CN118291523A - A method for producing cloned gametes in plants and its application - Google Patents

A method for producing cloned gametes in plants and its application Download PDF

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CN118291523A
CN118291523A CN202410483734.3A CN202410483734A CN118291523A CN 118291523 A CN118291523 A CN 118291523A CN 202410483734 A CN202410483734 A CN 202410483734A CN 118291523 A CN118291523 A CN 118291523A
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杨超
耿苗维
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Abstract

The invention relates to a method for producing cloned gametes in plants and application thereof, which converts meiosis of germ cells into similar mitosis by utilizing the technology such as gene mutation or genetic engineering technology editing to make proteins participating in meiosis DNA double strand break and mucin participating in meiosis specific lose function in plants, and produces non-meiotic cloned gametes with the same genotype as the parent. The subsequent development of cloned gametes into seeds or plants can be realized, so that the heterosis of the crop hybrid seeds can be fixed in a time-saving and convenient manner, the production efficiency of the hybrid seeds is greatly improved, the production cost is reduced, the influence on the plant nutrition growth is avoided, and the method has a wide application value.

Description

一种在植物中产生克隆配子的方法及其应用A method for producing cloned gametes in plants and its application

技术领域Technical Field

本发明属于生物技术技术领域,具体涉及一种在植物中产生克隆配子的方法及其应用。The invention belongs to the technical field of biotechnology, and specifically relates to a method for producing cloned gametes in plants and an application thereof.

背景技术Background technique

杂种优势是指两个遗传特性不同的亲本的杂交后代F1在生长势、产量、品质、抗逆性等方面优于双亲的现象(顾周琳等,2023)。长久以来,杂种优势在农业上被广泛利用,人们通过利用杂种优势,将小麦、水稻、油菜产量大幅度提高,为全球粮食安全做出了巨大的贡献(姜楠等,2023)。然而,由于杂交种在下一代会发生分离导致杂种优势无法保持。因此,人们需要不断的制备杂交种。Hybrid vigor refers to the phenomenon that the hybrid offspring F1 of two parents with different genetic characteristics is superior to both parents in terms of growth potential, yield, quality, stress resistance, etc. (Gu Zhoulin et al., 2023). Hybrid vigor has been widely used in agriculture for a long time. By utilizing hybrid vigor, people have greatly increased the yield of wheat, rice, and rapeseed, making a huge contribution to global food security (Jiang Nan et al., 2023). However, hybrid vigor cannot be maintained because hybrids will separate in the next generation. Therefore, people need to continuously prepare hybrids.

杂交种的生产可以分为“三系法”、“两系法”和“一系法”三个战略发展阶段(袁隆平,1987)。第一代的杂交种生产是以细胞质雄性不育系为遗传工具的三系法育种技术,通过细胞质雄性不育系、保持系和恢复系(简称三系)的配套来实现。三系法生产杂交种需要找到合适的不育系与优良的恢复系,受恢复基因的限制从而影响优良组合的选育(邓兴旺等,2013)。后来又提出了制种程序相对简便的两系法杂交育种技术,即光温敏核不育系法,在一定的光温条件下其花粉是可育的,通过它的这种可育性来繁殖种子;而在另一光温条件下其花粉是不育的,利用其不育性,与父本杂交可生产杂交种(付志远等,2018)。而光温敏核不育系统则会受到异常天气影响导致杂交种生产体系不稳定,大大影响了优良杂交种的生产进程,从而提高了生产成本,不利于商业推广(郑兴飞等,2021)。因此,一系法被认为是杂交育种的最高目标。通过一系法可以培育不分离的F1杂种,将植物的杂种优势固定下来,免除制种。The production of hybrids can be divided into three strategic development stages: the "three-line method", the "two-line method" and the "one-line method" (Yuan Longping, 1987). The first generation of hybrid production is a three-line breeding technology that uses cytoplasmic male sterile lines as genetic tools, which is achieved through the matching of cytoplasmic male sterile lines, maintenance lines and restorer lines (referred to as three lines). The three-line method of hybrid production requires finding suitable sterile lines and excellent restorer lines, which are restricted by the restorer gene and thus affect the selection of excellent combinations (Deng Xingwang et al., 2013). Later, a two-line hybrid breeding technology with a relatively simple seed production procedure was proposed, namely the photothermosensitive nuclear sterile line method. Under certain light and temperature conditions, its pollen is fertile, and seeds are propagated through its fertility; under another light and temperature condition, its pollen is sterile. Using its sterility, hybrids can be produced by hybridization with the male parent (Fu Zhiyuan et al., 2018). The photothermosensitive nuclear sterile system will be affected by abnormal weather, resulting in an unstable hybrid production system, which greatly affects the production process of excellent hybrids, thereby increasing production costs and being unfavorable for commercial promotion (Zheng Xingfei et al., 2021). Therefore, the one-line method is considered to be the highest goal of hybrid breeding. The one-line method can cultivate non-segregating F1 hybrids, fix the hybrid vigor of plants, and eliminate the need for seed production.

无融合生殖因其在杂种优势固定中的巨大潜力而受到广泛关注,人工创制无融合生殖是当前无融合生殖研究的重要方向,有丝分裂替代减数分裂(Mitosis instead ofMeiosis,MIME)能产生与母本遗传组成完全一致的二倍体配子,是人工创制无融合生殖的关键步骤。研究发现,拟南芥spo11-1/rec8双突变体的第一次减数分裂被一个类似于有丝分裂的过程所取代,随后是染色体不平衡分配的第二次分裂,导致配子严重不育。但将阻止第二次减数分裂的osd1突变与破坏第一次减数分裂的spo11-1/rec8相结合,OSD1基因突变抑制了spo11-1/rec8双突变体的染色体不平衡分配,使spo11-1/rec8/osd1三突变体中产生了减数分裂被有丝分裂取代且遗传信息与母本完全一致的可育无融合配子。在水稻中也有相似的发现。即在拟南芥和水稻中都需要同时突变三个基因才可产生遗传信息与母本完全一致的可育无融合配子,工作量较大且效率较低;此外,拟南芥和水稻里需要突变的第三个基因OSD1会参与到有丝分裂和细胞命运,会对植物生长带来负向的效应。Apomixis has attracted extensive attention due to its great potential in fixing heterosis. Artificial creation of apomixis is an important direction of current apomixis research. Mitosis instead of meiosis (MIME) can produce diploid gametes with completely identical genetic composition to the maternal parent, which is a key step in artificial creation of apomixis. Studies have found that the first meiosis of the Arabidopsis spo11-1/rec8 double mutant was replaced by a process similar to mitosis, followed by a second division with unbalanced chromosome allocation, resulting in severe gamete infertility. However, when the osd1 mutation that prevents the second meiosis is combined with the spo11-1/rec8 that destroys the first meiosis, the OSD1 gene mutation inhibits the unbalanced chromosome allocation of the spo11-1/rec8 double mutant, resulting in fertile apomixis gametes in which meiosis is replaced by mitosis and the genetic information is completely identical to that of the maternal parent in the spo11-1/rec8/osd1 triple mutant. Similar findings have also been made in rice. That is, three genes need to be mutated at the same time in both Arabidopsis and rice to produce fertile afusion gametes with genetic information that is completely consistent with the mother plant, which is a large workload and low efficiency. In addition, the third gene that needs to be mutated in Arabidopsis and rice, OSD1, is involved in mitosis and cell fate, and will have a negative effect on plant growth.

发明内容Summary of the invention

本发明目的在于提供一种在植物,尤其是多倍体植物中产生克隆配子的方法,其仅需使植物体内参与减数分裂DNA双链断裂的蛋白和参与减数分裂特异的黏连蛋白两类蛋白进行突变,使其丧失功能,即可将植株的生殖细胞减数分裂转化为类似有丝分裂,跳过第二次减数分裂,进而得到与亲本基因型相同的非减数的可育克隆配子,后续诱导克隆配子发育成种子或植株,进而利用植物的杂种优势,更加省时便捷地固定作物杂交种的杂种优势,极大提高了生产效率,并且不会对植物营养生长产生影响。The purpose of the present invention is to provide a method for producing cloned gametes in plants, especially polyploid plants, which only requires mutation of two types of proteins in the plant, namely, proteins involved in meiotic DNA double-strand breaks and meiotic specific adhesion proteins, to make them lose their functions, thereby converting the meiosis of the plant's reproductive cells into mitosis-like divisions, skipping the second meiosis, and obtaining non-reduced fertile cloned gametes with the same genotype as the parent, and subsequently inducing the cloned gametes to develop into seeds or plants, thereby utilizing the hybrid vigor of the plant, more time-saving and conveniently fixing the hybrid vigor of crop hybrids, greatly improving production efficiency, and not affecting the nutritional growth of the plant.

本发明提供了一种在植物中产生克隆配子的方法,令植物体内参与减数分裂DNA双链断裂的蛋白和参与减数分裂特异的黏连蛋白丧失功能,将生殖细胞的减数分裂转化为类似有丝分裂,产生与亲本基因型相同的非减数的克隆配子。The present invention provides a method for producing cloned gametes in plants, which causes the proteins involved in meiotic DNA double-strand breaks and the specific adhesion proteins involved in meiosis to lose their functions, convert the meiosis of reproductive cells into mitosis-like division, and produce non-reduced cloned gametes with the same genotype as the parent.

进一步地,所述参与减数分裂DNA双链断裂的蛋白包括:SPO11-1、MTOPVIB、PRD1、PRD2、PRD3和/或DFO,所述参与减数分裂特异的黏连包括:REC8。Furthermore, the proteins involved in meiotic DNA double-strand breaks include: SPO11-1, MTOPVIB, PRD1, PRD2, PRD3 and/or DFO, and the proteins involved in meiotic specific adhesion include: REC8.

进一步地,所述丧失功能可以通过基因突变或人工干预手段达到,所述基因突变包括自发突变和诱发突变,其中自发突变是机体内部自然产生的,诱发突变是外部环境诱导引发的;所述人工干预手段包括基因编辑技术,所述基因编辑技术包括:Cre-lox系统、锌指核酸内切酶系统、类转录激活因子效应物核酸酶系统、CRISPR-Cas9系统、RNAi和/或碱基编辑器系统。Furthermore, the loss of function can be achieved through gene mutation or artificial intervention, the gene mutation includes spontaneous mutation and induced mutation, wherein spontaneous mutation is naturally generated inside the body, and induced mutation is induced by the external environment; the artificial intervention means includes gene editing technology, and the gene editing technology includes: Cre-lox system, zinc finger nuclease system, transcription activator-like effector nuclease system, CRISPR-Cas9 system, RNAi and/or base editor system.

进一步地,所述SPO11-1蛋白的氨基酸序列如SEQ ID NO:1-2所示;所述MTOPVIB蛋白的氨基酸序列如SEQ ID NO:3-4所示;所述REC8蛋白的氨基酸序列如SEQ ID NO:5-6所示;所述PRD1蛋白的氨基酸序列如SEQ ID NO:7-8所示;所述PRD2蛋白的氨基酸序列如SEQID NO:9-12所示;所述PRD3蛋白的氨基酸序列如SEQ ID NO:13-16所示;所述DFO蛋白的氨基酸序列如SEQ ID NO:17-18所示。Furthermore, the amino acid sequence of the SPO11-1 protein is shown in SEQ ID NO:1-2; the amino acid sequence of the MTOPVIB protein is shown in SEQ ID NO:3-4; the amino acid sequence of the REC8 protein is shown in SEQ ID NO:5-6; the amino acid sequence of the PRD1 protein is shown in SEQ ID NO:7-8; the amino acid sequence of the PRD2 protein is shown in SEQID NO:9-12; the amino acid sequence of the PRD3 protein is shown in SEQ ID NO:13-16; and the amino acid sequence of the DFO protein is shown in SEQ ID NO:17-18.

进一步地,所述植物包括:油菜、白菜、甘蓝、小麦、燕麦、棉花、烟草、牧草、象草、苏丹草、鸭茅、梯牧草、甘蔗、香蕉、草莓、樱桃、苹果、葡萄、梨、西瓜、甜瓜、马铃薯、甘薯、木薯、甜菜、花生、咖啡、芝麻、苜蓿、芥菜、菜心、紫菜薹、红菜薹、西蓝花、菜花、卷心菜、萝卜、雪里蕻、番茄、茄子、芦笋、辣椒、黄瓜、冬瓜、丝瓜、南瓜、韭菜、山药、兰花、百合、水仙、菊、紫罗兰和/或郁金香。Furthermore, the plants include: rapeseed, cabbage, kale, wheat, oats, cotton, tobacco, forage grass, elephant grass, Sudan grass, orchard grass, timothy grass, sugarcane, banana, strawberry, cherry, apple, grape, pear, watermelon, melon, potato, sweet potato, cassava, beet, peanut, coffee, sesame, alfalfa, mustard, Chinese cabbage, purple cabbage, red cabbage, broccoli, cauliflower, cabbage, radish, Chinese mustard, tomato, eggplant, asparagus, pepper, cucumber, wax gourd, loofah, pumpkin, leek, yam, orchid, lily, daffodil, chrysanthemum, violet and/or tulip.

本发明还提供了上述方法在植物育种中应用。The invention also provides application of the method in plant breeding.

进一步地,所述植物包括:油菜、白菜、甘蓝、小麦、燕麦、棉花、烟草、牧草、象草、苏丹草、鸭茅、梯牧草、甘蔗、香蕉、草莓、樱桃、苹果、葡萄、梨、西瓜、甜瓜、马铃薯、甘薯、木薯、甜菜、花生、咖啡、芝麻、苜蓿、芥菜、菜心、紫菜薹、红菜薹、西蓝花、菜花、卷心菜、萝卜、雪里蕻、番茄、茄子、芦笋、辣椒、黄瓜、冬瓜、丝瓜、南瓜、韭菜、山药、兰花、百合、水仙、菊、紫罗兰和/或郁金香。Furthermore, the plants include: rapeseed, cabbage, kale, wheat, oats, cotton, tobacco, forage grass, elephant grass, Sudan grass, orchard grass, timothy grass, sugarcane, banana, strawberry, cherry, apple, grape, pear, watermelon, melon, potato, sweet potato, cassava, beet, peanut, coffee, sesame, alfalfa, mustard, Chinese cabbage, purple cabbage, red cabbage, broccoli, cauliflower, cabbage, radish, Chinese mustard, tomato, eggplant, asparagus, pepper, cucumber, wax gourd, loofah, pumpkin, leek, yam, orchid, lily, daffodil, chrysanthemum, violet and/or tulip.

本发明还提供了一种固定植物杂种优势的方法,包括以下步骤:The present invention also provides a method for fixing plant heterosis, comprising the following steps:

(1)采用上述方法得到与亲本基因型相同的非减数的克隆配子;(1) using the above method to obtain non-reduced clonal gametes with the same genotype as the parent;

(2)诱导非减数的克隆配子发育成种子或植株。(2) Induce non-reduced clonal gametes to develop into seeds or plants.

进一步地,诱导非减数的克隆配子发育成种子或植株的方法为如下任一或其组合:Further, the method of inducing non-reduced cloned gametes to develop into seeds or plants is any one of the following or a combination thereof:

A.通过参与孤雌生殖或单倍体诱导基因的突变相结合实现,其中孤雌生殖所需蛋白包括BBM1、BBM2以及PAR;单倍体诱导基因的突变所需蛋白包括CENH3、MTL、PLA1以及DMP。A. This is achieved through a combination of mutations in genes involved in parthenogenesis or haploid induction. The proteins required for parthenogenesis include BBM1, BBM2, and PAR; the proteins required for mutations in haploid induction genes include CENH3, MTL, PLA1, and DMP.

B.通过小孢子培养技术获得非减数克隆配子的单倍体植株;B. Obtaining haploid plants with non-reduced clonal gametes through microspore culture technology;

C.利用ROS诱导剂处理花粉产生单倍体。C. Treatment of pollen with ROS inducers produces haploids.

进一步地,所述植物包括油菜、白菜、甘蓝、小麦、燕麦、棉花、烟草、牧草、象草、苏丹草、鸭茅、梯牧草、甘蔗、香蕉、草莓、樱桃、苹果、葡萄、梨、西瓜、甜瓜、马铃薯、甘薯、木薯、甜菜、花生、咖啡、芝麻、苜蓿、芥菜、菜心、紫菜薹、红菜薹、西蓝花、菜花、卷心菜、萝卜、雪里蕻、番茄、茄子、芦笋、辣椒、黄瓜、冬瓜、丝瓜、南瓜、韭菜、山药、兰花、百合、水仙、菊、紫罗兰和郁金香。Further, the plants include rapeseed, cabbage, kale, wheat, oats, cotton, tobacco, forage grass, elephant grass, Sudan grass, orchard grass, timothy grass, sugarcane, banana, strawberry, cherry, apple, grape, pear, watermelon, melon, potato, sweet potato, cassava, beet, peanut, coffee, sesame, alfalfa, mustard, Chinese cabbage, purple cabbage, red cabbage, broccoli, cauliflower, cabbage, radish, Chinese mustard, tomato, eggplant, asparagus, pepper, cucumber, wax gourd, loofah, pumpkin, leek, yam, orchid, lily, daffodil, chrysanthemum, violet and tulip.

有益效果:本发明仅需对植物体内参与减数分裂DNA双链断裂的蛋白和参与减数分裂特异的黏连蛋白进行突变,使其丧失功能,即可将植株的生殖细胞减数分裂转化为类似有丝分裂,跳过第二次减数分裂,进而得到与亲本基因型相同的非减数的可育克隆配子,后续可结合孤雌生殖或单倍体诱导基因的突变诱导,或小孢子培养技术等,使克隆配子发育成种子或植株,进而省时便捷地固定作物杂交种的杂种优势,极大提高了生产效率,加快优良杂交种的育种进程、降低生产成本,并且不会对植物营养生长产生影响,具有广阔的应用价值。且该方法在其它多倍体植物如棉花、小麦等中均是保守的,对于多倍体作物具有重大意义。Beneficial effects: The present invention only needs to mutate the proteins involved in meiotic DNA double-strand breaks in the plant body and the specific adhesion proteins involved in meiosis to make them lose their functions, so as to convert the meiosis of the plant's reproductive cells into mitosis-like divisions, skip the second meiosis, and then obtain non-reduced fertile cloned gametes with the same genotype as the parents. Subsequently, the cloned gametes can be combined with parthenogenesis or mutation induction of haploid induction genes, or microspore culture technology, etc., so that the cloned gametes develop into seeds or plants, thereby saving time and conveniently fixing the hybrid vigor of crop hybrids, greatly improving production efficiency, accelerating the breeding process of excellent hybrids, reducing production costs, and not affecting plant nutritional growth, and having broad application value. Moreover, this method is conservative in other polyploid plants such as cotton, wheat, etc., and is of great significance for polyploid crops.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

图1为WT、spo11-1 rec8-1(Westar)、spo11-1 rec8-1(8x)(Westar)、spo11-1rec8(Westar x J9707 F1)和mtopVIB rec8(Westar x J9707 F1)突变体植株的花粉染色图,其中8x代表八倍体,F1代表杂种一代。Figure 1 shows the pollen staining images of WT, spo11-1 rec8-1(Westar), spo11-1 rec8-1(8x)(Westar), spo11-1rec8(Westar x J9707 F1) and mtopVIB rec8(Westar x J9707 F1) mutant plants, where 8x represents octoploid and F1 represents the first hybrid generation.

图2为Westar、spo11-1 rec8-1(Westar)、spo11-1 rec8-1(8x)(Westar)、Westarx J9707 F1、spo11-1 rec8(Westar x J9707 F1)和mtopVIB rec8(Westar x J9707 F1)突变体植株四分体时期地衣红染色情况。Figure 2 shows the orcein staining of Westar, spo11-1 rec8-1(Westar), spo11-1 rec8-1(8x)(Westar), Westarx J9707 F1, spo11-1 rec8(Westar x J9707 F1) and mtopVIB rec8(Westar x J9707 F1) mutant plants at the tetrad stage.

图3为WT、spo11-1 rec8-1(Westar)、spo11-1 rec8-1(8x)(Westar)、spo11-1rec8(Westar x J9707 F1)和mtopVIB rec8(Westar x J9707 F1)突变体植株花粉母细胞的染色体行为观察图。Figure 3 shows the observation of chromosome behavior in pollen mother cells of WT, spo11-1 rec8-1(Westar), spo11-1 rec8-1(8x)(Westar), spo11-1rec8(Westar x J9707 F1) and mtopVIB rec8(Westar x J9707 F1) mutant plants.

图4为WT和spo11-1 rec8-1突变体植株种子对比图。FIG. 4 is a comparison of seeds of WT and spo11-1 rec8-1 mutant plants.

具体实施方式Detailed ways

以下实施例仅用于更加清楚地说明本发明的技术方案,因此只是作为示例,而不能以此来限制本发明的保护范围。需要注意的是,除非另有说明,本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明,以下实施例所用试剂和材料均为市购。The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

实施例1甘蓝型油菜基因SPO11-1突变体的创建Example 1 Creation of the Brassica napus SPO11-1 mutant

一、构建CRISPR表达载体1. Construction of CRISPR expression vector

1、使用CRISPR-P v2.0网站针对如SEQ ID NO:1-2所示的BnaA01.SPO11-1与BnaC01.SPO11-1设计了两个靶点。1. Two targets were designed using the CRISPR-P v2.0 website for BnaA01.SPO11-1 and BnaC01.SPO11-1 as shown in SEQ ID NO: 1-2.

2、设计四条引物,如下:2. Design four primers as follows:

BnaSPO11-1DT1-BsF:BnaSPO11-1DT1-BsF:

ATATATGGTCTCGATTGATCTGCTTCGAAAGATCAAGTTATATATGGTCTCGATTGATCTGCTTCGAAAGATCAAGTT

BnaSPO11-1-DT1-F0:BnaSPO11-1-DT1-F0:

TGATCTGCTTCGAAAGATCAAGTTTTAGAGCTAGAAATAGCTGATCTGCTTCGAAAGATCAAGTTTTAGAGCTAGAAATAGC

BnaSPO11-1-DT2-R0:BnaSPO11-1-DT2-R0:

AACTCGACTTCCTCTACAAGAACAATCTCTTAGTCGACTCTACAACTCGACTTCCTCTACAAGAACAATCTCTTAGTCGACTCTAC

BnaSPO11-1-DT2-BsR:BnaSPO11-1-DT2-BsR:

ATTATTGGTCTCGAAACTCGACTTCCTCTACAAGAACAAATTATTGGTCTCGAAACTCGACTTCCTCTACAAGAACAA

3、以1ng/μl pCBC-DT1T2为模板进行四引物PCR扩增。BnaSPO11-1DT1BsF/BnaSPO11-1-DT2-BsR为正常引物浓度;BnaSPO11-1-DT1--F0/BnaSPO11-1-DT2--R0稀释20倍。3. Use 1 ng/μl pCBC-DT1T2 as template for four-primer PCR amplification. BnaSPO11-1DT1BsF/BnaSPO11-1-DT2-BsR is the normal primer concentration; BnaSPO11-1-DT1--F0/BnaSPO11-1-DT2--R0 is diluted 20 times.

4、纯化回收PCR产物,建立15μl酶切-连接反应体系。该反应体系包括2μl PCR片段、2μl pKSE401载体质粒、1.5μl 10xT4Buffer、1.5μl 10xBSA、1μl BsaI、1μl T4 Ligase和6μl ddH2O。4. Purify and recover the PCR product, and establish a 15μl restriction enzyme digestion-ligation reaction system, which includes 2μl PCR fragment, 2μl pKSE401 vector plasmid, 1.5μl 10xT4Buffer, 1.5μl 10xBSA, 1μl BsaI, 1μl T4 Ligase and 6μl ddH2O .

5、吸取5μl转化Top10大肠感受态,然后使用Kana板进行筛选。在37℃恒温培养箱放置约12h后,挑取菌斑进行菌落PCR鉴定。随后选取正确菌斑进行摇菌,第二天提取质粒得到CRISPR表达载体。5. Take 5 μl of Top10 colon competent cells and screen them using Kana plates. After placing in a 37°C constant temperature incubator for about 12 hours, pick the plaques for colony PCR identification. Then select the correct plaques for shaking, and extract the plasmid the next day to obtain the CRISPR expression vector.

二、突变体的获得2. Obtaining mutants

将CRISPR表达载体转化农杆菌GV301,然后以春性甘蓝型油菜Westar为受体材料对其下胚轴进行农杆菌侵染,后续转移到诱导愈伤组织的培养基中培养2到3周,挑选长势良好的愈伤组织进行2到3次继代培养。对已长出幼苗的愈伤组织进行切苗,转移到生根培养基中继续培养直至长出肉眼可见的根系。将生根的幼苗转入大田,然后进行栽培管理。The CRISPR expression vector was transformed into Agrobacterium GV301, and then the spring Brassica napus Westar was used as the recipient material to infect its hypocotyl with Agrobacterium. Subsequently, it was transferred to the medium for inducing callus tissue and cultured for 2 to 3 weeks. The callus tissue with good growth was selected for 2 to 3 subcultures. The callus tissue that has grown seedlings was cut and transferred to the rooting medium for continued culture until the roots visible to the naked eye grew. The rooted seedlings were transferred to the field and then cultivated and managed.

对T0代幼苗使用特异性的引物进行一代测序,最终得到Bnaspo11-1-1和Bnaspo11-1-2两个株系。The T0 generation seedlings were sequenced using specific primers, and eventually two strains, Bnaspo11-1-1 and Bnaspo11-1-2, were obtained.

所用引物如下:The primers used are as follows:

BnaSPO11-1-A1-T1-F2:BnaSPO11-1-A1-T1-F2:

GAGTGAAGTAATAAAACGGCGCGGAGTGAAGTAATAAAACGGCGCG

BnaSPO11-1-A1-T1-R1:BnaSPO11-1-A1-T1-R1:

GAACATAGCAGAAACGAACGGTAAACGAACATAGCAGAAACGAACGGTAAAC

BnaSPO11-1-A1-T2-F2:BnaSPO11-1-A1-T2-F2:

CTGTTATGAGGCATGTCCTTATTTCAAGCTGTTATGAGGCATGTCCTTATTTCAAG

BnaSPO11-1-A1-T2-R2:BnaSPO11-1-A1-T2-R2:

GTCCCGATCACAATATTGTCAGGTCCCGATCACAATATTGTCAG

BnaSPO11-1-C1-T1-F3:BnaSPO11-1-C1-T1-F3:

GCGAAGGTAGTAATAAAGCGGCACGGCGAAGGTAGTAATAAAGCGGCACG

BnaSPO11-1-C1-T1-R2:BnaSPO11-1-C1-T1-R2:

GCCCTTCCAAAATTTACCTTTGAATGCGCCCTTCCAAAATTTACCTTTGAATGC

BnaSPO11-1-C1-T2-F3:BnaSPO11-1-C1-T2-F3:

GGTGGGTACTTCTGACAGCGATTACGGTGGGTACTTCTGACAGCGATTAC

BnaSPO11-1-C1-T2-R3:BnaSPO11-1-C1-T2-R3:

CCAAGCTCCTGGAATTAAGTTATGCCAAGCTCCTGGAATTAAGTTATG

实施例2:甘蓝型油菜基因REC8突变体的创建Example 2: Creation of a mutant of the Brassica napus gene REC8

一、构建CRISPR表达载体1. Construction of CRISPR expression vector

1、使用CRISPR-P v2.0网站针对如SEQ ID NO:5-6所示的BnaA10.REC8与BnaC09.REC8设计了两个靶点。1. Two targets were designed using the CRISPR-P v2.0 website for BnaA10.REC8 and BnaC09.REC8 as shown in SEQ ID NO:5-6.

2设计四条引物,如下:2 Design four primers as follows:

BnaREC8DT1-BsF:BnaREC8DT1-BsF:

ATATATGGTCTCGATTGTAAAGGACGAACCCACGCCGTTATATATGGTCTCGATTGTAAAGGACGAACCCACGCCGTT

BnaREC8-DT1-F0:BnaREC8-DT1-F0:

TGTAAAGGACGAACCCACGCCGTTTTAGAGCTAGAAATAGCTGTAAAGGACGAACCCACGCCGTTTTAGAGCTAGAAATAGC

Bn REC8-DT2-R0:Bn REC8-DT2-R0:

AACTTCAAAGCGATCATACGTCCAATCTCTTAGTCGACTCTACAACTTCAAAGCGATCATACGTCCAATCTCTTAGTCGACTCTAC

BnaREC8-DT2-BsR:BnaREC8-DT2-BsR:

ATTATTGGTCTCGAAACTTCAAAGCGATCATACGTCCAAATTATTGGTCTCGAAACTTCAAAGCGATCATACGTCCAA

3、以1ng/μl pCBC-DT1T2为模板进行四引物PCR扩增。BnaSPO11-1DT1BsF/BnaSPO11-1-DT2-BsR为正常引物浓度;BnaSPO11-1-DT1--F0/BnaSPO11-1-DT2--R0稀释20倍。3. Use 1 ng/μl pCBC-DT1T2 as template for four-primer PCR amplification. BnaSPO11-1DT1BsF/BnaSPO11-1-DT2-BsR is the normal primer concentration; BnaSPO11-1-DT1--F0/BnaSPO11-1-DT2--R0 is diluted 20 times.

4、纯化回收PCR产物,建立15μl酶切-连接反应体系。该反应体系包括2μl PCR片段、2μl pKSE401载体质粒、1.5μl 10xT4Buffer、1.5μl 10xBSA、1μl BsaI、1μl T4 Ligase和6μl ddH2O。4. Purify and recover the PCR product, and establish a 15μl restriction enzyme digestion-ligation reaction system, which includes 2μl PCR fragment, 2μl pKSE401 vector plasmid, 1.5μl 10xT4Buffer, 1.5μl 10xBSA, 1μl BsaI, 1μl T4 Ligase and 6μl ddH2O .

5、吸取5μl转化Top10大肠感受态,然后使用Kana板进行筛选。在37℃恒温培养箱放置约12h后,挑取菌斑进行菌落PCR鉴定。随后选取正确菌斑进行摇菌,第二天提取质粒得到CRISPR表达载体。5. Take 5 μl of Top10 colon competent cells and screen them using Kana plates. After placing in a 37°C constant temperature incubator for about 12 hours, pick the plaques for colony PCR identification. Then select the correct plaques for shaking, and extract the plasmid the next day to obtain the CRISPR expression vector.

三、突变体的获得3. Obtaining mutants

将CRISPR表达载体转化农杆菌GV301,然后以春性甘蓝型油菜Westar为受体材料对其下胚轴进行农杆菌侵染,后续转移到诱导愈伤组织的培养基中培养2到3周,挑选长势良好的愈伤组织进行2到3次继代培养。对已长出幼苗的愈伤组织进行切苗,转移到生根培养基中继续培养直至长出肉眼可见的根系。将生根的幼苗转入大田,然后进行栽培管理。The CRISPR expression vector was transformed into Agrobacterium GV301, and then the spring Brassica napus Westar was used as the recipient material to infect its hypocotyl with Agrobacterium. Subsequently, it was transferred to the medium for inducing callus tissue and cultured for 2 to 3 weeks. The callus tissue with good growth was selected for 2 to 3 subcultures. The callus tissue that has grown seedlings was cut and transferred to the rooting medium for continued cultivation until the roots visible to the naked eye grew. The rooted seedlings were transferred to the field and then cultivated and managed.

对T0代幼苗使用特异性的引物进行一代测序,最终得到Bnarec8-1和Bnarec8-2两个株系。The T0 generation seedlings were sequenced using specific primers, and eventually two strains, Bnarec8-1 and Bnarec8-2, were obtained.

所用引物如下:The primers used are as follows:

BnaREC8-A10-T2-R1:BnaREC8-A10-T2-R1:

CTGCTGCAAGATGGAAAAACATAAATAGTGCTGCTGCAAGATGGAAAAACATAAATAGTG

BnaREC8-C9-T2-R1:BnaREC8-C9-T2-R1:

AAGATGGATGAACGAAAATACAGAAAGAGCAAGATGGATGAACGAAAATACAGAAAGAGC

BnaREC8-F1:BnaREC8-F1:

CGAGAGGAAAGTAAAGCTCCTATTCGGCGAGAGGAAAGTAAAGCTCCTATTCGG

BnaREC8-A10-T1-seqF1:BnaREC8-A10-T1-seqF1:

CCGTCAAAATTGATATTTCCCCGTCAAAATTGATATTTCC

BnaREC8-C9-T1-seqF1:BnaREC8-C9-T1-seqF1:

CTTCTGGTAGAAATTAATGGCTTCTGGTAGAAATTAATGG

实施例3:Bnaspo11-1 rec8双突变体的创建及细胞学分析Example 3: Creation and cytological analysis of the Bnaspo11-1 rec8 double mutant

使用杂交手段将Bnaspo11-1突变体和Bnarec8突变体聚合,得到Bnaspo11-1rec8突变体,并进行后续细胞学分析。The Bnaspo11-1 mutant and the Bnarec8 mutant were hybridized to obtain the Bnaspo11-1rec8 mutant, and subsequent cytological analysis was performed.

1、使用亚历山大染液对Bnaspo11-1-1rec8-1突变体花粉进行染色。首先,使用移液枪吸取15μl亚历山大染液滴在载玻片上,然后取新鲜的油菜花蘸取染液,随后立即盖上18×18cm盖玻片并在90℃加热板加热15min,最后在10×显微镜下观察花粉染色情况。结果如图1所示,Bnaspo11-1-1rec8-1突变体充满可育饱满的花粉粒,且与野生型相比均匀变大。1. Use Alexander stain to stain the pollen of the Bnaspo11-1-1rec8-1 mutant. First, use a pipette to draw 15μl of Alexander stain and drop it on a glass slide, then take a fresh rapeseed flower and dip it in the stain, then immediately cover it with an 18×18cm coverslip and heat it on a 90℃ heating plate for 15min, and finally observe the pollen staining under a 10× microscope. The results are shown in Figure 1. The Bnaspo11-1-1rec8-1 mutant is full of fertile and plump pollen grains, and is uniformly larger than the wild type.

2、使用地衣红染液对Bnaspo11-1-1rec8-1突变体四分体时期进行染色。首先,挑取出一枚适宜时期的花药,在1mol/L HCl中60℃加热1min。然后取出花药在水中浸泡1min,随后在地衣红染液中用弯钩形解剖针将花药捣碎,立即盖上18×18cm盖玻片并在40×显微镜下进行观察。结果如图2所示,野生型在四分体时期基本是平衡的四分体,而Bnaspo11-1-1rec8-1突变体在此时期大多为二分体。因此,Bnaspo11-1-1rec8-1突变体跳过了第二次减数分裂。2. Use orcein dye to stain the tetrad stage of the Bnaspo11-1-1rec8-1 mutant. First, pick out an anther of the appropriate period and heat it in 1mol/L HCl at 60℃ for 1min. Then take out the anther and soak it in water for 1min, then crush the anther with a hook-shaped dissecting needle in the orcein dye solution, immediately cover it with an 18×18cm coverslip and observe it under a 40× microscope. The results are shown in Figure 2. The wild type is basically a balanced tetrad during the tetrad period, while the Bnaspo11-1-1rec8-1 mutant is mostly dyads during this period. Therefore, the Bnaspo11-1-1rec8-1 mutant skipped the second meiotic division.

3、观察Bnaspo11-1-1rec8-1突变体减数分裂时期的染色体行为。操作步骤如下:3. Observe the chromosome behavior of the Bnaspo11-1-1rec8-1 mutant during meiosis. The steps are as follows:

(1)取新鲜且适宜时期的花蕾,用卡诺固定液(无水乙醇:冰醋酸=3:1;体积比)进行固定、脱色。(1) Take fresh flower buds of appropriate age and fix and decolorize them with Carnoy's fixative (absolute ethanol: glacial acetic acid = 3:1; volume ratio).

(2)取已完成固定、脱色的花蕾,使用卡宝品红染液进行减数分裂时期的挑选,然后将其在0.01mol/LPH=4.5的柠檬酸钠缓冲液中浸泡30-60min。(2) The fixed and decolorized flower buds were selected for meiosis using Carbofusin dye, and then immersed in 0.01 mol/LPH = 4.5 sodium citrate buffer for 30-60 min.

(3)将花药放入展片酶解液中37℃酶解50min,用移液枪吸取5μl H2O滴在载玻片中央,然后用镊子夹取两枚花药放入水中,随后用弯钩形解剖针将其敲成匀浆状,然后用移液枪吸取15μl 60%的醋酸滴入,继续敲成透明的细胞悬浮液,再加入15μl 60%的醋酸。随后,在45℃的加热板上进行展片。当载玻片上的液滴将要干时,用预冷的卡诺固定液进行冲洗。最后,放在45℃的加热板上风干。(3) Place the anthers in the enzymatic solution for enzymatic hydrolysis at 37°C for 50 min. Use a pipette to draw 5 μl of H 2 O and drop it in the center of the slide. Then use tweezers to pick up two anthers and put them in water. Then use a curved hook-shaped dissecting needle to knock them into a homogenate. Then use a pipette to draw 15 μl of 60% acetic acid and drop it in. Continue to knock it into a transparent cell suspension, and then add 15 μl of 60% acetic acid. Then, spread the slide on a heating plate at 45°C. When the droplets on the slide are about to dry, rinse them with pre-cooled Carnoy's fixative. Finally, place them on a heating plate at 45°C to air dry.

(4)待片子干燥后,滴加4’,6’-diamidino-2-phenylindole(DAPI)进行染色,然后用荧光显微镜观察染色体行为。(4) After the slide is dry, add 4’,6’-diamidino-2-phenylindole (DAPI) for staining, and then observe chromosome behavior using a fluorescence microscope.

结果如图3所示,在野生型花粉母细胞中,中期I19对二价体有序地排列在赤道板上。在后期I,同源染色体分离并沿相反地方向迁移到两极,导致在在末期I形成了各含19条染色体的二分体。经过第二次减数分裂,在末期Ⅱ形成含有19条染色体的四个子细胞。而Bnaspo11-1-1rec8-1突变体,在中期I,我们观察到38个单价体有序的排列在赤道板上。随后,在后期I单价体的姐妹染色单体提前分离平衡的移向两极。最终在末期I,形成各含38条染色体的二分体。并且,Bnaspo11-1-1rec8-1突变体雄性性母细胞没有进行第二次减数分裂。表明该突变体的减数分裂已转变为有丝分裂样分裂,产生了克隆的非减数配子。The results are shown in Figure 3. In the wild-type pollen mother cells, 19 pairs of bivalents are orderly arranged on the equatorial plate in metaphase I. In anaphase I, homologous chromosomes separate and migrate to the two poles in opposite directions, resulting in the formation of dyads containing 19 chromosomes each in telophase I. After the second meiotic division, four daughter cells containing 19 chromosomes are formed in telophase II. In the Bnaspo11-1-1rec8-1 mutant, we observed 38 univalents orderly arranged on the equatorial plate in metaphase I. Subsequently, the sister chromatids of the univalents separated and moved to the two poles in advance in anaphase I. Finally, in telophase I, dyads containing 38 chromosomes each are formed. In addition, the male mother cells of the Bnaspo11-1-1rec8-1 mutant did not undergo the second meiotic division. This indicates that the meiosis of the mutant has been transformed into mitotic-like division, producing clonal non-reduced gametes.

实施例4:Bnaspo11-1 rec8突变体自交后代细胞学分析Example 4: Cytological analysis of self-pollinated progeny of Bnaspo11-1 rec8 mutant

Bnaspo11-1-1rec8-1突变体充满可育饱满的花粉粒,且与野生型相比均匀变大(图1)。这也使Bnaspo11-1-1rec8-1突变体自交产生与野生型相比较大的种子(如图4所示)。后续将这些自交种种入温室,进行常规栽培管理。待植株现蕾后,取适宜时期的花蕾,用卡诺固定液(无水乙醇:冰醋酸=3:1;体积比)进行固定、脱色。The Bnaspo11-1-1rec8-1 mutant was full of fertile and plump pollen grains, and was uniformly larger than the wild type (Figure 1). This also caused the Bnaspo11-1-1rec8-1 mutant to produce larger seeds than the wild type after self-pollination (as shown in Figure 4). These self-pollinated seeds were subsequently planted in the greenhouse for conventional cultivation and management. After the plants budded, the flower buds at the appropriate period were taken and fixed and decolorized with Carnoy's fixative (anhydrous ethanol: glacial acetic acid = 3:1; volume ratio).

1、使用亚历山大染液对Bnaspo11-1 rec8-1(8x)突变体花粉进行染色,染色步骤参照实施例3。结果如图1所示,Bnaspo11-1 rec8-1(8x)突变体充满可育饱满的花粉粒,且与Bnaspo11-1-1rec8-1突变体相比均匀增大。1. The pollen of the Bnaspo11-1 rec8-1(8x) mutant was stained with Alexander stain, and the staining steps were as described in Example 3. As shown in FIG1 , the Bnaspo11-1 rec8-1(8x) mutant was full of fertile and plump pollen grains, and was uniformly enlarged compared with the Bnaspo11-1-1 rec8-1 mutant.

2、使用地衣红染液对Bnaspo11-1 rec8-1(8x)突变体四分体时期进行染色,实验步骤参照实施例3。结果如图2所示,野生型在四分体时期基本是平衡的四分体,而Bnaspo11-1 rec8-1(8x)突变体在此时期大多为二分体。因此,Bnaspo11-1rec8-1(8x)突变体也跳过了第二次减数分裂。2. The tetrad stage of the Bnaspo11-1 rec8-1(8x) mutant was stained with orcein dye, and the experimental steps were as described in Example 3. As shown in Figure 2, the wild type was basically a balanced tetrad during the tetrad stage, while the Bnaspo11-1 rec8-1(8x) mutant was mostly a dyad during this period. Therefore, the Bnaspo11-1rec8-1(8x) mutant also skipped the second meiotic division.

3、观察Bnaspo11-1 rec8-1(8x)突变体减数分裂时期的染色体行为。操作步骤同实施例3。3. Observe the chromosome behavior of the Bnaspo11-1 rec8-1 (8x) mutant during meiosis. The operation steps are the same as those in Example 3.

结果如图3所示,Bnaspo11-1 rec8-1(8x)突变体,在中期I,我们观察到76个单价体。随后,在后期I单价体的姐妹染色单体提前分离平衡的移向两极。最终在末期I,形成各含76条染色体的二分体。并且,Bnaspo11-1 rec8-1(8x)突变体雄性性母细胞没有进行第二次减数分裂。这也表明Bnaspo11-1-1rec8-1突变体自交后代倍性增加,间接证明Bnaspo11-1-1rec8-1突变体雌性性母细胞也可以产生克隆的非减数配子。The results are shown in Figure 3. In the Bnaspo11-1 rec8-1(8x) mutant, we observed 76 univalents in metaphase I. Subsequently, in anaphase I, the sister chromatids of the univalents separated and moved to the two poles in a balanced manner in advance. Finally, in telophase I, dyads containing 76 chromosomes were formed. In addition, the male meiocytes of the Bnaspo11-1 rec8-1(8x) mutant did not undergo the second meiotic division. This also indicates that the ploidy of the self-pollinated offspring of the Bnaspo11-1-1rec8-1 mutant is increased, indirectly proving that the female meiocytes of the Bnaspo11-1-1rec8-1 mutant can also produce cloned non-reduced gametes.

实施例5:Bnaspo11-1 rec8双突变体在Westar与J9707杂种一代中的应用Example 5: Application of the Bnaspo11-1 rec8 double mutant in the first generation hybrid of Westar and J9707

使用实例1和实例2中所涉及的CRISPR基因编辑技术构建同时含有敲除基因SPO11-1与REC8靶点的CRISPR表达载体,以Westar与J9707杂交种一代为受体进行组织培养,获得Westar与J9707杂交种背景下的spo11-1 rec8突变体。The CRISPR gene editing technology involved in Example 1 and Example 2 was used to construct a CRISPR expression vector containing both the knockout gene SPO11-1 and the REC8 target site, and tissue culture was performed using the first generation of a hybrid of Westar and J9707 as the recipient to obtain the spo11-1 rec8 mutant in the background of a hybrid of Westar and J9707.

所需引物如下:The primers required are as follows:

MiMe-BnaSPO11-1-DT1-BsFMiMe-BnaSPO11-1-DT1-BsF

ATATATGGTCTCGATTGTTCTTGTAGAGGAAGTCGAGTTATATATGGTCTCGATTGTTCTTGTAGAGGAAGTCGAGTT

MiMe-BnaSPO11-1-DT1-F0MiMe-BnaSPO11-1-DT1-F0

TGTTCTTGTAGAGGAAGTCGAGTTTTAGAGCTAGAAATAGCTGTTCTTGTAGAGGAAGTCGAGTTTTAGAGCTAGAAATAGC

MiMe-BnaREC8-DT2-R0MiMe-BnaREC8-DT2-R0

AACGGCGTGGGTTCGTCCTTTACAATCTCTTAGTCGACTCTACAACGGCGTGGGTTCGTCCTTTACAATCTCTTAGTCGACTCTAC

MiMe-BnaREC8-DT2-BsRMiMe-BnaREC8-DT2-BsR

ATTATTGGTCTCGAAACGGCGTGGGTTCGTCCTTTACAAATTATTGGTCTCGAAACGGCGTGGGTTCGTCCTTTACAA

1、使用亚历山大染液对Westar与J9707杂交种背景下的spo11-1 rec8突变体花粉进行染色。结果如图1所示,Westar与J9707杂种背景下的spo11-1 rec8突变体充满大量可育饱满的花粉粒,且与野生型相比均匀变大。1. The pollen of the spo11-1 rec8 mutant in the hybrid background of Westar and J9707 was stained with Alexander stain. As shown in Figure 1, the spo11-1 rec8 mutant in the hybrid background of Westar and J9707 was full of fertile and plump pollen grains, and was uniformly larger than the wild type.

2、使用地衣红染液对Westar与J9707杂交种背景下的spo11-1 rec8突变体四分体时期进行染色。结果如图2所示,Westar与J9707杂交种在四分体时期基本是平衡的四分体,而Westar与J9707杂种背景下的spo11-1 rec8突变体在此时期大多为二分体。因此,Westar与J9707杂交种背景下的spo11-1 rec8突变体跳过了第二次减数分裂。2. The spo11-1 rec8 mutant in the hybrid background of Westar and J9707 was stained with orcein red at the tetrad stage. As shown in Figure 2, the hybrid of Westar and J9707 was basically a balanced tetrad at the tetrad stage, while the spo11-1 rec8 mutant in the hybrid background of Westar and J9707 was mostly dyads at this stage. Therefore, the spo11-1 rec8 mutant in the hybrid background of Westar and J9707 skipped the second meiotic division.

3、观察Westar与J9707杂交种背景下的spo11-1 rec8突变体减数分裂时期的染色体行为。结果如图3所示,Westar与J9707杂种背景下的spo11-1 rec8突变体,在中期I,我们观察到38个单价体有序的排列在赤道板上。随后,在后期I单价体的姐妹染色单体提前分离平衡的移向两极。最终在末期I,形成各含38条染色体的二分体。并且,Westar与J9707杂交种背景下的spo11-1 rec8突变体雄性性母细胞没有进行第二次减数分裂。表明Westar与J9707杂交种背景下的spo11-1 rec8突变体的减数分裂已转变为有丝分裂样分裂,产生了克隆的非减数配子。3. Observe the chromosome behavior of the spo11-1 rec8 mutant during meiosis in the hybrid background of Westar and J9707. The results are shown in Figure 3. In the spo11-1 rec8 mutant in the hybrid background of Westar and J9707, we observed 38 univalents arranged in an orderly manner on the equatorial plate in metaphase I. Subsequently, in anaphase I, the sister chromatids of the univalents separated and moved to the two poles in a balanced manner in advance. Finally, in telophase I, dyads containing 38 chromosomes each were formed. In addition, the male meiocytes of the spo11-1 rec8 mutant in the hybrid background of Westar and J9707 did not undergo the second meiotic division. This shows that the meiosis of the spo11-1 rec8 mutant in the hybrid background of Westar and J9707 has been transformed into mitotic division, producing clonal non-reducing gametes.

实施例6:BnamtopVIB rec8双突变体的在Westar与J9707杂种一代中的应用Example 6: Application of the BnamtopVIB rec8 double mutant in the first generation hybrid of Westar and J9707

使用实例1和实例2中所涉及的CRISPR基因编辑技术构建同时含有敲除基因MTOPVIB与REC8靶点的CRISPR表达载体,以Westar与J9707杂交种一代为受体进行组织培养,获得Westar与J9707杂交种背景下的mtopVIB rec8突变体。The CRISPR gene editing technology involved in Example 1 and Example 2 was used to construct a CRISPR expression vector containing both the knockout gene MTOPVIB and REC8 targets, and tissue culture was performed using the first generation of a hybrid of Westar and J9707 as a recipient to obtain an mtopVIB rec8 mutant in the background of a hybrid of Westar and J9707.

所需引物,如下:The primers required are as follows:

MiMe-BnaMTOPVIB-DT1-BsFMiMe-BnaMTOPVIB-DT1-BsF

ATATATGGTCTCGATTGGGTGCCCTAGAGAGTTCAAGTTATATATGGTCTCGATTGGGTGCCCTAGAGAGTTCAAGTT

MiMe-BnaMTOPVIB-DT1-F0MiMe-BnaMTOPVIB-DT1-F0

TGGGTGCCCTAGAGAGTTCAAGTTTTAGAGCTAGAAATAGCTGGGTGCCCTAGAGAGTTCAAGTTTTAGAGCTAGAAATAGC

MiMe-BnaREC8-DT2-R0MiMe-BnaREC8-DT2-R0

AACGGCGTGGGTTCGTCCTTTACAATCTCTTAGTCGACTCTACAACGGCGTGGGTTCGTCCTTTACAATCTCTTAGTCGACTCTAC

MiMe-BnaREC8-DT2-BsRMiMe-BnaREC8-DT2-BsR

ATTATTGGTCTCGAAACGGCGTGGGTTCGTCCTTTACAAATTATTGGTCTCGAAACGGCGTGGGTTCGTCCTTTACAA

1、使用亚历山大染液对Westar与J9707杂交种背景下的mtopVIB rec8突变体花粉进行染色。结果如图1所示,Westar与J9707杂种背景下的mtopVIB rec8突变体也充满大量可育饱满的花粉粒,且与野生型相比均匀变大。1. Alexander stain was used to stain the pollen of the mtopVIB rec8 mutant in the hybrid background of Westar and J9707. As shown in Figure 1, the mtopVIB rec8 mutant in the hybrid background of Westar and J9707 was also full of fertile and plump pollen grains, and was uniformly larger than the wild type.

2、使用地衣红染液对Westar与J9707杂交种背景下的spo11-1 rec8突变体四分体时期进行染色。结果如图2所示,Westar与J9707杂交种在四分体时期基本是平衡的四分体,而Westar与J9707杂种背景下的mtopVIB rec8突变体在此时期大多为二分体。因此,Westar与J9707杂交种背景下的mtopVIB rec8突变体跳过了第二次减数分裂。2. The spo11-1 rec8 mutant in the hybrid background of Westar and J9707 was stained with orcein red at the tetrad stage. As shown in Figure 2, the hybrid of Westar and J9707 was basically a balanced tetrad at the tetrad stage, while the mtopVIB rec8 mutant in the hybrid background of Westar and J9707 was mostly dyads at this stage. Therefore, the mtopVIB rec8 mutant in the hybrid background of Westar and J9707 skipped the second meiotic division.

3、观察Westar与J9707杂交种背景下的mtopVIB rec8突变体减数分裂时期的染色体行为。结果如图3所示,Westar与J9707杂种背景下的mtopVIB rec8突变体,在中期I,我们观察到38个单价体有序的排列在赤道板上。随后,在后期I单价体的姐妹染色单体提前分离平衡的移向两极。最终在末期I,形成各含38条染色体的二分体。并且,Westar与J9707杂交种背景下的mtopVIB rec8突变体雄性性母细胞没有进行第二次减数分裂。表明Westar与J9707杂交种背景下的mtopVIB rec8突变体的减数分裂已转变为有丝分裂样分裂,产生了克隆的非减数配子。3. Observe the chromosome behavior of the mtopVIB rec8 mutant during meiosis in the hybrid background of Westar and J9707. The results are shown in Figure 3. In the mtopVIB rec8 mutant in the hybrid background of Westar and J9707, we observed 38 univalents arranged in an orderly manner on the equatorial plate in metaphase I. Subsequently, in anaphase I, the sister chromatids of the univalents separated and moved to the two poles in advance. Finally, in telophase I, dyads containing 38 chromosomes were formed. In addition, the male meiocytes of the mtopVIB rec8 mutant in the hybrid background of Westar and J9707 did not undergo the second meiotic division. This shows that the meiosis of the mtopVIB rec8 mutant in the hybrid background of Westar and J9707 has been transformed into mitotic division, producing clonal non-reduced gametes.

本发明仅以SPO11-1和MTOPVIB蛋白作为参与减数分裂DNA双链断裂蛋白的代表进行验证,而本领域公知的可以发挥相同功能的如PRD1、PRD2、PRD3和DFO等蛋白,都可用于实现本发明所述的将植物生殖细胞的减数分裂转化为类似有丝分裂,产生与亲本基因型相同的非减数的可育克隆配子。The present invention only uses SPO11-1 and MTOPVIB proteins as representatives of proteins involved in meiotic DNA double-strand breaks for verification, while proteins known in the art that can perform the same function, such as PRD1, PRD2, PRD3 and DFO, can all be used to achieve the conversion of plant reproductive cells' meiosis into mitosis-like divisions as described in the present invention to produce non-reduced fertile clonal gametes with the same genotype as the parent.

即本发明通过对参与减数分裂DNA双链断裂的蛋白(如SPO11-1、MTOPVIB、PRD1、PRD2、PRD3以及DFO)和减数分裂特异的黏连蛋白(如REC 8)进行编辑,进而将其植物生殖细胞的减数分裂转化为类似有丝分裂,产生与亲本基因型相同的非减数的可育克隆配子。后续可结合孤雌生殖或单倍体诱导基因的突变诱导非减数的克隆配子发育成种子或植株,进而用于植株杂种优势的固定,提高生产效率的同时不会对植物营养生长产生影响,具有广阔的应用价值。That is, the present invention edits proteins involved in meiotic DNA double-strand breaks (such as SPO11-1, MTOPVIB, PRD1, PRD2, PRD3 and DFO) and meiosis-specific adhesion proteins (such as REC 8), thereby converting the meiosis of plant germ cells into mitosis-like divisions, and producing non-reduced fertile cloned gametes with the same genotype as the parents. Subsequently, the non-reduced cloned gametes can be induced to develop into seeds or plants by combining parthenogenesis or mutation of haploid induction genes, and then used to fix plant heterosis, which improves production efficiency without affecting plant nutritional growth, and has broad application value.

以上具体实施方式详细描述了本发明的实施,但是,本发明并不限于上述实施方式中的具体细节。在本发明的权利要求书和技术构思范围内,可以对本发明的技术方案进行多种简单改型和改变,这些简单变型均属于本发明的保护范围。The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims (10)

1. A method for producing cloned gametes in plants, characterized in that proteins involved in meiosis DNA double strand breaks and mucins involved in meiosis specific in plants are rendered nonfunctional, the meiosis of germ cells is converted to similar mitosis, and non-meiotic cloned gametes of the same genotype as the parent are produced.
2. The method of claim 1, wherein the protein involved in a meiosis DNA double strand break comprises: SPO11-1, MTOPVIB, PRD1, PRD2, PRD3 and/or DFO, the involvement in meiosis specific adhesion comprising: REC8.
3. The method of claim 2, wherein the loss of function is achieved by genetic mutation or by means of human intervention, the genetic mutation comprising spontaneous mutation and induced mutation, wherein spontaneous mutation is naturally occurring in the body, and induced mutation is induced by external environment; the manual intervention means comprises a gene editing technique comprising: cre-lox system, zinc finger endonuclease system, transcription activator-like effector nuclease system, CRISPR-Cas9 system, RNAi and/or base editor system.
4. The method according to any one of claims 1-2, wherein the amino acid sequence of the SPO11-1 protein is as shown in SEQ ID No. 1-2; the amino acid sequence of MTOPVIB protein is shown as SEQ ID NO 3-4; the amino acid sequence of the REC8 protein is shown as SEQ ID NO. 5-6; the amino acid sequence of the PRD1 protein is shown in SEQ ID NO 7-8; the amino acid sequence of the PRD2 protein is shown as SEQ ID NO 9-12; the amino acid sequence of the PRD3 protein is shown as SEQ ID NO. 13-16; the amino acid sequence of the DFO protein is shown as SEQ ID NO. 17-18.
5. The method of any one of claims 1-4, wherein the plant comprises: rape, cabbage, wheat, oat, cotton, tobacco, pasture, elephant grass, sudan grass, fescue, timothy grass, sugarcane, banana, strawberry, cherry, apple, grape, pear, watermelon, melon, potato, sweet potato, cassava, beet, peanut, coffee, sesame, alfalfa, mustard, cabbage, lavender, red cabbage, broccoli, cauliflower, cabbage, radish, potherb mustard, tomato, eggplant, asparagus, chilli, cucumber, white gourd, towel gourd, pumpkin, leek, yam, orchid, lily, narcissus, chrysanthemum, violet and/or tulip.
6. Use of the method according to any one of claims 1-5 in plant breeding.
7. The use according to claim 6, wherein the plant comprises: rape, cabbage, wheat, oat, cotton, tobacco, pasture, elephant grass, sudan grass, fescue, timothy grass, sugarcane, banana, strawberry, cherry, apple, grape, pear, watermelon, melon, potato, sweet potato, cassava, beet, peanut, coffee, sesame, alfalfa, mustard, cabbage, lavender, red cabbage, broccoli, cauliflower, cabbage, radish, potherb mustard, tomato, eggplant, asparagus, chilli, cucumber, white gourd, towel gourd, pumpkin, leek, yam, orchid, lily, narcissus, chrysanthemum, violet and/or tulip.
8. A method for fixing plant hybrid vigour comprising the steps of:
(1) Obtaining non-subtractive cloned gametes of the same genotype as the parent using the method of any one of claims 1-5;
(2) Non-subtractive clonal gametes are induced to develop into seeds or plants.
9. The method of claim 8, wherein the method of inducing non-subtractive clonal gametes to develop into a seed or plant is any one or a combination of the following:
A. Through the combination of mutation of parthenogenesis or haploid induction genes, proteins required for parthenogenesis comprise BBM1, BBM2 and PAR; proteins required for haploid induction of mutation of genes include CENH3, MTL, PLA1 and DMP;
B. haploid plants of non-subtractive cloned gametes are obtained by microspore culture techniques;
C. treatment of pollen with ROS inducers produces haploids.
10. The method of claim 8, wherein the plant comprises canola, cabbage, wheat, oat, cotton, tobacco, pasture, grasses, sudan grass, cogongrass, timothy, sugarcane, banana, strawberry, cherry, apple, grape, pear, watermelon, melon, potato, sweet potato, cassava, beet, peanut, coffee, sesame, alfalfa, mustard, cabbage, laver, red tongue, broccoli, cauliflower, cabbage, radish, potherb mustard, tomato, eggplant, asparagus, capsicum, cucumber, white gourd, towel gourd, pumpkin, leek, yam, orchid, lily, narcissus, chrysanthemum, violet, and tulip.
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