WO2020006885A1 - 一种通过基因编辑创制雄性不育作物新种质的方法及其应用 - Google Patents

一种通过基因编辑创制雄性不育作物新种质的方法及其应用 Download PDF

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WO2020006885A1
WO2020006885A1 PCT/CN2018/106586 CN2018106586W WO2020006885A1 WO 2020006885 A1 WO2020006885 A1 WO 2020006885A1 CN 2018106586 W CN2018106586 W CN 2018106586W WO 2020006885 A1 WO2020006885 A1 WO 2020006885A1
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gene editing
sequence
gene
germplasm
cas9
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French (fr)
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王银磊
赵统敏
余文贵
赵丽萍
周蓉
宋刘霞
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江苏省农业科学院
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Priority to US16/612,764 priority Critical patent/US11236358B2/en
Publication of WO2020006885A1 publication Critical patent/WO2020006885A1/zh
Priority to US17/543,763 priority patent/US11820995B2/en

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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)
    • C12N15/8241—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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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

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  • the invention relates to the technical field of crop breeding by gene editing technology, in particular to a method for creating new germplasm of male sterile crops by gene editing and its application.
  • Crop male sterility is the phenomenon that pollen cannot be produced or aborted due to the abnormality of male organs caused by physiological or genetic reasons of sexually propagating crops, and normal pollination cannot be achieved. Because in crop heterosis breeding, artificial detasseling can be avoided during pollination, which saves a lot of labor input. At the same time, it can significantly improve the purity of hybrids and create varieties with heterosis.
  • Male sterility technology plays an important role in the use of heterosis, and obtaining stable male sterility materials has important application value.
  • Male sterility can be divided into nuclear male sterility and nucleoplasm interaction male sterility based on its genetic mode and localization in cells.
  • the application of male sterility has been carried out in rice, corn, wheat, cabbage, pepper and other crops.
  • male sterility is not used for production, and lack of male sterility resources and infertility of male sterility materials are the main problems it faces. It was found that male sterility material with natural mutation is the main source of male sterility material.
  • male sterility material can also be obtained through distant hybridization, artificial mutation, cell engineering and other methods. With the development of biotechnology, it has become possible to create male sterile materials through genetic engineering.
  • the gene Ty-5 is a tomato yellow leaf curl resistance gene in tomato. This gene is also a monitoring factor for monitoring peptide chain synthesis. The gene is present in all crops. In this case, through gene editing of this gene, a new germplasm of male sterility can be quickly created.
  • Current gene editing technologies include zinc finger nuclease technology, transcription-activator-like effector nuclease technology, and the latest CRISPR / CAS9 gene editing technology.
  • Gene editing realizes the recognition and shearing of specific DNA sequences, and introduces different types of mutations such as base deletions, substitutions, and insertions at double-strand breaks (DSBs) in DNA to achieve fixed-point DNA edit.
  • the invention aims at the problems of insufficient existing male sterility materials in crops, low purity of hybrids, and the current situation of conventional breeding with long time and high cost.
  • Gene editing is performed on the Ty-5 gene by means of gene editing to rapidly create male sterility New germplasm of breeding crops, while retaining the agronomic characteristics of the original male fertile material.
  • the gRNA target site is selected in the exon region of the Ty-5 gene. Based on the CRISPR / Cas9 target site anchoring principle, the protospacer-associated motif (PAM) upstream 18-20bp base is selected. Base as the target site, (5'-N18-20NGG-3 ', NGG is the PAM sequence, N18-20 represents the 18-20bp base recognition sequence);
  • Target-Sense 5’-TTG-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (N is the gRNAsense sequence)
  • Target-Anti 5’-AAC-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (N is the reverse complementary sequence of gRNAsense)
  • Plant cotyledons are used as explants, and plant regeneration is performed by the leaf disc method, and transformation is mediated by positive Agrobacterium GV3101, and the regenerated plants are obtained through hygromycin resistance screening;
  • design primers that can amplify the target site, extract the regenerated plant DNA, use it as a template for PCR amplification, sequence the amplified product, and determine whether gene regeneration occurs in the regenerated plant. And whether the editing type is homozygous for sequencing analysis;
  • the method for creating new germplasm of male-sterile crops provided by the present invention is not limited to crop types. After the Ty-5 gene of each crop is gene-edited, the materials before editing can be quickly obtained only during breeding. There are new germplasms with differences in sex and other agronomic traits, which effectively solve the problems of lack of male sterility materials and infertility in natural resources.
  • the method of the present invention can give the material male sterility more quickly than conventional breeding
  • the method of the present invention can better retain the agronomic traits of the recipient material than conventional breeding;
  • the hybrid seed production using the material created by the method for creating male sterility of the present invention can greatly reduce labor and improve the purity of the hybrid.
  • FIG. 1 is an amplification map of a regenerated plant after editing by a gene editing primer pair
  • Figure 2 shows the sequencing results of CRISPR5-F primers for each material amplification band.
  • Figure 3 is a comparison of pollen germination
  • a method for creating new germplasm of male sterile crops through gene editing including the following steps:
  • Tomato material moneymaker for tomato yellow leaf curl disease
  • CRISPR / Cas9 kit CRISPR / Cas9 kit VK005-14 from Beijing Weishidide Biotechnology Co., Ltd .;
  • T carrier Beijing Quanshijin Biotechnology Co., Ltd. (CT301-01);
  • DNA extraction kit Tiangen Biochemical Technology (Beijing) Co., Ltd. (DP305);
  • Agrobacterium competent GV3101;
  • the full-length sequence of Ty-5 gene (as shown in SEQ ID NO. 1) contains a total of 16 exons.
  • the second exon sequence is selected for the design of the gRNA target site.
  • the target sequence is 19 bp in length (as shown in SEQ ID NO. 2).
  • Target-F 5’-TTG-AGAAGAAGCTGATGATCTA-3 ’, as shown in SEQ ID NO.3,
  • Target-R 5'-AAC-TAGATCATCAGCTTCTTCT-3 ', as shown in SEQ ID NO.4;
  • Target primer Dilute the Target primer to 10 ⁇ m, take 5 ⁇ L of each of the primers, add 15 ⁇ L of water, and mix. Leave at 95 ° C for 3 minutes, then slowly cool to 25 ° C, and then 16 ° C for 5 minutes to complete the synthesis of oligo dimer;
  • the reaction system was: 1 ⁇ L of synthesized oligo dimer, 1 ⁇ L of Cas9 / gRNA carrier, 1 ⁇ L each of Solution and Solution 2, 6 ⁇ L of H 2 O added and mixed uniformly, and reacted at 16 ° C. for 2 h; the ligated vector was transformed into E. coli competent Trans1- T1, plate the plate overnight, pick a single colony, extract the plasmid, and perform sequencing analysis on the plasmid.
  • the sequencing primer is sqprimer: 5'-GATGAAGTGGACGGAAGGAAGGAGG-3 ', as shown in SEQ IN NO.5. Extract the plasmid from the bacteria with the correct sequencing result and transfer it into Agrobacterium GV3101 by freeze-thaw method.
  • Explant preparation Select cotyledons that have just emerged from the seed coat for transformation.
  • the cotyledons were cut and placed in 50-100 ml MS liquid medium.
  • the cotyledon's petiole and tip were excised and placed on D1 medium and cultured upside down.
  • the petri dish was pre-cultured in a 24 ° C constant temperature culture chamber (16L / 8D) for 2 days.
  • Co-culture Add 5ul of 0.074M acetylsyringone per 10ml of MS liquid medium. Aspirate 5 ml of MS liquid culture medium (+ AS) to rinse the Agrobacterium colonies, dilute the bacterial liquid OD600 to 0.3-0.4, and add the bacterial liquid to the pre-cultured (2days) cotyledons. 8-10 minutes after Agrobacterium infects the cotyledons, the excess bacterial liquid bacteria are aspirated. Cotyledons were transferred to this D1 medium containing filter paper. The cotyledons are inverted (the lower surface of the leaf is facing up). Incubate at 24 ° C for 2 days in the dark.
  • Rooting Remove the young shoots from the explants and place the young shoots in 100ml sterile bottles containing 40ml of MMSV medium (containing 0.5mg / L IBA, 15mg / L hygromycin and 200mg / L Temeter) . Roots began to appear around 2 weeks. When the plants are large enough, the seedlings can be transplanted into small plastic pots containing a mixture of vermiculite and soil, and irrigated with nutrient water. Through tomato transformation, a total of 2 transformed tomato seedlings were obtained.
  • MMSV medium containing 0.5mg / L IBA, 15mg / L hygromycin and 200mg / L Temeter
  • the leaf DNA of two regenerated plants and wild-type moneymaker materials were extracted, and the amplification primers were:
  • CRISPR5-R 5'-GCTAATAATGCTAAGCCCTCACA-3 ', as shown in SEQ ID NO.7.
  • the length of the amplified fragment was about 450bp.
  • Agarose electrophoresis was performed on the PCR amplified product to determine whether the band size was correct ( Figure 1).
  • the electrophoresis bands showed that 450bp bands could be amplified from both the regenerated plant and the wild type moneymaker material.
  • the 3 PCR products amplified from the regenerated plant and wild material were sequenced and analyzed with CRISPR5-F primers to detect gene editing. Variation of the site base sequence ( Figure 2).
  • the test results confirmed that compared with the wild type, the regenerated plants No. 1 and No. 2 had sequence variation at the gene editing site, but the No. 1 plant was heterozygous, and one of the double-stranded DNAs had a 13bp base. Base deletion; the mutation type of plant No. 2 is homozygous, and the double-stranded DNA has a 6bp base sequence deleted at the same site.
  • Pollens from 2 regenerated plants and wild-type moneymaker were collected and incubated in the dark on the germination medium for 4 h at 26 ° C. Observe under a microscope and compare the amount of pollen and germination of each material ( Figure 3). Through the germination comparison test, the pollen amount of the regenerated plant No. 1 was the same as that of the wild-type moneymaker, while the pollen number of the regenerated plant No. 2 was significantly reduced; Wild type moneymaker can see the pollen begins to germinate. It can be seen that the homozygous mutation (editing plant No. 2) can greatly reduce the pollen of the plant and stop the pollen germination.
  • the present invention provides a method for creating a new male sterility germplasm through gene editing means.
  • gene editing By performing gene editing on the ex-5 region of the Ty-5 gene, a homozygous edited plant is obtained, which is male. Sterile new germplasm.
  • the full-length Ty-5 gene sequence of tomato material moneymaker is the intron sequence; the shaded area is the gene recognition site.

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Abstract

一种通过基因编辑创制雄性不育作物新种质的方法及其应用，该方法通过对Ty-5外显子区域进行基因编辑，利用植物自身对DBS的修复机制，引入DNA序列的缺失，使Ty-5基因的功能丧失，从而获得雄性不育特性。

Description

一种通过基因编辑创制雄性不育作物新种质的方法及其应用 技术领域
本发明涉及基因编辑技术进行农作物育种技术领域，尤其是一种通过基因编辑创制雄性不育作物新种质的方法及其应用。
背景技术
作物雄性不育是由于有性繁殖作物生理上或遗传上的原因造成雄性器官异常而导致的不能产生花粉或花粉败育，无法正常授粉的现象。因其在作物杂种优势育种中可以避免授粉过程中人工去雄的环节，节约了大量的劳动力投入，同时也可以显著的提高杂交种的纯度，创制具有杂种优势的品种。
雄性不育技术对于杂种优势的利用发挥着重要的作用，获得稳定的雄性不育材料具有重要的应用价值。雄性不育根据其遗传方式和在细胞中的定位可将其分为细胞核雄性不育和核质互作雄性不育。目前在水稻、玉米、小麦、甘蓝、辣椒等作物中都已开展了对雄性不育的应用。但在很多作物中，雄性不育并未用于生产，缺乏雄性不育资源、雄性不育材料的不育性状不稳定是其面临的主要问题。通过发现自然突变的雄性不育系材料是雄性不育材料的主要来源，此外还可以通过远缘杂交、人工诱变、细胞工程等手段获得雄性不育材料。随着生物技术的发展，通过基因工程创制雄性不育材料已经成为可能。
基因Ty-5是番茄中的抗番茄黄化曲叶病基因，该基因也是监控肽链合成过程中的监控因子，该基因在所有作物中都存在。本案通过对该基因进行基因编辑，可以快速创制出雄性不育的新种质。基因编辑技术的发展，为Ty-5基因在杂种优势育种中的利用提供了有力的武器。目前的基因编辑技术主要有锌指核酸酶技术、类转录激活因子效应物核酸酶技术，以及最新的CRISPR/CAS9基因编辑技术。基因编辑实现了对特定DNA序列的识别、剪切，并在DNA双链断裂位点(double-strand breaks，DSBs)处引入碱基的缺失、替换、插入等不同突变类型，实现对DNA的定点编辑。
发明内容
本发明针对作物中现有雄性不育材料不足，杂交种纯度不高的问题，以及常规育种时 间长，成本高的现状，通过基因编辑的手段对Ty-5基因进行基因编辑，快速创制雄性不育作物新种质，同时保留原雄性可育材料的农艺性状不变。
为解决上述技术问题，本发明通过以下技术方案得以解决：
(1)已有基因编辑手段均可以用于本研究对Ty-5的编辑，本发明仅列出利用CRISPR/Cas9技术对Ty-5的基因编辑，利用植物自身对DBS的修复机制，引入DNA序列的缺失，使Ty-5基因功能的丧失，从而获得雄性不育特性；
(2)gRNA靶位点选择在Ty-5基因的外显子区域，根据CRISPR/Cas9靶位点锚定原理，选取原型间隔序列毗邻基序(protospacer-associated motif，PAM)上游18-20bp碱基作为靶位点，即(5'-N18-20NGG-3'，NGG为PAM序列，N18-20代表18-20bp碱基的识别序列)；
(3)根据识别序列设计oligo序列引物：
Target-Sense：5’-TTG-NNNNNNNNNNNNNNNNNNN(N表示gRNAsense序列)
Target-Anti：5’-AAC-NNNNNNNNNNNNNNNNNNN(N表示gRNAsense的反向互补序列)
将引物浓度稀释为10μm，取上述引物各5μl，加水15μl，混匀。95℃放置3分钟，之后慢慢冷却至25℃，之后16℃5分钟，完成oligo二聚体的合成；
(4)选用北京唯尚立德生物科技有限公司的CRISPR/Cas9试剂盒VK005-14，利用已合成的oligo二聚体1μl，Cas9/gRNA载体1μl，Solution和Solution 2各1μl，H 2O加入6μl混合均匀，16℃反应2h；
(5)将连接后的载体转入大肠杆菌感受态，挑取单克隆进行质粒测序分析，测序引物为sqprimer：5’-GATGAAGTGGACGGAAGGAAGGAG-3’，将测序结果阳性的质粒，转入农杆菌GV3101；
(6)以作物子叶为外植体，通过叶盘法进行植株再生，用阳性农杆菌GV3101介导转化，经过潮霉素抗性筛选，获得再生植株；
(7)根据靶位点上下游序列，设计可以扩增靶位点的引物，提取再生植株DNA，以其为模板进行PCR扩增，对扩增产物进行测序，对再生植株的是否发生基因编辑，以及编 辑类型是否为纯合突变进行测序分析；
(8)测序分析后，纯合基因编辑类型的株系进行花粉含量和萌发的鉴定，鉴定结果显示基因编辑创制的纯合编辑Ty-5的植株即为雄性不育新种质。
与现有技术相比，本发明的有益效果是：
(1)本发明所提供的创制雄性不育作物新种质的方法，不局限于作物类别，当对各作物的Ty-5基因进行基因编辑后，就可以快速获得与编辑前材料只在育性方面存在差异、其他农艺性状相同的新种质，有效解决了自然资源中雄性不育材料缺乏、育性不稳定的问题。
(2)本发明的方法较常规育种可以更快的赋予材料雄性不育的特性；
(3)本发明的方法较常规育种可以更好的保留受体材料的农艺性状；
(4)通过本发明创制的雄性不育新种质是对现有材料的有力补充；
(5)利用本发明创制雄性不育方法所创制的材料进行杂交制种，可以极大的减少用工，提高杂交种纯度。
附图说明
图1为基因编辑引物对编辑后再生植株扩增图谱；
(M：100bp Marker；1：基因编辑后1号再生植株；2：基因编辑后2号再生植株；3：野生型Money maker材料)；
图2为CRISPR5-F引物对各材料扩增条带测序结果
(a：1号再生植株；b：野生型moneymaker测序结果；c：2号再生植株；三角框分别表示1号和2号再生植株缺失的序列)；
图3为花粉萌发比较；
(a：1号再生植株；b：野生型moneymaker；c：2号再生植株)。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本 发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
通过基因编辑创制雄性不育作物新种质的方法，包括以下步骤：
(1)材料
番茄材料：感番茄黄化曲叶病材料moneymaker；
CRISPR/Cas9试剂盒：北京唯尚立德生物科技有限公司的CRISPR/Cas9试剂盒VK005-14；
T载体：北京全式金生物技术有限公司(CT301-01)；
DNA提取试剂盒：天根生化科技(北京)有限公司(DP305)；
农杆菌感受态：GV3101；
引物合成及测序均由南京金斯瑞生物科技有限公司完成。
(2)gRNA靶位点的选择
Ty-5基因全长序列(如SEQ ID NO.1所示)共包含16个外显子，本实施例选择第二个外显子序列进行gRNA靶位点的设计。靶序列长19bp(如SEQ IN NO.2所示)。根据靶序列，设计引物：
Target-F：5’-TTG-AGAAGAAGCTGATGATCTA-3’，如SEQ IN NO.3所示，
Target-R：5’-AAC-TAGATCATCAGCTTCTTCT-3’，如SEQ IN NO.4所示；
(3)oligo二聚体合成：
将Target引物稀释为10μm，取上述引物各5μL，加水15μL，混匀。95℃放置3分钟，之后慢慢冷却至25℃，之后16℃5分钟，完成oligo二聚体的合成；
(4)CRISPR/Cas9重组载体的构建及农杆菌转化
反应体系为：已合成的oligo二聚体1μL，Cas9/gRNA载体1μL，Solution和Solution2各1μL，H 2O加入6μL混合均匀，16℃反应2h；将连接后的载体转化大肠杆菌感受态Trans1-T1，涂板过夜，挑取单菌落，提取质粒，对质粒进行测序分析，测序引物为sqprimer：5’-GATGAAGTGGACGGAAGGAAGGAG-3’，如SEQ IN NO.5所示。将测序结果正确 的菌提取质粒，通过冻融法转入农杆菌GV3101中.
(5)番茄转化：
主要步骤如下：
外植体预培养准备：选择刚从种皮出现的子叶用于转化。将子叶切下并放入50-100ml MS液体培养基中。将子叶的柄端和尖端切除，放在D1培养基上颠倒放置培养。将培养皿放在24℃恒温培养室(16L/8D)预培养2天。
共培养：每10ml的MS液体培养基中加入5ul的0.074M乙酰丁香酮。吸取5ml的MS液体培养基(+AS)冲洗农杆菌菌落，将菌液OD600稀释至0.3-0.4，吸取菌液加入到预培养(2days)子叶中。农杆菌侵染子叶8-10分钟之后，将多余菌液菌吸出。将子叶转移到该含有滤纸的D1培养基上。子叶倒置(叶片下表面朝上)。24℃，黑暗培养2天。
筛选：将无菌子叶转移至50ml离心管中，无菌水洗涤2次，+Car洗涤一次；将子叶倒置(背面朝上)在分化培养基2Z上，每皿20-30片子叶，以保证子叶所需生长空间。双层封口膜封口，24℃培养10天(16L/8D)。在第10天，将子叶转移到新鲜2Z培养基平板。然后每2-3周继代培养一次。2-3周后会长愈伤。4-6周之内会出现初始芽。当芽开始出现后，每2周继代一次外植体，培养基为1Z选择性培养基。
生根：从外植体摘下幼芽，将幼芽放入含40ml的MMSV培养基(含0.5mg/L IBA，15mg/L潮霉素和200mg/L特美汀)的100ml无菌瓶中。2周左右开始出现根。当植物生长足够大时，可将幼苗移栽进含蛭石和土壤混合物的小塑料盆中，并用营养水浇灌。通过番茄转化，共获得2株转化番茄幼苗。
6.再生植株基因编辑检测：
分别提取2株再生植株和野生型moneymaker材料的叶片DNA，用扩增引物为：
CRISPR5-F：5’-TCCATTGAACTGAAGCAAATCTC-3’，如SEQ IN NO.6所示，
CRISPR5-R：5’-GCTAATAATGCTAAGCCCTCACA-3’，如SEQ IN NO.7所示。
扩增片段长度约为450bp，对PCR扩增后的产物进行琼脂糖电泳检测，判断条带大小是否正确(图1)。电泳条带显示，再生植株和野生型moneymaker材料均可以扩增出450bp 大小的条带，将再生植株和野生材料扩增后的3个PCR产物，用CRISPR5-F引物进行测序分析，检测基因编辑部位碱基序列的变异情况(图2)。检测结果证实，1号和2号再生植株与野生型相比，在基因编辑位点处均发生了序列的变异，但1号植株的变异为杂合类型，双链DNA其中一条发生了13bp碱基的缺失；2号植株的变异类型为纯合变异，DNA双链均在同一位点处缺失了6bp碱基序列。
7.花粉量及萌发比较
对2株再生植株及野生型moneymaker的花粉进行采集，26℃下在萌发培养基上黑暗孵育4h。显微镜下观察，比较各材料的花粉量及其萌发状况(图3)。通过萌发比较试验，1号再生植株和野生型moneymaker花粉量一致，而2号再生植株的花粉数明显减少；通过花粉萌发比较，2号花粉4h时未见花粉萌发现象，但1号再生植株和野生型moneymaker均可以看到花粉开始萌发。可见，纯合变异(2号编辑植株)可以使植株花粉大量减少，花粉停止萌发。
综上所述，本发明提供了一种通过基因编辑手段创制作物雄性不育新种质的方法，通过对Ty-5基因外显子区域，进行基因编辑，获得纯合编辑植株，即为雄性不育新种质。
SEQ ID NO.1
番茄材料moneymaker内Ty-5基因全长序列，下划线标出的序列为内含子序列；阴影标出的为基因识别位点。
Figure PCTCN2018106586-appb-000001
Figure PCTCN2018106586-appb-000002
Figure PCTCN2018106586-appb-000003
Figure PCTCN2018106586-appb-000004
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (5)

  1. 一种通过基因编辑创制雄性不育作物新种质的方法，其特征在于：通过对Ty-5外显子区域进行基因编辑，利用植物自身对DBS的修复机制，引入DNA序列的缺失，使Ty-5基因功能的丧失，从而获得雄性不育特性。
  2. 根据权利要求1所述的通过基因编辑创制雄性不育作物新种质的方法，其特征在于：利用CRISPR/Cas9技术对Ty-5的基因编辑。
  3. 根据权利要求2所述的通过基因编辑创制雄性不育作物新种质的方法，其特征在于，包括以下步骤：
    (1)gRNA靶位点的选择
    gRNA靶位点选择在Ty-5基因的外显子区域，根据CRISPR/Cas9靶位点锚定原理，选取原型间隔序列毗邻基序上游18-20bp碱基作为靶位点，即5'-N18-20NGG-3'，NGG为PAM序列，N18-20代表18-20bp碱基的识别序列；
    (2)根据识别序列设计oligo序列引物：
    Target-Sense：5’-TTG-NNNNNNNNNNNNNNNNNNN，其中，N表示gRNAsense序列；
    Target-Anti：5’-AAC-NNNNNNNNNNNNNNNNNNN，其中，N表示gRNAsense的反向互补序列；
    将引物浓度稀释为10μm，取上述引物各5μL，加水15μL，混匀；95℃放置3分钟，之后慢慢冷却至25℃，之后16℃5分钟，完成oligo二聚体的合成；
    (3)CRISPR/Cas9重组载体的构建及农杆菌转化
    选用北京唯尚立德生物科技有限公司的CRISPR/Cas9试剂盒VK005-14，利用已合成的oligo二聚体1μL，Cas9/gRNA载体1μL，Solution和Solution 2各1μL，H20加入6μL混合均匀，16℃反应2h；
    将连接后的载体转入大肠杆菌感受态，挑取单克隆进行质粒测序分析，测序引物为sqprimer：5’-GATGAAGTGGACGGAAGGAAGGAG-3’，如SEQ IN NO.5所示，将测序结果阳性的质粒，转入农杆菌GV3101；
    (4)作物转化
    以作物子叶为外植体，通过叶盘法进行植株再生，用阳性农杆菌GV3101介导转化，经过潮霉素抗性筛选，获得再生植株。
  4. 根据权利要求3所述的通过基因编辑创制雄性不育作物新种质的方法，其特征在于，还包括以下步骤：
    (1)再生植株基因编辑检测：根据靶位点上下游序列，设计可以扩增靶位点的引物，提取再生植株DNA，以其为模板进行PCR扩增，对扩增产物进行测序，对再生植株的是否发生基因编辑，以及编辑类型是否为纯合突变进行测序分析；
    (2)测序分析后，纯合基因编辑类型的株系进行花粉含量和萌发的鉴定，鉴定结果显示基因编辑创制的纯合编辑Ty-5的植株即为雄性不育新种质。
  5. 权利要求1-4任一所述的通过基因编辑创制雄性不育作物新种质的方法在创制雄性不育作物新种质中的应用。
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