Application of NaNAC72 gene in increasing nicotine content

Abstract

The invention discloses an application of a NaNAC72 gene in increasing nicotine content, and relates to the technical field of plant genetic engineering, cDNA (complementary deoxyribonucleic acid) of gradually narrow-leaf tobacco is used as a template, amplification is performed to obtain a nucleotide coding sequence of the NaNAC72 gene, the nucleotide coding sequence is shown as SEQ ID NO.1, and the sequence of coded amino acid is shown as SEQ ID NO.2. The invention further discloses an application of the NaNAC72 gene in increasing nicotine content. The transcription factor NaNAC72 with the function of negatively regulating and controlling the nicotine content of the tobacco is obtained from the gradually narrow-leaf tobacco, and the nicotine content in the tobacco can be increased without influencing plant growth through gene editing; the NaNAC72 gene provides a target gene for creating a plant material with improved tobacco nicotine content through methods such as gene editing or mutation; the important target gene is provided for directional improvement and cultivation of new high-quality tobacco varieties, and the important application value is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to an application of a NaNAC72 gene in increasing nicotine content. Background Art

[0002] Tobacco (Nicotiana) is a globally important economic crop, its commercial value primarily determined by the alkaloid content and quality of its leaves. Nicotine, as the core alkaloid component, accounts for over 90% of the total alkaloid content and is the key carrier of tobacco flavor. Therefore, precise regulation of nicotine biosynthesis is crucial for balancing tobacco quality and safety.

[0003] Nicotine is a plant's insect-resistant defense substance, and its biosynthesis involves multiple key enzymes, including PMT, MPO, BBL, and A622. Currently, several transcription factors have been shown to positively promote nicotine synthesis, including members of the ethylene response factor (ERF) family (e.g., NtORC1, NtJAP1, NtERF189, NtERF199) and basic helix-loop-helix (bHLH) proteins (e.g., NtMYC2). Although overexpressing these enzyme genes or regulatory factors can significantly increase nicotine content, there are "transgenic" barriers in breeding, and overexpression of these genes can affect plant growth and development.

[0004] Currently, our understanding of the negative regulatory mechanisms of nicotine synthesis is severely limited. Known negative regulators are scarce and their functions are unclear, with only reports suggesting that the transcription factor ARF can inhibit nicotine synthesis. If a series of negative nicotine regulators could be identified, approaches such as gene editing or EMS-induced mutagenesis could be used to screen for key regulatory factors that could increase nicotine content without affecting plant growth. Therefore, there is an urgent need to identify the negative "molecular switch" in tobacco that specifically regulates nicotine synthesis and to develop mutation-based technologies based on this target to overcome current technical barriers to quality improvement. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of the NaNAC72 gene in increasing nicotine content, and to obtain the transcription factor NaNAC72 from Nicotiana attenuata, which has the function of "negatively regulating" nicotine biosynthesis without affecting plant growth, thereby providing a target gene for increasing nicotine content through gene editing or mutation; the NaNAC72 gene provides an important target gene for the directed improvement and cultivation of high-quality new tobacco varieties, lays a theoretical foundation for subsequent molecular breeding work, and has important application value.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for cloning the tobacco NaNAC72 gene comprises amplifying the nucleotide coding sequence of the NaNAC72 gene using Nicotiana attenuata cDNA as a template, wherein the nucleotide coding sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0008] Further: the primer combination used to amplify the nucleotide coding sequence of the NaNAC72 gene is NaNAC72-F and NaNAC72-R;

[0009] NaNAC72-F is shown in SEQ ID NO. 3;

[0010] NaNAC72-F is shown in SEQ ID NO.4.

[0011] Further: NaNAC72 CRISPR-Cas9 gene editing was constructed, and a dual-target gene editing vector system was constructed based on the pHSE401 plasmid expression vector. The constructed knockout vector was pHSE401-NaNAC72;

[0012] The sgRNA target sequences for the first exon of the gene NaNAC72 include: NaNAC72-sgRNA1, NaNAC72-sgRNA2;

[0013] NaNAC72-sgRNA1 is shown in SEQ ID NO. 5;

[0014] NaNAC72-sgRNA2 is shown as SEQ ID NO.6.

[0015] Further: After constructing the knockout vector, the expression vector containing the target gene was introduced into the hypocotyl of Nicotiana attenuata through the Agrobacterium-mediated transformation method, and gene editing positive materials were obtained through tissue culture, antibiotic resistance screening and target site PCR detection.

[0016] A tobacco NaNAC72 gene cloning method is used in identifying tobacco mutants, the method comprising: using the genome of a transgenic plant as a template for PCR amplification, and then detecting by gel electrophoresis or sequencing to obtain a strain with a mutation in the NaNAC72 gene;

[0017] The primer combination used in the PCR amplification is Detect-F and Detect-R;

[0018] Detection-F is shown in SEQ ID NO.11;

[0019] Detection-R is shown as SEQ ID NO.12.

[0020] The invention discloses an application of a method for cloning the tobacco NaNAC72 gene, wherein the NaNAC72 gene is used to increase nicotine content.

[0021] Further: extracting nicotine from Nicotiana attenuata includes the following steps;

[0022] (1) Samples of Nicotiana attenuata leaves were collected, weighed, and stored in liquid nitrogen;

[0023] (2) Grind the sample thoroughly in liquid nitrogen until it becomes powdery;

[0024] (3) Add methanol / acetic acid extract, shake and centrifuge, aspirate the supernatant, and set aside for measurement.

[0025] Furthermore: a high performance liquid chromatography coupled with a triple quadrupole mass spectrometer system was used to calculate the nicotine content in tobacco based on the peak area.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) In the present invention, a NAC transcription factor NaNAC72 was cloned from Nicotiana attenuata, and a NaNAC72 CRISPR-Cas9 gene editing knockout vector was constructed to transform Nicotiana attenuata, thereby obtaining a homozygous NaNAC72 gene knockout mutant plant. Nicotiana attenuata was transformed by nicotine content determination. It was found that the homozygous NaNAC72 gene editing knockout mutant showed a higher nicotine content than the control, and its growth was not inhibited at all. The present invention provides an important target gene for the directional improvement and cultivation of high-quality new tobacco varieties, lays a theoretical foundation for subsequent molecular breeding work, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram showing the editing site and mutation information detection results of the NaNAC72 knockout mutant material gene of the present invention.

[0029] Figure 2 This is a diagram showing the amino acid information detection results of the NaNAC72 knockout mutant material after mutation.

[0030] Figure 3 This is a graph comparing the growth phenotypes of the NaNAC72 transgenic material and the wild-type plant in the present invention, as well as the results of measuring the nicotine content in the leaves of the NaNAC72 knockout mutant material, wherein WT represents the material without the NaNAC72 gene knockout, and nac72-1 and nac72-2 represent two different knockout mutant materials. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] Example 1:

[0033] 1. Cloning of tobacco NaNAC72 gene

[0034] Using the homologous cloning method, the cDNA of Nicotiana attenuata was used as a template to amplify the nucleotide coding sequence of the NaNAC72 gene. The nucleotide coding sequence is 1062 bp in length, as shown in SEQ ID NO.1, and encodes 353 amino acids. The sequence of the encoded amino acids is shown in SEQ ID NO.2.

[0035] The nucleotide sequence of NaNAC72 (SEQ ID NO. 1) is shown below:

[0036] Atgggtgttcaagaaaaagatcctcttttgcaattaagtttgccaccagggtttcgtttttatcctactgatgaagagctttta

[0037] gttcaatatttatgcaagaaagttgcaggccatgatttctctatgcaaatcattggagatattgatttgtacaaattcgaccct

[0038] tgggttcttccaagtaaggcactttttggtgagaaagaatggtatttttttagtccaagagataggaaatatccaaatggat

[0039] ctcgaccaaatagagtagctggtactggttattggaaagcaacaggaactgataagatcataacatcacaaggaagaa

[0040] aagttggaattaagaaagcacttgttttttatgtgggtaaagcaccaaaaggaaccaagaccaattggattatgcatgaat

[0041] atagactctttgaaccttcaaggaaaaatggaagtacaaagctagatgaatgggtgctctgtcgaatttataagaagaatt

[0042] caagtggacaaaagcctcatatgtccggtttgaacagcaaagaacacagccatggttcttcaacttcatcatcgtcccaa

[0043] ttcgatgatatgctcgaatcgttacctgagatggatcacaagttttctaacttgtcgagattgaactcgctcaagaacttcc

[0044] aacaaggtgaaaagtttaatcttgaacgcctggattctgcaaactttgattgggcaatccttgcggggctcaaaccaatg

[0045] ccagaattgggccccgcaaatcaagctccaggcattcaacctcaagctcaggggcatgttaacaacaacatacacaa

[0046] ccacaacaacaacaactatatgaattttctaaatgatgggtatgtccagcctacgaattttcgaggtaacaccaaggtag

[0047] aaagaattaatctggatgaagaagttgaaagcggaatcagaaatcaaggggttgataaatcaggattcttccaacagaa

[0048] tataaatggattttctcaaacctatacgaacactgtcgaccagtttggaattcagggtccaaaccaaacattaaatctggggttcaggccgtag。

[0049] NaNAC72(SEQ ID NO.2)的氨基酸序列如下所示:

[0050] MGVQEKDPLLQLSLPPGFRFYPTDEELLVQYLCKKVAGHDFSMQIIGDIDL

[0051] YKFDPWVLPSKALFGEKEWYFFSPRDRKYPNGSRPNRVAGTGYWKATGT

[0052] DKIITSQGRKVGIKKALVFYVGKAPKGTKTNWIMHEYRLFEPSRKNGSTKL

[0053] DEWVLCRIYKKNSSGQKPHMSGLNSKEHSHGSSTSSSSQFDDMLESLPEM

[0054] DHKFSNLSRLNSLKNFQQGEKFNLERLDSANFDWAILAGLKPMPELGPAN

[0055] QAPGIQPQAQGHVNNNIHNHNNNNYMNFLNDGYVQPTNFRGNTKVERIN

[0056] LDEEVESGIRNQGVDKSGFFQQNINGFSQTYTNTVDQFGIQGPNQTLNLGFRP*.

[0057] The primer combination NaNAC72-F and NaNAC72-R used to amplify the nucleotide coding sequence of the NaNAC72 gene is as follows:

[0058] NaNAC72-F (SEQ ID NO.3): atgggtgttcaagaaaaagatc;

[0059] NaNAC72-F (SEQ ID NO. 4): ctacggcctgaacccca.

[0060] 2. Construction of NaNAC72 CRISPR-Cas9 gene editing vector

[0061] A dual-target gene editing vector system was constructed based on the pHSE401 expression vector. The constructed knockout vector is pHSE401-NaNAC72.

[0062] The sgRNA target sequences for the first exon of the gene NaNAC72 include: NaNAC72-sgRNA1 and NaNAC72-sgRNA2, and their nucleotide sequences are as follows: NaNAC72-sgRNA1 (SEQ ID NO.5): GATTTCTCTATGCAAATCATTGG; NaNAC72-sgRNA2 (SEQ ID NO.6): TTGCAATTAAGTTTGCCACCAGG.

[0063] After constructing the knockout vector, the expression vector containing the target gene was introduced into the hypocotyl of Nicotiana attenuata through Agrobacterium-mediated transformation. After tissue culture, antibiotic resistance screening and target site PCR detection, gene editing positive materials were obtained.

[0064] The method for identifying tobacco mutants from transgenic plants includes: performing PCR amplification using the genome of the transgenic plant as a template, and then detecting by gel electrophoresis or sequencing to obtain a strain with a mutation in the NaNAC72 gene; the primer combination used in the PCR amplification is:

[0065] Detect-F (SEQ ID NO. 7): CGTCTGTTATTCTCTGAGCTTCT;

[0066] Detect-R (SEQ ID NO. 8): TCGGATTAAAGGCGAGACCA.

[0067] 3. Changes in nicotine content in NaNAC72 gene knockout materials Nicotine content

[0068] NaNAC72 was silenced using CRISPR / Cas9 technology, and two knockout mutants were obtained. The editing type of nac72a-1 is a 2-base deletion at the target site 1. The editing type of nac72a-2 is a 1-base deletion at the target site 1. Figure 1 Both of the above mutations lead to frameshift changes in translation and premature termination of translation ( Figure 2 The nicotine content in the leaves of NaNAC72 gene non-knockout material (WT) and knockout mutant material was determined ( Figure 3 )

[0069] The method for extracting nicotine from Nicotiana acuminate leaves comprises the following steps:

[0070] (1) Samples of Nicotiana attenuata leaves were taken, weighed, and stored in liquid nitrogen.

[0071] (2) Grind the sample thoroughly in liquid nitrogen until it becomes powder.

[0072] Add 1 mL of an extract containing 40% methanol and 0.1% acetic acid, shake for 10 min, centrifuge at 12000 rpm for 20 min, aspirate the supernatant, and place at 4°C for determination.

[0073] Method for determining nicotine content in Nicotiana attenuata:

[0074] A Shimadzu ultra-high performance liquid chromatograph (LC-20A) coupled to a triple quadrupole mass spectrometer (LCMS-8040) was used. The chromatographic column used was Shimadzu Shim-pack XR-ODS III, 2.0 mm ID × 75 mm L, 1.6 μm. Mobile phase A was 0.05% formic acid and mobile phase B was 0.05% acetonitrile. The flow rate was 0.3 ml / min. Nicotine content in tobacco was calculated based on peak area.

[0075] The test results showed that after the NaNAC72 gene was knocked out, the nicotine content increased by 36-42.5% compared with the wild-type control, and its growth was not significantly different from that of the plants without knockout ( Figure 3 ).

[0076] Although the present invention has been described herein with reference to a number of illustrative embodiments thereof, it will be understood that numerous other modifications and implementations may be devised by those skilled in the art that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure, the drawings, and the claims, numerous variations and modifications may be made to the components and / or layout of the subject combination arrangement. In addition to variations and modifications to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A method for cloning the tobacco NaNAC72 gene, characterized in that: The nucleotide coding sequence of the NaNAC72 gene was amplified using the cDNA of Nicotiana attenuata as a template. The nucleotide coding sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.

2.

2. The cloning method according to claim 1, characterized in that: The primer combination used to amplify the nucleotide coding sequence of the NaNAC72 gene is NaNAC72-F and NaNAC72-R; NaNAC72-F is shown in SEQ ID NO. 3; NaNAC72-F is shown in SEQ ID NO.

4.

3. The cloning method according to claim 1, wherein: Construct NaNAC72 CRISPR-Cas9 gene editing, and build a dual-target gene editing vector system based on the pHSE401 plasmid expression vector. The constructed knockout vector is pHSE401-NaNAC72; The sgRNA target sequences for the first exon of the gene NaNAC72 include: NaNAC72-sgRNA1, NaNAC72-sgRNA2; NaNAC72-sgRNA1 is shown in SEQ ID NO. 5; NaNAC72-sgRNA2 is shown as SEQ ID NO.

6.

4. The cloning method according to claim 3, characterized in that: After constructing the knockout vector, the expression vector containing the target gene was introduced into the hypocotyl of Nicotiana attenuata through Agrobacterium-mediated transformation. After tissue culture, antibiotic resistance screening and target site PCR detection, gene editing positive materials were obtained.

5. The method of claim 1 is used to identify tobacco mutants, characterized in that: The application is: using the genome of the transgenic plant as a template to perform PCR amplification, and then detecting by gel electrophoresis or sequencing to obtain a strain with a mutation in the NaNAC72 gene; The primer combination used in the PCR amplification is Detect-F and Detect-R; Detection-F is shown in SEQ ID NO.7; Detection-R is shown as SEQ ID NO.

8.

6. Application of the method according to claim 1, characterized in that: The NaNAC72 gene is used to increase nicotine content.

7. Application of the method according to claim 6, characterized in that: Extracting nicotine from Nicotiana attenuata comprises the following steps: (1) Samples of Nicotiana attenuata leaves were collected, weighed, and stored in liquid nitrogen; (2) Grind the sample thoroughly in liquid nitrogen until it becomes powdery; (3) Add methanol / acetic acid extract, shake and centrifuge, aspirate the supernatant, and set aside for measurement.

8. Application of the method according to claim 6, characterized in that: The nicotine content in tobacco was calculated based on the peak area using a high performance liquid chromatography coupled with a triple quadrupole mass spectrometer.

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