Application of NaARF5 gene in increasing nicotine content of tobacco leaves

Abstract

The invention discloses an application of a NaARF5 gene in improving nicotine content of tobacco leaves, which comprises the following steps: the NaARF5 gene is mutated after gene editing, and the nucleotide sequence of the NaARF5 gene is shown as SEQ ID NO. 1. The nicotine negative regulatory factor NaNaARF5 gene is obtained from tobacco with gradually narrow leaves, and by mutating the gene sequence, not only can the nicotine content be increased, but also an important regulatory factor for plant growth is not influenced.

Description

Technical Field

[0001] This application relates to the field of tobacco biotechnology, and in particular to a... NaARF5 Application of genes in increasing the nicotine content of tobacco leaves. Background Technology

[0002] Cultivated tobacco ( Nicotiana tabacum Tobacco is one of the world's most important economic crops. Nicotine, the core alkaloid component of tobacco, accounts for over 90% of the total alkaloids and is a key carrier of tobacco flavor. The commercial value of tobacco depends primarily on the content and quality of alkaloids in its leaves. Currently, it is relatively easy to create low-nicotine materials through bioengineering. However, the creation of high-nicotine materials remains a challenge.

[0003] Nicotine is the most important insect-resistant alkaloid in tobacco plants, and its biosynthesis involves several key enzymes, including PMT, QPT, BBL, and A622. Several transcription factors have been shown to positively regulate nicotine biosynthesis, such as members of the ethylene response factor (ERF) family (e.g., NtORC1, NtJAP1, NtERF189, NtERF199). Although overexpression of these regulatory factors or synthase genes can significantly increase the nicotine content in tobacco plants, there are "transgenic" barriers in breeding, and overexpression of these functional genes may affect plant growth and development.

[0004] Currently, our understanding of the negative regulatory mechanisms of nicotine synthesis is severely lacking, and known negative regulators are extremely scarce. Only reports suggest that the auxin-responsive factor NbARF1 in native tobacco and its ortholog NtARF6 in cultivated tobacco have negative regulatory functions on nicotine. Supporting evidence includes an increase in nicotine content after virus-mediated gene silencing of NbARF1, or a decrease in plants overexpressing NtARF6. However, there is currently no evidence to support the existence of mutant materials of the auxin-responsive factor NbARF1 and the NtARF6 gene that negatively regulate nicotine function.

[0005] There is an urgent need to identify the negative "molecular switch" that specifically regulates nicotine synthesis in tobacco or its closely related species, and to develop mutation technology based on this target to break through the current technical barriers to quality improvement.

[0006] Existing genes for increasing nicotine content are mainly concentrated in mutants, such as the NtJAZ1 gene mutant disclosed in CN202110917992.4, which increases the nicotine content of tobacco leaves. The NtJAZ1 gene mutant plant has an nicotine content in tobacco leaves that is about 40% higher than the control, but the growth of this mutant tobacco plant has not been studied.

[0007] The information disclosed in the background section is intended only to enhance the understanding of the general background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] This application provides a solution to the aforementioned technical problems. NaARF5 Application of genes in increasing nicotine content in tobacco leaves, nicotine negative regulators NaNaARF5 The gene was obtained from narrow-leaved tobacco. By mutating this gene, an important regulatory factor that can increase nicotine content without affecting plant growth can be obtained.

[0009] This application provides a NaARF5 The application of genes in increasing the nicotine content of tobacco leaves provides a basis for gene editing mutations. NaARF5 , NaARF5 The gene nucleotide sequence is shown in SEQ ID NO. 1; Under the same culture conditions, the resulting mutant tobacco plants have similar agronomic properties to wild-type narrow-leaved tobacco plants.

[0010] Another aspect of this application provides a method for increasing the nicotine content of tobacco leaves, comprising: gene editing mutation. NaARF5 , NaARF5 The gene nucleotide sequence is shown in SEQ ID NO. 1.

[0011] Preferably, mutation is performed using CRISPR / Cas9 technology. NaARF5 Gene.

[0012] Preferably, "mutation" NaARF5 "Genes" include the following steps: Build NaARF5 CRISPR-Cas9 gene editing: A dual-target gene editing vector system was constructed based on the pHSE401 plasmid expression vector, using the pHSE401-NaARF5 editing vector. The target sequences for editing are: NaARF5-sgRNA1 and NaARF5-sgRNA2; Na ARF5-sgRNA 1 is shown in SEQ ID NO. 3; Na ARF5-sgRNA 2 (as shown in SEQ ID NO. 4) was used to obtain the gene editing vector; The gene-editing vector was introduced into the hypocotyl of Nicotiana acuminate using Agrobacterium-mediated transformation, followed by tissue culture, antibiotic resistance screening, and target site PCR detection to obtain gene-editing positive materials as mutants.

[0013] Preferably, the tobacco plant is a wild-type narrow-leaved tobacco plant.

[0014] Another aspect of this application provides a mutant obtained by the method described above for increasing the nicotine content of tobacco leaves, a gene mutant for increasing the nicotine content of tobacco leaves. NaARF5 -1# Insert base A at position 142 of the wild-type narrow-leaved tobacco gene sequence; Gene mutants that increase nicotine content in tobacco leaves NaARF5 -2# is a deletion of bases 129-142 in the wild-type narrow-leaved tobacco gene sequence.

[0015] Preferably, gene mutants NaARF5 The nicotine content in the -1# tobacco leaves is 1.6 times that of wild-type narrow-leaf tobacco leaves.

[0016] Preferably, gene mutants NaARF5 The nicotine content in the -2# tobacco leaves is 2.5 times that of wild-type narrow-leaf tobacco leaves.

[0017] Preferably, under the same culture conditions, gene mutants NaARF5 The agronomic traits of the -1# tobacco plant are similar to those of wild-type narrow-leaf tobacco.

[0018] Preferably, under the same culture conditions, gene mutants NaARF5 The agronomic traits of the -2# tobacco plant are similar to those of wild-type narrow-leaf tobacco.

[0019] The beneficial effects that this application can produce include: 1) The method provided in this application for increasing the nicotine content of tobacco leaves NaARF5 Gene mutants, NaARF5 The amino acid sequence homology with the reported NtARF6 is only 40%, and its ortholog in cultivated tobacco is NtARF5. NaARF5 This provides target genes for upregulating nicotine content through gene editing or mutation, and provides important target genes for targeted improvement and breeding of high-quality new tobacco varieties, which has significant application value.

[0020] 2) The method provided in this application for increasing the nicotine content of tobacco leaves NaARF5 A gene mutant was cloned from Nicotiana acuminata, yielding an ARF transcription factor. NaARF5 By building NaARF5 A CRISPR-Cas9 gene-editing vector was used to transform narrow-leaved tobacco, resulting in homozygous individuals. NaARF5 Gene-edited plants; nicotinic content analysis revealed that they were homozygous. Figure 1The genetically edited plant shows higher nicotine content compared with the control, and its growth is not inhibited at all; the present application provides an important target gene for directional improvement and cultivation of new varieties of high-quality tobacco, lays a theoretical foundation for later molecular breeding work, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] NaARF5 The gene editing material obtained in Example 1 of the present application Figure 2 The editing site and mutation information detection result graph of the gene of the gene editing material.

[0022] NaARF5 The gene editing material obtained in Example 1 of the present application and NaARF5 -1# gene editing mutant, NaARF5 -2# gene editing mutant and wild type plant (WT) comparison graph; a is NaARF5 -1# gene editing mutant, NaARF5 -2# gene editing mutant and wild type leaf nicotine content determination result graph; b is NaARF5 -1# gene editing mutant, NaARF5 -2# gene editing mutant and wild type cultivation plant photos. DETAILED DESCRIPTION

[0023] The present application will be further described in detail below in combination with the drawings and examples, but in no way limits the present application, any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0024] The materials and instruments used in the following examples are obtained from commercial channels unless otherwise specified; the detection methods used are existing methods unless otherwise specified. The operations not detailed in the following examples of the present application are carried out according to the methods disclosed in CN202310679211.1.

[0025] Example 1: Cloning tobacco NaARF5 Gene Using homologous cloning method (the specific operation is the same as the existing method in the art, which is not repeated here), taking the cDNA of Nicotiana attenuata as a template, the nucleotide coding sequence of NaARF5 Gene nucleotide coding sequence, the nucleotide coding sequence is 2784 bp in length, as shown in SEQ ID NO. 1, encoding 353 amino acids, and the amino acid sequence encoded by it is as shown in SEQ ID NO. 2.

[0026] NaARF5 The nucleotide sequence of (SEQ ID NO. 1) is as follows: atgggttctg ttgaagaaaa gcacaagcca ggaagtttgg ttagtggagc acatactcta 60 cttgaggaaa tgaaattatt gaaagaaatg caggatcatt ctggaggaag gaagctaata 120 aattcagagc tgtggcatgc ttgtgctggc ccacttgtaa cattgcctca agttggaagc 180 cttgtgtatt acttcccaca ggggcacagt gaacaggtgg cagtttccac aaacagaaca 240 gcaacttccc agatacctaa ttatccaaat cttgcttctc agttgttgtg ccaagttcac 300 aatgttaccc tacatgcaga taaagaaaca gatgagatct atgcccaaat gagcctccaa 360 cctgtgaatt ctgagagaga tgtcttcccc attccagatt ttgggctgaa gcctagtaag 420 catccaactg agttcttttg caaaactttg actgctagtg atacgagcac acatggtgga 480 ttctcagttc ctagaagagc ggcagaaaag ctgtttccac ccttggatta ctccatgcaa 540 cctccaacac aagagcttgt tgtacgtgac ttgcacgata atacttggac attccgtcac 600 atataccgcg ggcagccaaa gcgacatctt ctcacaaccg gttggagcat gtttgttgga 660 gcaaaacgcc ttagagctgg ggattctgtt ctatttatca gagatgagaa gtcacagtta 720 atgttgggag tgaggcgtgc taaccgtcag cagacctcat tgccgtcttc agttctgtct 780 gctgatagta tgcatattgg agtacttgca gcagctgccc atgctgcagc taatagaagc 840 acattcacaa ttttctacaa tccgagggca tgcccttcag agtttgttat tcctttggcc 900 aaatatagaa aatctgttta tgggacacaa ctatcagttg gtatgaggtt tggaatgatg 960 tttgaaacag aggaatcggg taaacgcaga tatatgggca ctattgttgg cattagtgac 1020 ttggatccac tgagatggcc tggttccaag tggcggtgtc ttcaggcaga gtgggacgaa 1080 ccgggatgtg gagacaagca gagtagagtt agtccatggg aagttgagac accagaaagt 1140 cttttcatat ttccttctct gactgcaggc ctcaaacgac cttaccagtc ttcctttttg 1200 ggagcacaaa ctgagtggga cagtttgatg caccgccctt ttatgcgtgt tcctgaaaat 1260 gtatatggtg aacttcaaag ttcctcaatc tcaaacctat ggtcagaaca gttaatgaag 1320 atgctggcaa gacctcctgg tgttacaggt cttcaatgcg gtgtccccac agtacaagat 1380 attaaggttg cactacctca agaatctagg aatctaatac atgcagcagg caaccagaaa 1440 CCTGAGCTAA GTACC GTA GA AG AT AC AT CT GT AC AAA AT G AAAGAA AT CCT C AAGT CAT C 1500 CTCAATCAAC CTTGTGTTGT GAAC T CAATT CTTC ACC GC AAGC AACT CT GC AGCC AAAG 1560 TCTCAACCTC CAGAAAAAGT AGGCTCCGAC ACTGTAGGAA TAAGTTCTGAGCCTGCAA AA 1620 GAAACTAGTGT TTC AGTGGAT AG GTTAG AT C AGTGT C AGT CCCT AGGGC AGT GT AAT G AA 1680 GAAAAAGTGA CT AT AAAG CCT GCAAGT CC AC AT AT CTTC CGGCTGAT GC AAC AGCTTCC 1740 CACCAAAAC AGCTT AT CT CA AT TG CAAT CC AGT CC AT GGC GT C AAAT TG AGGCTGCT AGT 1800 GCCAAT AAT C CTTC AAT G CTCC AAT AAC AT G AGT GG AAT C TGCCCTC TTTC AGT CT 1860 ACTGCTGGAT TGCTC AGATC TCCTGTGTCG AACTCCACTT TGACGAAGCACGAGC ATTC T 1920 TTTATGTTGC CTGATTCAGT TATTGGT CCT GGACT AGCTC CAAT AGG C C AGGT TTGT GG 1980 GATC AT C AGTT G AAT G AT GT G AAGT G CTTTTCCCAAT C AAACCTTC AGGTT CCGCT GTTA 2040 ACGCAAGAC AT AACT AAC AT GC AGT CTTC CCTGATTC AT ATGGTTTAA AGACTTGTCA 2100 GAGGAGAGCC AT AACCAAAG CG AC AT AT AT AGTTGTCTT AATTTTGAT AGT AAC AGT GGG 2160 agcacagtga ttgaaattc tgtttcaagc acagtgttag atgagttctg cactttaaaa 2220 cataccgatt tccagagtcc ttcagattt ctattaggca acttcagttc tagtcaagac 2280 gttcagtctc agattacatc agctagtctc gcagattctc agaatttctc tgtacaagat 2340 tttgccgaca actcaggtgg tgcatcgtca agcaatgtaa atttcgatga aagtaatctt 2400 ttgcagaata gctcatggca gcaagtagct ccacgtgtgc gaacatatac aaagattcaa 2460 aaggctggat ctgtaggaag gtcaattgat gtctccagtt ttaaaaccta cgatgaactg 2520 cgatctgaaa ttgaaagaat gtttggactt gaggggctgc tgaatgacac gagaggctcg 2580 ggatggaagc tggtatacgt tgattatgag aatgatgtac tccttgtcgg ggatgatcct 2640 tgggaggaat ttgttggttg tgttcgatgc atcagaatct tgtcgcctac agaagtgcag 2700 caaatgggag aagaaggaat gcagctcttg aactcggccg gactgcaggg cataaatggc 2760

[0027] NaARF5 (SEQ ID NO .2) is the following: MGSVEEKHKP GSLVSGAHTL LEEMKLLKEM QDHSGGRKLI NSELWHACAG PLVTLPQVGS 60 LVYYFPQGHS EQVAVSTNRT ATSQIPNYPN LASQLLCQVH NVTLHADKET DEIYAQMSLQ 120 PVNSERDVFP IPDFGLKPSK HPTEFFCKTL TASDTSTHGG FSVPRRAAEK LFPPLDYSMQ 180 PPTQELVVRD LHDNTWTFRH IYRGQPKRHL LTTGWSMFVG AKRLRAGDSV LFIRDEKSQL 240 MLGVRRANRQ QTSLPSSVLS ADSMHIGVLA AAAHAAANRS TFTIFYNPRA CPSEFVIPLA 300 KYRKSVYGTQ LSVGMRFGMM FETEESGKRR YMGTIVGISD LDPLRWPGSK WRCLQAEWDE 360 PGCGDKQSRV SPWEVETPES LFIFPSLTAG LKRPYQSSFL GAQTEWDSLM HRPFMRVPEN 420 VYGELQSSSI SNLWSEQLMK MLARPPGVTG LQCGVPTVQD IKVALPQESR NLIHAAGNQK 480 PELSTVEDTS VQNERNPQVI LNQPCVVNSI SSPQATLQPK SQPPEKVGSD TVGISSEPAK 540 ETSVSVDRLD QCQSLGQCNE EKVTIKPASP HILPADATAS HQNSLSQLQS SPWRQIEAAS 600 ANNILQCSNN NEWNLPSFQS TAGLLRSPVS NSTLTKHEHS FMLPDSVIGP GLAPIGQDLW 660 DHQLNDVKCF SQSNLQVPLL TQDITNMQFL PDSYGFKDLS EESHNQSDIY SCLNFDSNSG 720 STVIDNSVSS TVLDEFCTLK HTDFQSPSDF LLGNFSSSQD VQSQITSASL ADSQNFSVQD 780 FADNSGGASS SNVNFDESNL LQNSSWQQVA PRVRTYTKIQ KAGSVGRSID VSSFKTYDEL 840 RSEIERMFGL EGLLNDTRGS GWKLVYVDYE NDVLLVGDDP WEEFVGCVRC IRILSPTEVQ 900 QMGEEGMQLL NSAGLQGING STSEFPN 927. Example 2 Obtain NaARF5 CRISPR-Cas9 gene editing mutant material 1, based on the expression vector pHSE401, a double-target gene editing vector system was constructed. The editing vector constructed is pHSE401- NaARF5 .

[0028] For the gene NaARF5 The sgRNA target sequence of the first exon includes: NaARF5 -sgRNA1 and NaARF5 -sgRNA2, the nucleotide sequences are as follows: NaARF5 -sgRNA 1 (SEQ ID NO. 3): AAGCACAAGCCAGGAAGTTTGG; NaARF5 -sgRNA 2 (SEQ ID NO. 4): CTTGTGTATTACTTCCCACAGG.

[0029] Then the gene editing vector was introduced into the hypocotyl of Nicotiana attenuata by Agrobacterium-mediated transformation method, and the gene editing positive material was obtained through tissue culture, hygromycin resistance screening and target site PCR detection.

[0030] 2, NaARF5 Detection of nicotine content changes in gene editing material Using CRISPR / Cas9 technology to silence NaARF5 , two gene editing mutants were obtained, 1# and 2#. NaARF5 -1# and NaARF5 -2#. NaARF5 -1# editing type is 1 base insertion at target 1 position; Figure 1 -2# 13 base deletion at target 1 position NaARF5 ). NaARF5 -1# gene editing mutant inserts base A at position 142 of the wild type Nicotiana attenuata gene sequence. NaARF5The -2# gene edited mutant has a deletion of the wild type N. sylvestris gene sequence from base 129 to 142. Both mutations result in a frame shift change and premature translation termination.

[0031] Example 3: Nicotine content determination of mutant and wild type materials The wild type N. sylvestris material (WT) and NaARF5 Gene editing NaARF5 the -1# mutant, Figure 2 the -2# mutant Figure 2 a. 1. Sample preparation: (1) Sample tobacco leaves were taken, weighed, and stored in liquid nitrogen.

[0032] (2) The sample was ground into powder in liquid nitrogen. 1 mL of extraction solution containing 40% methanol and 0.1% acetic acid was added, shaken for 10 min, and then centrifuged at 12000 rpm for 20 min. The supernatant was collected and stored at 4°C for determination.

[0033] 2. Determination of nicotine content in N. sylvestris: A Shimadzu ultra-high performance liquid chromatograph LC-20A was used in combination with a triple quadrupole mass spectrometer LCMS-8040. The chromatographic column was Shimadzu Shim-pack XR-ODS III 2.0 mm I.D. x 75 mm L., 1.6 μm. The mobile phase A was 0.05% formic acid, and the mobile phase B was 0.05% acetonitrile. The flow rate was 0.3 ml / min. The nicotine content in tobacco was calculated according to the peak area.

[0034] 3. The results are shown in NaARF5 a. NaARF5 After gene editing, NaARF5 the nicotine content of the -1# gene edited mutant was increased by 1.6 times compared with the wild type control; NaARF5 the nicotine content of the -2# gene edited mutant was increased by 2.5 times compared with the wild type control.

[0035] Example 4: Effect of mutation on plant growth and development Under greenhouse conditions at 25-30°C, wild type N. sylvestris plants were cultivated according to the cultivation method (the specific cultivation method is not repeated here). Meanwhile, NaARF5 the -1# gene edited mutant tobacco plant, Figure 2 the -2# gene edited mutant and the wild type N. sylvestris plant were cultivated according to the cultivation method (the specific cultivation method is not repeated here). The obtained tobacco plants are shown in NaARF5 b. NaARF5 the -1# gene edited mutant tobacco plant,​ - The growth of the 2# gene edited mutant was not significantly different from the wild type plant that was not edited.

[0036] Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified, or some of the technical features can be replaced by equivalent features, by those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A kind NaARF5 The application of genes in increasing the nicotine content of tobacco leaves is characterized by, Gene editing mutation NaARF5 Gene, NaARF5 The gene nucleotide sequence is shown in SEQ ID NO. 1; Under the same culture conditions, the resulting mutant tobacco plants have similar agronomic properties to wild-type narrow-leaved tobacco plants.

2. A method for increasing the nicotine content of tobacco leaves, characterized in that, include: Gene editing mutation NaARF5 , NaARF5 The gene nucleotide sequence is shown in SEQ ID NO.

1.

3. The method according to claim 2, characterized in that, Mutated using CRISPR / Cas9 technology NaARF5 Gene.

4. The method according to claim 2, characterized in that, "mutation NaARF5 "Genes" include the following steps: Build NaARF5 CRISPR-Cas9 gene editing: A dual-target gene editing vector system was constructed based on the pHSE401 plasmid expression vector, using the pHSE401-NaARF5 editing vector. The target sequences for editing are: NaARF5-sgRNA1 and NaARF5-sgRNA2; Na ARF5-sgRNA 1 is shown in SEQ ID NO. 3; Na ARF5-sgRNA 2 (as shown in SEQ ID NO. 4) was used to obtain the gene editing vector; The gene-editing vector was introduced into the hypocotyl of Nicotiana acuminate using Agrobacterium-mediated transformation, followed by tissue culture, antibiotic resistance screening, and target site PCR detection to obtain gene-editing positive materials as mutants.

5. The method according to claim 2, characterized in that, The tobacco plant in question is a wild-type narrow-leaved tobacco plant.

6. A mutant obtained by the method for increasing the nicotine content of tobacco leaves as described in any one of claims 2 to 5, characterized in that, Gene mutants that increase nicotine content in tobacco leaves NaARF5 -1# Insert base A at position 142 of the wild-type narrow-leaved tobacco gene sequence; Gene mutants that increase nicotine content in tobacco leaves NaARF5 -2# is a deletion of bases 129-142 in the wild-type narrow-leaved tobacco gene sequence.

7. The mutant according to claim 6, characterized in that, Gene mutants NaARF5 The nicotine content in the -1# tobacco leaves is 1.6 times that of wild-type narrow-leaf tobacco leaves.

8. The mutant according to claim 6, characterized in that, Gene mutants NaARF5 The nicotine content in the -2# tobacco leaves is 2.5 times that of wild-type narrow-leaf tobacco leaves.

9. The mutant according to claim 6, characterized in that, Under the same culture conditions, gene mutants NaARF5 The agronomic traits of the -1# tobacco plant are similar to those of wild-type narrow-leaf tobacco.

10. The mutant according to claim 6, characterized in that, Under the same culture conditions, gene mutants NaARF5 The agronomic traits of the -2# tobacco plant are similar to those of wild-type narrow-leaf tobacco.

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