WO2019196738A1 - 一种克服二倍体马铃薯自交不亲和的方法 - Google Patents

一种克服二倍体马铃薯自交不亲和的方法 Download PDF

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WO2019196738A1
WO2019196738A1 PCT/CN2019/081515 CN2019081515W WO2019196738A1 WO 2019196738 A1 WO2019196738 A1 WO 2019196738A1 CN 2019081515 W CN2019081515 W CN 2019081515W WO 2019196738 A1 WO2019196738 A1 WO 2019196738A1
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plant
potato
sequence
gene
rnase
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PCT/CN2019/081515
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French (fr)
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黄三文
张春芝
彭真
叶明旺
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中国农业科学院农业基因组研究所
中国农业科学院深圳农业基因组研究所
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Priority to US16/960,292 priority Critical patent/US20210054391A1/en
Priority to EP19784808.8A priority patent/EP3719132A4/en
Publication of WO2019196738A1 publication Critical patent/WO2019196738A1/zh
Priority to US18/448,621 priority patent/US20240018532A1/en

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    • A01H6/827—Solanum tuberosum [potato]
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    • 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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    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the technical field of biotechnology and genetic breeding, and is specifically a method for overcoming the self-incompatibility of diploid potato.
  • Diploid potato is a self-incompatible type of gametophyte, and its pollen tube can germinate on the stigma and elongate into the style, but its growth is subsequently inhibited.
  • This trait is controlled by the S-RNase gene, and the expression of this gene inhibits the elongation of the pollen tube, making it difficult for the potato to obtain an inbred line.
  • Sli S-locus inhibitor
  • the present invention provides a method for overcoming the self-incompatibility of the diploid potato, and the purpose of the invention is to find a kind of Simple, accurate and efficient method to overcome the self-incompatibility of diploid potato, thus overcoming the self-incompatibility barrier of diploid potato, providing core technical support for the creation of self-compatible affinity material and homozygous inbred line. .
  • the present invention provides a method for overcoming the self-incompatibility of diploid potato, comprising the following steps:
  • step (3) introducing the recombinant vector obtained in the step (2) into potato cells, inducing that the guide RNA expression cassette of the target fragment and the Cas9 nuclease expression cassette are co-expressed in the cell, and cleavage of the double-stranded target fragment of the S-RNase gene , triggering the DNA repair function of the potato cell itself, randomly inserting or deleting the base at the target site, and realizing the functional deletion mutation of the intracellular S-RNase gene;
  • the gene region of the potato self-incompatibility determining gene S-RNase genes S p3 and S p4 includes an exon and a promoter, or a portion thereof.
  • the target fragment is located on the target gene S-RNase, and one strand of the target fragment has the nucleic acid sequence structure as shown in SEQ ID No. 1.
  • one strand of the target fragment has a 5'-(N) X- NGG-3' structure, and (N) X represents a base sequence of number X Each of ⁇ N 1 , N 2 ... N X ⁇ , N 1 , N 2 ...
  • N X represents any one of bases A, G, C, T, and N in NGG is A, G, C Any one of T; X is an integer between 15 and 25, preferably, said X is an integer between 17 and 23; more preferably, said X is 18, 19, 20, 21.
  • the recombinant vector in the step (2) comprises the target fragment, and the target fragment is a nucleic acid sequence of SEQ ID No. 1 or a sequence complementary thereto.
  • Another aspect of the present invention provides a potato plant, a plant part, a tuber or tuber part thereof, a plant cell, a pollen or a seed, which comprises a S-RNase gene function deletion mutation, and the nucleotide sequence of the S-RNase protein is SEQ ID NO: 2 (Sp3), or its complement, degenerate sequence, homologous sequence; and/or sequence as shown in SEQ ID NO: 3 (Sp4), or its complement, degenerate Sequence, homologous sequence.
  • the homologous sequence of the nucleotide sequence of the S-RNase protein may be a nucleotide in SEQ ID NO: 2 and/or SEQ ID NO: 3 under stringent conditions. a sequence or a complement thereof that hybridizes to a polynucleotide that does not express a polynucleotide of the S-RNase protein or a fragment thereof;
  • the "stringent conditions” described herein may be any of low stringent conditions, medium stringent conditions, and high stringency conditions, preferably high stringency conditions.
  • the "low stringency conditions” may be 30 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “medium stringent conditions” may be 40 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “high stringency conditions” may be 50 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide.
  • the higher the temperature the more highly homologous polynucleotides can be obtained.
  • one skilled in the art can select a comprehensive result of a plurality of factors affecting the stringency of hybridization, probe concentration, probe length, ionic strength, time, salt concentration, etc. to achieve a corresponding stringency.
  • hybridizable polynucleotide may be, when calculated by a system-set default parameter by a homology search software such as FASTA or BLAST, having about 30% or more of the polynucleotide encoding the present invention.
  • the S-RNase gene function is deleted by adding, deleting or replacing nucleotides (one or more) in a gene expressing the S-RNase protein.
  • nucleotides one or more
  • one, two or more nucleotides are added to a gene expressing an S-RNase protein; one, two or more nucleotides are deleted; one, two or more are substituted; Nucleotide.
  • nucleotide sequence of the S-RNase protein is as shown in SEQ ID NO: 2 (Sp3), or its complement, degenerate sequence, homologous sequence; and/or as SEQ ID NO: The sequence shown in 3 (Sp4), or its complement, degenerate sequence, homologous sequence.
  • the loss of function of the S-RNase gene is achieved by adding, deleting or replacing nucleotides (one or more) in ACGATTCACGGGCTTTGGCC or its complement. For example, 1-5 nucleotides are added, 1-10 nucleotides are deleted, or 1-8 nucleotides are replaced.
  • the addition, deletion or replacement of the nucleotide is achieved by a CRISPR/Cas9 recombinant vector.
  • the CRISPR/Cas9 recombinant vector is capable of targeting a gene localized to an S-RNase protein
  • the nucleotide sequence of the S-RNase protein is the sequence shown in SEQ ID NO: (Sp3), or its complement, degenerate sequence, homologous sequence; and/or the sequence set forth in SEQ ID NO: 3 (Sp4), or its complement, degenerate sequence, homologous sequence.
  • the CRISPR/Cas9 recombinant vector is capable of targeting the first exon region of the S-RNase gene.
  • the CRISPR/Cas9 recombinant vector is capable of targeting ACGATTCACGGGCTTTTGGCCGG or its complement of the S-RNase gene.
  • the nucleotide sequence of the sgRNA in the CRISPR/Cas9 recombinant vector is:
  • S-RNase P3 (ie Seq ID No. 4): xxxx ACGATTCACGGGCTTTGGC
  • S-RNase P4 (ie Seq ID No. 5): xxxx GCCAAAGCCCGTGAATCGT
  • the portion not underlined is the sequence or complementary sequence in which the NGG is removed from the above target site, and the underlined portion is the sticky end for ligation of the vector.
  • the present invention provides a CRISPR/Cas9 recombinant vector targeting a knockout S-RNase gene
  • the nucleotide sequence of the S-RNase gene targeted by the CRISPR/Cas9 recombinant vector is set forth in SEQ ID NO: 2.
  • the CRISPR/Cas9 recombinant vector is capable of targeting the first exon region of the S-RNase gene.
  • the CRISPR/Cas9 recombinant vector is capable of targeting ACGATTCACGGGCTTTTGGCCGG or its complement of the S-RNase gene.
  • the nucleotide sequence of the sgRNA in the CRISPR/Cas9 recombinant vector is:
  • S-RNase P3 (ie Seq ID No. 4): xxxx ACGATTCACGGGCTTTGGC
  • S-RNase P4 (ie Seq ID No. 5): xxxx GCCAAAGCCCGTGAATCGT
  • the portion not underlined is the sequence or complementary sequence in which the NGG is removed from the above target site, and the underlined portion is the sticky end for ligation of the vector.
  • the construction of the CRISPR/Cas9 recombinant vector comprises the following steps:
  • BsaI endonuclease cleaves the pKSE401 vector as a backbone fragment of the framework recombinant vector
  • sequences of the primers S-RNase P3 and S-RNase P4 are as follows:
  • S-RNase P3 (ie Seq ID No. 4): xxxx ACGATTCACGGGCTTTGGC
  • S-RNase P4 (ie Seq ID No. 5): xxxx GCCAAAGCCCGTGAATCGT
  • the portion not underlined is the sequence or complementary sequence in which the NGG is removed from the above target site, and the underlined portion is the sticky end for ligation of the vector.
  • Another aspect of the invention provides the use of the above CRISPR/Cas9 recombinant vector for the preparation of a knockout S-RNase protein gene.
  • Another aspect of the present invention provides a method for breeding a self-compatible potato comprising not expressing or inactivating an S-RNase gene in a potato, wherein the S-RNase gene is the sequence shown in SEQ ID NO: Sp3), or its complement, degenerate sequence, homologous sequence; and/or the sequence set forth in SEQ ID NO: 3 (Sp4), or its complement, degenerate sequence, homologous sequence.
  • the breeding method specifically includes:
  • step (1) (2) introducing the CRISPR/Cas9 recombinant vector in step (1) into potato cells, inducing that the target RNA expression cassette and the Cas9 nuclease expression cassette of the target fragment are co-expressed in the cell, and the double strand of the S-RNase gene is cleaved.
  • the target fragment triggers the DNA repair function of the potato cell itself, causing the target site to randomly insert, delete or replace the base, thereby realizing the functional deletion mutation of the intracellular S-RNase gene;
  • Another aspect of the present invention provides a method of breeding a potato comprising self-crossing a potato plant obtained by using the potato plant described above, a plant part, a tuber or tuber part, a plant cell, a pollen or a seed, or the above breeding method.
  • Another aspect of the present invention provides a method of producing a commercial plant product, comprising obtaining the above-described plant or a part thereof, and manufacturing the commercial plant product from the plant or a plant part thereof, wherein the plant product is selected from the group consisting of Groups: fresh whole potatoes, French fries, potato chips, dehydrated potato material, potato flakes and potato granules.
  • Another aspect of the present invention provides a food product made from the above-mentioned potato plant, a plant part, a tuber or a tuber part thereof, or a potato plant, tuber or tuber part produced by the growth of the plant cell, pollen or seed.
  • the food product is a sliced potato tuber food.
  • the food product is a group consisting of French fries, potato chips and baked potatoes.
  • the present invention adopts the above technical solution, and the present invention includes the following beneficial effects: Compared with the conventional method for solving the self-incompatibility of potato, the present invention has the following advantages:
  • the invention directly edits the self-incompatible gene, constructs a vector, can simultaneously target the target genes of the two target genes, and creates a plurality of new self-compatible breeding materials;
  • FIG. 1 shows a result of detecting a target mutation according to an embodiment of the present invention.
  • Figure 2 shows the ploidy detection of regenerated plants provided by an embodiment of the present invention.
  • Figure a is a diploid;
  • Figure b is a tetraploid.
  • FIG. 3 is a phenotype diagram showing elongation of a wild type material (A) and a gene editing material (B) pollen tube in a style according to an embodiment of the present invention.
  • Figure 4 is a diagram showing the self-pollination fruit set phenotype of wild-type and genetically edited lines according to an embodiment of the present invention.
  • Embodiment 1 The present invention provides a method for overcoming the self-incompatibility of diploid potato, comprising the following steps:
  • the target fragment is located on the target gene S-RNase.
  • One strand of the target fragment has a nucleic acid sequence structure as shown in SEQ ID No. 1.
  • One strand of the target fragment has a nucleic acid sequence structure as shown in SEQ ID No. 1.
  • one strand of the target fragment has a 5'-(N) X- NGG-3' structure, and (N) X represents a base sequence of number X
  • ⁇ N 1 , N 2 ... N X ⁇ , N 1 , N 2 ... N X represents any one of bases A, G, C, T, and N in NGG is A, G, C Any one of T and T.
  • S-RNase P3 (ie Seq ID No. 4): xxxx ACGATTCACGGGCTTTGGC;
  • S-RNase P4 (ie Seq ID No. 5): xxxx GCCAAAGCCCGTGAATCGT
  • portion not underlined is a sequence or a complementary sequence in which the NGG is removed from the target site, and the underlined portion is a sticky end for attaching the carrier;
  • the pKSE401 vector was digested with BsaI endonuclease at 50 °C for 12 hours, and the enzyme digestion system was inactivated at 65 °C for 10 min, as a framework fragment of the framework recombinant vector;
  • the recombinant vector backbone fragment and the insert were ligated with T4 ligase and transferred to E. coli. After sequencing, the positive transformants were extracted to form a recombinant vector plasmid for the diploid potato S-RNase gene CRISPR/Cas9 targeting;
  • the recombinant vector plasmid was transferred into Agrobacterium strain EHA105, and after sequencing, the positive transformed strain was extracted.
  • Fig. 4A is a wild-type strain
  • Fig. 4B is a genetically edited strain. It can be seen from Fig. 4 that the wild type cannot be self-sufficient, and the mutant type can be self-crossing, indicating that the mutant strain is a self-compatible new material, and successfully overcomes the self-incompatibility disorder;
  • a slash before the detected number represents the T 1 of Cas9 number-generation plant
  • a slash is detected number represents the number of T-generation plant. 1
  • b 1-generation plant Cas9 type of separation of S-RNase T ;
  • c indicates the type of S-RNase mutation.

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Abstract

本发明公开的一种克服二倍体马铃薯自交不亲和的方法，包括(1)选取靶标片段；(2)构建基因打靶的重组载体；(3)实现细胞内S-RNase基因的功能缺失突变；(4)再生若干个马铃薯植株；(5)特异性扩增再生植株中S-RNase基因包含靶标片段的DNA区段；(6)选择S-RNase基因被编辑的再生植株；(7)对所选基因编辑植株进一步筛选出二倍体的基因编辑株系；(8)扩繁并定植所选基因编辑株系，于开花期进行自交亲和性表型鉴定；(9)对收获自交亲和性植株后代扩增产物测序，检测被编辑靶标基因在后代的遗传和分离。本发明的优点在于，找到一种简便、精准、高效的方法克服二倍体马铃薯自交不亲和。

Description

一种克服二倍体马铃薯自交不亲和的方法 技术领域
本发明属于生物技术及遗传育种技术领域，具体为一种克服二倍体马铃薯自交不亲和的方法。
背景技术
二倍体马铃薯为配子体自交不亲和类型，其花粉管可以在柱头上萌发并伸长到花柱中，但其生长随后被抑制。这种性状受S-RNase基因控制，该基因的表达抑制了花粉管的伸长，导致马铃薯难以获得自交系。
前人研究发现烟草S-RNase基因在花芽至蕾期阶段仅有微量表达，而在开花期检测到较高的S-RNase蛋白富集。我们对马铃薯雌蕊蛋白的研究，亦得到类似结论，这就为马铃薯通过蕾期自交克服自交不亲和创造了可能。然而，蕾期自交对授粉时期和环境要求较高，结果率低，种子量少，而且自交种子成苗后仍然是自交不亲和植株。该方法费时费力，成本极高，且自交亲和性仅在蕾期自交当代表现，不具有可遗传性。蕾期自交的方法不能实现自交亲和材料的创制。
Hosaka和Hanneman在1998年定位了一个来源于野生种S.chacoense的基因Sli(S-locus inhibitor)基因位点，该基因赋予二倍体马铃薯自交亲和性。然而，将Sli基因渐渗入栽培种马铃薯会不可避免的带入不利性状，如匍匐茎变长、薯块变小、甾类糖苷生物碱含量增加等。
因此，人们希望通过更好的方式创制马铃薯自交亲和材料。但是目前，还没有研究机构在这方面取得突破。
发明内容
为解决现有技术中没有更好的方法能够创制出马铃薯自交亲和材料的缺陷，本发明提供了一种克服二倍体马铃薯自交不亲和的方法，实现的目的为寻找到一种简便、精准、高效的方法克服二倍体马铃薯自交不亲和，从而克服二倍体马铃薯自交不亲和障碍，为创制自交亲和材料及纯合自交系的创制提供核心技术支持。
为了实现上述目的，本发明提供以下技术方案：本发明提供的一种克服 二倍体马铃薯自交不亲和的方法，包括如下步骤：
(1)在马铃薯自交不亲和决定基因S-RNase基因S p3和S p4的基因区域区域选取靶标片段；
(2)根据步骤(1)得到的靶标片段的核酸排列顺序，构建用于二倍体马铃薯S-RNase基因打靶的CRISPR/Cas9重组载体；
(3)将步骤(2)得到的重组载体导入到马铃薯细胞，诱导所述靶标片段的向导RNA表达框和Cas9核酸酶表达框在细胞中共同表达，剪切S-RNase基因的双链靶标片段，触发马铃薯细胞自身的DNA修复功能，在靶标位点随机***或缺失碱基，实现细胞内S-RNase基因的功能缺失突变；
(4)用导入了所述重组载体的马铃薯细胞再生若干个马铃薯植株，对所选再生植株进行标记基因筛选；
(5)通过基因组PCR方法特异性扩增所选再生植株中S-RNase基因包含靶标片段的DNA区段，并对扩增产物测序；
(6)选择S-RNase基因被编辑的再生植株；
(7)检测所选基因编辑植株的染色体倍性，以筛选出二倍体的基因编辑株系；
(8)扩繁并定植所选基因编辑株系，于开花期进行自交亲和性表型鉴定；
(9)收获自交亲和性株系所结种子，提取后代基因组DNA，通过PCR方法特异性扩增所选后代中S-RNase基因包含靶标片段的DNA区段，并对扩增产物测序，检测被编辑靶标基因在后代的遗传和分离。
其中所述马铃薯自交不亲和决定基因S-RNase基因S p3和S p4的基因区域包括外显子和启动子，或其部分。
进一步的，所述步骤(1)中靶标片段位于目的基因S-RNase上，所述靶标片段的一条链具有如SEQ ID No.1所示的核酸序列结构。例如，其中所述靶标片段位于目的基因S-RNase上，所述靶标片段的一条链具有5’-(N) X-NGG-3’结构，(N) X表示数目为X的一条碱基序列{N 1,N 2……N X}，N 1,N 2……N X中的每一个表示碱基A、G、C、T中的任意一个，NGG中的N为A、G、C、T中的任意一个；X为15-25之间的整数，优选地，所述X 为17-23之间的整数；更优选地，所述X为18、19、20、21。
进一步的，所述步骤(2)中所述重组载体包括具有所述靶标片段，所述靶标片段为SEQ ID No.1核酸序列或与之互补的序列。
本发明另一方面提供一种马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，其包含的S-RNase基因功能缺失突变，所述S-RNase蛋白的核苷酸序列如SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
在本发明的一个具体实施方式中，所述S-RNase蛋白的核苷酸序列的同源序列可为在严谨条件下与SEQ ID NO：2和/或SEQ ID NO：3中的核苷酸序列或其互补序列进行杂交，其不表达S-RNase蛋白的多核苷酸或其片段的多核苷酸；
本文所述的“严谨条件”，可以为低严谨条件、中严谨条件、高严谨条件中的任一种，优选为高严谨条件。示例性地，“低严谨条件”可为30℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“中严谨条件”可为40℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“高严谨条件”可为50℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件。本领域技术人员应当理解温度越高越能得到高同源性的多核苷酸。另外，本领域技术人员可以选择影响杂交的严谨度的温度、探针浓度、探针长度、离子强度、时间、盐浓度等多个因素形成的综合结果来实现相应的严谨度。
除此之外可杂交的多核苷酸还可以为，通过FASTA、BLAST等同源性检索软件用***设定的默认参数进行计算时，与编码本发明的多核苷酸具有约30％或以上、40％或以上、50或以上、60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％ 或以上、或99.9％或以上同源性的多核苷酸。
核苷酸序列的同源性，可以使用Karlin及Altschul的算法规则BLAST(Proc.Natl.Acad.Sci.USA 87：2264-2268,1990；Proc.Natl.Acad.Sci.USA 90:5873,1993)来确定。基于BLAST算法规则的程序BLASTN、BLASTX已被开发(Altschul SF,et al:J Mol Biol 215:403,1990)。使用BLASTN分析碱基序列时，如使参数为score＝100、wordlength＝12；使用BLAST和Gapped BLAST程序时，采用各程序的***可设定默认参数值。
在本发明的一个具体实施方式中，所述S-RNase基因功能缺失是通过在表达S-RNase蛋白的基因中添加、缺失或替换核苷酸(1个或多个)而实现。例如在表达S-RNase蛋白的基因中添加1个、2个或2个以上的核苷酸；缺失1个、2个或2个以上的核苷酸；替换1个、2个或2个以上的核苷酸。
示例性地，所述S-RNase蛋白的核苷酸序列如SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
在本发明的一个具体实施方式中，所述S-RNase基因功能缺失是通过在ACGATTCACGGGCTTTGGCC或其互补序列中添加、缺失或替换核苷酸(1个或多个)实现。例如，添加1-5个核苷酸，缺失1-10个核苷酸，或替换1-8个核苷酸等。
在本发明的一个具体实施方式中，所述添加、缺失或替换核苷酸是通过CRISPR/Cas9重组载体实现的。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体能够靶向定位于S-RNase蛋白的基因，所述S-RNase蛋白的核苷酸序列如SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体能够靶向定位于S-RNase基因的第一个外显子区域。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体能够靶向定位于S-RNase基因的ACGATTCACGGGCTTTTGGCCGG或其互补序列。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体中sgRNA的核苷酸序列为：
S-RNase P3(即Seq ID No.4)： xxxxACGATTCACGGGCTTTGGC
S-RNase P4(即Seq ID No.5)： xxxxGCCAAAGCCCGTGAATCGT
其中未被下划线标注的部分为上述靶位点中去除NGG的序列或互补序列，下划线部分为用于连接载体的粘性末端。
本发明另一方面提供一种靶向敲除S-RNase基因的CRISPR/Cas9重组载体，所述CRISPR/Cas9重组载体靶向的S-RNase基因的核苷酸序列如SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体能够靶向定位于S-RNase基因的第一个外显子区域。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体能够靶向定位于S-RNase基因的ACGATTCACGGGCTTTTGGCCGG或其互补序列。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体中sgRNA的核苷酸序列为：
S-RNase P3(即Seq ID No.4)： xxxxACGATTCACGGGCTTTGGC
S-RNase P4(即Seq ID No.5)： xxxxGCCAAAGCCCGTGAATCGT
其中未被下划线标注的部分为上述靶位点中去除NGG的序列或互补序列，下划线部分为用于连接载体的粘性末端。
在本发明的一个具体实施方式中，所述CRISPR/Cas9重组载体的构建包括如下步骤：
(1)依据靶标序列设计引物S-RNase P3和S-RNase P4；
(2)将S-RNase P3和S-RNase P4形成形成具有粘性末端的双链DNA，作为构建重组载体的***片段；
(3)BsaI内切酶酶切pKSE401载体作为构架重组载体的骨架片段；
(4)T4连接酶将重组载体骨架片段和***片段相连，转入大肠杆菌，筛选出CRISPR/Cas9重组载体。
示例性地，所述引物S-RNase P3和S-RNase P4的序列如下：
S-RNase P3(即Seq ID No.4)： xxxxACGATTCACGGGCTTTGGC
S-RNase P4(即Seq ID No.5)： xxxxGCCAAAGCCCGTGAATCGT
其中未被下划线标注的部分为上述靶位点中去除NGG的序列或互补序列，下划线部分为用于连接载体的粘性末端。
本发明另一方面提供上述CRISPR/Cas9重组载体在制备敲除S-RNase蛋白基因中的应用。
本发明另一方面提供自交亲和的马铃薯的育种方法，包括使马铃薯中的S-RNase基因不表达或者被失活，所述的S-RNase基因为SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
在本发明的一个具体实施方式中，所述育种方法具体包括：
(1)构建CRISPR/Cas9重组载体；
(2)将步骤(1)中的CRISPR/Cas9重组载体导入马铃薯细胞，诱导所述靶标片段的向导RNA表达框和Cas9核酸酶表达框在细胞中共同表达，剪切S-RNase基因的双链靶标片段，触发马铃薯细胞自身的DNA修复功能，导致靶标位点随机***、缺失或替换碱基，实现细胞内S-RNase基因的功能缺失突变；
(3)筛选S-RNase基因发生突变的植株。
本发明另一方面提供马铃薯的育种方法，包括使用上述所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，或上述的育种方法获得的马铃薯植物进行自交。
本发明另一方面提供一种制造商业植物产品的方法，其包括获得上述的植物或其部分及由所述植物或其植物部分制造所述商业植物产品，其中所述植物产品是选自由以下组成的群组：新鲜的的完整马铃薯、法式炸薯条、马铃薯片、脱水马铃薯材料、马铃薯雪片及马铃薯颗粒。
本发明另一方面提供一种由上述的马铃薯植物，其植物部分，块茎或块茎部分，或其植物细胞、花粉或种子生长产生的马铃薯植物，块茎或块茎部分制成的食品。
在本发明的一个具体实施方式中，所述食品为切片的马铃薯块茎食品。
在本发明的一个具体实施方式中，其中所述食品为法式炸薯条、薯片及烘焙马铃薯组成的群组。
本发明采用上述技术方案，本发明包括以下有益效果：相对于传统的解决马铃薯自交不亲和方法，本技术发明有如下优点：
1),本发明定向编辑自交不亲和基因，构建一种载体，即可同时靶向2条目标基因的靶点，而且创制出多种新的自交亲和育种材料；
2),育种周期短，整个材料定向创制过程约12个月；
3),人工自交授粉受开花时期和环境影响较小，省时省力，可操作性强，坐果率高，种子多，而蕾期自交局限性大、坐果率低，种子少；
4),本发明创制的自交亲和材料的自交亲和性具有可遗传性，是从根本上解决了二倍体马铃薯自交不亲和障碍。
附图说明
图1所示为本发明实施例提供的靶点突变检测结果。
图2所示为本发明实施例提供的再生植株的倍性检测。其中图a为二倍体；图b为四倍体。
图3所示为本发明实施例提供的野生型材料(A)和基因编辑材料(B)花粉管在花柱中伸长的表型图。
图4所示为本发明实施例提供的野生型和基因编辑株系自花授粉坐果表型图。
具体实施方式
下面通过具体的实施例并结合附图对本发明做进一步的详细描述。
实施例一：本发明提供的一种克服二倍体马铃薯自交不亲和的方法，包括如下步骤：
(1)在马铃薯自交不亲和决定基因S-RNase基因S p3和S p4第一个外显子区域选取靶标片段；所述步骤(1)中靶标片段位于目的基因S-RNase上，所述靶标片段的一条链具有如SEQ ID No.1所示的核酸序列结构。所述靶标片段的一条链具有如SEQ ID No.1所示的核酸序列结构。例如，其中所述靶标片段位于目的基因S-RNase上，所述靶标片段的一条链具有5’-(N) X-NGG-3’结构，(N) X表示数目为X的一条碱基序列{N 1,N 2……N X}，N 1,N 2……N X中的每一个表示碱基A、G、C、T中的任意一个，NGG中的N为A、G、C、T中的任意一个。
(2)根据步骤(1)得到的靶标片段的核酸排列顺序，构建用于二倍体马铃薯S-RNase基因打靶的CRISPR/Cas9重组载体，所述步骤(2)中所述重组载体包括具有所述靶标片段，所述靶标片段为SEQ ID No.1核酸序列或与之互补的序列；
具体操作为：2.1选择二倍体马铃薯S.phureja CIP 703541两条S-RNase基因(S p3和S p4)第一个外显子上完全保守的一段核苷酸序列ACGATTCACGGGCTTTGGCCGG，(所述最末位CGG部分为所述5’-(N) X-NGG-3’结构中NGG部分)，作为打靶位点。S p3核苷酸序列如Seq ID No.2所示，S p4核苷酸序列如Seq ID No.3所示，所述S p3靶标核苷酸序列如Seq ID No.2第154-172位所示，所述S p4靶标核苷酸序列如Seq ID No.3第157-175位所示；
2.2按所选择靶位点合成正向寡核苷酸链(S-RNase P3)和可与之互补的反向寡核苷酸链(S-RNase P4)，
具体序列为：
S-RNase P3(即Seq ID No.4)： xxxxACGATTCACGGGCTTTGGC；
S-RNase P4(即Seq ID No.5)： xxxxGCCAAAGCCCGTGAATCGT
其中未被下划线标注的部分为上述靶位点中去除NGG的序列或互补序列，下划线部分为用于连接载体的粘性末端；
2.3对引物S-RNase P3和S-RNase P4进行退火程序，将S-RNase P3和S-RNase P4两链退火形成具有粘性末端的双链DNA，作为构建重组载体的***片段；
2.4用BsaI内切酶在50℃酶切pKSE401载体12小时，65℃失活酶切体系10min，作为构架重组载体的骨架片段；
2.5用T4连接酶将重组载体骨架片段和***片段相连，转入大肠杆菌。经测序验证后，提取其阳性转化子，构成用于二倍体马铃薯S-RNase基因CRISPR/Cas9打靶的重组载体质粒；
2.6将重组载体质粒转入农杆菌EHA105菌株，经测序验证后，提取其阳性转化菌株。
(3)将步骤(2)得到的重组载体导入到马铃薯细胞，诱导所述靶标片 段的向导RNA表达框和Cas9核酸酶表达框在细胞中共同表达，剪切S-RNase基因的双链靶标片段，触发马铃薯细胞自身的DNA修复功能，导致靶标位点随机***或缺失碱基，实现细胞内S-RNase基因的功能缺失突变；
(4)用导入了所述重组载体的马铃薯细胞再生若干个马铃薯植株，对所选再生植株进行标记基因筛选；
(5)通过基因组PCR方法特异性扩增所选再生植株中S-RNase基因包含靶标片段的DNA区段，并对扩增产物测序；
(6)选择S-RNase基因被编辑的再生植株；
(7)检测所选基因编辑植株的染色体倍性，以筛选出二倍体的基因编辑株系；
(8)扩繁并定植所选基因编辑株系，进行自交亲和性表型鉴定；
(9)收获自交亲和性株系所结种子，提取后代基因组DNA，通过PCR方法特异性扩增所选后代中S-RNase基因包含靶标片段的DNA区段，并对扩增产物测序，检测被编辑靶标基因在后代的遗传和分离。
上述步骤中马铃薯S-RNase基因编辑检测的具体步骤：
3.1无菌保存的供体马铃薯材料S.phureja CIP 703541的茎尖在MS30基础培养基上培养3周后，取节间为外植体平铺在P-MS20平板培养基(预先于培养基表面放置2片灭菌滤纸，添加2mL PACM溶液)上预培养2天，基础培养基配方如MS30所述，预培养基配方如P-MS20所述，PACM溶液配方如PACM所述；
3.2活化农杆菌EHA105阳性转化菌株，摇菌至OD 0.5，然后浸染2.1所述预培养的外植体15分钟，然后将外植体平铺在C-MS20平板培养基(预先于培养基表面放置1片灭菌滤纸)上黑暗共培养2天，共培养基配方如C-MS20所述；
3.3将结束3.2所述共培养的外植体转入D-MS20平板分化培养基培养，每隔14天更换一次培养基，分化培养基配方如D-MS20所述；
3.4切取外植体上分化产生的能够伸长的芽，接入组培瓶R-MS30培养基进行阳性转化株抗性筛选，抗性筛选培养基配方如R-MS30所述；
3.5提取阳性转化株基因组DNA作为模板，分别用特异性引物对S p3-F: GGGGAAACTGGAAAATGGTT(即Seq ID No.6)、S p3-R:ATGTGAAGTTGTTCAGCGAAA(即Seq ID No.7)和S p4-F:CAACAAAATGGCTAAATCGCAG(即Seq ID No.8)、S p4-R:GGTTTTCTGTTGGGTGGCAT(即Seq ID No.9)扩增2条S-RNase基因S p3和S p4的基因全长，用Sanger测序法检测目标基因序列的靶点突变，试验结果如图1所示；
从图1中可以看出，本实施例中获得了5种靶点突变的植株，其S-RNase蛋白均发生移码突变。
3.6利用流式细胞仪检测上述靶点突变的阳性转化株，筛选出仍然保持二倍体染色体的马铃薯S-RNase基因编辑材料。其试验结果如图2所示，其中图a为二倍体；图b为四倍体。本实施例中获得了5种靶点突变的植株，其染色体倍性均为图A所示的二倍体类型。
4、二倍体马铃薯S-RNase基因编辑材料表型鉴定：
4.1扩繁并定植二倍体马铃薯S-RNase基因编辑株系，于开花期进行人工自花授粉；
4.2授粉48小时后分别取野生型和突变型株系雌蕊组织，用3:1 95％EtOH:glacial acetic acid固定24小时，经5M NaOH软化24小时并ddH 2O漂洗后，0.005mg·mL -1苯胺蓝溶液染色24小时，在荧光显微镜下检测花粉管染色。检测结果如图3所示。根据检测结果显示，一组为野生型材料S.phureja CIP 703541自花授粉48小时后花粉管不能进入胚株，即野生型的材料为自交不亲和，另一组为供体材料(S-RNase突变株)经定向突变后自花授粉48小时后花粉管成功进入胚株，表明突变型株系自交亲和；
4.3鉴定野生型和突变型株系自花授粉坐果表型，试验结果如图4所示。图4A为野生型株系，图4B为基因编辑株系。从图4中可以看出，野生型不能自交结实，而突变型能够自交结实，表明突变型株系是自交亲和新材料，成功克服了自交不亲和障碍；
4.4收获自交亲和株系所结种子，种子播种于穴盘，待其长出真叶后，提取所有幼苗基因组DNA，通过PCR方法特异性扩增所选幼苗中2条S-RNase基因包含靶标片段的DNA区段，并对扩增产物测序，检测被编辑 靶标基因在后代的遗传和分离，经验证通过S-RNase定点基因编辑创制的新材料的自交亲和性能够遗传给后代。验证结果如表1所示。
表1基因编辑材料的T0和T1代株系的突变模式
Figure PCTCN2019081515-appb-000001
注： a斜杠前数字代表检测出的T 1代无Cas9单株个数，斜杠后数字代表被检测T 1代单株数量； bT 1代无Cas9单株的S-RNase类型分离情况； c指示S-RNase突变类型。
以上所述用到的培养基配方如下表所示：
MS30(1L)：
MS 4.43g
sucrose 30g
pH 5.8
agar 8g
P-MS20(1L)：
MS 4.43g
sucrose 20g
pH 5.8
agar 8g
PA-MS20(1L)：
MS 4.43g
sucrose 20g
caseine hydrolysate 2g
2,4-D 1mg/L
KT 0.5mg/L
pH 6.5
C-MS20(1L)：
MS 4.43g
sucrose 20g
pH 5.8
agar 8g
a-napthaleneacetic acid 2mg·L -1
trans-zeatin 1mg·L -1
AS 40mg·L -1
D-MS20(1L)：
MS 4.43g
sucrose 20g
pH 5.8
agar 8g
a-napthaleneacetic acid 0.01mg·L -1
trans-zeatin 2mg·L -1
kanamycin 100mg·L -1
temetine 200mg·L -1
R-MS30(1L)：
MS 4.43g
sucrose 30g
pH 5.8
agar 8g
kanamycin 50mg·L -1
temetine 200mg·L -1
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Figure PCTCN2019081515-appb-000002
Figure PCTCN2019081515-appb-000003
Figure PCTCN2019081515-appb-000004
Figure PCTCN2019081515-appb-000005

Claims (17)

  1. 一种克服二倍体马铃薯自交不亲和的方法，其特征在于，该方法包括如下步骤：
    (1)在马铃薯自交不亲和决定基因S-RNase基因S p3和S p4的基因区域选取靶标片段；
    (2)根据步骤(1)得到的靶标片段的核酸排列顺序，构建用于二倍体马铃薯S-RNase基因打靶的CRISPR/Cas9重组载体；
    (3)将步骤(2)得到的重组载体导入到马铃薯细胞，诱导所述靶标片段的向导RNA表达框和Cas9核酸酶表达框在细胞中共同表达，剪切S-RNase基因的双链靶标片段，触发马铃薯细胞自身的DNA修复功能，导致靶标位点随机***或缺失碱基，实现细胞内S-RNase基因的功能缺失突变；
    (4)用导入了所述重组载体的马铃薯细胞再生若干个马铃薯植株，对所选再生植株进行标记基因筛选；
    (5)通过基因组PCR方法特异性扩增所选再生植株中S-RNase基因包含靶标片段的DNA区段，并对扩增产物测序；
    (6)选择S-RNase基因被编辑的再生植株；
    (7)检测所选基因编辑植株的染色体倍性，以筛选出二倍体的基因编辑株系；
    (8)扩繁并定植所选基因编辑株系，于开花期进行自交亲和性表型鉴定；
    (9)收获自交亲和性株系所结种子，提取后代基因组DNA，通过PCR方法特异性扩增所选后代中S-RNase基因包含靶标片段的DNA区段，并对扩增产物测序，检测被编辑靶标基因在后代的遗传和分离。
  2. 根据权利要求1所述的克服二倍体马铃薯自交不亲和的方法，其特征在于，所述步骤(1)中靶标片段位于目的基因S-RNase上，所述靶标片段的一条链具有如SEQ ID No.1所示的核酸序列结构。
  3. 根据权利要求2所述的克服二倍体马铃薯自交不亲和的方法，其特征在于，所述步骤(2)中所述重组载体包括具有所述靶标片段，所述靶标片段为SEQ ID No.1核酸序列或与之互补的序列。
  4. 马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种 子，其特征在于，其包含的S-RNase基因功能缺失突变，所述S-RNase基因的核苷酸序列如SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
  5. 如权利要求4所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，其特征在于，所述同源序列的同源性在30％或以上、40％或以上、50或以上、60％或以上、70％或以上、80％或以上、90％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.5％或以上、或99.9％或以上。
  6. 如权利要求4或5所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，其特征在于，所述S-RNase基因功能缺失突变是通过在表达S-RNase蛋白的基因中添加和/或缺失核苷酸(1个或多个)实现。
  7. 如权利要求6所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，其特征在于，通过在5’-(N) X-NGG-3’结构序列或其互补序列中添加、缺失或替换核苷酸(1个或多个)而实现。
  8. 如权利要求7所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，其特征在于，通过在ACGATTCACGGGCTTTGGCC或其互补序列中添加、缺失或替换核苷酸(1个或多个)实现。
  9. 如权利要求6所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，其特征在于，所述添加和/或缺失核苷酸是通过CRISPR/Cas9重组载体实现的。
  10. 如权利要求9所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，其特征在于，所述CRISPR/Cas9重组载体中sgRNA的核苷酸序列为：
    S-RNase P3(即Seq ID No.4)： xxxxACGATTCACGGGCTTTGGC
    S-RNase P4(即Seq ID No.5)： xxxxGCCAAAGCCCGTGAATCGT
    其中未被下划线标注的部分为上述靶位点中去除NGG的序列或互补序列，下划线部分为用于连接载体的粘性末端。
  11. 一种靶向敲除S-RNase蛋白基因的CRISPR/Cas9重组载体，其特征在于：所述CRISPR/Cas9重组载体靶向的S-RNase蛋白的核苷酸序列如SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
  12. 如权利要求11所述的重组载体，所述CRISPR/Cas9重组载体中sgRNA的核苷酸序列为：
    S-RNase P3(即Seq ID No.4)： xxxxACGATTCACGGGCTTTGGC；
    S-RNase P4(即Seq ID No.5)： xxxxGCCAAAGCCCGTGAATCGT；
    其中未被下划线标注的部分为上述靶位点中去除NGG的序列或互补序列，下划线部分为用于连接载体的粘性末端。
  13. 权利要求11或12所述的CRISPR/Cas9重组载体在制备敲除S-RNase蛋白基因中的应用。
  14. 自交亲和的马铃薯的育种方法，其特征在于，包括使马铃薯中的S-RNase基因不表达或者被失活，所述的S-RNase基因为SEQ ID NO:2所示的序列(Sp3)，或其互补序列，简并序列，同源序列；和/或如SEQ ID NO:3所示的序列(Sp4)，或其互补序列，简并序列，同源序列。
  15. 马铃薯的育种方法，其特征在于，包括使用权利要求4-10中任一项所述的马铃薯植物，其植物部分，块茎或块茎部分，植物细胞、花粉或种子，或权利要求14所述的育种方法获得的马铃薯植物进行自交。
  16. 一种制造商业植物产品的方法，其包括获得权利要求4-10中任一项所述的植物或其部分及由所述植物或其植物部分制造所述商业植物产品，其中所述植物产品是选自由以下组成的群组：新鲜的的完整马铃薯、法式炸薯条、马铃薯片、脱水马铃薯材料、马铃薯雪片及马铃薯颗粒。
  17. 一种由权利要求4-10中任一项所述的马铃薯植物，其植物部分，块茎或块茎部分，或其植物细胞、花粉或种子生长产生的马铃薯植物，块茎或块茎部分制成的食品。
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