Application of NtESR2b gene in reduction of growth of tobacco axillary buds

Abstract

The invention discloses application of an NtESR2b gene in reduction of growth of axillary buds of tobacco, and relates to the technical field of genetic engineering, and the technical key point is that nucleotide coded by the NtESR2b gene is mutated through a gene editing technology, so that the function of the NtESR2b gene is lost, and the tobacco with reduced growth of the axillary buds is obtained. Growth of the tobacco axillary buds is reduced from the source through a gene editing technology, the tobacco yield and the tobacco quality are improved, growth of the tobacco axillary buds can be inhibited, and a high-efficiency and good-effect method for reducing growth of the tobacco axillary buds is provided for tobacco production.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to application of NtESR2b gene in reducing the growth of tobacco axillary buds. Background Art

[0002] Tobacco (Nicotiana tabacum L.), an annual or limited perennial plant of the Solanaceae family, is an important cash crop in my country, primarily harvested for its leaves. After a period of growth, tobacco plants transition from vegetative to reproductive growth. While the leaves retain only a portion of nutrients for their own metabolism, the majority of nutrients are transported to the reproductive organs, promoting their development. Therefore, to ensure tobacco leaf quality, topping is a common practice during tobacco production to ensure a sufficient nutrient supply. However, topping eliminates apical dominance, and photosynthetic products from the tobacco leaves are preferentially transported to new axillary buds, promoting their growth. This depletes nutrients from the leaves, leading to reduced tobacco yield and quality. Therefore, after topping, it is important to promptly remove any germinated axillary buds. Failure to do so will result in underdeveloped, lighter, less oily, and less elastic lower leaves, diminishing flavor and aroma, and smaller, thinner upper leaves, resulting in overall reduced tobacco yield and quality. If the topping is not controlled and the axillary buds are allowed to grow, the yield will be lost by 1% every day and the aroma of the tobacco leaves will also decrease.

[0003] Artificial branch removal or the use of bud inhibitors are important measures to ensure the quality of tobacco leaves after topping the tobacco plants. However, artificial branch removal and the use of bud inhibitors will undoubtedly increase the cost of tobacco production. In order to reduce costs and increase efficiency, cultivating tobacco materials with few or no axillary buds is of great significance in tobacco production. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems and provide an application of the NtESR2b gene in reducing the growth of tobacco axillary buds.

[0005] In order to achieve the above object, the present invention provides the use of NtESR2b gene in reducing the growth of tobacco axillary buds.

[0006] Furthermore, the present invention utilizes CRISPR / Cas9 gene editing technology to mutate the NtESR2b gene, wherein the sequence information of the NtESR2b gene comes from cultivated tobacco K326 (Nicotiana tabacum L.cv K326), the CDS sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0007] Furthermore, the "Application of NtESR2b Gene in Reducing the Growth of Tobacco Axillary Buds" specifically uses CRISPR / Cas9 gene editing technology to disable the function of the NtESR2b gene, thereby reducing the growth of tobacco axillary buds.

[0008] Compared with the existing technology, the beneficial effects of this solution are: the present invention reduces the growth of tobacco axillary buds from the root through gene editing technology, improves tobacco yield and tobacco quality, and provides a method for reducing the growth of tobacco axillary buds with high efficiency and good effect for tobacco production. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the CRISPR / Cas9-NtESR2b vector construction in the embodiment of the present invention;

[0010] Figure 2 This is a PCR test of a T0 generation CRISPR / Cas9-NtESR2b vector-positive plant in the embodiment of the present invention;

[0011] Figure 3 It is the edited form of the homozygous mutant of ntesr2b in the embodiment of the present invention;

[0012] Figure 4 The PCR test of T1 generation T-DNA-eliminated plants in the embodiment of the present invention;

[0013] Figure 5 The growth conditions of tobacco ntesr2b mutant and WT in the examples of the present invention are shown;

[0014] Figure 6 The agronomic traits of tobacco ntesr2b mutant and WT were measured in the examples of the present invention;

[0015] Figure 7 The leaf number statistics of tobacco ntesr2b mutant and WT in the examples of the present invention are shown;

[0016] Figure 8 This is the weight measurement of tobacco ntesr2b mutant and WT axillary buds in the examples of the present invention. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0018] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0019] The present invention provides the use of the NtESR2b gene to reduce tobacco axillary bud growth. Loss of NtESR2b gene function reduces tobacco axillary bud growth. ntesr2 mutant material is obtained using CRISPR / Cas9 gene editing technology. A binary vector containing the target sequence sgRNA is first constructed, and cultivated tobacco K326 is transformed using Agrobacterium-mediated leaf disc transformation to achieve mutation of the NtESR2b gene. Plants with homozygous mutations in the NtESR2b gene and T-DNA deletion are screened from the progeny. The axillary bud growth weight of the obtained ntesr2 mutant tobacco is significantly reduced compared to the K326 control. The tobacco NtESR2b gene CDS coding sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2:

[0020] Example 1 Construction of CRISPR / CAS9-NtESR2b gene editing vector

[0021] (1) Using tobacco NtESR2b as the target gene, the CRISPR-P online analysis platform (http: / / crispr.hzau.edu.cn / CRISPR2 / ) was used to perform blast sequence alignment of the NtESR2 gene to determine the optimal target site, and target primers were designed. 5'-GTCCTTTAGAGCAGCCTGATAGG-3' was selected as the sgRNA target site, where the AGG at the 3' end was the PAM sequence.

[0022] (2) The designed sgRNA sequence was constructed into the CRISPR / Cas9 editing vector. The pCAMBIA1300-pYAO:Cas9 vector used was a publicly published or sold vector. The method for constructing the target sequence sgRNA into the vector was referred to the vector manual, and the vector CRISPR / Cas9-NtESR2b was successfully constructed.

[0023] (3) To further verify the successful connection of the target sequence, PCR amplification and identification were performed on the transformed E. coli single colony. The identification primers were 1300-gRNA-F: 5'-CCAGTCACGACGTTGTAAAAC-3', 1300-gRNA-R: 5'-CAATGAATTTCCCATCGTCGAG-3'. The primer amplification band was 750 bp. The amplified band is shown in the attached figure. Figure 1 As shown, the bands in lanes 1, 2, 3, 5, 6, 7, and 8 are at 750 bp, indicating that the target sequence has been successfully connected to the pCAMBIA1300-pYAO:Cas9 vector.

[0024] Example 2 Genetic transformation of tobacco K326 using CRISPR / CAS9-NtESR2b gene editing vector

[0025] (1) The constructed CRISPR / Cas9-NtESR2b gene editing vector was transformed into Agrobacterium competent cells GV3101.

[0026] (2) Select a successfully transformed monoclonal colony and inoculate it into YEP medium containing the corresponding antibiotics for expansion culture. Cultivate the culture at 200 rpm and 28°C until the OD600 of the culture solution is between 0.6 and 0.8.

[0027] (3) Collect the cells by centrifugation at 6000 rpm for 10 min, remove the supernatant medium and resuspend the cells in an equal volume of sterile water.

[0028] (4) A 1×1 cm sterile leaf disc was placed in the Agrobacterium bacterial solution and infected for 10 min. After infection, the bacterial solution on the surface of the leaf disc was dried with sterile absorbent paper and then placed on the co-culture medium for 2 days (28°C).

[0029] (5) Rinse the leaf disc three times with sterile water containing 160 mg / L Timentin, dry the leaf disc with sterile absorbent paper, and transfer it to the screening medium containing the corresponding antibiotic.

[0030] (6) After the leaf disc forms adventitious buds, cut off the adventitious buds of appropriate size and transfer them to the rooting medium for further cultivation.

[0031] (7) After the seedlings have formed a complete root system, they are hardened and transplanted into the substrate.

[0032] Note:

[0033] Co-culture medium: MS medium + 1.5 mg / L 6-BA

[0034] Screening medium: MS culture + 1.5 mg / L 6-BA + 10 mg / L hygromycin + 160 mg / L timentin

[0035] Rooting medium: 1 / 2MS medium + 0.1mg / L NAA + 10mg / L hygromycin + 160mg / L timentin

[0036] After the above culture medium is prepared, the pH value is adjusted to 6.2-6.3 and sterilized at 121℃ for 20 minutes.

[0037] Example 3 Identification of T0 Generation Edited Plants

[0038] (1) Extract DNA from leaves of T0 generation plants.

[0039] (2) Specific primers were designed based on the detection of eukaryotic hygromycin resistance genes. The primer sequences were HYG-F: 5'-TTGACATTGGGGAGTTTAGCG-3', HYG-R: 5'-CAGGACATTGTTGGAGCCG-3'. The transformation of T0 generation plants was identified by PCR amplification. The PCR reaction system was 20 μL, including: 10 μL Max enzyme, 1 μL HYG-F, 1 μL HYG-R, 6 μL Nuclease-free Water and 2 μL DNA. The PCR program was: pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 30 seconds, annealing at 63°C for 30 seconds, extension at 72°C for 1 minute, for a total of 35 cycles; extension at 72°C for 5 minutes. If a specific band of 383 bp was amplified, it indicated that a positive plant was successfully obtained, as shown in the attached figure. Figure 2 shown.

[0040] (3) Detection of T0 generation gene editing: Specific primers were designed at both ends of the sgRNA sequence. The primer sequences were NtESR2b-F: 5'-CATCTGTCGGGGCGGTGAGGTAC-3', NtESR2b-R: 5'-CAAAGAATCATTGCTTTTCTGA-3'. The PCR reaction system was 40 μL, including: 20 μL ExTaq enzyme, 2 μL NtESR2b-F, 2 μL NtESR2b-R, 12 μL Nuclease-free Water, and 4 μL DNA. The PCR program was as follows: 94°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 63°C annealing for 30 s, and 72°C extension for 1 min 30 s, for a total of 35 cycles; 72°C extension for 5 min.

[0041] (4) The PCR product of step (3) was sent to a biological company for sequencing. The editing form of the T0 generation positive plants was determined by the sequencing peak diagram, and the NtESR2b mutant plants were screened. The editing form of the homozygous mutant was as shown in the attached Figure 3 As shown, an A base was inserted after the 416th base of the CDS of the NtESR2b gene, resulting in a frameshift mutation and premature termination of protein translation. The original protein had a total of 399 amino acids, which became 147 amino acids after the mutation.

[0042] Example 4: NtESR2b gene loss in tobacco reduces axillary bud growth

[0043] (1) Using the conventionally cultivated K326 as a control, the ntesr2b mutant was sown with three biological replicates (30 plants / replicate), with a row spacing of 1.1 m and a plant spacing of 0.55 m. A nitrogen application rate of 6.5 g / plant was applied. Agronomic traits such as natural plant height, natural leaf number, and effective leaf number of tobacco plants were measured at the peak flowering stage. Tobacco plants were toppled when 50% of the central flowers were open, and axillary buds were removed simultaneously. Thirty days after topping, the axillary buds of the tobacco plants were collected and weighed.

[0044] (2) The plant height, waist leaf length, waist leaf width and stem girth of ntesr2b and K326 were measured and there was no significant difference between the two materials. Figure 6 The natural leaf number and effective leaf number of tobacco ntesr2b and K326 were statistically analyzed and there was no significant difference between the two. Figure 7 shown.

[0045] (3) From the observation of tobacco growth, there is no significant difference in growth between the ntesr2b mutant and the wild-type tobacco K326. However, from the observation of axillary bud growth, the axillary bud growth of the ntesr2b mutant is significantly less than that of K326. Figure 5 The weight of the axillary buds of tobacco ntesr2b mutant and tobacco K326 was measured before and after topping. The weight of the axillary buds of tobacco ntesr2b mutant was significantly lower than that of tobacco K326 30 days after topping, as shown in the attached figure. Figure 8 shown.

[0046] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. Application of NtESR2b gene in reducing tobacco axillary bud growth.

2. The use of the NtESR2b gene as claimed in claim 1 in reducing the growth of tobacco axillary buds, characterized in that: Loss of function of the NtESR2b gene reduces the growth of tobacco axillary buds.

3. The use of the NtESR2b gene as claimed in claim 1 in reducing the growth of tobacco axillary buds, characterized in that: Through gene editing technology, the nucleotide encoded by the NtESR2b gene was mutated, causing the NtESR2b gene function to be lost, and tobacco with reduced axillary bud growth was obtained.

4. The use of the NtESR2b gene in reducing tobacco axillary bud growth as claimed in claim 3, wherein: The CDS sequence of the edited NtESR2b gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

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