WO2017059582A1 - 一种抗烟草花叶病毒的N'au基因及其克隆方法和应用 - Google Patents

一种抗烟草花叶病毒的N'au基因及其克隆方法和应用 Download PDF

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WO2017059582A1
WO2017059582A1 PCT/CN2015/091560 CN2015091560W WO2017059582A1 WO 2017059582 A1 WO2017059582 A1 WO 2017059582A1 CN 2015091560 W CN2015091560 W CN 2015091560W WO 2017059582 A1 WO2017059582 A1 WO 2017059582A1
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gene
mosaic virus
tobacco
tmv
tobacco mosaic
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French (fr)
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刘勇
袁欣婕
黄昌军
李永平
于海芹
陈学军
肖炳光
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云南省烟草农业科学研究院
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Priority to US15/545,946 priority Critical patent/US10487340B2/en
Priority to PCT/CN2015/091560 priority patent/WO2017059582A1/zh
Priority to BR112017011621-9A priority patent/BR112017011621B1/pt
Priority to CN201510768990.8A priority patent/CN105274120B/zh
Publication of WO2017059582A1 publication Critical patent/WO2017059582A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/823—Nicotiana, e.g. tobacco
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/10—Chemical features of tobacco products or tobacco substitutes
    • A24B15/12—Chemical features of tobacco products or tobacco substitutes of reconstituted tobacco
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the invention belongs to the technical field of plant protection, and further belongs to the technical field of tobacco virus control, and particularly relates to a N'au gene against tobacco mosaic virus and a cloning method and application thereof.
  • TMV Tobacco mosaic virus
  • Zhu Xianchao 2002
  • TMV virus is differentiated from strains such as TMV-Cg strain and TMV-U1 strain, among which TMV-U1 strain is the main harmful strain on tobacco.
  • the main varieties of Chinese flue-cured tobacco, K326 and Yunyan 87, are not resistant to TMV-U1 strains. Generally, they mainly adopt measures such as breeding non-toxic seedlings, chemical control and destroying disease residues in the field, and have certain control on the occurrence and prevalence of TMV control.
  • TMV resistant varieties are still the most fundamental and most cost-effective means of preventing and controlling TMV; while the requirements for disease-resistant varieties are high resistance, no yield disadvantage, and no agronomic traits.
  • the TMV resistance of flue-cured tobacco is mainly derived from Nicotiana glutinosa, its resistance is controlled by a dominant single gene (N), and the N gene is resistant to TMV-U1 strain.
  • the N gene was cloned in 1994 and is the first NBS resistance gene cloned in plants (Whitham, 1994).
  • the genomic sequence size of the N gene is 6656 bp, including 5 exons and 4 introns, belonging to the TIR-NBS-LRR type disease resistance gene.
  • N-terminus of its encoded protein encodes a structure similar to the extracellular domain of the Drosophila Toll protein and the mammalian interleukin-Ireceptor (TIR), and also encodes a nucleotide binding site (nucleotide). -binding-site, NBS) and the leucine-rich repeat (LRR) domain (Whitham, 1994).
  • NBS nucleotide binding site
  • LRR leucine-rich repeat
  • the representative variety is the earlier commercialized planted anti-TMV flue-cured tobacco varieties Coker 176 and Speight H20 carrying the N gene introgression fragment. Due to the low yield and the slow chaining of the upper leaves, the flue-cured tobacco varieties carrying the N gene cannot meet the urgent needs of production.
  • the chromosomal fragments derived from the heart-smoke, which are closely linked to the N gene, are linked to each other, the genetic background of the TMV resistance source that has been used for breeding, and the limitations of conventional breeding methods, leading to a lack of breakthrough in anti-TMV flue-cured tobacco breeding. Therefore, screening and identifying new resistances in the tobacco germplasm resource bank The TMV gene is of great significance.
  • the corresponding avirulence gene is the coat protein gene (Coat protein of CPA) belonging to the tobacco mosaic virus, belonging to the CC-NBS-LRR disease resistance gene (Sekine et Al., 2012).
  • the N' gene is resistant to the TMV-Cg strain but not to the TMV-U1 strain.
  • the present invention contemplates the search for a novel gene that is resistant to the TMV virus.
  • the object of the present invention is to provide a transgenic cell line of the N'au gene against tobacco mosaic virus; and a tenth object is to provide a recombinant strain comprising the
  • the first object of the present invention is achieved by the base sequence of the N'au gene against tobacco mosaic virus as shown in SEQ ID No. 1.
  • the second object of the present invention is achieved by the method for cloning the N'au gene of the tobacco mosaic virus comprising the following steps:
  • PCR amplification was carried out using the total DNA of Nicotiana alata as a template, and the upstream and downstream primers used were:
  • N'-H8-F 5'-ATGGAGATTGGCTTAGCAGT-3'
  • N'-H8-R 5'-TCACAGGCATTCACAATCGA-3';
  • the third object of the present invention is achieved by the amino acid sequence of the polypeptide encoded by the N'au gene of the tobacco mosaic virus as shown in SEQ ID No. 2.
  • a fourth object of the present invention is achieved by the transient expression vector of the N'au gene against tobacco mosaic virus comprising the N'au gene and the vector pHellsgate 8.
  • the fifth object of the present invention is achieved by the method for constructing the transient expression vector of the N'au gene against tobacco mosaic virus by restriction endonuclease XhoI and XbaI digestion vector pHellsgate 8, N-recovery of gum
  • the au PCR amplification product was ligated to the linear pHellsgate 8 vector using a one-step seamless cloning kit.
  • the sixth object of the present invention is achieved by the N'au gene of the tobacco mosaic virus
  • the application is to obtain a tobacco plant containing the N'au gene by chromosomal fragment introduction, gene introduction, or gene editing.
  • the seventh object of the present invention is achieved by the tobacco variety, and the seed thereof and the asexual propagule obtained according to the application of the N'au gene against tobacco mosaic virus.
  • An eighth object of the present invention is achieved by the expression cassette of the N'au gene against tobacco mosaic virus.
  • a ninth object of the present invention is achieved by the transgenic cell line of the N'au gene against tobacco mosaic virus.
  • the tenth object of the present invention is achieved by the recombinant strain of the N'au gene against tobacco mosaic virus.
  • the N'au gene which is resistant to the TMV-U1 and TMV-Cg strains provided by the invention has important application value, and a broad-spectrum resistant tobacco variety is cultivated by means of cross breeding, transgenic, gene mutation and the like.
  • Conventional breeding is easy to obtain an anti-TMV variety that carries the N'au gene and has a small linkage.
  • the study found that the N'au gene has similar homologous sequences in cultivated tobacco varieties such as Yunyan 87.
  • the introgression fragments carrying the N'au gene are easily exchanged with cultivars, and it is easy to obtain N'au.
  • the introgression of a single plant with a short fragmentation therefore, conventional breeding is easy to obtain an anti-TMV variety carrying the N'au gene and having a small linkage.
  • the introgression fragment of tobacco wild-type leaf tobacco carrying the N gene is longer, and the nucleotide homology with the cultivated tobacco variety such as Yunyan 87 is low, and the introgression fragment carrying the N gene is difficult.
  • conventional breeding is difficult to obtain a single introgression fragment, so it is difficult to obtain an anti-TMV variety that carries the N gene and has a small linkage.
  • Figure 1 shows four tobacco varieties inoculated with TMV-U1 strain virus
  • A is N.alata (PI42334), showing dead spots; B is Coker176, showing dead spots; C is N.sylverstris (PI555569), showing mosaics, no dead spots; D is K326, showing pulsed mosaics No dead spots.
  • FIG. 2 shows the detection of the N gene using molecular markers N1/N2 and E1/E2;
  • 1 is N. alata (PI42334); 2 is Coker 176; 3 is N. sylverstris (PI555569); 4 is K326; M is 2000 bp molecular weight standard.
  • Figure 3 shows four tobacco varieties inoculated with TMV-U1CP, TMV-Cg CP and blank transient expression vector Agrobacterium;
  • A is N.alata (PI42334); B is Coker176; C is N.sylverstris (PI555569); D is K326; 1 is a transient expression vector Agrobacterium inoculated with TMV-U1CP; 2 is a transient expression vector Agrobacterium inoculated with TMV-Cg CP; 3 is a transient expression vector Agrobacterium inoculated with blank.
  • Figure 4 is a PCR amplification result of the N'au gene
  • 1 is ddH 2 O (negative control); 2 is N. sylvestris DNA (positive control); 3 is N. alata (PI42334) DNA; 5K is 5 kb molecular weight standard.
  • Figure 5 is a diagram showing the inoculation of N. benthamiana with the transient expression vector Agrobacterium containing the N'au and N' genes.
  • A is inoculated with N'au+TMV-U1CP transient expression vector Agrobacterium; B is inoculated with N'au+TMV-Cg CP transient expression vector Agrobacterium; C is inoculated with N'au+BLK transient expression vector Agrobacterium; D is inoculated with N'+TMV-U1CP transient expression vector Agrobacterium; E is N'+TMV-Cg CP transient expression vector Agrobacterium; F is inoculated with N'+BLK (F) combination transient expression vector Agrobacterium.
  • the N'au gene against tobacco mosaic virus of the present invention has a base sequence as shown in SEQ ID No. 1.
  • the N'au gene is a CC-NBS-LRR gene which is resistant to not only the TMV-U1 strain of tobacco mosaic virus but also the TMV-Cg strain.
  • nucleotide sequence information shown by SEQ ID NO. 1 provided by the present invention, a person skilled in the art can easily obtain a functionally equivalent gene by (1) obtaining by genomic database search; (2) using SEQ ID NO.1 is obtained by screening a tobacco genomic library or cDNA library for a probe; (3) designing an oligonucleotide primer according to the sequence information of SEQ ID NO. 1, and obtaining the genomic and cDNA of tobacco by PCR amplification method; (4) It was obtained by genetic editing based on the N' gene sequence; (5) the gene was obtained by chemical synthesis. (6) Obtained by mutation of one or several amino acid residues, and/or mutation of one or several base pairs.
  • a variant having a significant sequence identity to the polynucleotide represented by the SEQ ID NO. 1 of the present invention and the polypeptide of SEQ ID NO. 2 may be present in nature. These variants may be naturally occurring or artificially produced. One or more nucleotides are deleted and/or added and/or substituted at one or more positions in the natural variant compared to the sequence set forth in SEQ ID NO. Due to the degeneracy of the genetic code, conservative variants of the polynucleotide also include coding for SEQ ID NO. Those sequences of the amino acid sequences of the polypeptides shown.
  • Naturally occurring variants can be identified by well-known molecular biology techniques, such as by polymerase chain reaction (PCR) and hybridization techniques known in the art.
  • Anthropogenic variants also include synthetic-derived polynucleotides, such as variant polynucleotides that are generated by site-directed mutagenesis but which share significant sequence identity with the naturally occurring sequences disclosed herein, and thus obtain an anti-TMV-U1 strain. Resistance.
  • these variants have a sequence identity of more than 95% with the sequence set forth in SEQ ID NO.
  • Polynucleotide variants can also be assessed by comparing the amino acid sequences of the polypeptides encoded by the sequences and variants set forth in SEQ ID NO.
  • sequence identity rate between any two polypeptides can be calculated using sequence alignment programs and parameters to compare the percent sequence identity shared by the two polypeptides. In general, the sequence identity between the two encoded polypeptides should be above 95%.
  • sequence identity can be calculated by molecular biological methods such as MEGA, BLAST, and the like.
  • sequence set forth in SEQ ID NO. 1 can be isolated from other members of the genus Nicotiana and homologously identified by PCR, hybridization and other methods.
  • Such sequences include orthologue sequences of the sequence set forth in SEQ ID NO. "Orthologs" are genes that are derived from a common ancestral gene and are found in different species due to speciation. It has been found in different species that a gene is considered to be an ortholog when its nucleotide sequence and/or its encoded protein sequence has more than 95% sequence identity. The function of orthologs is usually highly conserved across species.
  • the method for cloning the N'au gene against tobacco mosaic virus according to the present invention comprises the following steps:
  • PCR amplification was carried out using the total DNA of Nicotiana alata as a template, and the upstream and downstream primers used were:
  • N'-H8-F 5'-ATGGAGATTGGCTTAGCAGT-3'
  • N'-H8-R 5'-TCACAGGCATTCACAATCGA-3';
  • the total reaction volume of the PCR system was 50 ⁇ L, of which 4.0 ⁇ L of 100 ng/ ⁇ L DNA sample, 10.0 ⁇ L of 5 ⁇ PCR buffer, 4 ⁇ L of dNTPs (2.5 mmol/L each), and 10 ⁇ mol/L of primers N′-H8-F and N. '-H8-R each 2.0 ⁇ L, PrimeSTAR GXL DNA Polymerase 1 ⁇ L, ddH 2 O 27 ⁇ L.
  • the reaction conditions of the PCR were: 98 ° C for 2 min, 98 ° C for 10 s, 52 ° C for 15 s, 68 ° C for 5 min, 38 cycles, and 68 ° C for 10 min.
  • the manner of sequencing can be performed by direct sequencing or by using TA vector clone sequencing.
  • a polypeptide encoded by the N'au gene of tobacco mosaic virus according to the present invention the amino group thereof The acid sequence is shown in SEQ ID No. 2.
  • the transient expression vector of the N'au gene against tobacco mosaic virus of the present invention comprises the N'au gene and the vector pHellsgate 8.
  • the transient expression vector is constructed by using restriction endonuclease XhoI and XbaI digestion vector pHellsgate 8, PCR-recovered PCR product of N'au gene, using one-step seamless cloning kit and linear pHellsgate 8 vector connection.
  • the one-step process for the seamless cloning kit One Step Cloning KitClonExpress TM II.
  • the use of the N'au gene against tobacco mosaic virus according to the present invention is to obtain a tobacco plant containing the N'au gene by chromosomal fragment introduction, gene introduction and/or gene editing.
  • the method of introducing a chromosome fragment includes cross breeding, protoplast fusion and/or introduction of a chromosomal fragment into a substitution line or a introduced line to a target tobacco, and a new tobacco variety resistant to TMV virus is obtained.
  • a germplasm resource comprising a N'au gene is screened from a Nicotiana plant by using a functional molecular marker or a linked molecular marker encoding the sequence of SEQ ID NO.
  • the quality resources include tobacco wild species, cultivars, and hybrids of wild species and cultivars. Then, through the breeding methods such as hybridization and backcrossing, the tobacco material with improved resistance is bred into a commercial variety, and the resistance of the main tobacco variety to TMV is improved.
  • Gene introduction is the introduction of exogenous resistance genes into the target tobacco, including the introduction of foreign genes into (ie, transgenic) and direct introduction.
  • the most commonly used method for transgene transformation is Agrobacterium transformation; direct introduction methods include microscopy.
  • Conventional biological methods such as injection, pollen tube pathway, conductance, and gene gun are used to transform tobacco cells or tissues, and the transformed tissues are grown into plants.
  • Gene editing is a technology that has been developed in recent years to accurately modify the genome. It can perform gene-specific InDel mutations, knock-in, simultaneous multi-site mutations and censoring of small fragments. It can be accurately performed at the genome level. Gene editing.
  • the edited homologue acquires the functions of the anti-TMV-U1 and anti-TMV-Cg strains by precise gene editing of the homolog of the N'au gene.
  • N'au gene against tobacco mosaic virus
  • a new anti-TMV tobacco variety and its seeds and vegetative propagules can be obtained.
  • some genetic engineering products can be developed, including the expression cassette of the N'au gene against tobacco mosaic virus, transgenic cell lines and recombinant bacteria.
  • test materials used are purchased from conventional biochemical reagent companies.
  • the tobacco material is N. Sylverstris (PI555569), N.alata (PI42334), N. benthamiana, Coker 176, and K326 are all from the Yunnan Academy of Tobacco Agricultural Sciences.
  • the TMV-U1 strain and the TMV-Cg strain virus were from the Yunnan Academy of Tobacco Agricultural Sciences.
  • the cDNA of TMV-U1 and TMV-Cg was extracted by total RNA from virus-infected tobacco leaves by conventional methods, and total RNA was obtained by a conventional reverse transcription method.
  • Gateway LR clonase Enzyme Mix kit pENTR 2B vector was purchased from Invitrogen, and Agrobacterium GV3101 was purchased from Invitrogen.
  • the pHellsgate 8 carrier was purchased from Thermofisher.
  • a plasmid DNA extraction kit, an agarose gel DNA recovery kit, and a DNA fragment purification kit were purchased from QIAGEN.
  • Escherichia coli DH5 ⁇ ; restriction endonuclease, reverse transcription kit, DNA Marker, PrimeSTAR GXL DNA Polymerase, T4 DNA polymerase and T4 DNA ligase, spectinomycin were purchased from Dalian Biotech and Roche.
  • the RNA extraction kit Trizol was purchased from Invitrogen, and the ELISA kit and test strip for the detection of TMV were purchased from Agdia.
  • transient expression vector plasmid was transformed into Agrobacterium tumefaciens GV3101. Positive clones were cultured in 2 mL LB antibiotics (50 mg/L rifampicin, 50 mg/L spectinomycin) in a culture medium, 28 ° C, 210 r / min, 30 h. Take 150 ⁇ L of activated bacterial solution to 10 mL of LB medium (containing 10 mmol/L morpholine ethanesulfonic acid (MES) (pH 5.6), 40 ⁇ mol/L ether acetosyringone, 50 mg/L rifampicin, 50 mg/L Prime).
  • MES mmol/L morpholine ethanesulfonic acid
  • Example 1 Discovery and functional verification of the N'au gene of N.alata (PI42334) anti-TMV-U1 strain
  • N.alata (PI42334) anti-TMV-U1 strain resistance is different from N gene
  • N.sylverstris PI555569, N.alata (PI42334), Coker176, K326 and other four tobacco products were planted in 15 plants (potted plants), and 4 to 5 leaves were inoculated with TMV-U1 strain and blank control. The symptoms were recorded on the 5th, 7th, and 14th day after the inoculation.
  • HR is the disease resistance reaction
  • the mosaic is the symptom of the disease.
  • TMV-U1CP and TMV-Cg CP gene transient expression vectors The coat protein gene (abbreviated as CP) transient expression vector of TMV-U1 and TMV-Cg strains was modified pHellsgate 8 vector construction.
  • the CP genes of the TMV-U1 and TMV-Cg strains were amplified with the following primer pairs, respectively.
  • U1-CP-F 5'-AAAAAGCAGGCTATGTCTTACAGTATCACTACTCCATCTC-3';
  • U1-CP-R 5'-AGAAAGCTGGGTTCAAGTTGCAGGACCAGAGG-3';
  • Cg-CP-F 5'-AAAAAGCAGGCTATGTCTTACAACATCACGAGCTCG-3';
  • Cg-CP-R 5'-AGAAAGCTGGGTCTATGTAGCTGGCGCAGTAGTCC-3'.
  • the amplified fragment was inserted into the pHellsgate 8 vector according to the pHellsgate 8 vector kit instructions.
  • the attB site was amplified using the following primer pairs.
  • AttB1_adapter 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’;
  • AttB2_adapter 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'.
  • the amplified fragment was inserted into the pDONR221 (Invitrogen) vector by the BP reaction pDONR221 (Invitrogen) vector kit method, and then used.
  • the technical LR reaction was inserted into the expression vector pHellsgate 8.
  • N. alata (PI42334) inoculated with TMV-U1CP showed a HR response, indicating that the avirulence gene interacting with TMV-U1 strain in N. alata (PI42334) is CP.
  • the Agrobacterium transient expression vector of Coker176 inoculated with TMV-U1CP had no HR response, indicating that the avirulence gene of Coker176 interacting with TMV-U1 strain was not CP, further indicating that the anti-TMV-U1 strain in N.alata (PI42334)
  • the disease resistance gene is different from the N gene in Coker176.
  • Agrobacterium from N.alata (PI42334), N.sylverstris (PI555569) and K326 inoculated with TMV-Cg CP showed HR response, indicating 3 parts of tobacco material
  • the disease resistance gene interacting with tobamovirus CP in N. sylverstris (PI555569) is N' (Sekine, 2012), and the N' gene is also present in K326.
  • the test results show that N. alata (PI42334) does have a new gene different from the N. sylverstris (PI555569) N' gene.
  • the total reaction volume of the PCR reaction system was 50 ⁇ L, of which 4.0 ⁇ L of 100 ng/ ⁇ L DNA sample, 10.0 ⁇ L of 5 ⁇ PCR buffer, 4 ⁇ L of dNTPs (2.5 mmol/L each), and 10 ⁇ mol/L of primers N′-H8-F and N′- 2.0 ⁇ L of each of H8-R, 1 ⁇ L of PrimeSTAR GXL DNA Polymerase, and 27 ⁇ L of ddH 2 O.
  • the reagents used were purchased from Bao Bio.
  • the reaction conditions of the PCR were: 98 ° C for 2 min, 98 ° C for 10 s, 52 ° C for 15 s, 68 ° C for 5 min, 38 cycles, and 68 ° C for 10 min.
  • PCR product recovery and purification the PCR product was electrophoresed on a 1.5% agarose gel, the electrophoresis buffer was 1 ⁇ TAE, and the electrophoretic indicator bromophenol blue migrated to a sufficient DNA fragment at 120V for 60 min. At the time, the gel was removed and the results were recorded using a gel image analysis system as shown in FIG. The DNA fragment gel was cut under a UV lamp. The DNA was recovered using a gel recovery kit (QIAGEN).
  • Example 3 Polypeptide sequence encoded by the N'au gene
  • the amino acid sequence of the polypeptide encoded by the N'au gene was deduced by the molecular biology software MEGA6 as shown in SEQ ID No. 2.
  • PHellsgate 8 vector using restriction enzymes XhoI and XbaI digested and gel recovered N'au N 'amplification product was purified using One Step Cloning Kit ClonExpress TM II ( Vazyme, Nanjing, China) pHellsgate 8 attached to a linear vector.
  • Example 5 Verification of the biological function of the N'au gene against TMV-U1 and TMV-Cg
  • N'au and N' Agrobacterium transient expression vectors were constructed. Among them, the transient expression vector of N'au was constructed in the same manner as in Example 4, and the construction of the transient expression vector of N' was the same as that of Example 4.
  • transient expression vector combinations were inoculated using Agrobacterium infiltration: N'au+U1CP; N'au+CgCP; N'au+BLK. N'+U1CP; N'+CgCP; N'+BLK, wherein BLK is a blank transient expression vector.
  • Inoculation of tobacco wild N. benthamiana N. benthamiana.
  • Ten strains of N. benthamiana were inoculated in 4 to 5 episodes, and each plant was inoculated with 2 largest leaves.
  • the HR response was investigated on the 7th and 10th day after inoculation.
  • a chromosomal fragment comprising the N'au gene of SEQ ID NO. 1 in a Nicotiana plant is transferred to a tobacco of interest by conventional breeding means.
  • the N'au gene germplasm resources are screened from Nicotiana plants by using N'au functional molecular markers or linked molecular markers, or by artificial inoculation of the TMV method.
  • Germplasm resources include hybrids and cultivars of tobacco wild species, wild species and cultivated tobacco.
  • the chromosomal fragments containing the N'au gene in the germplasm resources are introduced into the target tobacco by conventional hybrid breeding or protoplast fusion or introduction of chromosomal fragments to obtain non-transgenic tobacco materials with improved TMV resistance. Through the breeding methods such as hybridization and backcrossing, the tobacco material with improved resistance is bred into a commercial variety, and the resistance of the main tobacco variety to TMV is improved.
  • the homolog of the N'au gene (Homology) in the target tobacco is modified by biotechnology to obtain a function equivalent to N'au, and tobacco having improved disease resistance is obtained.
  • the homologue of the N'au gene in the target tobacco was obtained by cloning, and the nucleotide sequence and amino acid sequence of the homologue of the N'au gene were sequenced, and the homology of the N'au gene was found by sequence alignment analysis. Amino group with N'au Acid sequence and nucleotide differences.
  • the key differences in the determination of the N'au anti-TMV-U1 strain and the homologue of the N'au gene against the TMV-U1 strain were identified by PCR mutation and co-infiltration with TMV-U1CP Agrobacterium transient expression vector.
  • Nucleotide Modification of the key differential nucleotides of the N'au gene homologue to the corresponding nucleotide sequence of the N'au gene by molecular biology techniques such as mutagenesis and gene editing, so that the modified N'au gene is identical.
  • the source body acquires the function of the TMV-U1 strain.
  • the N'au gene is a new disease resistance gene that is different from the N gene and the N' gene. It can simultaneously resist TMV-U1 and TMV-Cg strains, and has a wide range in actual production. Application prospects.
  • Bagley C A.2002 Controlling tobacco mosaic virus in tobacco throughresistance.M.S.thesis.Virginia Polytechnic Inst.and State Univ.,Blacksburg,VA.

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Abstract

提供了烟草花叶病毒(TMV)抗性基因N'au的分离克隆与育种应用。烟草抗TMV基因N'au的核苷酸序列如SEQ ID No.1所示，其编码的多肽的氨基酸如SEQ ID NO.2所示。

Description

一种抗烟草花叶病毒的N′au基因及其克隆方法和应用 技术领域
本发明属于植物保护技术领域，进一步属于烟草病毒防治技术领域，具体涉及一种抗烟草花叶病毒的N'au基因及其克隆方法和应用。
背景技术
烟草花叶病毒病(Tobacco mosaic virus,TMV)是中国重要的烟草病害，每年造成的损失位列十大烟草侵染性病害名单前列(朱贤朝，2002)。TMV病毒存在TMV-Cg株系和TMV-U1株系等株系分化，其中，TMV-U1株系是烟草上的主要危害株系。中国烤烟主栽品种K326和云烟87等均不抗TMV-U1株系，一般主要采取培育无毒苗、药剂防治和销毁田间病残体等措施防治，对控制TMV的发生和流行上有一定的效果，但TMV在局部田块爆发的情况仍然时有发生，造成较大的经济损失(朱贤朝，2002)。因此，种植TMV抗病品种依然是防控TMV最根本、最经济有效的手段；而对抗病品种的要求是抗性高、无产量劣势、无农艺性状劣势。
目前，烤烟的TMV抗源主要来源于烟草野生种心叶烟(Nicotianaglutinosa)，其抗性由一个显性单基因(N)控制，N基因抗TMV-U1株系。N基因于1994年被克隆，是植物中克隆的第一个NBS类抗病基因(Whitham,1994)。N基因的基因组序列大小为6656bp，包括5个外显子和4个内含子，属于TIR-NBS-LRR类型抗病基因。在其编码蛋白的N端编码一个与果蝇Toll蛋白及哺乳动物白细胞介素-1受体(Toll/interleukin-Ireceptor，TIR)的胞外域相似的结构，还编码核苷酸结合位点(nucleotide-binding-site，NBS)和富含亮氨酸的重复序列(1eucine-richrepeat，LRR)结构域(Whitham,1994)。N基因的抗病机理为在病毒侵染位点出现过敏性坏死斑(枯斑)，通过诱导产生的细胞过敏性死亡限制TMV在植物体内的移动。在介导了过敏反应后，烟草植株能够获得系统性抗性，对TMV或其它类似病原的再次入侵产生广谱抗性(Whitham,1994)。通过一系列的常规杂交和回交转育，N基因的抗性从心叶烟先转育至香料烟中，后转育至烤烟品种中(Bagley,2002)。采用杂交育种转育N基因的抗性，实际上是转育携带N基因的染色体片段(简称为N导入片段)。世界上抗TMV烤烟育种利用的抗TMV基因几乎都是N基因。代表性品种是较早商业化种植的携带N基因渐渗片段的抗TMV烤烟品种Coker176和Speight H20。由于产量较低、上部叶落黄较慢等连锁累赘，携带N基因的烤烟品种不能满足生产的迫切需要。与N基因紧密连锁的来源于心叶烟的染色体片段存在连锁累赘、已被育种利用的TMV抗源的遗传背景狭窄和常规育种手段的局限性，导致抗TMV烤烟育种缺乏突破性进展。因此，在烟草种质资源库中筛选、鉴定新的抗 TMV基因具有重大意义。
另外，烟草野生种存在一种N'基因，对应的无毒基因为烟草花叶病毒属病毒的外壳蛋白基因(Coat protein，简写为CP)，属于CC-NBS-LRR类抗病基因(Sekine et al.，2012)。N'基因抗TMV-Cg株系但不抗TMV-U1株系。
为此，本发明希望寻找到一种能抗TMV病毒的新基因。
发明内容
本发明第一目的在于提供一种抗烟草花叶病毒的N'au基因；第二目的在于提供一种所述抗烟草花叶病毒的N'au基因的克隆方法；第三目的在于提供一种所述抗烟草花叶病毒的N'au基因编码的多肽；第四目的在于提供一种所述抗烟草花叶病毒的N'au基因的瞬时表达载体；第五目的在于提供一种所述抗烟草花叶病毒的N'au基因的瞬时表达载体的构建方法；第六目的在于提供一种所述抗烟草花叶病毒的N'au基因的应用；第七目的在于提供一种根据所述抗烟草花叶病毒的N'au基因的应用所得到的烟草品种、及其种子和无性繁殖体；第八目的在于提供一种所述抗烟草花叶病毒的N'au基因的表达盒；第九目的在于提供一种所述抗烟草花叶病毒的N'au基因的转基因细胞系；第十目的在于提供一种含所述抗烟草花叶病毒的N'au基因的重组菌。
本发明第一目的是这样实现的，所述抗烟草花叶病毒的N'au基因的碱基序列如SEQ ID No.1所示。
本发明第二目的是这样实现的，所述抗烟草花叶病毒的N'au基因的克隆方法包括如下步骤：
(1)以Nicotiana alata的总DNA为模板，进行PCR扩增，使用的用上下游引物分别为：
N'-H8-F：5’-ATGGAGATTGGCTTAGCAGT-3’，
N'-H8-R：5’-TCACAGGCATTCACAATCGA-3’；
(2)将得到的PCR产物进行回收和纯化；
(3)测序。
本发明第三目的是这样实现的，所述抗烟草花叶病毒的N'au基因编码的多肽的氨基酸序列如SEQ ID No.2所示。
本发明第四目的是这样实现的，所述抗烟草花叶病毒的N'au基因的瞬时表达载体包括N'au基因和载体pHellsgate 8。
本发明第五目的是这样实现的，所述抗烟草花叶病毒的N'au基因的瞬时表达载体的构建方法为采用限制性内切酶XhoI和XbaI酶切载体pHellsgate 8，胶回收的N'au PCR扩增产物,使用一步法无缝克隆试剂盒与线性pHellsgate 8载体连接。
本发明第六目的是这样实现的，所述抗烟草花叶病毒的N'au基因的 应用是通过染色体片段导入、或基因导入、或基因编辑得到含N'au基因的烟草植株。
本发明的第七目的是这样实现的，根据所述抗烟草花叶病毒的N'au基因的应用所得到的烟草品种、及其种子和无性繁殖体。
本发明的第八目的是这样实现的，所述抗烟草花叶病毒的N'au基因的表达盒。
本发明的第九目的是这样实现的，所述抗烟草花叶病毒的N'au基因的转基因细胞系。
本发明的第十目的是这样实现的，所述抗烟草花叶病毒的N'au基因的重组菌。
本发明提供的对TMV-U1和TMV-Cg株系均有抗性的N'au基因具有重要的应用价值，通过杂交育种、转基因、基因突变等手段培育出广谱抗性的烟草品种。常规育种容易获得携带N'au基因且连锁累赘较小的抗TMV品种。经研究发现：N'au基因在栽培烟草品种如云烟87等中存在相似性较高的同源序列，携带N'au基因的渐渗片段容易与栽培品种发生交换，容易获得携带N'au的渐渗片段较短的单株，因此，常规育种容易获得携带N'au基因且连锁累赘较小的抗TMV品种。与之相比，携带N基因的烟草野生种心叶烟的渐渗片段较长、且与在栽培烟草品种如云烟87的核苷酸同源性较低，携带N基因的渐渗片段很难与栽培品种发生交换，常规育种难以获得较短的渐渗片段单株，因此，难以获得携带N基因且连锁累赘较小的抗TMV品种。
附图说明
图1为4种烟草品种接种TMV-U1株系病毒；
图中：A为N.alata(PI42334)，表现枯斑；B为Coker176，表现枯斑；C为N.sylverstris(PI555569)，表现花叶、无枯斑；D为K326，表现脉明花叶、无枯斑。
图2为使用分子标记N1/N2和E1/E2检测N基因；
图中：1为N.alata(PI42334)；2为Coker176；3为N.sylverstris(PI555569)；4为K326；M为2000bp分子量标准。
图3为4种烟草品种分别接种TMV-U1CP、TMV-Cg CP和空白的瞬时表达载体农杆菌；
图中：A为N.alata(PI42334)；B为Coker176；C为N.sylverstris(PI555569)；D为K326；1为接种TMV-U1CP的瞬时表达载体农杆菌； 2为接种TMV-Cg CP的瞬时表达载体农杆菌；3为接种空白的瞬时表达载体农杆菌。
图4为N'au基因PCR扩增结果；
图中：1为ddH2O(阴性对照)；2为N.sylvestris DNA(阳性对照)；3为N.alata(PI42334)DNA；5K为5kb分子量标准。
图5为将含N'au和N'基因的瞬时表达载体农杆菌接种本氏烟(N.benthamiana)。
图中：A为接种N'au+TMV-U1CP瞬时表达载体农杆菌；B为接种N'au+TMV-Cg CP瞬时表达载体农杆菌；C为接种N'au+BLK瞬时表达载体农杆菌；D为接种N'+TMV-U1CP瞬时表达载体农杆菌；E为N'+TMV-Cg CP瞬时表达载体农杆菌；F为接种N'+BLK(F)组合瞬时表达载体农杆菌。
具体实施方式
下面结合附图对本发明作进一步说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换，均落入本发明保护范围。
本发明所述的一种抗烟草花叶病毒的N'au基因，其碱基序列如SEQID No.1所示。所述N'au基因是一种CC-NBS-LRR基因，不仅能抗烟草花叶病毒的TMV-U1株系，同时还能抗TMV-Cg株系。
根据本发明提供的SEQ ID NO.1所示的核苷酸序列信息，本领域技术人员可以通过以下方法容易地获得功能等同的基因：(1)通过基因组数据库检索获得；(2)以SEQ ID NO.1为探针筛选烟草基因组文库或cDNA文库获得；(3)根据SEQ ID NO.1序列信息设计寡核苷酸引物，用PCR扩增方法从烟草的基因组和cDNA中获得；(4)在N’基因序列的基础上用基因编辑方法改造获得；(5)用化学合成的方法获得该基因。(6)通过将缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的突变得到。
另外，在自然界可能存在与本发明SEQ ID NO.1所示的多核苷酸和SEQ ID NO.2所示的多肽共有显著序列相同性的变体。这些变体可以是天然存在的，也可能是由人为产生。与SEQ ID NO.1所示的序列相比，天然变体内一个或多个位置处删除和/或添加和/或取代一种或多种核苷酸。由于遗传密码的简并性，多核苷酸的保守变体也包括编码SEQ ID NO.2 所示多肽的氨基酸序列的那些序列。天然产生的变体可通过用熟知的分子生物学技术鉴定，例如用聚合酶链式反应(PCR)和本领域已知杂交技术。人为产生的变体还包括合成来源的多核苷酸，如用定点诱变生成但仍与本文公开的天然产生序列共有显著序列相同性的变体多核苷酸，且因此获得抗TMV-U1株系抗性。通常，这些变体与SEQ ID NO.1所示的序列具有95％以上的序列一致率。
多核苷酸变体还可通过比较SEQ ID NO.2所示的序列与变体所编码的多肽的氨基酸序列评估。任何两种多肽之间的序列一致率可用序列比对程序和参数计算，比较两种多肽共有的序列相同性百分比。一般来说，两种编码的多肽之间的序列一致率应在95％以上。
所述序列一致率可采用MEGA,BLAST等分子生物学方法计算。
此外，SEQ ID NO.1所示的序列可从烟草属的其他成员中分离，并通过PCR、杂交和其他方法来进行同源性鉴定。根据与SEQ ID NO.1所示的核苷酸序列或其变体和片段的序列相同性，分离出且具有抗TMV-U1株系功能的序列。此类序列包括SEQ ID NO.1所示序列的直系同源物序列。“直系同源物”是源自共同祖先基因且由于物种形成而在不同物种中发现的基因。在不同物种中发现：基因在其核苷酸序列和/或其编码蛋白序列具有95％以上的序列相同性时被认为是直系同源物。直系同源物的功能通常在物种间高度保守。
本发明所述的一种抗烟草花叶病毒的N'au基因的克隆方法包括如下步骤：
(1)以Nicotiana alata的总DNA为模板，进行PCR扩增，使用的用上下游引物分别为：
N'-H8-F：5’-ATGGAGATTGGCTTAGCAGT-3’，
N'-H8-R：5’-TCACAGGCATTCACAATCGA-3’；
(2)将得到的PCR产物进行回收和纯化；
(3)测序。
所述PCR的反应体系总体积为50μL，其中100ng/μL DNA样品4.0μL，5×PCR buffer 10.0μL，dNTPs(2.5mmol/L each)4μL，10μmol/L的引物N'-H8-F和N'-H8-R各2.0μL，PrimeSTAR GXL DNA Polymerase1μL，ddH2O 27μL。所述PCR的反应条件为：98℃2min，98℃10s，52℃15s，68℃5min，38个循环，68℃10min。
所述测序的方式可以采用直接测序，也可使用TA载体克隆测序。
本发明所述的一种抗烟草花叶病毒的N'au基因编码的多肽，其氨基 酸序列如SEQ ID No.2所示。
本发明所述的一种抗烟草花叶病毒的N'au基因的瞬时表达载体包括N'au基因和载体pHellsgate 8。所述瞬时表达载体的构建方法为采用限制性内切酶XhoI和XbaI酶切载体pHellsgate 8，胶回收的N'au基因的PCR扩增产物,使用一步法无缝克隆试剂盒与线性pHellsgate 8载体连接。作为本发明的一种优选，所述一步法无缝克隆试剂盒为One Step Cloning KitClonExpressTM II。
本发明所述的一种抗烟草花叶病毒的N'au基因的应用为通过染色体片段导入、基因导入和/或基因编辑得到含N'au基因的烟草植株。其中，将染色体片段导入的方法包括杂交育种、原生质体融和/或染色体片段导入代换系或导入系转育至目的烟草中，得到抗TMV病毒的烟草新品种。首先，通过利用编码SEQ ID NO.1所示序列的功能分子标记或连锁的分子标记，或者人工接种TMV方法，从烟草属植物中筛选获得包含N'au基因的种质资源；所述的种质资源包含烟草野生种、栽培种、以及野生种与栽培种的杂交种。然后，通过杂交、回交等育种手段，将该抗性提高的烟草材料育成商业品种，改良烟草主栽品种对TMV的抗性。
基因导入是将外源的抗性基因导入目的烟草中，包括将外源基因转入后导入(即转基因)和直接导入，转基因最常用的方法为农杆菌转化法；直接导入的方法包括显微注射、花粉管通道法、电导、基因枪等常规生物学方法转化烟草细胞或组织，并将转化的组织培育成植株。
基因编辑是近年来发展起来的可以对基因组完成精确修饰的一种技术，可完成基因定点InDel突变、敲入、多位点同时突变和小片段的删失等，可在基因组水平上进行精确的基因编辑。通过对N'au基因的同源体(Homology)进行精确的基因编辑，使编辑后的同源体获得抗TMV-U1和抗TMV-Cg株系的功能。
根据所述抗烟草花叶病毒的N'au基因的应用可以得到新的抗TMV烟草品种、及其种子和无性繁殖体。另外，还可以开发一些基因工程产品，包括所述抗烟草花叶病毒的N'au基因的表达盒，转基因细胞系和重组菌等。
下面结合实施例进行进一步的说明和验证。
如无特殊说明，下述各实施例使用的均为常规方法；如无特殊说明，所用的试验材料均为自常规生化试剂公司购买得到的。烟草材料为N. sylverstris(PI555569)、N.alata(PI42334)、本氏烟(N.benthamiana)、Coker176、K326，均来自云南省烟草农业科学研究院。TMV-U1株系和TMV-Cg株系病毒来自云南省烟草农业科学研究院。TMV-U1和TMV-Cg的cDNA通过常规方法提取病毒侵染的烟草叶片总RNA，总RNA通过常规反转录方法获得。
Gateway LR clonase Enzyme Mix kit、pENTR 2B载体购自Invitrogen公司，农杆菌GV3101购自Invitrogen公司。pHellsgate 8载体购自thermofisher公司。质粒DNA提取试剂盒、琼脂糖凝胶DNA回收试剂盒、DNA片段纯化试剂盒购自QIAGEN公司。大肠杆菌(Escherichia coli)DH5α；限制性内切酶、反转录试剂盒、DNA Marker、PrimeSTAR GXL DNAPolymerase、T4DNA聚合酶及T4DNA连接酶、壮观霉素均购自大连宝生物公司和Roche公司。RNA提取试剂盒Trizol购自Invitrogen公司，检测TMV的ELISA试剂盒和试纸条购自Agdia公司。
农杆菌培养与接种方法：瞬时表达载体质粒转化农杆菌(Agrobacterium tumefaciens)GV3101。阳性克隆在2mL LB抗生素(50mg/L利福平，50mg/L壮观霉素)培养基震荡培养活化，28℃,210r/min，30h。取150μL活化菌液至10mL LB培养基(含10mmol/L吗啉乙磺酸(MES)(pH 5.6),40μmol/L乙醚丁香酮(acetosyringone)，50mg/L利福平，50mg/L壮观霉素)。28℃,210r/min，培养16h后，4700r/min，离心5min收集菌体。用浸润缓冲液(10mmol/LMgCl2,10mmol/L MES,200μmol/L acetosyringone)悬浮调整至菌液OD600＝0.6。室温放置3h,用2mL注射器浸润4周大小的烟苗叶片。烟苗接种后25～28℃的光照培养室中培养7d，调查观察过敏坏死(HR反应)。
实施例1：N.alata(PI42334)抗TMV-U1株系的N'au基因的发现与功能验证
(1)N.alata(PI42334)抗TMV-U1株系，抗性不同于N基因
取N.sylverstris(PI555569)，N.alata(PI42334)，Coker176，K326等4种烟草品各栽15株(盆栽)，4～5片叶时，分别接种TMV-U1株系和空白对照。接种后的第5、7、14天调查记录症状。
病毒接种第7天的结果表明：N.alata(PI42334)和Coker176抗TMV-U1株系，N.sylverstris和K326感TMV-U1株系(表1，图1)。这说明N.alata(PI42334)和Coker176中存在抗TMV-U1株系的抗病基因。
表1 4种烟草品种接种TMV-U1株系的结果
Figure PCTCN2015091560-appb-000001
注：HR为抗病反应；花叶为染病症状。
已有文献报道：Coker176中存在抗TMV-U1株系的N基因，并开发了检测N基因的特异分子标记N1/N2和E1/E2(Lewis,2005)。为了验证N.alata(PI42334)中抗病基因与N基因的关系，取4种烟草品种叶片，用QIAGEN试剂盒提取DNA，采用N基因的特异分子标记N1/N2和E1/E2进行PCR检测。结果(表1，图2)显示：除Coker176外，其他3种烟草品种的检测结果均为阴性。表明N.alata(PI42334)对TMV-U1株系的抗性基因并非N基因，N.alata(PI42334)中存在一种抗TMV-U1株系的新抗性基因，命名为N'au基因。
(2)N.alata(PI42334)抗TMV-U1的无毒基因确定
构建TMV-U1CP和TMV-Cg CP基因瞬时表达载体：TMV-U1和TMV-Cg株系的外壳蛋白基因(缩写为CP)瞬时表达载体采用修饰的 pHellsgate 8载体构建。TMV-U1和TMV-Cg株系的CP基因分别采用如下引物对扩增病毒的cDNA。
U1-CP-F：5’-AAAAAGCAGGCTATGTCTTACAGTATCACTACTCCATCTC-3’；
U1-CP-R：5’-AGAAAGCTGGGTTCAAGTTGCAGGACCAGAGG-3’；
Cg-CP-F：5’-AAAAAGCAGGCTATGTCTTACAACATCACGAGCTCG-3’；
Cg-CP-R：5’-AGAAAGCTGGGTCTATGTAGCTGGCGCAGTAGTCC-3’。
扩增片段按照pHellsgate 8载体试剂盒说明书方法插入pHellsgate 8载体。attB位点采用如下引物对进行扩增。
attB1_adapter：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’；
attB2_adapter：5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’。
扩增片段利用BP反应pDONR221(Invitrogen)载体试剂盒说明书方法插入pDONR221(Invitrogen)载体，然后采用
Figure PCTCN2015091560-appb-000002
技术的LR反应插入到表达载体pHellsgate 8上。
为了确定N.alata(PI42334)中与TMV-U1株系互作的无毒基因，采用TMV-U1CP和TMV-Cg CP的瞬时表达载体农杆菌和空白载体农杆菌对照，用2mL注射器接种烟草。接种前用注射器针头在合适的烟叶上均匀打孔，浸润接种4～5片叶的N.sylverstris(PI555569)，N.alata(PI42334)，Coker176和K326各5株，每株接种最大的2片叶，接种后避光培养1～2d后，在28℃光照培养室培养第7、10天调查HR反应。
结果(表2，图3)表明：N.alata(PI42334)接种TMV-U1CP的农杆菌表现为HR反应，这说明N.alata(PI42334)中与TMV-U1株系互作的无毒基因为CP。Coker176接种TMV-U1CP的农杆菌瞬时表达载体无HR反应，这说明Coker176与TMV-U1株系互作的无毒基因并非CP，进一步表明：N.alata(PI42334)中的抗TMV-U1株系的抗病基因不同于Coker176中的N基因。N.alata(PI42334)、N.sylverstris(PI555569)和K326接种TMV-Cg CP的农杆菌都表现为HR反应，这表明3份烟草材料 中均存在与TMV-Cg CP互作的抗病基因，Coker176接种TMV-Cg CP的农杆菌的HR反应不典型。根据已有文献报道：N.sylverstris(PI555569)中与tobamovirus CP互作的抗病基因为N'(Sekine，2012)，K326中也存在N'基因。试验结果证明：N.alata(PI42334)的确存在与N.sylverstris(PI555569)N'基因功能不同的新基因。
表2 N.alata(PI42334)中与TMV-U1株系互作的无毒基因
Figure PCTCN2015091560-appb-000003
实施例2：N.alata(PI42334)的N'au基因克隆与序列分析
(1)烟草总DNA的提取：取烟草新鲜嫩叶，采用QIAGEN DNeasyPlant Mini试剂盒提取烟草总基因组DNA。采用紫外分光光度法(Nanodrop)和琼脂糖凝胶电泳法初步检测DNA质量。质量合格的DNA样品，用0.5×TE溶液稀释至100ng/μL，保存备用。
(2)N'au基因的克隆：以N.alata(PI42334)和N.sylvestris(PI555569)的DNA为模板，用引物N'-H8-F(5’-ATGGAGATTGGCTTAGCAGT-3’)和引物N'-H8-R(5’-TCACAGGCATTCACAATCGA-3’)进行PCR扩增。PCR的反应体系总体积为50μL，其中100ng/μL DNA样品4.0μL，5×PCR buffer 10.0μL，dNTPs(2.5mmol/L each)4μL，10μmol/L的引物N'-H8-F和N'-H8-R各2.0μL，PrimeSTAR GXL DNA Polymerase 1μL，ddH2O 27μL。所用试剂购自宝生物公司。PCR的反应条件为：98℃2min，98℃10s，52℃15s，68℃5min，38个循环，68℃10min。
(3)PCR产物回收与纯化：将PCR产物在1.5％的琼脂糖凝胶上电泳，电泳缓冲液为1×TAE，当电泳指示剂溴酚蓝在120V，60min条件下迁移至足够分离DNA片段时，取下凝胶，用凝胶图像分析系统记录结果，如图4所示。在紫外灯下割下DNA片段凝胶。用胶回收试剂盒(QIAGEN公司出品)回收DNA。
(4)PCR产物测序：胶回收的PCR产物，送宝生物公司测序。N'au基因的DNA序列如序列表的SEQ ID No.1所示，开放阅读框为序列表SEQ ID No.1的序列自5’端第1位至第4143位。
实施例3：N'au基因所编码的多肽序列
根据N'au基因的核苷酸序列，采用分子生物学软件MEGA6推导出N'au基因所编码的多肽的氨基酸序列如SEQ ID No.2所示。
实施例4：N'au和N'基因瞬时表达载体构建
载体pHellsgate 8采用限制性内切酶XhoI和XbaI酶切，胶回收的N'au和N'扩增产物采用One Step Cloning Kit ClonExpressTM II(Vazyme，中国南京)与线性pHellsgate 8载体连接。
实施例5：验证N'au基因具有抗TMV-U1和TMV-Cg的生物学功能
为了确定N.alata(PI42334)中的N'au基因具有抗TMV-U1和TMV-Cg的生物学功能，构建N'au和N'的农杆菌瞬时表达载体。其中，N'au的瞬时表达载体构建与实施例4相同，N'的瞬时表达载体的构建与实施例4相同。
构建完成后，使用农杆菌浸润接种以下瞬时表达载体组合：N'au+U1CP；N'au+CgCP；N'au+BLK。N'+U1CP；N'+CgCP；N'+BLK，其中BLK为空白瞬时表达载体。浸润接种烟草野生种本氏烟(N.benthamiana)。接种4～5片时期的本氏烟10株，每株接种2片最大叶片。接种后第7、10天调查HR反应。
结果表明(表3，图5)：N'au同时与TMV-U1CP和TMV-Cg CP浸润时在本氏烟上产生HR反应，而N'与TMV-U1CP共浸润本氏烟无HR反应。这表明N'au具有抗TMV-U1和TMV-Cg株系的生物学功能，N'不具有抗TMV-U1株系的生物学功能。在抗TMV-U1株系的生物学功能上，N'au和N'具有明显差异。
表3 N'au与TMV-U1CP和TMV-Cg CP浸润产生HR反应
Figure PCTCN2015091560-appb-000004
实施例6：N'au基因的常规育种应用
将烟草属植物中包含SEQ ID NO.1所示N'au基因的染色体片段，通过常规育种手段转育至目的烟草中。通过利用N'au的功能分子标记或连锁的分子标记，或者人工接种TMV方法，从烟草属植物中筛选获得包含N'au基因种质资源。种质资源包含烟草野生种、野生种与栽培烟草的杂交种和栽培种。利用常规杂交育种或者原生质体融合或者染色体片段导入等技术手段，将种质资源中包含N'au基因的染色体片段导入目的烟草，获得TMV抗性提高的非转基因烟草材料。通过杂交和回交等育种手段，将该抗性提高的烟草材料育成商业品种，改良烟草主栽品种对TMV的抗性。
实施例7：N'au基因的基因编辑育种应用
将目的烟草中N'au基因的同源体(Homology)，通过生物技术修饰获得等同于N'au的功能，得到抗病性提高的烟草。通过克隆获得目的烟草中的N'au基因的同源体，测序获得N'au基因的同源体的核苷酸序列和氨基酸序列，通过序列比对分析，找出N'au基因的同源体与N'au的氨基 酸序列和核苷酸差异。通过PCR突变、与TMV-U1CP农杆菌瞬时表达载体共浸润接种等方法，找出决定N'au抗TMV-U1株系而N'au基因的同源体不抗TMV-U1株系的关键差异核苷酸。通过诱变、基因编辑等分子生物学技术，将N'au基因的同源体的关键差异核苷酸修饰为N'au基因对应的多核苷酸序列，使修饰后的N'au基因的同源体获得抗TMV-U1株系的功能。
综上所述，N'au基因是一种既不同于N基因，也不同于N'基因的新抗病基因，能够同时抗TMV-U1和TMV-Cg株系，在实际生产中具有广泛的应用前景。
参考文献：
朱贤朝,王彦亭,王智发.2002.中国烟草病害.北京:中国农业出版社,152-162.
Bagley C A.2002.Controlling tobacco mosaic virus in tobacco throughresistance.M.S.thesis.Virginia Polytechnic Inst.and State Univ.,Blacksburg,VA.
Lewis R S,Milla S R,Levin J S.2005.MolecuLar and geneticcharacterization of Nicotiana glutinosa L.chromosome segments in tobaccomosaic virus-resistant tobacco accessions.Crop Sci,45:2355-2362.
Whitham S,Dinesh-Kumar S P,Choi D,et al..1994.The product of thetobacco mosaic virus resistance gene N:Similarity to Toll and theInterleukin-1receptor.Cell,78:1101-1115.
Sekine K T,Tomita R,Takeuchi S,et al.2012.Functional differentiationin the leucine-rich repeat domains of closely related plant virus-resistanceproteins that recognize common avr proteins.Mol Plant Microbe Interact.,25(9):1219-1229.

Claims (16)

  1. 一种抗烟草花叶病毒的N'au基因，其特征在于所述的抗烟草花叶病毒的的N'au基因的碱基序列如SEQ ID No.1所示。
  2. 根据权利要求1所述的一种抗烟草花叶病毒的N'au基因，其特征在于所述烟草花叶病毒的株系为TMV-U1、TMV-Cg。
  3. 一种根据权利要求1所述的抗烟草花叶病毒的N'au基因的克隆方法，其特征在于包括如下步骤：
    (1)以Nicotiana alata总DNA为模板，进行PCR扩增，使用的用上下游引物分别为：
    N'-H8-F：5’-ATGGAGATTGGCTTAGCAGT-3’，
    N'-H8-R：5’-TCACAGGCATTCACAATCGA-3’；
    (2)将得到的PCR产物进行回收和纯化；
    (3)测序。
  4. 根据权利要求3所述的抗烟草花叶病毒的N'au基因的克隆方法，其特征在于所述PCR的反应体系总体积为50μL，其中100ng/μL DNA样品4.0μL，5×PCR buffer 10.0μL，dNTPs(2.5mmol/L each)4μL，10μmol/L的引物N'-H8-F和N'-H8-R各2.0μL，PrimeSTAR GXL DNA Polymerase 1μL，ddH2O 27μL。
  5. 根据权利要求3所述的抗烟草花叶病毒的N'au基因的克隆方法，其特征在于所述PCR的反应条件为：98℃2min，98℃10s，52℃15s，68℃5min，38个循环，68℃10min。
  6. 一种根据权利要求1所述的抗烟草花叶病毒的N'au基因编码的多肽，其特征在于其氨基酸序列如SEQ ID No.2所示。
  7. 一种根据权利要求1所述的抗烟草花叶病毒的N'au基因的瞬时表达载体，其特征在于所述表达载体包括N'au基因和载体pHellsgate 8。
  8. 一种根据权利要求7所述的抗烟草花叶病毒的N'au基因的瞬时表达载体的构建方法，其特征为采用限制性内切酶XhoI和XbaI酶切载体pHellsgate 8，胶回收的N'au PCR扩增产物,使用一步法无缝克隆试剂盒与线性pHellsgate 8载体连接。
  9. 一种根据权利要求1所述的抗烟草花叶病毒的N'au基因的应用，其特征在于通过染色体片段导入、基因导入和/或基因编辑得到含N'au基因的烟草植株。
  10. 根据权利要求9所述的抗烟草花叶病毒的N'au基因的应用，其特征在于通过染色体片段导入得到含N'au基因的烟草植株的方法为：将含有N'au基因的染色体片段通过杂交育种、原生质体融和/或染色体片段导入代换系或导入系转育至目的烟草中，得到抗TMV病毒的烟草新品种。
  11. 根据权利要求9所述的抗烟草花叶病毒的N'au基因的应用，其特征在于通过基因导入得到含N'au基因的烟草植株的方法为：将外源的N'au基因导入目的烟草中，得到抗TMV病毒的转基因烟草。
  12. 根据权利要求9所述的抗烟草花叶病毒的N'au基因的应用，其特征在于通过基因编辑得到含N'au基因的烟草植株的方法为将目的烟草中存在的N'au基因同源体，通过对其特定基因位点的缺失、增加和/替换实现与SEQ ID No.1所示序列具有相同的功能。
  13. 一种根据权利要求9～12任一所述的抗烟草花叶病毒的N'au基因的应用所得到的烟草品种、及其种子和无性繁殖体。
  14. 一种根据权利要求1所述的抗烟草花叶病毒的N'au基因的表达盒。
  15. 一种根据权利要求1所述的抗烟草花叶病毒的N'au基因的转基因细胞系。
  16. 一种含有根据权利要求1所述的抗烟草花叶病毒的N'au基因的重组菌。
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US11421238B2 (en) 2018-02-20 2022-08-23 Longas Technologies Pty Ltd Method for introducing mutations
CN108754028A (zh) * 2018-06-26 2018-11-06 云南省烟草农业科学研究院 一种检测抗烟草花叶病毒Np基因的分子标记及其应用
CN109355419A (zh) * 2018-11-20 2019-02-19 云南省烟草农业科学研究院 一组打破烟草tmv抗性基因n下游（3’端）连锁累赘的分子标记及其应用
CN109439789A (zh) * 2018-11-20 2019-03-08 云南省烟草农业科学研究院 一组打破烟草tmv抗性基因n上游（5’端）连锁累赘的分子标记及其应用
CN109355419B (zh) * 2018-11-20 2021-05-18 云南省烟草农业科学研究院 一组打破烟草tmv抗性基因n下游（3’端）连锁累赘的分子标记及其应用

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