Tobacco salt-tolerant gene Nt-zfhd-6 and application thereof

Abstract

The application discloses a tobacco salt-tolerant gene Nt-zfhd-6 and application thereof. The nucleotide sequence of the tobacco salt-tolerant gene Nt-zfhd-6 comprises a sequence as shown in SEQ ID No. 1. The amino acid sequence coded by the tobacco salt-tolerant gene Nt-zfhd-6 is shown in SEQ ID No. 2. Under a salt stress condition, the expression amount of the Nt-zfhd-6 gene in tobacco seedling leaves is increased by nearly 40 times. The gene is amplified, enzyme-cut, and constructed into an expression vector, and then is infected into tobacco by means of agrobacterium, and a tobacco with the Nt-zfhd-6 gene is obtained. Under a salt stress treatment condition, the root length of the tobacco is obviously increased compared with that of a wild type, and it is proved that the Nt-zfhd-6 gene is a salt-tolerant gene. The functional identification of the tobacco Nt-zfhd-6 gene enriches the gene resources of tobacco salt-tolerant breeding, and has important theoretical significance and application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a tobacco salt-tolerant gene Nt-zfhd-6 and application thereof. BACKGROUND

[0002] Tobacco (Nicotiana tabacum L.) belongs to Solanaceae and Nicotiana L., is an annual herb, and is one of important economic crops in China, and plays an important role in the national economy. Non-biological stress caused by the external environment has an important influence on the quality and yield of tobacco, especially salt stress causes physiological and biochemical reactions in the body of tobacco to be disordered, seriously affects the growth and development of tobacco, and causes a large amount of reduction in yield in agricultural production. At the same time, due to thin tobacco leaves and much water, it is difficult to fix the color during conditioning, and after conditioning, the color is dark, the oil content is low, the elasticity is poor, the taste is weak, and the influence on aroma quality and aroma amount of tobacco is particularly serious, so that the quality of tobacco is rapidly reduced. Therefore, drought seriously restricts the further development of the tobacco industry in China.

[0003] Although China has made a breakthrough in the technology of flue-cured tobacco drought cultivation through long-term technical research, thereby reducing the dependence on water in the cultivation of flue-cured tobacco to a certain extent, the construction of water conservancy facilities is relatively slow, the investment is large, and after investigation in various tobacco producing areas, it is found that the construction of water conservancy facilities in various places is obviously lagging behind, so that the production of tobacco lacks sufficient guarantee. Due to drought, the upper leaves cannot be fully expanded, and the loss of tobacco caused by water irrigation is still very large every year. How to deal with the adverse weather conditions of drought, improve the yield and quality of tobacco, has become a problem to be solved at present. Therefore, cultivating a new salt-tolerant tobacco variety is of great importance to the development of the tobacco industry. Compared with traditional breeding, using molecular biology technology to cultivate a new salt-tolerant tobacco variety has the advantages of short cycle, high efficiency and strong pertinence. The mining and utilization of salt-tolerant genes have important practical significance for ensuring the quality and yield of tobacco.

[0004] At present, there are few genes with salt-tolerant function in tobacco, and it is urgent to mine and provide a gene related to drought resistance of tobacco. SUMMARY

[0005] In view of the deficiencies of the prior art and actual needs, the present application provides a tobacco salt-tolerant gene Nt-zfhd-6 and application thereof. The transgenic line obtained after transforming the Nt-zfhd-6 gene into tobacco is obviously superior to the wild type line in root length under salt stress treatment conditions, which proves that the gene can effectively improve the salt tolerance of plants.

[0006] To achieve the purpose of the application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a tobacco salt-tolerant gene Nt-zfhd-6, wherein the nucleotide sequence of the tobacco salt-tolerant gene Nt-zfhd-6 comprises a sequence as shown in SEQ ID No. 1.

[0008] The present application screens a tobacco gene Nt-zfhd-6 with significantly increased expression under salt stress through transcriptome sequencing analysis, and verifies that the expression of the gene in tobacco seedling leaves under salt stress is increased by nearly 40 times through fluorescent quantitative PCR. After the gene is cloned, an overexpression vector is constructed, a recombinant strain is overexpressed, and the recombinant strain is transformed into tobacco, a transgenic tobacco plant Nt-zfhd-6-OE is obtained, and it is confirmed that the root length of Nt-zfhd-6-OE under salt stress is significantly increased compared with that of the wild type. It is proved that the Nt-zfhd-6 gene is a salt-tolerant gene, and the functional identification of the tobacco Nt-zfhd-6 gene not only enriches the gene resources of tobacco salt-tolerant breeding, but also is beneficial to analyzing the diversity function of the ZFHD transcription factor in tobacco, and has important theoretical significance and application value.

[0009] SEQ ID No. 1:

[0010] ATGGCATTAGCTGGTGAGGATAAGGAAATGAGAATGCAAGGGTCATTAGGGTATCATTCACTTGATCAAGGTAACCAACACCACCACCAACCCAACAACAATAATCTTCAGCCACAACAAGATAATGAAAAATCATCCTCGGGTGGTACTGGTGCAGCACCTCCAGTAGTTGTACCATATTCCAGTGGCGGATCTACTAATAATAAATTTAAAATAACATCACGAGCAAGATATCGTGAATGTCTGAAGAATCATGCCGCTAGCATTGGTGGGAATGTTACAGACGGATGTGGTGAGTTTATGCCTAGTGGAGAAGAAGGAACGCTTGAGGCGTTGAAATGTGCTGCTTGTAATTGCCACCGTAATTTTCACAGAAAAGAACAACCAAATGTTGATAATAATAATGCTGGTATTATGGTTGTACATCCGCTTCAGCTTCCTCAACCACTGCCTTCTCCGCTTCCGTCGATGAACCATCACCATCAACACGGTCGCTCAGTTTGGAGCACAATGCCACCTCAGCCTGTCAAAATGGCCTTCGGAGGTAGCGGCGGAGGTGGAAGTGGAGCTACGGACTCATCAAGTGAAGAACTTAACTTCAACACTTATCAGCAGGCAACCTCAGTGCCTCCACAACCGCAACCACCATTTATGTTGGCGAAGAAACGGTTTCGGACAAAGTTCACTCAAGACCAGAAGGAAAAAATGTTGGAATTTGCGGAGAAATTAGGGTGGAGAATTCCAAGAGAAGATGACACTGAAGTACAAAGATTCTGCTCTCAAGTTGGGGTGAAGAGACAGGTTTTTAAGGTTTGGATGCATAATAATAAGAATCCGTCTGCTAAAAAGAACCCACAAGAAGAACCTTAA.

[0011] It can be understood that the functionally similar genes obtained by certain base mutations (such as base substitution, deletion, etc.) based on the tobacco salt-tolerant gene Nt-zfhd-6 discovered in the application shall be within the protection scope of the application.

[0012] Preferably, the amino acid sequence of the protein encoded by the tobacco salt-tolerant gene Nt-zfhd-6 is shown as SEQ ID No. 2.

[0013] SEQ ID No. 2:

[0014] MALAGEDKEMRMQGSLGYHSLDQGNQHHHQPNNNNLQPQQDNEKSSSGGTGAAPPVVVPYSSGGSTNNKFKITSRARYRECLKNHAASIGGNVTDGCGEFMPSGEEGTLEALKCAACNCHRNFHRKEQPNVDNNNAGIMVVHPLQLPQPLPSPLPSMNHHHQHGRSVWSTMPPQPVKMAFGGSGGGGSGATDSSSEELNFNTYQQATSVPPQPQPPFMLAKKRFRTKFTQDQKEKMLEFAEKLGWRIPREDDTEVQRFCSQVGVKRQVFKVWMHNNKNPSAKKNPQEEP.

[0015] In a second aspect, the present application provides the use of the tobacco salt-tolerant gene Nt-zfhd-6 and / or the reagent for overexpressing the tobacco salt-tolerant gene Nt-zfhd-6 in the first aspect in improving the salt tolerance of plants.

[0016] In a third aspect, the present application provides a method for improving the salt tolerance of plants, the method comprising the following steps:

[0017] (1) amplifying the tobacco salt-tolerant gene Nt-zfhd-6 in the first aspect using primers;

[0018] (2) cloning the tobacco salt-tolerant gene Nt-zfhd-6, and after enzyme digestion and purification, connecting the gene to an expression vector to obtain an overexpression recombinant vector;

[0019] (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0020] (4) transforming the overexpression recombinant strain into plants, and screening and obtaining Nt-zfhd-6 transgenic lines.

[0021] Preferably, the nucleic acid sequence of the primers in step (1) comprises the sequences shown as SEQ ID No. 3 and SEQ ID No. 4.

[0022] SEQ ID No. 3: GGATCCATGGCATTAGCTGGTGAGGA.

[0023] SEQ ID No. 4: GAGCTCTTAAGGTTCTTCTTGTGGGTTCT.

[0024] Preferably, the conditions of the amplification in step (1) are: 96-98℃ (for example 96℃, 97℃, 98℃) pre-denaturation for 20-30 s (for example 20 s, 25 s, 30 s); 96-98℃ (for example 96℃, 97℃, 98℃) denaturation for 10-20 s (for example 10 s, 15 s, 20 s), 56-58℃ (for example 56℃, 57℃, 58℃) annealing for 10-20 s (for example 10 s, 15 s, 20 s), 70-72℃ (for example 70℃, 71℃, 72℃) for 40-50 s (for example 40 s, 45 s, 50 s), 30-40 cycles (for example 30 cycles, 35 cycles, 40 cycles); 70-72℃ (for example 96℃, 97℃, 98℃) post-extension for 10-15 min (for example 10 min, 13 min, 15 min).

[0025] Preferably, the expression vector in step (2) comprises a pCambia2300EC vector containing a 35S promoter and a NOS terminator.

[0026] Preferably, the plant in step (4) comprises tobacco.

[0027] In a fourth aspect, the present application provides an application of the tobacco salt-tolerant gene Nt-zfhd-6 in breeding salt-tolerant plant varieties.

[0028] Preferably, the plant comprises tobacco.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The present application screens a tobacco gene Nt-zfhd-6 with significantly increased expression under salt stress through transcriptome sequencing analysis, and verifies that the expression of the gene in tobacco seedling leaves under salt stress increases by nearly 40 times through fluorescence quantitative PCR. After the gene is cloned, an overexpression vector is constructed, a recombinant strain is overexpressed, and the recombinant strain is transformed into tobacco, a transgenic tobacco plant Nt-zfhd-6-OE is obtained, and it is confirmed that the root length of Nt-zfhd-6-OE under salt stress is significantly increased compared with the wild type. It is proved that the Nt-zfhd-6 gene is a salt-tolerant gene, and the functional identification of the tobacco Nt-zfhd-6 gene not only enriches the gene resources of tobacco salt-tolerant breeding, but also is beneficial to analyzing the diversity function of the ZFHD transcription factor in tobacco, which has important theoretical significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure for the expression quantity result of Nt-zfhd-6 gene under salt stress;

[0032] Figure 2 Figure for the salt tolerance result of overexpression line;

[0033] Figure 3 Figure for the malondialdehyde content detection result of overexpression line and wild type tobacco;

[0034] Figure 4 Figure for the superoxide dismutase activity detection result of overexpression line and wild type tobacco. DETAILED DESCRIPTION

[0035] In order to further illustrate the technical means adopted by the present application and its effects, the present application will be further described below in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

[0036] The specific techniques or conditions not specified in the embodiments are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not specified by the manufacturer are all conventional products that can be commercially available through regular channels.

[0037] The present application screens a tobacco gene Nt-zfhd-6 with significantly improved expression under salt stress through transcriptome sequencing analysis, the nucleotide sequence of which is shown as SEQ ID No. 1, and the encoded amino acid sequence is shown as SEQ ID No. 2. It is found by fluorescence quantitative PCR verification that the expression quantity of Nt-zfhd-6 gene in tobacco seedling leaves is improved by nearly 40 times under salt stress, and it is preliminarily determined that the gene plays an important role in the response of tobacco to salt stress. Further, the Nt-zfhd-6 gene fragment is obtained by PCR amplification using tobacco leaf cDNA as the test material; after the expression vector is constructed by enzyme digestion and ligation, it is transferred into tobacco by using Agrobacterium infection, and transgenic tobacco plants of Nt-zfhd-6 gene are obtained, and it is confirmed that the gene can significantly improve the salt stress resistance of transgenic tobacco plants, thereby proving that Nt-zfhd-6 gene is a salt-tolerant gene. Therefore, the functional identification of tobacco Nt-zfhd-6 gene not only enriches the gene resources of tobacco salt-tolerant breeding, but also is beneficial to analyzing the diversity function of ZFHD transcription factor in tobacco, which has important theoretical significance and application value.

[0038] Example 1

[0039] The present embodiment provides a method for cloning tobacco salt-tolerant gene Nt-zfhd-6.

[0040] (1) Taking the coding region sequence of tobacco Nt-zfhd-6 gene as reference, specific primers Nt-zfhd-6-F and Nt-zfhd-6-R are designed, and the primer sequences are as follows:

[0041] Nt-zfhd-6-F (SEQ ID No. 3):

[0042] GGATCCATGGCATTAGCTGGTGAGGA;

[0043] Nt-zfhd-6-R (SEQ ID No. 4):

[0044] GAGCTCTTAAGGTTCTTCTTGTGGGTTCT.

[0045] (2) The mRNA of tobacco seedling leaf of tobacco cultivar "K326" is extracted, and is reversely transcribed into cDNA, which is used as a PCR amplification template. The primers in step (1) are used for PCR amplification, and an amplification fragment of Nt-zfhd-6 gene is obtained.

[0046] The PCR amplification system is as follows: ddH2O 31 μL, 5×HF buffer containing Mg2+ 10 μL, dNTP with a concentration of 2.5 mM 2 μL, Nt-zfhd-6-F and Nt-zfhd-6-R each 2 μL with a concentration of 5 μM, DMSO 0.6 μL, Phusion enzyme 0.5 μL, and cDNA template 2 μL. 2+

[0047] The PCR reaction conditions are as follows: 98 ℃ pre-denaturation for 30 s; 98 ℃ denaturation for 10 s, 58 ℃ annealing for 10 s, 72 ℃ for 50 s, 30 cycles; and 72 ℃ post-extension for 10 min.

[0048] (3) The overexpression vector of Nt-zfhd-6 is constructed: the PCR amplification fragment obtained in step (2) is connected to the cloning vector pEASY-Blunt simple, and after sequencing, the plasmid is extracted, and is double-enzyme cut by BamH I and Sac I, and after purification, is connected to the pCambia2300EC vector (the vector contains 35S promoter and NOS terminator) which is enzyme cut by BamH I and Sac I, and after sequencing, the agrobacterium GV3101 is transformed by freeze-thaw method, and the tobacco is genetically transformed by immersion staining method, and the transgenic plant Nt-zfhd-6 is obtained.

[0049] Example 2

[0050] The embodiment provides an application of a tobacco salt-tolerant gene Nt-zfhd-6.

[0051] ​(1) Analysis of expression pattern in tobacco seedling leaves under salt stress

[0052] The seedlings of “K326” were treated with 200 mM NaCl solution, and the dynamic changes of Nt-zfhd-6 gene expression at 0 h, 6 h, 24 h and 48 h were analyzed by using the fluorescence quantitative PCR technology.

[0053] The primer sequence of the fluorescence quantitative PCR is as follows:

[0054] Nt-zfhd-6-qF (SEQ ID No. 5): ATCAACACGGTCGCTCAGTT;

[0055] Nt-zfhd-6-qR (SEQ ID No. 6): GTCCGTAGCTCCACTTCCAC.

[0056] The results are shown in Figure 1 After the salt stress treatment, the expression amount of Nt-zfhd-6 was obviously increased, and reached the peak at 48 h, which was increased by nearly 40 times. It is preliminarily verified that the Nt-zfhd-6 gene responds to salt stress, and further indicates that the Nt-zfhd-6 gene is involved in the salt tolerance reaction of tobacco.

[0057] (2) Transgenic tobacco salt tolerance identification

[0058] The T3 generation seeds of the transgenic plant Nt-zfhd-6-OE and the wild type (K326) tobacco seeds were uniformly sown in the soil mixture and kept under normal conditions. After growing for 6 weeks, the wild type tobacco seeds (WT) were used as a control and treated with 200 mM NaCl solution for 1 week. At the same time, the superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content of the tobacco seedlings were detected by using the nitrogen blue tetrazolium (NBT) photochemical reduction method and the TBARS-TCA method, respectively.

[0059] The results show that after the salt treatment, the salt tolerance of the transgenic tobacco OE2 and OE3 plants is obviously increased compared with the wild type tobacco ( Figure 2 ), and the MDA content after the salt treatment is significantly lower than that of WT ( Figure 3 ), and the SOD activity is significantly higher than that of WT ( Figure 4 ). Therefore, the salt tolerance of the transgenic tobacco is better than that of the wild type, that is, the Nt-zfhd-6 gene can improve the salt tolerance of the transgenic tobacco.

[0060] In summary, by transcriptome sequencing analysis, the present application screens a tobacco gene Nt-zfhd-6 which has a significantly increased expression under salt stress, and the fluorescence quantitative PCR verifies that the expression of the gene in tobacco seedling leaves under salt stress is increased by nearly 40 times, then the gene is cloned, an overexpression vector is constructed, a recombinant strain is overexpressed, and is transformed into tobacco to obtain a transgenic tobacco plant Nt-zfhd-6-OE, and it is confirmed that the root length of Nt-zfhd-6-OE under salt stress is significantly increased compared with the wild type. It is proved that the Nt-zfhd-6 gene is a salt-tolerant gene, and the functional identification of the tobacco Nt-zfhd-6 gene not only enriches the gene resources of tobacco salt-tolerant breeding, but also is beneficial to analyzing the diversity function of the ZFHD transcription factor of tobacco, and has important theoretical significance and application value.

[0061] The applicant declares that the detailed method of the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the present application must rely on the above-mentioned detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A use of a tobacco salt-tolerant gene Nt-zfhd-6 in improving plant salt tolerance, wherein the nucleotide sequence of the tobacco salt-tolerant gene Nt-zfhd-6 is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the tobacco salt-tolerant gene Nt-zfhd-6 is shown in SEQ ID No. 2; and the plant is tobacco.

2. A method for increasing salt tolerance in plants, characterized in that, The method for improving plant salt tolerance comprises the following steps: (1) amplifying the tobacco salt-tolerant gene Nt-zfhd-6 using primers, wherein the nucleotide sequence of the tobacco salt-tolerant gene Nt-zfhd-6 is shown in SEQ ID No. 1; (2) cloning the tobacco salt-tolerant gene Nt-zfhd-6, and then connecting the tobacco salt-tolerant gene Nt-zfhd-6 to an expression vector after enzyme digestion and purification to obtain an overexpression recombinant vector; (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) transforming the overexpression recombinant strain into a plant, and screening and obtaining an Nt-zfhd-6 transgenic line, wherein the plant is tobacco.

3. The method of increasing salt tolerance in plants according to claim 2, wherein, The nucleic acid sequence of the primers in step (1) is shown in SEQ ID No. 3 and SEQ ID No.

4.

4. The method of increasing salt tolerance in plants according to claim 2, wherein, The amplification conditions in step (1) are as follows: 96-98 ℃ pre-denaturation for 20-30 s; 96-98 ℃ denaturation for 10-20 s, 56-58 ℃ annealing for 10-20 s, 70-72 ℃ extension for 40-50 s, 30-40 cycles; and 70-72 ℃ post-extension for 10-15 min.

5. The method of increasing salt tolerance in plants of claim 2, wherein, The expression vector in step (2) comprises a pCambia2300EC vector, and the pCambia2300EC vector contains a 35S promoter and a NOS terminator. 6.A use of a tobacco salt-tolerant gene Nt-zfhd-6 in breeding salt-tolerant plant varieties, wherein the nucleotide sequence of the tobacco salt-tolerant gene Nt-zfhd-6 is shown in SEQ ID No. 1, and the plant is tobacco.

Images