPTD1 and application thereof.
  • the abnormal function of the gene can produce male sterility of gametophytes, so that it can be used to change pollen fertility, control transgene drift, prepare male gametophyte sterility and reproduce Sporophyte male sterility, etc.
  • Plant male sterility plays an important role in pollen development research and heterosis utilization.
  • Male sterility includes sporophyte and gametophyte sterility.
  • the sporophyte sterility is controlled by the genotype of the plant itself, and all pollen in the anthers are sterile. In the field, the anthers are usually abnormal in shape, color, and size, and are not strong, so it is easy to find and identify; the sporophyte sterility gene can be obtained through Female gametes are passed from generation to generation, and can also be passed from generation to generation through male gametes.
  • the gametophyte sterility is controlled by the pollen genotype, half of the pollen in the anther is fertile, and the other half is sterile. There is no significant difference between the wild type and the wild type in the field. Gamete transmission.
  • gametophyte sterility Due to the above differences, the preparation, discovery and identification of gametophyte sterility is very difficult. It needs to be combined with indoor microscopic observation, PCR and electrophoresis. Objectively, gametophyte sterility materials are scarce, and relevant basic and applied research lags behind sporophyte. Infertility.
  • the environment-insensitive type in sporophytic nuclear sterility has great potential in the utilization of heterosis, but it is difficult to reproduce completely sterile groups by itself, and the sterile plant rate is 100%, which makes its application limited; While gametophytic male sterility can be used to reproduce sporophytic male sterility. Therefore, it is necessary to discover and verify some gametophytic male sterility-related genes, and to create some gametophytic male sterility materials.
  • Plant multi-copper oxidase including laccase and ascorbate oxidase, is involved in regulating plant growth and development, reproduction, senescence and response to environmental stress, and can regulate flowering period (Shen et al. 2009).
  • one of the objects of the present invention is to provide the gene OsPTD1 for controlling rice pollen tube development
  • the second object of the present invention is to provide the application of the rice pollen tube growth-related gene OsPTD1 in restoring the pollen fertility of mutants.
  • the present invention The third purpose of the invention is to provide an application of inhibiting the expression of the rice pollen tube growth-related gene OsPTD1 in the preparation of gametophytic male sterility; the fourth purpose of the present invention is to provide an application of inhibiting the expression of the rice pollen tube growth-related gene OsPTD1 to prepare gametophytic male sterility;
  • the fifth object of the present invention is to provide a method and application for utilizing the rice pollen tube growth-related gene OsPTD1 to down-regulate and express the reproductive sporophytic male sterility (the gene encoding amino acid sequence is an amino acid sequence such as SEQ ID NO.1, the gene The CDS nucleotide sequence is such as SEQ ID NO.2, and the genomic nucleotide sequence of this gene is such as SEQ ID NO.3).
  • the present invention provides the following technical solutions:
  • a rice pollen tube growth-related gene OsPTD1 the amino acid sequence encoded by the rice pollen tube growth-related gene is as shown in SEQ ID NO.1; or the amino acid sequence shown in SEQ ID NO.1 is substituted, deleted or added at least One amino acid and has the same amino acid sequence as the OsPTD1 protein function;
  • the CDS nucleotide sequence of the rice pollen tube growth-related gene OsPTD1 is shown in SEQ ID NO.2; or the sequence shown in SEQ ID NO.2 is substituted and deleted Or add at least one nucleotide and have the same nucleotide sequence as the rice pollen tube growth-related gene OsPTD1;
  • the genomic nucleotide sequence of the rice pollen tube growth-related gene OsPTD1 is shown in SEQ ID NO.3; or The sequence shown in SEQ ID NO.3 is substituted, deleted or added at least one nucleotide and has the same nucleotide sequence as the rice pollen tube growth-related gene Os
  • the method for down-regulating the expression of the rice pollen tube growth-related gene OsPTD1 is to interfere with the expression of the rice pollen tube growth-related gene OsPTD1.
  • the method for interfering with the expression of the rice pollen tube growth-related gene OsPTD1 is to construct a vector for interfering with the expression of the rice pollen tube growth-related gene OsPTD1, and then transfect the plants through Agrobacterium-mediated transfection, and then screen the transgenic positive plants.
  • the expression vector contains an interference stem loop, an interference left arm and an interference right arm, and the sequence of the interference stem loop is the interference stem loop shown in SEQ ID NO. 8; the interference left arm is represented by SEQ ID NO. 27 and the sequences shown in SEQ ID NO.28 are primers, amplified from Zhonghua 11; the interference right arm is primers with the sequences shown in SEQ ID NO.29 and SEQ ID NO.30, amplified from Zhonghua 11. increase gain.
  • a method for preparing gametophyte male sterility by down-regulating the expression of the rice pollen tube growth-related gene OsPTD1, comprising the following steps:
  • the down-regulated element Xi of the pollen-specific promoter regulating rice pollen tube growth-related gene OsPTD1 was transfected into plants through Agrobacterium-mediated transfection, and the transgenic positive strains were screened.
  • the transgenic positive strains were gametophytic male sterility.
  • a method for preparing sporophyte nuclear male sterility by down-regulating the expression of the rice pollen tube growth-related gene OsPTD1, comprising the following steps: linking the sporophytic male fertility gene Y with the OsPTD1 gene interference fragment regulated by the promoter POsPTD1, and then transferring it into the sporophyte
  • For the sterile mutant of the male fertile gene Y screen the transgenic positive plants, and use the positive plant as the male parent to pollinate the sterile mutant of the sporophytic male fertile gene Y, and the sporophytic nuclear male sterility can be propagated.
  • the sporophytic male fertile gene Y can be any sporophytic male fertile gene, such as the sporophytic male fertile gene OsABCG15, and the sterile mutant of the sporophytic male fertile gene Y is Zhongjiu B-osabcg15 : Taking the pollen-free male sterile mutant of OsABCG15 gene as the non-recurrent female parent, the sterile Zhongjiu B-osabcg15 was obtained by backcrossing. Other sporophyte male fertility genes can also be selected.
  • the promoters, interference fragments, terminators, species and varieties to be transformed can be flexibly selected in the present invention; spore sports genes can be flexibly selected; gene expression inhibition methods can be interference, antisense and miRNA etc.; the loading sequence and relative position of various segments can be adjusted according to specific conditions.
  • the carrier names used in the method statement are only used for convenience of presentation, and do not limit the use of other symbols to represent related carriers in practical applications. Therefore, the actual use of the above optional contents does not indicate any substantial difference from the present invention, and thus does not affect the protection of the present invention.
  • OsPTD1 is an endogenous gene of rice, and by down-regulating it, it is possible to produce gametophyte sterility without resorting to exogenous genes or fragments, thereby cultivating plant varieties with transgenic components that do not drift with pollen (if in If this method is used in other species, the gametophyte sterility-related genes in the target species can be selected); it can also be used to reproduce sporophyte nuclear sterility to solve the reproductive problem of sporophyte sterility, thereby facilitating the free selection of male parents and making full use of Heterosis; the method for preparing and identifying male sterility of gametophytes has the advantages of intuition, simplicity, accuracy, time saving, economy and the like.
  • Figure 1 shows the expression profiling analysis of OsPTD1 gene (A is RT-qPCR analysis of OsPTD1 gene expression in wild-type organs and flower development stages and at the later stage of flower development of interference homozygous progeny; B is promoter POsPTD1 driving GUS gene in pollen GUS staining detection of specific expression, showing GUS staining of transgenic hybrid pollen; C is the GUS staining detection of specific expression of GUS gene in pollen driven by promoter POsPTD1, showing GUS staining of transgenic homozygous pollen);
  • Figure 2 is the electrophoresis diagram of RACE analysis of OsPTD1 gene and full-length cDNA amplification (A and B are 3 rounds of amplification at the 5 and 3 ends, respectively, and C is full-length cDNA amplification);
  • Fig. 3 is a vector transformation map
  • Figure 4 is a graph of pollen germination (A is wild-type pollen, B is interference T 0 generation pollen (about half of the pollen ruptured in advance));
  • Figure 5 is a photo of the transmission of the purple line in F 1 of the transgenic T 0 generation positive strain.
  • various vectors known in the art can be selected, such as commercially available vectors, including plasmids, cosmids, and the like.
  • the full-length nucleotide sequences or fragments thereof can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequences disclosed in this example, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art, or genomic DNA as template, and the related sequences are amplified. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • the rice Loc_Os05g40740 gene is specifically highly expressed in late flowers, and its Arabidopsis homologous gene is specifically expressed in pollen.
  • the Loc_Os05g40740 gene was initially listed as a male gametophyte development-related gene, named OsPTD1, and the amino acid sequence is as SEQ ID NO. 1, the nucleotide sequence is shown in SEQ ID NO.2, and the genome sequence is shown in SEQ ID NO.3.
  • the spatiotemporal expression pattern of this gene was analyzed by RT-qPCR.
  • the qPCR primers of the OsPTD1 gene used were: upstream primer MQF2: 5'-ggcgtcttcaggtacaaccag-3' (SEQ ID NO. 9), downstream primer MQR25'-ggtacacctggaccgagtgc-3 ' (SEQ ID NO. 10).
  • the internal reference primers used were: upstream primer OsActin2F: 5'-ctctgtatgccagtggtcgt-3' (SEQ ID NO.11), downstream primer OsActin2R: 5'-ccgttgtggtgaatgagtaac-3' (SEQ ID NO.12).
  • qPCR results showed that OsPTD1 gene was not expressed in roots, stems, leaves, sheaths and seeds, and was highly expressed in flowers in the middle and late stages of development (Fig. 1). Further analysis of the expression of the gene in the later stage of flower development of the interference homozygous plants found that the expression of the gene was severely reduced by the interference.
  • the promoter of this gene was cloned, placed in front of the GUS gene of pCAMBIA1301, and investigated by transgene and GUS staining.
  • the specific operations are:
  • promoter upstream primer P5F1 5'-gcgtcgacgtcccatgtcaccgacagtact-3' (SEQ ID NO. 13, with restriction site SalI) and downstream primer P5R1: 5'-catgccatggcgtggaaatgtgatcgctaggct-3' (SEQ ID NO. 14, with restriction enzyme cut
  • the site NcoI expands the promoter of Loc_Os05g40740 (referred to as POsPTD1) from Zhonghua 11, and its nucleotide sequence is shown in SEQ ID NO.6. It was loaded in front of the GUS gene of vector pCAMBIA1301 with SalI and NcoI (incomplete digestion) to obtain pPOsPTD1-GUS.
  • the pPOsPTD1-GUS was transformed into Agrobacterium LBA4404, and then the positive Agrobacterium was transfected into Zhonghua 11. GUS staining was performed on the roots, leaves and flowers of the transgenic positive progeny at the flowering stage. The results showed that the roots, leaves, glumes, filaments and pistils were not stained, but the anthers were stained blue. The anthers were further crushed and observed under a microscope: the GUS-stained pollen of the transgenic heterozygous plants was only about 50%, and the pollen of the homozygous plants was almost all stained by GUS (Fig. 1). The above results indicate that the promoter POsPTD1 is indeed a pollen-specific promoter.
  • the RACE kit of Bao Bio was used for analysis: the reverse transcription product of total RNA of rice anther was used as the template, the universal primers provided by the kit and the 5'RACE primers and 3'RACE primers designed by our were used to carry out the analysis.
  • Three rounds of PCR amplification ( Figure 2), the product of the last round was recovered and connected to the pMD18-T vector, and the 3'UTR, 5'UTR and CDS of the OsPTD1 gene cDNA were finally determined after PCR detection of bacterial liquid and sample sequencing.
  • the 5'UTR of this gene is 201bp
  • the 3'UTR is 261bp
  • the full length of CDS is 1671bp
  • the full length of cDNA is 2338bp.
  • the 5'RACE specific primers are shown in SEQ ID NO.15-17 respectively
  • the 3'RACE specific primers are respectively shown in SEQ ID NO.18-20
  • the full-length amplification primers are shown in SEQ ID NO.21-22.
  • the POsPTD1 promoter was used to drive the interference fragment of the OsPTD1 gene, and the creation and identification of male gametophyte sterility were performed:
  • Vector modification In order to facilitate the loading of relevant target genes or DNA fragments, in order to accurately and directly identify transgenic positive strains and male gametophyte sterility, the modified gene OsMYB76R of rice anthocyanin synthesis-related genes was used to replace the GUS gene, and The multiple cloning site of the vector pCAMBIA1301 has been transformed, and the specific operations are as follows:
  • OsMYB76 gene modification and replacement of GUS OsMYB76 gene is an essential gene for anthocyanin synthesis in rice and can be used as an endogenous reporter gene.
  • the common restriction sites in the OsMYB76 gene were removed by base substitution, and the CDS sequence of the OsMYB76R gene modified by the OsMYB76 gene was obtained and synthesized artificially. The sequence is shown in SEQ ID NO.4.
  • OsMYB76RF 5'-ggactcttgaccatgatgggacgcagggcttgct-3' (SEQ ID NO. 23, with a homologous fragment near BglII of pCAMBIA1301)
  • OsMYB76RR 5'-attcgagctggtcactcacgcacacaggttccaag-3' (SEQ ID NO.
  • upstream primer LF2 5'-gaagatctacgagctggtgagctagcta-3' (SEQ ID NO.25, with restriction site BglII) and downstream primer LR2: 5'-ctggtcacccctcaaacctgaaaattcag-3' (SEQ ID NO.26 , with an enzyme cleavage site BstEII), the first intron of the OsMYB76 gene was expanded from R25 as an interfering stem loop (SEQ ID NO. 8), and loaded into pOsMYB76RM to obtain the vector pOsMYB76RL.
  • SEQ ID NO. 8 interfering stem loop
  • the pXi-OsMYB76RM was transformed into Agrobacterium LBA4404, and then the positive Agrobacterium was transfected into the colorless rice variety ZhongjiuB in all organs.
  • OsPTD1 gene is related to the development of male gametophyte pollen. By interfering with it, and by means of the genetic investigation of linked purple line traits, the male gametophyte sterile material was obtained and the genetic identification was successfully carried out.
  • Example 4 Using the OsPTD1 gene to down-regulate the expression of nine B-osabcg15 in reproductive sporophyte nuclear male sterility
  • the nine B-osabcg15 in the sterile material produced by the OsABCG15 gene was used as the object, and the interferometric fragment of the OsPTD1 gene was linked to the OsABCG15 gene , carried out the breeding practice of Zhongjiu B-osabcg15, as follows:
  • Zhongjiu B-osabcg15 Male fertile, no purple
  • OsMYB76 abnormal OsMYB76 gene
  • a pollen-free male sterile mutant of OsABCG15 gene was used (Wu et al., 2014 ) is a non-recurrent female parent, and the non-purple and sterile Zhongjiu B-osabcg15 was obtained by backcrossing.
  • the pOsABCG15-Xi-OsMYB76RM was transferred into Zhongjiu B-osabcg15, and 11 positive strains were screened according to the presence or absence of purple. All the purple strains showed fertile pollen.
  • the mutant Zhongjiu B-osabcg15 was pollinated with the positive strain as the male parent, and a total of 429 F 1 seeds were obtained. After the hybrid seeds were sown and transplanted, a total of 386 flowering plants were obtained, of which 376 were colorless and sterile. There were 10 fertile plants, colorless and sterile co-separate, purple and fertile co-separation, and the sterile plant rate was 97.4%.
  • the method with the highest sterile plant rate of Nine B-osabcg15 in reproduction is sibling cross, the highest sterile plant rate is 50%, and the sterile plant rate of our method is over 95%. It is indicated that the method of the present invention can be used to reproduce sporophyte nuclear male sterility.

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Abstract

提供了一个水稻花粉管生长相关基因OsPTD1(Pollen Tube Development)及应用。该基因编码的蛋白为SEQ ID NO.1所示氨基酸序列，或经过取代、缺失或添加至少一个氨基酸且具有与OsPTD1蛋白功能相同的蛋白质；SEQ ID NO.1所示氨基酸序列组成的蛋白质，其核苷酸序列如SEQ ID NO.2所示。所述OsPTD1基因编码一个抗坏血酸氧化酶，为花粉管正常生长的必需基因，该基因功能的异常会导致花粉管提前破裂，不能完成授精，产生配子体雄性不育，可用于制备配子体雄性不育，在防止转基因随花粉漂移和繁殖孢子体雄性核不育等方面具有重要作用。

Description

一种植物花粉管生长基因及应用 技术领域
本发明属于基因工程领域，涉及一种水稻花粉管生长相关基因OsPTD1及应用，该基因功能异常会产生配子体雄性不育，从而可用来改变花粉育性，控制转基因漂移、制备雄配子体不育和繁殖孢子体雄性核不育等。
背景技术
植物雄性核不育在花粉发育研究和杂种优势利用中具有重要的作用。雄性不育包括孢子体不育和配子体不育。
孢子体不育受植株本身的基因型控制，花药中的所有花粉都不育，在田间通常表现花药形态、颜色、大小异常，不结实，很容易发现和鉴定；孢子体不育基因既可通过雌配子在世代间传递，也可通过雄配子在世代间传递。而配子体不育受花粉基因型控制，花药中一半花粉可育一半花粉不育，在田间同野生型比无明显差异，结实也正常，难以直接发现和鉴定；雄配子体不育基因只能通过雌配子传递。由于上述差异，使得配子体不育的制备、发现和鉴定非常困难，需要结合室内的显微观察、PCR和电泳等手段进行，客观上造成配子体不育材料稀少，相关基础和应用研究滞后于孢子体不育。孢子体核不育中的环境不敏感型在杂种优势利用中有极大潜力，但难以单靠自身繁殖出不育彻底，不育株率为100％的群体，使得其在应用中受到限制；而配子体雄性不育可用来繁殖孢子体雄性不育。因此，很有必要发现和验证一些配子体雄性不育相关基因，创制一些配子体雄性不育材料。
植物多铜氧化酶包括漆酶和抗坏血酸氧化酶，参与调控植物的生长发育、繁殖、衰老以及环境胁迫的响应，可调控花期(Shen et al.2009)。水稻中有10多个编码多铜氧化酶的基因，但它们的生物学功能很不清楚，从中找到配子体雄性不育相关基因并创制配子体雄性不育，将其用于繁殖孢子体不育以及减少转基因飘逸等具有重要意义。
发明内容
有鉴于此，本发明的目的之一在于提供控制水稻花粉管发育基因OsPTD1，本发明的目的之二在于提供所述水稻花粉管生长相关基因OsPTD1在恢复突变体花粉育性中的应用，本发明的目的之三在于提供抑制所述水稻花粉管生长相关基因OsPTD1表达在制备配子体雄性不育中的应用；本发明的目的之四在于提供抑制水稻花粉管生长相关基因OsPTD1基因表达制备配子体雄性不育的方法；本发明的目的之五在于提供利用水稻花粉管生长相关基因OsPTD1下调表达繁殖孢子体核雄性不育的方法及应用(该基因编码氨基酸序列为氨基酸序列如SEQ ID NO.1，该基因CDS核苷酸序列如SEQ ID NO.2，该基因的基因组核苷酸序列如SEQ ID NO.3)。
为达到上述目的，本发明提供如下技术方案：
1、一种水稻花粉管生长相关基因OsPTD1，所述水稻花粉管生长相关基因编码的氨基酸序列如SEQ ID NO.1所示；或者SEQ ID NO.1所示氨基酸序列经取代、缺失或添加至少一个氨基酸且具有与OsPTD1蛋白功能相同的氨基酸序列；所述水稻花粉管生长相关基因OsPTD1的CDS核苷酸序列如SEQ ID NO.2所示；或SEQ ID NO.2所示序列经取代、缺失或添加至少一个核苷酸且具有与水稻花粉管生长相关基因OsPTD1功能相同的核苷酸序列；所述水稻花粉管生长相关基因OsPTD1的基因组核苷酸序列如SEQ ID NO.3所示； 或SEQ ID NO.3所示序列经取代、缺失或添加至少一个核苷酸且具有与水稻花粉管生长相关基因OsPTD1功能相同的核苷酸序列。
2、所述水稻花粉管生长相关基因OsPTD1在恢复突变体花粉育性中的应用，所述突变体为OsPTD1基因或同源基因变异产生的花粉不育突变体。
3、下调或突变所述水稻花粉管生长相关基因OsPTD1表达在制备配子体雄性不育或繁殖孢子体核不育中的应用。
优选的，下调水稻花粉管生长相关基因OsPTD1表达的方法为干扰水稻花粉管生长相关基因OsPTD1表达。
优选的，所述干扰水稻花粉管生长相关基因OsPTD1表达的方法是先构建干涉水稻花粉管生长相关基因OsPTD1表达的载体，通过农杆菌介导转染植物，筛选转基因阳性植株即可。
优选的，所述表达载体含有干涉茎环、干涉左臂和干涉右臂，所述干涉茎环的序列如SEQ ID NO.8所示的干涉茎环；所述干涉左臂以SEQ ID NO.27和SEQ ID NO.28所示序列为引物，从中花11中扩增获得；所述干涉右臂是以SEQ ID NO.29和SEQ ID NO.30所示序列为引物，从中花11中扩增获得。
4、下调水稻花粉管生长相关基因OsPTD1表达制备配子体雄性不育的方法，包括如下步骤：
将花粉特异启动子调控水稻花粉管生长相关基因OsPTD1的下调元件Xi通过农杆菌介导转染植物，筛选转基因阳性株，转基因阳性株为配子体雄性不育。
5、下调水稻花粉管生长相关基因OsPTD1表达制备孢子体核雄性不育的方法，包括如下步骤：将孢子体雄性可育基因Y与启动子POsPTD1调控的OsPTD1基因干扰片段连锁，然后转入孢子体雄性可育基因Y的不育突变体，筛选转基因阳性植株，以阳性植株为父本给孢子体雄性可育基因Y的不育突变体授粉，即可繁殖该孢子体核雄性不育。
本发明中，所述孢子体雄性可育基因Y可以为任何孢子体雄性可育基因，如孢子体雄性可育基因OsABCG15，孢子体雄性可育基因Y的不育突变体为中九B-osabcg15：以OsABCG15基因的无花粉雄性不育突变体为非轮回母本，通过回交获得不育的中九B-osabcg15。也可以选择其他的孢子体雄性可育基因。
作为一个总的技术方案，本发明所述启动子、干涉片段、终止子和待转物种、品种可灵活选用；孢子体育性基因可灵活选用；基因抑制表达的方式可为干涉、反义和miRNA等；各种片段载入的先后顺序和相对位置可根据具体情况调整。另外，在方法陈述中所用的载体名称仅仅是为了陈述方便而使用，并不限制在实际应用中使用其他符号来表示相关载体。因而，上述可选内容的实际采用不表明同本发明有实质性差异，从而不影响本发明的保护。
本发明的有益效果在于：OsPTD1是水稻内源性基因，通过对其进行下调表达，无需借助外源基因或片段，即可产生配子体不育，从而培育转基因成分不随花粉漂移的植物品种(如果在其他物种中使用本方法，可选用目标物种中的配子体不育相关基因进行)；还可用来繁殖孢子体核不育，解决孢子体不育的繁殖问题，从而有利于自由选用父本，充分利用杂种优势；所述制备和鉴定配子体雄性不育的方法具有直观，简单，准确，省时，经济等优点。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为OsPTD1基因表达谱分析(A为OsPTD1基因在野生型各器官和花发育各时期和在干涉纯合后代花发育后期表达的RT-qPCR分析；B为启动子POsPTD1驱动GUS基因在花粉中特异表达的GUS染色检测，示转基因杂合体花粉的GUS染色；C为启动子POsPTD1驱动GUS基因在花粉中特异表达的GUS染色检测，示转基因纯合体花粉的GUS染色)图；
图2为OsPTD1基因的RACE分析和cDNA全长扩增(A和B分别为5和3端的3轮扩增，C为cDNA全长扩增)电泳图；
图3为载体改造图谱；
图4为花粉萌发情况(A为野生型花粉，B为干涉T 0代花粉(大约一半花粉提前破裂))图；
图5为转基因T 0代阳性株杂交F 1中紫线的传递情况照片。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南(第四版，冷泉港实验室出版)或精编分子生物学实验指南(第五版，科学出版社)中所述的条件，或按照制造厂商所建议的条件。
实施例中，可选用本领域已知的各种载体，如市售的载体，包括质粒，粘粒等。
相关核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本实施例所公开的有关核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库，或基因组DNA作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
实施例1、雄配子体功能相关基因OsPTD1的选定：
为了找到一些配子体发育相关基因，我们对BAR水稻基因表达数据库(http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi)进行分析，发现很多在中后期花中表达的基因，经RT-qPCR验证，选出一批可能同雄配子体发育相关的基因。其中，水稻Loc_Os05g40740基因在后期花中特异高表达，其拟南芥的同源基因在花粉中特异表达，初步将Loc_Os05g40740基因列为雄配子体发育相关基因，命名为OsPTD1，氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示，基因组序列如SEQ ID NO.3所示。
1、RT-qPCR分析
用RT-qPCR对该基因的时空表达模式进行了分析，所用OsPTD1基因的qPCR引物为：上游引物MQF2：5'-ggcgtcttcaggtacaaccag-3'(SEQ ID NO.9)，下游引物MQR25'-ggtacacctggaccgagtgc-3'(SEQ ID NO.10)。所用内参引物为：上游引物OsActin2F：5'-ctctgtatgccagtggtcgt-3'(SEQ ID NO.11)，下游引物OsActin2R：5'-ccgttgtggtgaatgagtaac-3'(SEQ ID NO.12)。qPCR结果表明：OsPTD1基因在根、茎、 叶、叶鞘和种子中均不表达，在发育中后期的花中高表达(图1)。进一步分析干涉纯合植物花发育后期该基因的表达，发现干涉造成该基因的表达严重降低。
2、GUS染色分析
为了确定该基因是否在花粉中特异表达，克隆了该基因的启动子，将其置于pCAMBIA1301的GUS基因前面，并进行转基因和GUS染色调查。具体操作为：
用启动子上游引物P5F1：5'-gcgtcgacgtcccatgtcaccgacagtact-3’(SEQ ID NO.13，带酶切位点SalI)和下游引物P5R1：5'-catgccatggcgtggaaatgtgatcgctaggct-3’(SEQ ID NO.14，带酶切位点NcoI)从中花11中扩出Loc_Os05g40740的启动子(简称POsPTD1)，其核苷酸序列如SEQ ID NO.6所示。用SalI和NcoI(不完全酶切)将其载入载体pCAMBIA1301的GUS基因前面得p POsPTD1-GUS。
将pPOsPTD1-GUS转入农杆菌LBA4404，再将阳性农杆菌转染中花11。开花期对转基因阳性后代的根、叶和花进行GUS染色观察，结果表明：根、叶、颖壳、花丝、雌蕊等均不染色，而花药染成蓝色。进一步捣碎花药，在显微镜下观察发现：转基因杂合的植株的GUS染色花粉仅有50％左右，纯合的植株的花粉几乎全部能被GUS染色(图1)。上述结果说明启动子POsPTD1确实为花粉特异启动子。
实施例2、OsPTD1基因的克隆及结构分析
为了获得OsPTD1的基因结构，采用宝生物的RACE试剂盒进行分析：以水稻花药总RNA反转录产物为模板，利用试剂盒提供的通用引物和自己设计的5’RACE引物和3’RACE引物进行PCR三轮扩增(图2)，将最后一轮的产物回收连接到pMD18-T载体，经过菌液PCR检测和送样测序，最终确定了OsPTD1基因cDNA的3’UTR、5’UTR和CDS：该基因的5’UTR有201bp，3’UTR有261bp，CDS全长为1671bp，cDNA全长为2338bp。5’RACE特异引物分别如SEQ ID NO.15-17所示，3’RACE特异引物分别如SEQ ID NO.18-20所示，全长扩增引物如SEQ ID NO.21-22所示。
实施例3、抑制OsPTD1基因表达制备雄配子体不育
为了确定OsPTD1基因是否调控花粉育性，能否用于制备和鉴定雄性配子体不育材料，用POsPTD1启动子驱动OsPTD1基因的干涉片段，实施了雄性配子体不育创造和鉴定：
1、载体改造：为了便于载入相关目的基因或DNA片段，为了准确和直接进行转基因阳性株以及雄配子体不育的鉴定，用水稻花青素合成相关基因的改造基因OsMYB76R替换GUS基因，并对载体pCAMBIA1301的多克隆位点进行了改造，具体操作为：
(1)OsMYB76基因改造并代替GUS：OsMYB76基因在水稻中为花青素合成必须基因，可用作内源性报告基因。为了避免OsMYB76基因本身带有的酶切位点影响载入目的片段，通过碱基替换，除去OsMYB76基因中的常用酶切位点，获得OsMYB76基因改造后的OsMYB76R基因的CDS序列并人工合成，其序列如SEQ ID NO.4所示。以合成的OsMYB76R基因为模板，用引物OsMYB76RF：5'-ggactcttgaccatgatgggacgcagggcttgct-3'(SEQ ID NO.23，带pCAMBIA1301的BglII附近同源片段)和OsMYB76RR：5'-attcgagctggtcactcacgcacacaggttccaag-3'(SEQ ID NO.24，带pCAMBIA1301的BstEII附近同源片段)扩出目的片段，用同源重组方式将目的片段连入pCAMBIA1301，得载体pOsMYB76R。
(2)多克隆位点改造：对前述载体pOsMYB76R的多克隆位点的酶切位点种类和顺序进行改造。先人工合成新的多克隆位点序列，其核苷酸序列如SEQ ID NO.5所示。然后通过同源重组将合成序列替换原有多克 隆位点的序列，新的多克隆酶切位点如图3所示，多克隆位点改造后的载体命名为pOsMYB76RM。
2、OsPTD1基因的抑制元件Xi的载入
(1)用茎环结构上游引物LF2：5'-gaagatctacgagctggtgagctagcta-3’(SEQ ID NO.25，带酶切位点BglII)和下游引物LR2：5'-ctggtcacccctcaaacctgaaaattcag-3’(SEQ ID NO.26，带酶切位点BstEII)从R25中扩出OsMYB76基因的第一内含子作干涉茎环(SEQ ID NO.8)，载入pOsMYB76RM得载体pOsMYB76RL。
(2)用干涉左臂上游引物I5F1：5'-cggaattcgcagaaggtgatcctgatcaac-3’(SEQ ID NO.27，带酶切位点EcoRI)和下游引物I5R1：5'-gaagatctgcaggcggctgacgacgctgat-3’(SEQ ID NO.28，带酶切位点BglII)从中花11中扩出OsPTD1的干涉左臂；用干涉右臂上游引物I5F2：5'-gacctgcagggcagaaggtgatcctgatcaac-3’(SEQ ID NO.29，带酶切位点SbfI)和下游引物I5R2：5'-ctggtcaccgcaggcggctgacgacgctgat-3’(SEQ ID NO.30，带酶切位点BstEII)从中花11中扩出Loc_Os05g40740的干涉右臂。将左右臂分别载入载体pOsMYB76RL中的茎环两边，并用SbfI和AscI将Tnos终止子载入干涉右臂后面，得载体pOsMYB76RLi；干涉片段的核苷酸序列为SEQ ID NO.7所示。
(3)用启动子上游引物P5F1：5'-gcgtcgacgtcccatgtcaccgacagtact-3’(SEQ ID NO.13，带酶切位点SalI)和下游引物P5R1：5'-catgccatggcgtggaaatgtgatcgctaggct-3’(SEQ ID NO.14，带酶切位点EcoRI)从中花11中扩出Loc_Os05g40740的启动子，其核苷酸序列如SEQ ID NO.6所示。将其载入载体pOsMYB76RLi得雄性配子体不育载体pXi-OsMYB76RM。
3、将pXi-OsMYB76RM转入农杆菌LBA4404，再将阳性农杆菌转染所有器官无色的水稻品种中九B。
4、在T 0代根据茎基部紫色有无选出阳性株，调查其花粉萌发和紫色传递情况发现：
(1)阳性株的花粉在培养基上约有一半花粉的花粉管不能正常生长，表现提前破裂(图4)，说明对OsPTD1基因的干涉造成花粉管生长异常，OsPTD1基因参与调控花粉育性。
(2)让阳性株自交，并用阳性株作父本，给前述未转基因的中九B授粉。对自交和杂交种子进行紫线调查，发现自交F 2种子紫线有无分离比例符合1:1，不符合3:1，96.4％的杂交F 1种子表现无紫线(图5)，这说明转基因成分难以随花粉在代间进行传递，但随雌配子在代间进行传递，这正是雄性配子体不育的特征。
(3)因此，OsPTD1基因与雄配子体花粉发育有关，通过对其进行干涉，借助连锁的紫线性状的遗传调查，获得了雄性配子体不育材料并成功进行了遗传鉴定。
实施例4、利用OsPTD1基因下调表达繁殖孢子体核雄性不育中九B-osabcg15
为了确定能否利用OsPTD1基因繁殖孢子体核雄性不育，以OsABCG15基因(Wu et al.,2014)产生的不育材料中九B-osabcg15为对象，用OsPTD1基因的干涉等片段同OsABCG15基因连锁，进行了中九B-osabcg15的繁殖实践，具体如下：
1、中九B-osabcg15的选育：以OsMYB76基因异常的中九B(雄性可育，无紫色)为轮回父本，以OsABCG15基因的无花粉雄性不育突变体(Wu et al.,2014)为非轮回母本，通过回交获得无紫色且不育的中九B-osabcg15。
2、将正常的OsABCG15基因连入pXi-OsMYB76RM，获得载体p OsABCG15-Xi-OsMYB76RM。
3、将pOsABCG15-Xi-OsMYB76RM转入中九B-osabcg15，根据紫色有无共筛出阳性株11株，所有紫色株均表现有可育花粉。
4、以阳性株为父本给突变体中九B-osabcg15授粉，共获得F 1种子429粒，将杂交种子播种移栽后共获得开花植株386株，其中无色不育376株，紫色可育10株，无色同不育共分离，紫色同可育共分离，不育株率97.4％。
自然条件下，繁殖中九B-osabcg15的不育株率最高的方法是兄妹交，不育株率最高为50％，而通过我们这种方法其不育株率达95％以上。说明利用本发明所述方法能用于繁殖孢子体核雄性不育。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
Figure PCTCN2021114625-appb-000001
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Figure PCTCN2021114625-appb-000004
Figure PCTCN2021114625-appb-000005
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Claims (10)

  1. 一种水稻花粉管生长相关基因OsPTD1，其特征在于：所述水稻花粉管生长相关基因OsPTD1编码的氨基酸序列如SEQ ID NO.1所示；或者SEQ ID NO.1所示氨基酸序列经取代、缺失或添加至少一个氨基酸且具有与OsPTD1蛋白功能相同的氨基酸序列。
  2. 根据权利要求1所述水稻花粉管生长相关基因OsPTD1，其特征在于：所述水稻花粉管生长相关基因OsPTD1的CDS核苷酸序列如SEQ ID NO.2所示；或SEQ ID NO.2所示序列经取代、缺失或添加至少一个核苷酸且具有与水稻花粉管生长相关基因OsPTD1功能相同的核苷酸序列。
  3. 根据权利要求1所述水稻花粉管生长相关基因OsPTD1，其特征在于：所述水稻花粉管生长相关基因OsPTD1的基因组核苷酸序列如SEQ ID NO.3所示；或SEQ ID NO.3所示序列经取代、缺失或添加至少一个核苷酸且具有与水稻花粉管生长相关基因OsPTD1功能相同的核苷酸序列。
  4. 权利要求1～3任一项所述水稻花粉管生长相关基因OsPTD1在恢复突变体花粉育性中的应用，所述突变体为OsPTD1基因或同源基因变异产生的花粉不育突变体。
  5. 下调或突变权利要求1～3任一项所述水稻花粉管生长相关基因OsPTD1表达在制备配子体雄性不育或繁殖孢子体核不育中的应用。
  6. 根据权利要求5所述的应用，其特征在于：下调水稻花粉管生长相关基因OsPTD1表达的方法为干涉水稻花粉管生长相关基因OsPTD1表达。
  7. 根据权利要求6所述的应用，其特征在于：所述干涉水稻花粉管生长相关基因OsPTD1表达的方法是先构建干涉水稻花粉管生长相关基因OsPTD1表达的载体，通过农杆菌介导转染植物，筛选转基因阳性植株即可。
  8. 根据权利要求7所述的应用，其特征在于：所述表达载体含有干涉茎环、干涉左臂和干涉右臂，所述干涉茎环的核苷酸序列如SEQ ID NO.8所示的干涉茎环；所述干涉左臂以SEQ ID NO.27和SEQ ID NO.28所示序列为引物，从中花11中扩增获得；所述干涉右臂是以SEQ ID NO.29和SEQ ID NO.30所示序列为引物，从中花11中扩增获得。
  9. 下调水稻花粉管生长相关基因OsPTD1表达以制备配子体雄性不育的方法，其特征在于，包括如下步骤：
    将花粉特异启动子调控水稻花粉管生长相关基因OsPTD1的下调元件Xi通过农杆菌介导转染植物，筛选转基因阳性株，转基因阳性株即为配子体雄性不育。
  10. 下调水稻花粉管生长相关基因OsPTD1表达以繁殖孢子体核雄性不育的方法，其特征在于：
    将孢子体雄性可育基因Y与启动子POsPTD1调控的OsPTD1基因干涉片段连锁，然后转入孢子体雄性可育基因Y的不育突变体，筛选转基因阳性植株，以所得的转基因阳性植株为父本给孢子体雄性可育基因Y的不育突变体授粉，即可繁殖该孢子体核雄性不育。
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