Method for obtaining tobacco variety for preventing and controlling tobacco mosaic virus

Abstract

The invention belongs to the technical field of crop breeding, and particularly relates to a method for obtaining a tobacco variety for preventing and controlling tobacco mosaic disease, manual intervention mutation is carried out by using the 120th to 142 basic groups of a tobacco NtFd gene shown as SEQ ID NO.1, and the sequence of a mutated nucleotide fragment is shown as SEQ ID NO.2. The ntfd editing strain obtained on the basis of manual intervention mutation of the NtFd gene sequence shows a remarkable effect of preventing and controlling the tobacco common mosaic disease, and a gene resource and a material basis are provided for cultivation of a tobacco disease-resistant strain.

Description

Technical Field

[0001] The invention belongs to the technical field of crop breeding, and in particular relates to a method for obtaining a tobacco variety capable of preventing and controlling tobacco common mosaic disease. Background Art

[0002] Tobacco mosaic virus (TMV) is a tobacco disease caused by tobacco mosaic virus (TMV). It is one of the common diseases in tobacco production and occurs in tobacco-producing areas around the world. At present, the prevention and control measures for tobacco mosaic virus mainly include breeding disease-resistant varieties, strengthening field management, and using chemical or biological agents for prevention and control. Among them, breeding tobacco varieties with strong disease resistance is the most effective prevention and control method. Ferredoxin (Fd) is a type of acidic, low molecular weight iron-sulfur protein that is commonly found in plants, animals, bacteria and algae. In plants, Fd is generally composed of 95 to 100 amino acid residues, contains a redox center of the [2Fe-2S] cluster, has a relatively negative redox potential, and exists not only in plastids, but also in non-photosynthetic organs such as roots. The main functions of plant Fd include participating in photosynthesis, nitrogen fixation, and disease resistance response. For example, Zhu Li et al. found that rice OsFD1 is involved in the assimilation of carbon, nitrogen and sulfur in rice (CN111100867B); Lai Zhongxiong et al. introduced the longan ferredoxin gene into Oncidium, and the transformed plants not only had a higher survival rate after transplantation, but also had a higher resistance to soft rot (CN103898156B); Cheng Yuqin et al. overexpressed FdV in corn protoplasts to effectively inhibit the replication of sugarcane mosaic virus SCMV (CN102379305B); Sun Xiangchao et al. used antibody detection to show that tomato mosaic virus ToMV infection can reduce the expression of tobacco ferredoxin Fdn I (Chinese Agricultural Science, 2010, 43(19):3981-3987); Deng Lele et al. found that after the expression of tobacco ferredoxin gene Ntab0899580 was reduced, the height of the edited plants decreased, the number of leaves decreased, and the total sugar and reducing sugar content of the leaves increased (CN117024541A). The above studies show that the functions of Fd in plants, including tobacco, are diverse. In-depth research and exploration of the potential functions of different Fd genes is one of the key points of plant gene function research. However, the existing technology is still blank in the research on the regulation of tobacco NtFd gene to obtain strains that prevent and control tobacco common mosaic disease. Summary of the invention

[0003] The purpose of the present invention is to provide a method for obtaining a tobacco variety capable of preventing and controlling tobacco common mosaic disease, so as to fill the gap in the existing tobacco NtFd gene technology for preventing and controlling common mosaic disease and provide a material basis for the cultivation of tobacco disease-resistant strains.

[0004] To achieve the above objectives, this application is implemented through the following technical solutions:

[0005] A method for obtaining a tobacco variety for preventing and controlling tobacco common mosaic disease, using the 120th to 142nd bases of the tobacco NtFd gene shown in SEQ ID NO.1 to perform artificial intervention mutation, and the sequence of the mutated nucleotide fragment is shown in SEQ ID NO.2.

[0006] Furthermore, the protein encoding sequence of the tobacco NtFd gene shown in SEQ ID NO.1 is shown in SEQ ID No.3.

[0007] Furthermore, the mutated tobacco NtFd gene was named tobacco ferredoxin gene ntfd.

[0008] Furthermore, the tobacco ferredoxin gene ntfd was obtained from the tobacco NtFd gene obtained from the tobacco safflower Dajinyuan sample through gene editing technology.

[0009] Furthermore, the PCR amplification primer sequences of the tobacco ferredoxin gene ntfd were designed as follows:

[0010] F: 5'-AACTGGTGAATCCTGCTAAG-3',

[0011] R: 5'-AGACTGGTCCGTACTTGTTT-3'.

[0012] Furthermore, the reaction conditions for PCR amplification were: 95°C 10 sec, 52°C 30 sec, 72°C 2.5 min, for a total of 30 cycles; 72°C 10 min; 12°C Hold.

[0013] The beneficial effects of the present invention are:

[0014] 1. The tobacco NtFd mutant gene sequence obtained by the present invention has a significant effect in the prevention and control of tobacco common mosaic disease.

[0015] 2. The present invention provides a new possibility for the prevention and control of common mosaic disease by using the tobacco NtFd mutant gene, filling the gap in the relevant technical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a phylogenetic tree of a group of NtFd genes of the present invention.

[0017] Figure 2 Shown are the leaf conditions of ntfd tobacco plants and control tobacco plants at full flowering stage. ntfd is the NtFd mutant, and ck is the control sample Honghua Dajinyuan. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is described in detail below through examples. The following examples are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as limiting the technical solution of the present invention.

[0019] In the various embodiments of the present application, if no specific technology or conditions are specified, the existing technology or conditions in the field are used, and the materials or equipment used without indicating the manufacturer are all conventional products that can be purchased.

[0020] Unless otherwise specified in the present invention, percentages refer to volume percentages and ratios refer to volume ratios.

[0021] The tobacco variety used in this application is Honghua Dajinyuan, a commercial tobacco variety.

[0022] Example 1

[0023] The key to this technical solution is the process of obtaining the mutated tobacco NtFd gene, i.e., the ferredoxin gene ntfd. The specific steps are as follows:

[0024] This technical solution uses the leaves of the conventional tobacco cultivar Honghua Dajinyuan as samples, uses an RNA extraction kit to extract total RNA from tobacco leaves, and reversely transcribes it into cDNA for standby use:

[0025] The total RNA of the leaves of S. cyrtonema was extracted according to the instructions of the plant RNA extraction kit.

[0026] 1 μg of total RNA was extracted from the leaves of safflower Dajinyuan for reverse transcription. The transcription system was as follows:

[0027] Total RNA 1 μg;

[0028] Oligo(dT) (10 μM) 1.5 μL;

[0029] ddH2O up to 15μL.

[0030] Mix the above system and place it in PCR, keep it at 70℃ for 5 minutes, remove it and immediately place it on ice for 5 minutes, then add the following reagents to the system:

[0031] M-MLV Buffer (5X) 5 μL;

[0032] M-MLV reverse transcriptase 0.5 μL;

[0033] RNase inhibitor 0.5 μL;

[0034] dNTP Mixture 4 μL;

[0035] ddH2O up to 25μL.

[0036] The above system was placed in a PCR instrument, incubated at 42°C for 65 min, 65°C for 10 min, kept at 4°C, and then stored in a -20°C refrigerator for use.

[0037] By homology comparison, referring to the Arabidopsis gene sequence and the known tobacco partial gene sequence, the amplification primer sequences were designed as follows:

[0038] F: 5'-AACTGGTGAATCCTGCTAAG-3', (SEQ ID No. 4);

[0039] R: 5'-AGACTGGTCCGTACTTGTTT-3'; (SEQ ID No. 5).

[0040] Using the above prepared cDNA as template, PCR amplification was performed using the above primers:

[0041] Amplification system (50 μL):

[0042] cDNA 0.5 μL;

[0043] 5× Reaction Buffer 10 μL;

[0044] Upstream primer (10mmol / L) 2.5μL;

[0045] Downstream primer (10mmol / L) 2.5μL;

[0046] dNTP (10 mM) 5 μL;

[0047] Phusion DNA Polymerase 0.5μL;

[0048] ddH2O up to 50μL.

[0049] After mixing and centrifuging, PCR amplification was performed. The PCR reaction conditions were: 95°C 10 sec, 52°C 30 sec, 72°C 2.5 min, for a total of 30 cycles; 72°C 10 min; 12°C Hold.

[0050] The amplified product was purified and sequenced to obtain the tobacco NtFd gene sequence, whose base sequence is shown in SEQ ID No.1, with a total length of 435 bp, and the encoded protein sequence is shown in SEQ ID No.2, including 144 aa.

[0051] The CDS gene sequence of tobacco NtFd provided in this example was compared with the Chinese tobacco database and the result was gene ID: Ntab0493020.

[0052] NCBI sequence alignment was used to obtain 21 Fd amino acid sequences, and a phylogenetic tree was drawn using MEGA 11.0 ( Figure 1 ), indicating that NtFd in cultivated tobacco has high homology with forest tobacco NsFd (XP_009796636), Nicotiana acuminate NaFd (XP_019249985) and Nicotiana benthamiana NbFd (AKQ98361). The tobacco NtFd (Ntab0493020) of this example has a similarity of 67.48% with tobacco Fdn I (GenBank No.: AY864890) published by Sun Xianchao et al., and a similarity of 42.38% with tobacco ferredoxin gene Ntab0899580 published by Deng Lele et al.

[0053] Example 2

[0054] The gene mutant tobacco seeds and tobacco plants are respectively the control Honghua Dajinyuan (referred to as ck) and the NtFd gene mutant (referred to as ntfd). Among them, ntfd is obtained from Honghua Dajinyuan by gene editing technology. Ntfd is artificially intervened to mutate the 120-142 bases in the tobacco NtFd gene as shown in the nucleotide sequence of SEQID NO.1, and the sequence of the mutated nucleotide fragment is shown in SEQ ID NO.2: GGGTGGTAGGATTGCATGG, lacking ACTT nucleotide. Specifically, the tobacco plant and the obtained tobacco seeds are obtained after the ACTT base is knocked out in the 120-142 bases in the NtFd gene of Honghua Dajinyuan.

[0055] Example 3

[0056] NTFD tobacco plants and CK control tobacco plants were planted, 60 plants each, to observe the incidence of tobacco mosaic disease. The results showed that tobacco leaves of NTFD tobacco plants did not suffer from tobacco mosaic disease, with a natural incidence of 0%, while the natural incidence of tobacco mosaic disease in CK control red large tobacco leaves was 70%.

[0057] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for obtaining a tobacco variety for preventing and controlling tobacco common mosaic disease, characterized in that: The 120-142 bases of the tobacco NtFd gene shown in SEQ ID NO.1 were artificially mutated, and the sequence of the nucleotide fragment after the mutation was shown in SEQ ID NO.

2.

2. The method for obtaining a tobacco variety for preventing and controlling tobacco common mosaic disease according to claim 1, characterized in that: The protein sequence encoded by the tobacco NtFd gene shown in SEQ ID NO.1 is shown in SEQ ID No.

3.

3. The method for obtaining a tobacco variety for preventing and controlling tobacco common mosaic disease according to claim 1, characterized in that: The mutated tobacco NtFd gene was named tobacco ferredoxin gene ntfd.

4. The method for obtaining a tobacco variety for preventing and controlling tobacco common mosaic disease according to claim 3, characterized in that: The tobacco ferredoxin gene ntfd was obtained from the tobacco NtFd gene obtained from the tobacco safflower Dajinyuan sample through gene editing technology.

5. The method for obtaining a tobacco variety for preventing and controlling tobacco common mosaic disease according to claim 4, characterized in that: The PCR amplification primer sequences of tobacco ferredoxin gene ntfd were designed as follows: F: 5'-AACTGGTGAATCCTGCTAAG-3', R: 5'-AGACTGGTCCGTACTTGTTT-3'.

6. The method for obtaining a tobacco variety for preventing and controlling tobacco common mosaic disease according to claim 4, characterized in that: The reaction conditions for PCR amplification were as follows: 95°C for 10 sec, 52°C for 30 sec, 72°C for 2.5 min, for a total of 30 cycles; 72°C for 10 min; and 12°C Hold.

Images