Tobacco mosaic virus resistant N'au gene and cloning methods and applications thereof
10487340 ยท 2019-11-26
Assignee
Inventors
- Yong Liu (Yunnan, CN)
- Xinjie Yuan (Hubei, CN)
- Changjun Huang (Yunnan, CN)
- Yongping Li (Yunnan, CN)
- Haiqin Yu (Yunnan, CN)
- Xuejun Chen (Yunnan, CN)
- Bingguang Xiao (Yunnan, CN)
Cpc classification
International classification
C12N15/82
CHEMISTRY; METALLURGY
A01H1/04
HUMAN NECESSITIES
Abstract
The invention relates to the isolation and the cloning and breeding application of a tobacco mosaic virus (TMV) resistant Nau gene. The invention discloses the nucleotide sequence of the TMV resistant Nau gene shown as SEQ ID NO.1. The amino acids of polypeptide encoded by the TMV resistant Nau gene are shown as SEQ ID NO.2. A non-transgenic TMV resistant tobacco variety can be obtained by transferring an Nau gene which is contained by germplasm resources into a TMV infected popular tobacco variety by conventional breeding means. The Nau gene of the invention has a homologous sequence with high identity rate of nucleotides in the popular tobacco variety, so that it is easy to obtain a shorter introgression segment single plant carrying the Nau gene by conventional breeding, and thereby to obtain a TMV resistant variety with lower linkage drag. The gene can resist both U1 strain and Cg strain of TMV. The novel antiviral gene Nau of the invention has great application prospect in cultivation of a TMV resistant tobacco.
Claims
1. A method for producing a tobacco plant resistant to a tobacco mosaic virus, comprising introducing a tobacco mosaic virus resistant Nau gene as shown in SEQ ID NO: 1 into a tobacco plant by a chromosome segment introgression, a gene introduction and/or gene editing.
2. The method according to claim 1, wherein the chromosome segment introgression includes: transferring a chromosome segment comprising the Nau gene to the tobacco plant by hybridization breeding, protoplast fusion and/or introgression of the chromosome segment into substitution lines or introgression lines.
3. The method according to claim 1, wherein the gene introduction includes: introducing the Nau gene to the tobacco plant.
4. A tobacco variety, a seed and an asexual propagule thereof obtained by the method according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWING
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EMBODIMENTS OF THE INVENTION
(6) A further illustration of the present invention will be described below in conjunction with the accompanying drawings; however, it's not intended to limit the present invention in any manner. Any change made based on the teachings of the present invention would fall within the protection scope of the present invention.
(7) The base sequence of the tobacco mosaic virus resistant Nau gene of the present invention is shown as SEQ ID No.1. The Nau gene is a CC-NBS-LRR gene, not only resistant to TMV-U1 strain of tobacco mosaic virus, but also resistant to TMV-Cg strain.
(8) According to the nucleotide sequence information shown as SEQ ID No.1 provided by the present invention, those skilled in the art can easily obtain a functionally equivalent gene by the following method: (1) by a genomic database search; (2) by screening the genomic library or cDNA library of tobacco with SEQ ID NO.1 as a probe; (3) by PCR amplification method from the genomic library or cDNA library of tobacco with oligonucleotide primers designed according to the sequence information of SEQ ID NO.1; (4) by modification via gene editing method on the basis of the sequence of N gene; (5) by a chemical synthesis method; and (6) by a deletion of codons of one or several amino acid residues and/or a mutation of one or several base pairs.
(9) In addition, there may exist variants in nature having significant sequence identity with the polynucleotide as shown in SEQ ID NO.1 or the polypeptide as shown in SEQ ID NO.2 of the present invention. These variants may exist naturally, or may be artificially produced. Compared with the sequence as shown in SEQ ID NO.1, one or more nucleotides are deleted and/or added and/or substituted at one or more sites within the naturally occurring variants. Due to the degeneracy of genetic code, a conservative variant of polynucleotides also includes those sequences encoding the amino acid sequence of the polypeptide as shown in SEQ ID NO.2. The naturally occurring variants could be identified by the well-known molecular biology techniques, for example, by polymerase chain reaction (PCR) and hybridization technique known in the art. The variants produced artificially further include polynucleotides from the synthetic resources, such as a polynucleotide variant produced by site-directed mutagenesis, which still shares significant sequence identity with the naturally occurring sequence disclosed herein. As a result the resistance to the TMV-U1 strain is acquired. Typically, these variants have an identity rate of more than 95% with the sequence shown in SEQ ID NO.1.
(10) The polynucleotide variant can also be evaluated by comparing the sequence shown as SEQ ID NO.2 with the amino acid sequence of the polypeptide encoded by the variant. The sequence identity rate between any two polypeptides could be calculated by sequence alignment programs and parameters. The identity percentage of the consensus sequence of two polypeptides is compared. In general, the sequence identity rate between two polypeptides encoded thereby should be above 95%.
(11) The sequence identity rate may be calculated by using molecular biology methods such as MEGA, BLAST, etc.
(12) In addition, the sequence shown as SEQ ID NO.1 can be isolated from other species of Nicotiana. The homology may be identified by PCR, hybridization, and other methods. According to the sequence identity with the nucleotide sequence shown as SEQ ID NO.1, or variants and segments thereof, sequences with the function of TMV-U1 strain resistance are isolated. Such kinds of sequences include an ortholog sequence of the sequence shown as SEQ ID NO.1. Ortholog is a gene derived from a common ancestral gene and found in different species as a result of the species formation. It is found in different species that genes which have a sequence identity of more than 95% in the nucleotide sequence thereof and/or the protein encoded thereby are considered as orthologs. The function of orthologs is often highly conservative among species.
(13) The method of cloning the tobacco mosaic virus resistant Nau gene of the present invention comprising following steps: (1) using a total DNA of Nicotiana alata as a template for PCR amplification, wherein the upstream primer and downstream primers used are as follows:
(14) TABLE-US-00002 N-H8-F: (SEQIDNO.3) 5-ATGGAGATTGGCTTAGCAGT-3, and N-H8-R: (SEQIDNO.4) 5-TCACAGGCATTCACAATCGA-3,
respectively; (2) recovering and purifying the resulting PCR product; and (3) sequencing.
(15) The total volume of the PCR reaction system is 50 L, containing 4.0 L of 100 ng/L DNA sample, 10.0 L of 5PCR buffer, 4 L of dNTPs (2.5 mmol/L each), 2.0 L of 10 mol/L primer N-H8-F and N-H8-R each, 1 L of PrimeSTAR GXL DNA Polymerase, and 27 L of ddH.sub.2O. The reaction condition of the PCR is 98 C., 2 min; 38 cycles of 98 C., 10 s, 52 C., 15 s, 68 C., 5 min; and 68 C., 10 min.
(16) The method of the sequencing can be direct sequencing, and can also be the cloning sequencing by TA vector.
(17) The amino acid sequence of the polypeptide encoded by the tobacco mosaic virus resistant Nau gene of the present invention is shown as SEQ ID No.2.
(18) The transient expression vector of the tobacco mosaic virus resistant Nau gene of the present invention comprises Nau gene and vector pHellsgate 8. The method for constructing the transient expression vector is such that the vector pHellsgate 8 is digested by using restriction enzymes XhoI and XbaI, and a PCR amplification product of Nau gene recovered from gel is connected to the linear pHellsgate 8 vector using a one-step seamless cloning kit. As a preferred embodiment of the invention, the one-step seamless cloning kit is One Step Cloning Kit ClonExpress II.
(19) An application of the tobacco mosaic virus resistant Nau gene of the present invention is to obtain a tobacco plant comprising Nau gene by a chromosome segment introgression, a gene introduction and/or gene editing. The method of chromosome segment introgression comprises hybridization breeding, protoplasts fusion and/or introgression of chromosome segment into substitution lines or introgression lines to transfer into the target tobacco, thus obtaining a new Tobacco mosaic virus resistant variety. Firstly, a germplasm resource comprising Nau gene is obtained by screening from Nicotiana plants, by using a functional molecular marker or a linked molecular marker associated with the sequence shown as SEQ ID NO.1, or by artificial inoculation of TMV; the germplasm resource comprises wild species of cultivated tobacco, a cultivated cultivar of tobacco, and a hybrid cultivar of wild species and a cultivated cultivar of tobacco. Then, such tobacco material having increased-resistance is bred to be a commercial variety to improve the TMV resistance of main tobacco cultivars by hybridization, backcrossing and other breeding means.
(20) The gene introgression relates to introducing an exogenous resistance gene into a target tobacco, including an introduction after the transferring of the exogenous gene (i.e., transgene) and a direct introduction. The most commonly used method for transgene is Agrobacterium transformation method. The method of direct introduction includes conventional biological methods such as microscope injection, pollen tube pathway, conductivity, gene gun, and the like to transform tobacco cells or tissues. The transformed tissues are cultivated into a plant.
(21) Gene editing is a recently developed technique which can accomplish the accurate modification to a genome, which can accomplish site-directed InDel mutation, knock-in, simultaneous mutation on multiple sites, and deletion of small segments of gene, etc., and can perform accurate gene editing at genomic level. Accurate gene editing performed on the homologue of Nau gene allows the edited homologue to obtain the function of resistance to TMV-U1 and TMV-Cg strain.
(22) A new TMV resistant tobacco variety, seeds and asexual propagules thereof can be obtained, depending on the application of the tobacco mosaic virus resistant Nau gene. In addition, some genetic engineering products including an expression cassette, a transgenic cell line and a recombinant strain, etc., of the resistant tobacco mosaic virus can also be developed.
(23) Further explanation and verification will be given below in combination with examples.
(24) Unless otherwise specified, the methods used in the following examples were all conventional methods. If there is no special specification, the experimental materials used were all purchased from conventional biochemical reagent companies. The tobacco materials were N. sylverstris (PI555569), N. alata (PI42334), N. benthamiana, Coker176, and K326, which are all from Yunnan Tobacco Agricultural Science Research Institute. TMV-U1 and TMV-Cg strain virus were from Yunnan Tobacco Agricultural Science Research Institute. The cDNA of TMV-U1 and TMV-Cg were obtained by a conventional method of the reverse transcription of total RNA extracted from virus-infected tobacco leaves by conventional methods.
(25) Gateway LR clonase Enzyme Mix kit and pENTR2B vector were purchased from Invitrogen Corporation, and Agrobacterium GV3101 was purchased from Invitrogen Corporation. The pHellsgate8 vector was purchased from Thermofisher Company. Plasmid DNA extraction kit, Agarose gel DNA recovery kit, and DNA fragment purification kit were purchased from QIAGEN Company. Escherichia coli DH5a, restriction enzymes, reverse transcription kit, DNA Marker, PrimeSTAR GXL DNA Polymerase, T4 DNA polymerase and T4 DNA ligase, and spectinomycin were all purchased from Takara (Dalian) Company and Roche Company. RNA extraction kit Trizol was purchased from Invitrogen Corporation, and ELISA kits for detecting TMV and Immunostrips were purchased from Agdia Company.
(26) The culture and inoculation method of Agrobacterium: Agrobacterium tumefaciens GV3101 was transformed with a transient expression vector plasmid. The positive clones were activated by a shake culture in 2 mL LB antibiotics (50 mg/L rifampicin, and 50 mg/L spectinomycin) medium, at 28 C., at 210 r/min for 30 hours. 150 L activated bacteria solution was added to 10 mL LB medium (containing 10 mmol/L morpholine ethane sulfonic acid (MES) (pH 5.6), 40 mol/L acetosyringone, 50 mg/L rifampicin, and 50 mg/L spectinomycin). After cultivated at 28 C., at 210 r/min for 16 hours, the bacteria was collected by a centrifugation at 4700 r/min for 5 minutes. The bacteria was resuspended and adjusted to a bacteria solution with an OD.sub.600=0.6 by using infiltration buffer (10 mmol/L MgCl.sub.2, 10 mmol/L MES, 200 mol/L acetosyringone). After kept at room temperature for 3 hours, the 4-week old tobacco seedling leaf was infiltrated with a 2 mL syringe. After inoculation, the tobacco seedling was cultivated in a light culturing room at 2528 C. for 7 days and the hypersensitive response (HR reaction) was surveyed and observed.
Example 1: Discovery and Function Verification of TMV-U1 Strain Resistant NAu Gene of N. alata (PI42334)
(27) (1) TMV-U1 Strain Resistant N. alata (PI42334), with a Resistance Different from N Gene
(28) 15 plants for each of the four kinds of tobaccos variety such as N. sylverstris (PI555569), N. alata (PI42334), Coker176, and K326 were planted (potted). When there were 4-5 leaves, each was inoculated with TMV-U1 strain and a blank control. The symptoms on the 5.sup.th, 7.sup.th and 14.sup.th day after inoculation were surveyed and recorded.
(29) The result on 7.sup.th day after inoculation of virus shows that N. alata (PI42334) and Coker176 are TMV-U1 strain resistant, and N. sylverstris and K326 are susceptible to TMV-U1 strain (Table 1,
(30) TABLE-US-00003 TABLE 1 Results of four kinds of tobacco varieties inoculated with TMV-U1 strain Detection of Detection TMV-U1 Blank N1N2 of E1E2 Tobacco variety strain control marker marker N. alata HR No Negative Negative (PI42334) symptom Coker176 HR No Positive Positive symptom N. sylverstris mosaic No Negative Negative (PI555569) symptom K326 mosaic No Negative Negative symptom Notes: HR is the disease-resistant reaction; and mosaic is an infected symptom.
(31) It has been reported in the literature that there is a TMV-U1 strain resistant N gene in Coker176, and specific molecular markers N1/N2 and E1/E2 for detecting N gene have been developed (Lewis, 2005). In order to verify the relationship between the disease-resistant genes in N. alata (PI42334) and N gene, leaves from the four kinds of tobacco varieties were taken, the DNA from each was extracted by QIAGEN kit, and PCR detection was performed by using specific molecular markers N1/N2 and E1/E2 of N gene. The result (Table 1,
(32) (2) Confirmation of TMV-U1 Resistant Avirulence Gene of N. alata (PI42334)
(33) Construction of a transient expression vector of TMV-U1 and TMV-Cg CP gene: transient expression vectors of coat proteins (abbreviated as CP) gene of TMV-U1 and TMV-Cg strain were constructed by using a modified vector pHellsgate8. The following primer pairs were used to amplify the viral cDNA of the CP genes of TMV-U1 and TMV-Cg strain:
(34) TABLE-US-00004 U1-CP-F: (SEQIDNO.5) 5-AAAAAGCAGGCTATGTCTTACAGTATCACTACTCCATCTC-3; U1-CP-R: (SEQIDNO.6) 5-AGAAAGCTGGGTTCAAGTTGCAGGACCAGAGG-3; Cg-CP-F: (SEQIDNO.7) 5-AAAAAGCAGGCTATGTCTTACAACATCACGAGCTCG-3; and Cg-CP-R: (SEQIDNO.8) 5-AGAAAGCTGGGTCTATGTAGCTGGCGCAGTAGTCC-3.
(35) The amplified fragment is inserted into pHellsgate8 vector according to the method of pHellsgate8 vector kit protocol. The attB sites are amplified by using the following primer pairs:
(36) TABLE-US-00005 attB1_adapter: (SEQIDNO.9) 5-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3; and attB2_adapter: (SEQIDNO.10) 5-GGGGACCACTTTGTACAAGAAAGCTGGGT-3.
(37) The amplified fragment is inserted into pDONR221 vector (Invitrogen) according to the method of pDONR221 vector (Invitrogen) kit for BP reaction protocol, and then inserted into expression vector pHellsgate8 by using LR reaction of Gateway technique.
(38) To determine the avirulence gene in N. alata (PI42334) interacting with TMV-U1 strain, the transient expression vectors Agrobacterium of TMV-U1 CP and TMV-Cg CP and a blank vector Agrobacterium control were used to inoculate the tobacco with a 2 mL syringe. Before inoculation, a suitable tobacco leaf was uniformly punched with a syringe needle. Five plants for each of the N. sylverstris (PI555569), N. alata (PI42334), Coker176 and K326 during 45 leaves period were infiltrated and inoculated. The largest 2 leaves of each plant were inoculated. The plants were cultivated in dark for 12 days after inoculation, and HR reaction was investigated on the 7.sup.th, 10.sup.th day at 28 C. in a light culturing room.
(39) The result (Table 2,
(40) TABLE-US-00006 TABLE 2 Avirulence gene interacting with TMV-U1 strain in N. alata (PI42334) TMV-U1 CP TMV-Cg CP Blank vector BLK (number of leaves (number of leaves (number of leaves exhibiting HR/ exhibiting HR/ exhibiting HR/ Tobacco number of leaves number of leaves number of leaves variety inoculated) inoculated) inoculated) N. alata 10/10 10/10 0/10 (PI42334) Coker176 0/10 0/10 0/10 N. sylverstris 0/10 10/10 0/10 (PI555569) K326 0/10 6/10 0/10
Example 2: Cloning and Sequence Analysis of the NAu Gene of N. alata (PI42334)
(41) (1) Extraction of the total DNA of tobacco: a fresh tobacco leaf was taken, and the total genomic DNA of tobacco was extracted by using QIAGEN DNeasy Plant Mini kit. DNA quality was preliminarily detected by using UV spectrophotometry (Nanodrop) and Agarose gel electrophoresis method. DNA samples with acceptable quality were diluted to 100 ng/L by using 0.5TE solution, and preserved until ready for use.
(42) (2) Cloning of Nau gene: PCR amplification was performed by using the DNA of N. alata (PI42334) or N. sylvestris (PI555569) as a template, and using primer N-H8-F (5-ATGGAGATTGGCTTAGCAGT-3 (SEQ ID NO.3)) and primer N-H8-R (5-TCACAGGCATTCACAATCGA-3 (SEQ ID NO.4)). The total volume of PCR reaction system is 50 L, containing 4.0 L of 100 ng/L DNA sample, 10.0 L of 5PCR buffer, 4 L of dNTPs (2.5 mmol/L each), 2.0 L of 10 mol/L primer N-H8-F and N-H8-R each, 1 L of PrimeSTAR GXL DNA Polymerase, and 27 L of ddH.sub.2O. The reagents used were purchased from Takara Bio. The reaction condition for PCR is 98 C., 2 min; 38 cycles of 98 C., 10 s, 52 C., 15 s, 68 C., 5 min; and 68 C., 10 min.
(43) (3) Recovery and purification of PCR product: The PCR product was electrophoresed through 1.5% Agarose gel. The electrophoresis buffer was 1TAE. When the electrophoresis indicator bromophenol blue migrated sufficiently to separate DNA fragments under the condition of 120V for 60 minutes, the gel was taken down. The result was recorded by using gel image analysis system, as shown in
(44) (4) PCR product sequencing: The PCR product from gel recovery was sent to Takara Bio for sequencing. The DNA sequence of Nau gene is shown as SEQ ID No.1 in the sequence list, and the open reading frame is from the Pt to the 4143.sup.th position at the 5 end of the sequence of SEQ ID No.1 in the sequence list.
Example 3: Polypeptide Sequence Encoded by NAu Gene
(45) According to the nucleotide sequence of Nau gene, the amino acid sequence of the polypeptide encoded by the Nau gene, which is deduced by using molecular biology software MEGA6, is shown in SEQ ID No.2.
Example 4: Construction of a Transient Expression Vector of NAu and N Gene
(46) Vector pHellsgate 8 was digested by using restriction enzymes XhoI and XbaI. The amplification products of Nau and N recovered from gel were linked to linear vector pHellsgate 8 by using One Step Cloning Kit ClonExpress II (Vazyme, Nanjing, China).
Example 5: Verifying that NAu Gene Possesses the Biological Function of TMV-U1 and TMV-Cg Resistance
(47) To determine that the Nau gene in N. alata (PI42334) has the biological function of TMV-U1 and TMV-Cg resistance, the Agrobacterium transient expression vector of Nau and N were constructed. The construction of transient expression vector of Nau was the same as that in Example 4, and the construction of the transient expression vector of N was the same as that in Example 4.
(48) After the accomplishment of construction, a combination of the following transient expression vectors was infiltrated and inoculated by using Agrobacterium: Nau+U1 CP; Nau+CgCP; Nau+BLK. N+U1CP; N+CgCP; N+BLK, wherein BLK is a blank transient expression vector. N. benthamiana, a wild tobacco variety was infiltrated and inoculated. 10 plants of N. benthamiana were inoculated during 45 leaves period, and the largest 2 leaves of each plant were inoculated. The HR reaction was investigated on the 7.sup.th and the 10.sup.th day.
(49) The result suggests that (Table 3,
(50) TABLE-US-00007 TABLE 3 Infiltration with Nau in combination with TMV-U1CP and TMV-CgCP results in HR reaction. TMV-U1 CP TMV-Cg CP Blank vector (BLK) (number of HR (number of HR (number of HR leaves/number of leaves/number of total leaves/number of Gene total leaves) leaves) total leaves) Nau 20/20 20/20 0/20 N 0/20 9/20 0/20
Example 6: Conventional Breeding Application of NAu Gene
(51) The chromosome segments comprising Nau gene shown as SEQ ID NO.1 in Nicotiana plants were transferred into the target tobacco by conventional breeding method. By using a functional molecular marker or a linked molecular marker of Nau, or method of artificial inoculation of TMV, the germplasm resource comprising Nau gene was obtained by screening from Nicotiana plants. The germplasm resource comprises wild species of cultivated tobacco, a hybrid cultivar of wild species and a cultivated cultivar of tobacco, and a cultivated cultivar of tobacco. The chromosome segments comprising Nau gene in the germplasm resource were introduced into the target tobacco to obtain the non-transgenic tobacco materials with increased TMV resistance by using conventional crossbreeding or protoplast fusion or introduction of chromosome segments or other technical means. Such resistance-increased tobacco materials were bred to be a commercial variety to improve the TMV resistance in a main cultivated variety by hybridization, backcrossing and other breeding means.
Example 7: Breeding Application of Gene Editing of NAu Gene
(52) The homologue of Nau gene in the target tobacco acquired a function equivalent to Nau by biotechnology modification. Tobacco with increased resistance was obtained. The homologue of Nau gene in the target tobacco was obtained by cloning. The nucleotide sequence and amino acid sequence of the homologue of Nau gene were obtained by sequencing. The difference between the homologue of Nau gene and the Nau in the nucleotide and amino acid sequence was found by sequence alignment analysis. The key different nucleotides which determine that the Nau is TMV-U1 strain resistant while the homology of Nau gene is TMV-U1 strain susceptible were found by means such as PCR mutation, and inoculation by co-infiltrating with TMV-U1 CP Agrobacterium transient expression vector, etc. By using molecular biology techniques such as mutagenesis, gene editing and the like, the key different nucleotides of the homologue of Nau gene were modified to be the polynucleotide sequence corresponding to Nau gene, such that the modified homologue of Nau gene acquires the function of TMV-U1 strain resistance.
(53) In summary, Nau gene is a new disease-resistant gene which is not only different from N gene, but also different from N gene, and it can be both TMV-U1 strain resistant and TMV-Cg strain resistant. Thus, there is broad application prospect in actual production.
REFERENCES
(54) Zhu Xianchao, Wang Yanting, Wang Zhifa. 2002. Chinese tobacco diseases. Beijing: China Agriculture Press, 152-162. Bagley C A. 2002. Controlling tobacco mosaic virus in tobacco through resistance. M. S. thesis. Virginia Polytechnic Inst. and State Univ., Blacksburg, Va. Lewis R S, Milla S R, Levin J S. 2005. MolecuLar and genetic characterization of Nicotianaglutinosa L. chromosome segments in tobacco mosaic virus-resistant tobacco accessions. CropSci, 45: 2355-2362. Whitham S, Dinesh-Kumar S P, Choi D, et al. 1994. The product of the tobacco mosaic virus resistance gene N: Similarity to Toll and the Interleukin-1 receptor. Cell, 78: 1101-1115. Sekine K T, Tomita R, Takeuchi S, et al. 2012. Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins. Mol Plant Microbe Interact., 25(9):1219-1229.