WO2016141893A1 - 一种提高植物对入侵的dna病毒的抵御能力的方法 - Google Patents

一种提高植物对入侵的dna病毒的抵御能力的方法 Download PDF

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WO2016141893A1
WO2016141893A1 PCT/CN2016/076246 CN2016076246W WO2016141893A1 WO 2016141893 A1 WO2016141893 A1 WO 2016141893A1 CN 2016076246 W CN2016076246 W CN 2016076246W WO 2016141893 A1 WO2016141893 A1 WO 2016141893A1
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dna
plant
virus
vector
sequence
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高彩霞
姬祥
张华伟
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中国科学院遗传与发育生物学研究所
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Priority to KR1020177029290A priority Critical patent/KR101994953B1/ko
Priority to BR112017016423A priority patent/BR112017016423A2/pt
Priority to EA201791991A priority patent/EA201791991A1/ru
Priority to JP2017548173A priority patent/JP2018507705A/ja
Priority to CA2979292A priority patent/CA2979292A1/en
Priority to AU2016228599A priority patent/AU2016228599A1/en
Priority to EP16761120.1A priority patent/EP3309255A4/en
Priority to US15/557,306 priority patent/US20180195084A1/en
Publication of WO2016141893A1 publication Critical patent/WO2016141893A1/zh

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Definitions

  • the invention belongs to the field of plant genetic engineering and relates to a method for improving the resistance of plants to invading DNA viruses.
  • DNA viruses are widely found in nature and have a broad host of bacteria, animals, and plants. Most of the known DNA viruses cause serious infectious diseases and bring huge losses to economic crops and mammals, including humans.
  • geminiviruses are the only single-stranded DNA viruses found in plants that have a twin particle morphology and are the largest family of single-stranded DNA viruses known to date. Its genomic type can be divided into one-component and two-component, and the size is about 2.5-3.1 kb. It spreads through the insect mediator in a persistent manner, most infecting the phloem tissue of parasitic plants. It has been reported that the geminivirus mainly causes huge economic losses to crops such as tomatoes, cassava and cotton.
  • the virus only encodes the replication protein Rep and the replication-enhancing protein Ren, and the Rep protein binds to the double
  • the strand DNA is the origin of replication and is cleaved in its conserved sequence TAATATTAC to create a nick, thereby initiating rolling circle replication.
  • Rep protein By transferring a full-length or partial exogenous Rep protein, it can compete with the Rep protein encoded by the geminivirus itself, thereby inhibiting replication of the geminivirus.
  • high expression levels, interference with host cell growth, and non-universality make this technology a huge disadvantage.
  • RNAi technology is used to interfere with the expression of viral pathogenic proteins for antiviral purposes. This technique is also not universal because of its homologous dependence.
  • Takashi Sera developed a method for inhibiting replication of geminiviruses using zinc finger binding proteins to specifically bind to DNA duplexes. It uses Beet severe curly top virus (BSCTV) as a model to compete with the viral replication initiation protein Rep by transferring exogenous artificial zinc finger protein (AZP) and binds to the virus in the middle of the double strand. The body "copyes the starting point, thus hindering its copying.
  • BSCTV Beet severe curly top virus
  • AZP exogenous artificial zinc finger protein
  • this method is only directed to a type of DNA geminivirus in which the geminivirus has the Rep protein as its replication initiation protein, and does not have broad-spectrum resistance to the DNA virus.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat sequences
  • tracrRNA Trans-acting CRISPR RNA
  • CRISPR-associated genes CRISPR-associated. Genes, Cas gene). Certain DNA fragments of the virus will be recorded between the CRISPR repeats. The CRISPR RNA containing the viral sequence is then expressed.
  • CRISPR RNA under the synergy of tracrRNA, directs the endonuclease encoded by the Cas gene to recognize and eliminate the double-stranded DNA of the foreign virus.
  • the CRISPR/Cas immune system is divided into three types according to the sequence of the core elements of the Cas gene: type I, type II and type III.
  • type I and type III need to be composed of a plurality of proteins encoded by the Cas gene to complete the cleavage of double-stranded DNA; and type II only requires Cas9 protein to cleave double-stranded DNA. Therefore, the most widely used type is the Type II CRISPR/Cas system.
  • the artificial synthesis method can be used to transform the crRNA and tracrRNA into a single-guide RNA (sgRNA), and the sgRNA can be used to successfully guide the Cas9 endonuclease to perform DNA spotting.
  • sgRNA single-guide RNA
  • the eukaryote can have a system similar to the bacterial CRISPR/Cas, it will be able to significantly improve the antiviral ability of eukaryotes.
  • the method for cultivating a plant having improved resistance to DNA viruses provided by the present invention, specifically It can include the following steps:
  • the target sequence is a sequence conforming to a 5'-N X -NGG-3' or 5'-CCN-N X -3' sequence alignment rule;
  • N represents any one of A, G, C and T, 14 ⁇ X ⁇ 30, and X is an integer, and N X represents X consecutive deoxyribonucleotides;
  • the recombinant vector is capable of transcribing a guide RNA and expressing a Cas9 protein;
  • the guide RNA is a palindrome-structured RNA formed by base-pairing of a crRNA and a tracrRNA;
  • the crRNA comprises an RNA transcribed from the DNA fragment Fragment
  • the DNA virus is a double-stranded DNA virus or a single-stranded DNA virus having a DNA double strand as an intermediate.
  • the present invention also provides a method of cultivating a plant having improved resistance to DNA viruses, comprising the steps of:
  • N represents a deoxyribonucleotide selected from any one of A, G, C and T; 14 ⁇ X ⁇ 30, and X is an integer; N X represents X consecutive deoxyribonucleotides;
  • the DNA virus is a double-stranded DNA virus or a single-stranded DNA virus having a DNA duplex as an intermediate.
  • the recombinant vector for expression of CRISPR/Cas9 nuclease is capable of transcription of a guide RNA and expression of a Cas9 protein.
  • the guide RNA is an RNA having a palindrome structure in which a crRNA and a tracrRNA are combined by base pairing.
  • Another object of the invention is to provide a method of increasing the ability of a plant to resist DNA viruses.
  • the method for improving the ability of a plant to resist DNA viruses may specifically include the following steps:
  • the target sequence is a sequence conforming to a 5'-N X -NGG-3' or 5'-CCN-N X -3' sequence alignment rule;
  • N represents any one of A, G, C and T, 14 ⁇ X ⁇ 30, and X is an integer, and N X represents X consecutive deoxyribonucleotides;
  • step (a2) constructing several of the DNA fragments obtained in the step (a1) into a vector for expressing a CRISPR/Cas9 nuclease to obtain a plurality of recombinant vectors;
  • the recombinant vector is capable of transcribing a guide RNA and expressing a Cas9 protein;
  • the guide RNA is a palindrome-structured RNA formed by base-pairing of a crRNA and a tracrRNA;
  • the crRNA comprises an RNA transcribed from the DNA fragment Fragment
  • the DNA fragment carried on the introduced recombinant vector is the DNA fragment of interest
  • the DNA virus is a double-stranded DNA virus or a single-stranded DNA virus having a DNA double strand as an intermediate;
  • the recipient plant 1 and the recipient plant 2 may be the same recipient plant or a different species of recipient plant.
  • the "plant which obtains an increase in the resistance against the DNA virus from the recipient plant (or the recipient plant 1) introduced into the recombinant vector" is specifically obtained by a method comprising the following steps:
  • the empty vector is the vector for expressing a CRISPR/Cas9 nuclease (an empty vector corresponding to the recombinant vector in which the DNA fragment is not inserted).
  • the "selecting the individual whose DNA virus content is significantly lower than the DNA virus content in the plant B from the plurality of plants A” further comprises: selecting the DNA from the plurality of plants A The virus content is extremely significantly lower than (P ⁇ 0.01) the individual of the DNA virus content in the plant B; further: an individual without any pathological phenotype is selected from the plurality of plants A.
  • the RNA fragment is capable of complementary binding to a target fragment;
  • the target fragment is in the genome of a double-stranded DNA virus or in a DNA double-stranded intermediate of a single-stranded DNA virus a double-stranded DNA corresponding to the sequence of "Nx" in the target sequence described in the step (1) in the gene sequence;
  • the guide RNA is transcribed from the recombinant vector, and the Cas9 protein is expressed; formed by the guide RNA and the Cas9 protein
  • the CRISPR/Cas9 nuclease is capable of inhibiting the replication of the DNA virus in the recipient plant 2, thereby increasing the resistance of the recipient plant 2 to the DNA virus.
  • the CRISPR/Cas9 nuclease formed by the guide RNA and the Cas9 protein is capable of inhibiting replication of the DNA virus in the recipient plant 2 by: action of the CRISPR/Cas9 nuclease The target fragment is cleaved, thereby inhibiting replication of the DNA virus in the recipient plant 2.
  • the vector for expressing a CRISPR/Cas9 nuclease is specifically a pHSN 401 vector.
  • the recombinant vector is the two cleavage sites Bsa I in the pHSN401 vector.
  • the X is specifically 20.
  • the plant is a dicot.
  • the plant is a tobacco, such as Nicotiana benthamiana.
  • the DNA virus is a geminivirus, specifically a sugar beet severe top virus (BSCTV).
  • the DNA fragments designed for synthesis against several of the target sequences are any of Table 2.
  • the target fragment is any one of Table 2 (ie, the DNA fragment shown in any one of the reverse complements of the sequence 1-15 in the sequence listing), and further excludes any of V4 and V9 (ie, the sequence in the sequence listing) a DNA fragment of any of the reverse complements of 1-3 and 4-8 and 10-15), further V7 and V8 (ie, the DNA fragment shown in the reverse complement of sequence 7 or 8 in the sequence listing) ).
  • the expression vector (i.e., "expression vector expressing the DNA virus” in the foregoing) is a pCambiaBSCTV1.8 plasmid; the pCambiaBSCTV1.8 plasmid is prepared according to a method comprising the following steps: (a1) in the sequence listing The DNA fragment shown in SEQ ID NO:16 is inserted positively between the restriction sites EcoRI and BamHI of the pCambia1300 vector to obtain an intermediate vector; (a2) an enzyme which positively inserts the DNA fragment shown in SEQ ID NO: 17 in the sequence table into the intermediate vector The pCambiaBSCTV1.8 plasmid was obtained by cleavage site EcoRI.
  • the method provided by the present invention simulates the corresponding immune system of bacteria, expresses sgRNA specifically recognized by the viral target site in the plant, and guides the Cas9 protein to specifically remove the viral DNA duplex in the body.
  • the present invention uses the sugar beet severe top virus (BSCTV) as a model virus, and for the first time realizes the use of the CRISPR/CAS9 system to effectively inhibit the expansion of BSCTV in plants, thereby improving the disease resistance of plants. Since the design of this method is based solely on the genomic sequence of the virus and does not require knowledge of the specific function of the viral gene, the method can be widely applied to various double-stranded DNA viruses known to be resistant to sequences and to DNA double-stranded intermediates. Single-stranded DNA virus.
  • Figure 1 shows the results of relative detection of BSCTV virus in each group of tobacco plants.
  • the relative content of BSCTV virus in the pHSN401 empty vector control group was recorded as 1, and the remaining groups were compared with the pHSN401 empty vector control group.
  • Figure 2 shows the phenotype of each group of tobacco plants.
  • A is a pHSN 401 empty vector control group
  • B is a V7 group
  • C is a V8 group
  • the D group is V4.
  • the pHSN401 vector is described in "Hui-Li Xing et al., A CRISPR/Cas9toolkit for multiplex genome editing in plants. BMC plant biology 2014.” The public is available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.
  • Nicotiana benthamiana recorded in "Cui Haitao, Li Chunxia, Wang Hongyan, etc. The establishment of tissue culture and genetic transformation system of the present tobacco. Shandong Science, 2006-01", the public can be from the Chinese Academy of Sciences Genetics and Developmental Biology Institute obtained.
  • pCambia1300 carrier Youbao Biological Products, product number: VT1375.
  • Agrobacterium strain EHA105 Youbao Biotech product, product number: ST1140.
  • Example 1 Establishment of a method for improving the ability of plants to resist DNA viruses
  • the beet severe topping virus (BSCTV) is selected as the DNA virus to be resisted, and Nicotiana benthamiana is selected as the plant body attacked by BSCTV, indicating a method for improving the ability of the plant to resist DNA virus.
  • BSCTV is a single-stranded DNA virus, but a large amount of amplification of BSCTV needs to be performed by rolling circle replication.
  • the specific process is to first synthesize the complementary strands of the virus single-stranded DNA as a template, form a circular double-stranded DNA intermediate, form a single-stranded nick on the double strand, and synthesize a large number of viral single-stranded genomes using the complementary strand as a template.
  • double-stranded intermediates play an important role in the large-scale amplification of viral genomes.
  • the present invention designs multiple specific sgRNAs for viral double-stranded intermediates, and directs Cas9 to clear double-stranded intermediates of BSCTV.
  • a single-stranded primer with a sticky end (underlined portion) was synthesized according to each target fragment designed in step 2 (Table 2).
  • a double-stranded DNA having a sticky end (which encodes an RNA complementary to the target fragment) is inserted through a primer annealing procedure, and inserted into the two restriction sites BsaI of the pHSN401 vector to obtain a recombinant plasmid.
  • Sequencing revealed that the recombinant plasmid obtained by inserting the reverse complement of the target fragment in Table 1 between the two restriction sites BsaI of the pHSN401 vector was positive, and was designated as pHSN401-sgRNA.
  • the positive recombinant plasmid transcribes a guide RNA (sgRNA) specific for the corresponding target fragment and expresses the Cas9 protein.
  • sgRNA guide RNA
  • the present invention utilizes tobacco for the first time to establish an instantaneous system for screening active sgRNAs that are resistant to BSCTV.
  • the Nicotiana benthamiana was directly sown in the nutrient soil. After two weeks of growth, the seedlings were transplanted, and a seedling was transplanted in each 9 ⁇ 9 cm culture, grown for about two weeks, and grown to 8-9 leaves. Two laterally similar leaves were selected for injection. Greenhouse culture conditions: 16h light and 8h dark, the temperature is 24 °C.
  • the pCFH vector containing BSCTV is from the American Type Culture Collection (ATCC, http://www.lgcstandardsatcc.org/, Number: PVMC-6 TM ) obtained.
  • ATCC American Type Culture Collection
  • a 0.8 copy BSCTV fragment (SEQ ID NO: 16) was obtained by double digestion of the pCFH vector by EcoRI and BamHI and cloned into the pCambia1300 vector at the restriction sites EcoRI and BamHI, and the resulting recombinant plasmid was named pCambiaBSCTV0.8.
  • a 1 copy BSCTV fragment (SEQ ID NO: 17) was then digested by EcoRI and constructed into the EcoRI site of pCambiaBSCTV0.8 (forward insertion), and the resulting recombinant vector was named pCambiaBSCTV1.8.
  • the literature for this recombinant vector construction method is Chen et al., BSCTV C2 Attenuates the Degradation of SAMDC1 to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis.
  • the virus Since the virus has 1.8 copies integrated into the binary vector with pCambia1300 as the backbone (1.8 copies refers to a sequence other than a copy of itself, plus 0.8 copies of the sequence to allow the virus integrated in the binary vector to form in the plant. Ring-shaped individuals), so the virus can be integrated into the genome of Nicotiana benthamiana by Agrobacterium injection to complete self-replication.
  • One thousandth of a volume of 200 mM acetosyringone (AS) was added and allowed to stand for two hours, and then injected into two pieces of tobacco to be injected.
  • AS mM acetosyringone
  • the pHSN401 plasmid was set as a control group. At least one tobacco was selected for the experiment.
  • the phenotypic differences of the tobacco plants of each group were recorded, and the injected leaves were sampled. It was ground to a powder with liquid nitrogen, 100 mg per sample, and DNA was extracted using a TIANGEN DNA quick Plant System.
  • the PPR (Pentatricopeptide repeat containing protein) gene in Nicotiana benthamiana was selected as the internal reference (Accession number: GO602734) gene, and a suitable fragment was selected as 133 bp.
  • the upstream and downstream primers were:
  • PPR-F 5'-CTCGGCCAAGAAGATCAACCATAC-3';
  • PPR-R 5'-GGTGCTTTATGTGGTTGTAGTTATGC-3'.
  • the BSCTV virus amplified fragment is 76 bp in size, and its upstream and downstream primers:
  • BSCTV-F 5'-CAGGGATTTTCGCACAGAGGAAC-3';
  • BSCTV-R 5'-GATTCGGTACCAAGTCCACGGG-3'.
  • the reagent used for qPCR was Roche Lightcycler@480 SYBR Green I Master.
  • the qPCR reaction system was: 2 ⁇ mix 10 ⁇ l; 1 ⁇ l of each of the upstream and downstream primers; 4 ⁇ l of ddH 2 O; 4 ⁇ l of the DNA template.
  • Virus content 2 (CT (internal reference PPR)-CT (BSCTV))
  • the virus content of the experimental group relative to the control group was the experimental group /viral content control group of the virus content, and an Excel spreadsheet was constructed based on the results, and the difference statistical analysis was performed.
  • the relative content of BSCTV virus in each group of tobacco plants is shown in Fig. 1. It can be seen from the figure that the relative content of BSCTV virus in each group is lower than that of the pHSN401 empty vector control group. After statistical analysis of differences, except for V4 and V9, the experimental groups were significantly different (P ⁇ 0.05), and the other groups were significantly different from the no-load control group (P ⁇ 0.01). The tobacco in the empty vector control group of pHSN401 The plants showed obvious BSCTV pathology. The V4 and V9 plants showed slightly better phenotype than the empty vector control group, but they also showed obvious pathological conditions. The V7 and V8 experimental groups with the most obvious phenotype were normal and showed strong performance. Resist the phenotype of the virus. Moreover, it was found that the relative content of BSCTV virus in each group was consistent with the trend of phenotypic morbidity of tobacco plants (the representative tobacco plant phenotypic results are shown in Fig. 2).

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Abstract

本发明提供了一种提高植物对DNA病毒的抵御能力的方法，包括如下步骤：1）从DNA病毒的基因组序列中选取符合5'-N X-NGG-3'或5'-CCN-N X-3'排列规则的序列作为靶序列，合成与之反向互补的DNA片段；2）将所述DNA片段构建到用于表达CRISPR/Cas9核酸酶的载体中，得到能转录向导RNA和表达Cas9蛋白的重组载体；3）将所述重组载体导入受体植物。本发明的方法可广泛应用于序列已知的双链DNA病毒及以双链DNA为中间体的单链DNA病毒。

Description

一种提高植物对入侵的DNA病毒的抵御能力的方法 技术领域
本发明属于植物基因工程领域，涉及一种提高植物对入侵的DNA病毒的抵御能力的方法。
背景技术
DNA病毒广泛存在于自然界中，并在细菌，动物以及植物中具有广范性的宿主。已知的DNA病毒中多数会引起严重的传染性疾病，并给经济性作物和哺乳动物(包括人类)带来巨大的损失。
以双生病毒为例，双生病毒是存在于植物中的唯一一类具有孪生颗粒形态的单链DNA病毒，也是目前已知的最大的单链DNA病毒家族。其基因组类型可分为单组份和双组份，大小约2.5-3.1kb。其通过昆虫介体以持久性的方式传播，大多数侵染寄生植物的韧皮部组织。目前已报道双生病毒主要对番茄、木薯、棉花等作物造成巨大的经济损失。1991-1992年，美国佛罗里达番茄受该病毒感染损失1.4亿美元；1992-1997年，棉花曲叶病毒造成巴基斯坦损失50亿美元；全球木薯每年损失达10亿英镑；1999年，Science杂志专题报告“双生病毒正在成为作物的严重威胁”来阐明双生病毒的危害性。
传统的抵御双生病毒的方式主要针对于阻断DNA病毒的复制或干扰其致病蛋白的表达。以双生病毒为例，其复制方式是较为保守的。在侵染宿主细胞后，病毒DNA会表达一个与复制相关的蛋白，该蛋白会结合在病毒基因组复制起点并起始DNA病毒的复制。当双生病毒的单链DNA进入到宿主细胞后，首先会依据单链DNA合成互补链，并形成双链中间体dsDNA，而病毒只编码了复制蛋白Rep和复制增强蛋白Ren，Rep蛋白结合在双链DNA复制起点，并在其保守序列TAATATTAC进行切割产生切口，从而起始滚环复制。因此，通过转入全长或部分外源Rep蛋白可与双生病毒自身编码的Rep蛋白进行竞争，从而抑制双生病毒的复制。然而，高表达量、干扰宿主细胞生长、非普适性使得该技术具有巨大的劣势。另外有 报道利用RNAi技术来干扰病毒致病蛋白的表达从而达到抗病毒的目的。该技术也因为其同源依赖性的特点而不具有普适性。2005年，Takashi Sera利用锌指结合蛋白特异性结合DNA双链的特性，研发了一种抑制双生病毒复制的方法。其以甜菜严重曲顶病毒(Beet severe curly top virus，BSCTV)为模式，通过转入外源人工锌指蛋白(AZP)，与病毒复制起始蛋白Rep进行竞争，并结合于病毒“双链中间体”复制起点，从而阻碍其复制。然而该方法只针对于双生病毒具有Rep蛋白作为其复制起始蛋白为特性的一类DNA双生病毒，而并不对DNA病毒具有广谱抗性。
近些年来的研究发现，细菌中存在一种适应性免疫反应系统，能够特异性的清除入侵细菌的外源双链DNA片段，包括噬菌体和质粒。这种免疫系统由成簇的规律间隔短回文重复序列(Clustered Regularly Interspaced Short Palindromic Repeat sequences,CRISPR)、反式作用CRISPR RNA(trans-acting CRISPR RNA,tracrRNA)基因和CRISPR相关基因(CRISPR-associated genes,Cas gene)组成。病毒的某些DNA片段会被记录在CRISPR重复序列之间。继而表达出包含病毒序列的CRISPR RNA。当病毒再次入侵时，CRISPR RNA在tracrRNA的协同作用下，引导Cas基因编码的核酸内切酶识别并清除外源病毒双链DNA。
根据Cas基因核心元件序列的不同，CRISPR/Cas免疫系统被分为三种类型：I型、II型和III型。其中I型和III型需要由多个Cas基因编码的蛋白组成复合体，完成对双链DNA的切割；而II型只需要Cas9蛋白即可切割双链DNA。所以，目前应用最广泛的是II型CRISPR/Cas系统。最近有研究发现，利用人工合成的方法，可以将crRNA和tracrRNA改造成一个融合的向导RNA(single-guide RNA,sgRNA)，并利用这种sgRNA成功引导Cas9内切酶实现对DNA的定点切割。
如果能使真核生物具备与细菌的CRISPR/Cas类似的这套系统，将能够显著提高真核生物的抗病毒能力。
发明内容
本发明的一个目的是提供一种培育对DNA病毒的抵御能力提高的植物的方法。
本发明所提供的培育对DNA病毒的抵御能力提高的植物的方法，具体 可包括如下步骤：
(1)从待抵御的DNA病毒的基因组序列中选取若干靶序列；针对各所述靶序列，分别设计合成与所述靶序列反向互补的DNA片段(双链)；
所述靶序列为符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的序列；
N表示A、G、C和T中的任一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸；
(2)将步骤(1)获得的若干个所述DNA片段分别构建到用于表达CRISPR/Cas9核酸酶的载体中，得到若干个重组载体；
所述重组载体能转录向导RNA和表达Cas9蛋白；所述向导RNA为由crRNA和tracrRNA通过碱基配对结合而成的具有回文结构的RNA；所述crRNA含有由所述DNA片段转录所得的RNA片段；
(3)将若干个所述重组载体分别导入受体植物，从导入所述重组载体的受体植物中获得对所述DNA病毒抵御能力提高的植物；
所述DNA病毒为双链DNA病毒或以DNA双链为中间体的单链DNA病毒。
本发明还提供了一种培育对DNA病毒的抵御能力提高的植物的方法，其包括如下步骤：
1)从所述DNA病毒的基因组序列中选择靶序列，所述靶序列具有5’-NX-NGG-3’或5’-CCN-NX-3’的序列，
其中N表示选自A、G、C和T中任一种的脱氧核糖核苷酸；14≤X≤30，且X为整数；NX表示X个连续的脱氧核糖核苷酸；
2)构建用于表达CRISPR/Cas9核酸酶的重组载体，所述载体能够转录向导RNA和表达Cas9蛋白；所述向导RNA特异性靶向步骤1)所述的靶序列；
3)将所述重组载体导入所述植物，从而获得对所述DNA病毒抵御能力提高的植物。
在一些实施方式中，所述DNA病毒为双链DNA病毒或以DNA双链为中间体的单链DNA病毒。所述用于表达CRISPR/Cas9核酸酶的重组载体能转录向导RNA和表达Cas9蛋白。所述向导RNA为由crRNA和tracrRNA通过碱基配对结合而成的具有回文结构的RNA。
本发明的另一个目的是提供一种提高植物抵御DNA病毒的能力的方法。
本发明所提供的提高植物抵御DNA病毒的能力的方法，具体可包括如下步骤：
(a)按照包括如下步骤的方法获得目的DNA片段：
(a1)从待抵御的DNA病毒的基因组序列中选取若干靶序列；针对各所述靶序列，分别设计合成与所述靶序列反向互补的DNA片段(双链)；
所述靶序列为符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的序列；
N表示A、G、C和T中的任一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸；
(a2)将步骤(a1)获得的若干个所述DNA片段分别构建到用于表达CRISPR/Cas9核酸酶的载体中，得到若干个重组载体；
所述重组载体能转录向导RNA和表达Cas9蛋白；所述向导RNA为由crRNA和tracrRNA通过碱基配对结合而成的具有回文结构的RNA；所述crRNA含有由所述DNA片段转录所得的RNA片段；
(a3)将若干个所述重组载体分别导入受体植物1，从导入所述重组载体的受体植物1中获得对所述DNA病毒抵御能力提高的植物，记为目的植物；
在所述目的植物中，被导入的所述重组载体上携带的所述DNA片段即为目的DNA片段；
(b)按照步骤(a2)和(a3)的方法将所述目的DNA片段导入所述用于表达CRISPR/Cas9核酸酶的载体，将所得重组载体导入受体植物2，从而提高所述受体植物2对所述DNA病毒的抵御能力；
所述DNA病毒为双链DNA病毒或以DNA双链为中间体的单链DNA病毒；
所述受体植物1和所述受体植物2既可为同一种受体植物，也可为不同种受体植物。
在上述方法中，所述“从导入所述重组载体的受体植物(或受体植物1)中获得对所述DNA病毒抵御能力提高的植物”具体是通过包括如下步骤的方法完成的：
(I)在进行如上步骤(3)的过程中，设置向所述受体植物(或所述受体植物1)中导入空载体的对照组；
所述空载体即为所述用于表达CRISPR/Cas9核酸酶的载体(与所述重组载体对应的未插入所述DNA片段的空载体)。
(II)向若干导入所述重组载体的受体植物(或受体植物1)中分别导入表达所述DNA病毒的表达载体，得到若干植物A；向导入所述空载体的受体植物中导入所述表达载体，得到植物B；检测所述若干植物A和所述植物B中所述DNA病毒的含量，从所述若干植物A中选出所述DNA病毒含量显著低于(P<0.05)所述植物B中所述DNA病毒含量的个体，记为植物A’，所述植物A’对应的导入所述重组载体的受体植物(或受体植物1)即为对所述DNA病毒抵御能力提高的植物。
其中，所述“从所述若干植物A中选出所述DNA病毒含量显著低于所述植物B中所述DNA病毒含量的个体”进一步为：从所述若干植物A中选出所述DNA病毒含量极显著低于(P<0.01)所述植物B中所述DNA病毒含量的个体；更进一步为：从所述若干植物A中选出无任何病态表型的个体。
在上述方法的步骤(2)和(a2)中，所述RNA片段能够与靶标片段互补结合；所述靶标片段为在双链DNA病毒的基因组或在单链DNA病毒的DNA双链中间体的基因序列中对应于步骤(1)中所述靶序列中的“Nx”所示序列的双链DNA；
在上述方法的步骤(b)中，在所述受体植物2中，由所述重组载体转录出所述向导RNA，并表达出所述Cas9蛋白；由所述向导RNA和所述Cas9蛋白形成的CRISPR/Cas9核酸酶能够抑制所述DNA病毒在所述受体植物2体内的复制，从而提高所述受体植物2对所述DNA病毒的抵御能力。进一步，所述由向导RNA和所述Cas9蛋白形成的CRISPR/Cas9核酸酶是通过如下实现抑制所述DNA病毒在所述受体植物2体内的复制的：在所述CRISPR/Cas9核酸酶的作用下，剪切所述靶标片段，从而实现抑制所述DNA病毒在所述受体植物2体内的复制。
在本发明一些实施方式中，所述用于表达CRISPR/Cas9核酸酶的载体具体为pHSN401载体。
相应的，所述重组载体为在所述pHSN401载体的两个酶切位点Bsa I 之间正向插入所述DNA片段后得到的重组质粒。
在本发明一些实施方式中，所述X具体为20。
在本发明一些实施方式中，所述植物为双子叶植物。
在本发明一些实施方式中，所述植物为烟草，如本生烟(Nicotiana benthamiana)。
在本发明一些实施方式中，所述DNA病毒为双生病毒，具体为甜菜严重曲顶病毒(BSCTV)。相应的，针对若干所述靶序列(表1)设计合成的所述DNA片段为表2中的任一种。所述目的片段为表2任一种(即序列表中序列1-15的反向互补序列中任一所示DNA片段)，进一步为排除V4和V9后的任一种(即序列表中序列1-3和4-8和10-15的反向互补序列中任一所示DNA片段)，更进一步为V7和V8(即序列表中序列7或8的反向互补序列中所示DNA片段)。所述表达载体(即前文中的“表达所述DNA病毒的表达载体”)为pCambiaBSCTV1.8质粒；所述pCambiaBSCTV1.8质粒是按照包括如下步骤的方法制备获得的：(a1)将序列表中序列16所示DNA片段正向插入到pCambia1300载体的酶切位点EcoRI和BamHI之间，得到中间载体；(a2)将序列表中序列17所示DNA片段正向插入到所述中间载体的酶切位点EcoRI处，得到所述pCambiaBSCTV1.8质粒。
本发明所提供的方法是模拟细菌的相应免疫系统，在植物中表达与病毒靶位点特异识别的sgRNA，引导Cas9蛋白特异性的清除机体内的病毒DNA双链。本发明以甜菜严重曲顶病毒(BSCTV)为模式病毒，首次实现了利用CRISPR/CAS9系统有效的抑制BSCTV在植物中的扩增，从而提高植物的抗病能力。鉴于该方法的设计仅仅基于病毒的基因组序列，而不需要了解病毒基因的具体功能，所以该方法可以广泛地应用于抵御序列已知的各种双链DNA病毒及以DNA双链为中间体的单链DNA病毒。
附图说明
图1为各组烟草植株中BSCTV病毒的相对含量检测结果。以pHSN401空载体对照组中BSCTV病毒的相对含量记为1，其余各组为与pHSN401空载体对照组相比后的比值。
图2为各组烟草植株的表型。其中，A为pHSN401空载体对照组；B为V7组；C为V8组；D组为V4。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
pHSN401载体：记载于“Hui-Li Xing et al.,A CRISPR/Cas9toolkit for multiplex genome editing in plants.BMC plant biology 2014.”一文，公众可从中国科学院遗传与发育生物学研究所获得。
本生烟(Nicotiana benthamiana)：记载于“崔海涛,李春霞,王红艳等.本生烟组织培养及遗传转化体系的建立.山东科学,2006年01期”一文，公众可从中国科学院遗传与发育生物学研究所获得。
pCambia1300载体：优宝生物公司产品，产品编号：VT1375。
农杆菌菌株EHA105：优宝生物公司产品，产品编号：ST1140。
实施例1、提高植物抵御DNA病毒的能力的方法的建立
本实施例选用甜菜严重曲顶病毒(BSCTV)作为待抵御的DNA病毒，选用本生烟(Nicotiana benthamiana)作为BSCTV攻击的植物体，说明建立提高植物抵御DNA病毒的能力的方法。
BSCTV是单链DNA病毒，但是BSCTV的大量扩增需要通过滚环复制进行。具体过程是，首先以病毒单链DNA为模板，合成其互补链，形成环状双链DNA中间体，再在双链上形成单链切口，以互补链为模板，合成大量病毒单链基因组。由此可见，双链中间体在病毒基因组大量扩增过程中起着重要的作用。本发明针对病毒双链中间体设计了多个特异性的sgRNA，引导Cas9清除BSCTV的双链中间体。
一、双子叶Crispr/Cas9系统双元载体的构建
1、依据对甜菜严重曲顶病毒(BSCTV)的二代测序结果(序列16和序列17)绘制其结构图谱
2、依据其结构图谱，任意挑选一个区域依据位置命名为V区，该区域300bp密集覆盖设计靶标片段(表1)。另外构建一个非病毒序列的靶标片段作为对照。
表1 V区靶标片段设计
Figure PCTCN2016076246-appb-000001
注：表1中下划线部分为PAM序列。
3、根据步骤2设计的各靶标片段，合成带有粘性末端(下划线部分)的单链引物(表2)。经过引物退火程序形成有粘性末端的双链DNA(可编码与所述靶标片段互补结合的RNA)，正向插入到pHSN401载体的两个酶切位点BsaI之间，得到重组质粒。经测序表明在pHSN401载体的两个酶切位点BsaI之间正向插入表1中靶标片段的反向互补序列后得到的重组质粒为阳性，记为pHSN401-sgRNA。阳性重组质粒可转录出特异于相应靶标片段的向导RNA(sgRNA)，并表达出Cas9蛋白。
表2 对应于V区靶标片段的单链引物
Figure PCTCN2016076246-appb-000002
Figure PCTCN2016076246-appb-000003
二、烟草瞬时系统筛选活性sgRNA
本发明首次利用烟草建立了瞬时系统筛选对BSCTV具有抗性的活性sgRNA。
1、烟草植株准备
将本生烟(Nicotiana benthamiana)直接播种在营养土中，生长两周后移栽幼苗，每个9×9cm的培养格移栽一棵小苗，生长约两周，生长至8-9片叶片，选取2片大小相似的侧生叶片待注射。温室培养条件：16h光照和 8h黑暗，温度是24℃。
2、抗性载体以及病毒接种实验
含有BSCTV的pCFH载体从American Type Culture Collection(ATCC,http://www.lgcstandardsatcc.org/，
Figure PCTCN2016076246-appb-000004
Number:PVMC-6TM)获得。首先通过EcoRI和BamHI双酶切pCFH载体获得0.8copy BSCTV片段(序列16)并克隆进入pCambia1300载体的酶切位点EcoRI和BamHI之间，所得重组质粒命名为pCambiaBSCTV0.8。然后通过EcoRI酶切获得1copy BSCTV片段(序列17)并构建进入pCambiaBSCTV0.8的酶切位点EcoRI处(正向插入)，所得重组载体命名为pCambiaBSCTV1.8。该重组载体构建方法的文献出处：Chen et al.,BSCTV C2Attenuates the Degradation of SAMDC1to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis.2011。
由于病毒有1.8拷贝整合进入以pCambia1300为骨架的双元载体(1.8拷贝是指除了本身一个拷贝的序列，另外需加上0.8拷贝的序列从而使得整合在双元载体上的病毒可以在植物体内形成环形个体)，因此病毒可通过农杆菌注射法整合进入本生烟(Nicotiana benthamiana)基因组来完成自我复制。
液氮冻融法将pHSN401-sgRNA质粒转化进入农杆菌菌株EHA105。接单斑至1ml含kana和rif抗生素的LB中小摇过夜，而后接菌至5ml含kana和rif抗生素的LB中大摇过夜，3600rpm/10mins离心，用10mM MgCl2重悬并将之稀释至OD600＝1.5。加入千分之一体积的200mM乙酰丁香酮(AS)静置两小时，而后将之注射入烟草2片待注射叶片。
两天后用同样方法将pCambiaBSCTV1.8质粒转入农杆菌菌株EHA105，大摇后用10mM MgCl2重悬并将之稀释至OD600＝0.5。注射入先前注入pHSN401-sgRNA质粒的2片注射叶片。
实验设置pHSN401质粒为对照组。至少选择1株烟草进行实验。
10天后，记录各组烟草植株的表型差异，并取样注射叶片。用液氮研磨至粉末，每个样品取100mg，用TIANGEN DNA quick Plant System(非离心柱型)提取DNA。选取本生烟(Nicotiana benthamiana)中PPR(Pentatricopeptide repeat containing protein)基因作为内参(Accession number：GO602734)基因，选取一合适片段为133bp，其上下游引物为：
PPR-F：5’-CTCGGCCAAGAAGATCAACCATAC-3’；
PPR-R：5’-GGTGCTTTATGTGGTTGTAGTTATGC-3’。
BSCTV病毒扩增片段大小为76bp，其上下游引物：
BSCTV-F：5’-CAGGGATTTTCGCACAGAGGAAC-3’；
BSCTV-R：5’-GATTCGGTACCAAGTCCACGGG-3’。
qPCR所用试剂为Roche Lightcycler@480 SYBR Green I Master。qPCR反应体系为：2×mix10μl；上下游引物各1μl；ddH2O 4μl；DNA模板4μl。
根据2-△△CT法，计算各实验组烟草中BSCTV病毒相对于空载体对照的相对含量：
(1)将各组BSCTV含量归一化。病毒含量＝2(CT(内参PPR)-CT(BSCTV))
(2)实验组相对于对照组的病毒含量为病毒含量实验组/病毒含量对照组，并依据结果构建Excel表格，并进行差异统计分析。
各组烟草植株中BSCTV病毒的相对含量结果如图1所示，由图可以看出，与pHSN401空载体对照组相比，各组中的BSCTV病毒的相对含量均较低。经过差异统计分析，各实验组除了V4，V9为显著差异(P<0.05)之外，其余各组都与空载对照组具有极显著差异(P<0.01)，pHSN401空载体对照组中的烟草植株呈现明显的BSCTV病态，V4、V9组植株表型略好于空载体对照组，但也表现出较为明显的病态，而差异最为明显的V7、V8实验组其表型正常，表现出极强的抵御病毒的表型。并且，发现各组中的BSCTV病毒的相对含量与烟草植株的表型病态程度趋势一致(具有代表性的烟草植株表型结果见图2)。

Claims (9)

  1. 一种培育对DNA病毒的抵御能力提高的植物的方法，包括如下步骤：
    (1)从待抵御的DNA病毒的基因组序列中选取若干靶序列；针对各所述靶序列，分别设计合成与所述靶序列反向互补的DNA片段；
    所述靶序列为符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的序列；
    N表示A、G、C和T中的任一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸；
    (2)将步骤(1)获得的若干个所述DNA片段分别构建到用于表达CRISPR/Cas9核酸酶的载体中，得到若干个重组载体；
    所述重组载体能转录向导RNA和表达Cas9蛋白；所述向导RNA为由crRNA和tracrRNA通过碱基配对结合而成的具有回文结构的RNA；所述crRNA含有由所述DNA片段转录所得的RNA片段；
    (3)将若干个所述重组载体分别导入受体植物，从导入所述重组载体的受体植物中获得对所述DNA病毒抵御能力提高的植物；
    所述DNA病毒为双链DNA病毒或以DNA双链为中间体的单链DNA病毒。
  2. 一种提高植物抵御DNA病毒的能力的方法，包括如下步骤：
    (a)按照包括如下步骤的方法获得目的DNA片段：
    (a1)从待抵御的DNA病毒的基因组序列中选取若干靶序列；针对各所述靶序列，分别设计合成与所述靶序列反向互补的DNA片段；
    所述靶序列为符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的序列；
    N表示A、G、C和T中的任一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸；
    (a2)将步骤(a1)获得的若干个所述DNA片段分别构建到用于表达CRISPR/Cas9核酸酶的载体中，得到若干个重组载体；
    所述重组载体能转录向导RNA和表达Cas9蛋白；所述向导RNA为由crRNA和tracrRNA通过碱基配对结合而成的具有回文结构的RNA；所述 crRNA含有由所述DNA片段转录所得的RNA片段；
    (a3)将若干个所述重组载体分别导入受体植物1，从导入所述重组载体的受体植物1中获得对所述DNA病毒抵御能力提高的植物，记为目的植物；
    在所述目的植物中，被导入的所述重组载体上携带的所述DNA片段即为目的DNA片段；
    (b)按照步骤(a2)和(a3)的方法将所述目的DNA片段导入所述用于表达CRISPR/Cas9核酸酶的载体，将所得重组载体导入受体植物2，从而提高所述受体植物2对所述DNA病毒的抵御能力；
    所述DNA病毒为双链DNA病毒或以DNA双链为中间体的单链DNA病毒；
    所述受体植物1和所述受体植物2为同一种受体植物或不同种受体植物。
  3. 根据权利要求1或2所述的方法，其特征在于：所述用于表达CRISPR/Cas9核酸酶的载体为pHSN401载体。
  4. 根据权利要求3所述的方法，其特征在于：所述重组载体为在所述pHSN401载体的两个酶切位点BsaI之间插入所述DNA片段后得到的重组质粒。
  5. 根据权利要求1-4中任一所述的方法，其特征在于：所述X为20。
  6. 根据权利要求1-5中任一所述的方法，其特征在于：所述植物为双子叶植物或单子叶植物。
  7. 根据权利要求6所述的方法，其特征在于：所述双子叶植物为烟草。
  8. 根据权利要求1-7所述的方法，其特征在于：所述DNA病毒为双生病毒。
  9. 根据权利要求8所述的方法，其特征在于：所述双生病毒为甜菜严重曲顶病毒。
PCT/CN2016/076246 2015-03-12 2016-03-14 一种提高植物对入侵的dna病毒的抵御能力的方法 WO2016141893A1 (zh)

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KR101994953B1 (ko) 2019-07-01
CN105969792A (zh) 2016-09-28
AU2016228599A1 (en) 2017-08-17
US20180195084A1 (en) 2018-07-12
JP2018507705A (ja) 2018-03-22
EP3309255A1 (en) 2018-04-18
EP3309255A4 (en) 2018-08-01
EA201791991A1 (ru) 2018-07-31

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