CN112143738B - Tobacco receptor protein gene and cloning method and application thereof - Google Patents
Tobacco receptor protein gene and cloning method and application thereof Download PDFInfo
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Abstract
The invention discloses a tobacco receptor protein gene and a cloning method and application thereof. The tobacco receptor protein gene comprisesNtCOI1‑1AndNtCOI1‑2the nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO. 2. The cloning procedure involved tobacco root cDNA synthesis: extracting total RNA of tobacco roots, and performing reverse transcription to obtain first-strand cDNA;NtCOI1‑1、2PCR amplification of the genes: using tobacco root cDNA as templateNtCOI1‑1、2Designing primers according to the gene sequence, carrying out PCR amplification, recovering and purifying PCR amplification products, and sequencing. The application is that the tobacco receptor protein geneNtCOI1‑1、2The application in the transgenic tobacco plant for regulating and controlling the nicotine content of tobacco. Cloning using CRISPR/CAS9 technology of the present inventionNtCOI1‑1、2The gene is functionally identified. TobaccoNtCOI1‑1、2The cloning of the gene is identified to provide a new target gene for regulating the nicotine content of the tobacco.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a tobacco receptor protein gene as well as a cloning method and application thereof.
Background
Nicotine is a characteristic compound in cultivated tobacco, and deeply influences the quality of cigarettes. The synthesis of nicotine is regulated by plant hormones such as auxin, ethylene and jasmonic acid. The research of regulating nicotine synthesis pathway by jasmonic acid is clear. In the absence of jasmonic acid, the JAZ protein binds to the MYC2 protein thereby inhibiting the MYC2 protein from activating the function of the nicotine synthesis pathway gene. In the presence of jasmonic acid, jasmonic acid activates the degradation of JAZ protein (through 26S protease pathway) by combining with receptor protein COI1, and the degradation of JAZ protein releases MYC2, so that genes related to nicotine synthesis pathway are activated, and the nicotine content of tobacco leaves is increased.
The regulation and control of nicotine content by gene means have been studied more, for example, the nicotine content of tobacco leaves can be obviously improved by over-expressing transcription factor genes, such as ERF, MYC2 and the like; the nicotine synthesis pathway gene is reduced through an RNA interference technology, such as QPT and the like, so that the nicotine content of tobacco leaves can be obviously reduced. In recent years, genome editing technology is also applied to regulate and control nicotine content, for example, BBL family genes are knocked out to obtain tobacco with extremely low nicotine content.
With the rapid development of novel tobacco products, the market has increasingly increased specialization requirements on tobacco leaves with different nicotine contents, including low nicotine content tobacco.
Disclosure of Invention
The first purpose of the invention is to provide a tobacco receptor protein gene; the second purpose is to provide a cloning method of the tobacco receptor protein gene; the third purpose is to provide the application of the tobacco receptor protein gene.
The first purpose of the invention is realized by that the tobacco receptor protein gene comprises NtCOI1-1 and NtCOI1-2, and the nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO. 2.
The second object of the present invention is achieved by comprising the steps of:
A. extracting RNA of tobacco root tissue, and performing reverse transcription to obtain first-chain cDNA;
B. using cDNA as a template, designing primers according to the sequence of a tobacco receptor protein gene NtCOI1-1 and a tobacco receptor protein gene NtCOI1-2, carrying out PCR amplification, and recovering and purifying PCR amplification products;
C. linking the purified amplification product with a vector, and specifically carrying out the following processes: 4 μ L of purified product, 1 μ L of salt solution, 1 μ L
-Blunt II-TOPO (Invitrogen) and water bath at 25 deg.C for 30min; and transforming the connected vector into escherichia coli DH5 alpha through heat shock, adding a liquid culture medium, performing shake culture, coating the obtained product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting a bacterial colony, performing bacterial liquid culture, performing plasmid extraction and PCR (polymerase chain reaction) detection, screening positive clones, and sequencing the positive clones.
The third purpose of the invention is realized by the application of the tobacco receptor protein gene in obtaining a transgenic tobacco plant for regulating and controlling the nicotine content of tobacco.
The invention also provides a method for carrying out function identification on the NtCOI1-1 and NtCOI 2 genes of tobacco by using the CRSIPR/CAS9 editing technology, which specifically comprises the following steps:
(1) Construction of CRISPR/CAS9 vectors
A. The CRISPR/CAS9 target site (PAM), namely GAATTGGTTGCGATGAATCGGGG, is designed according to NtCOI1-1 and NtCOI 2 genome sequences.
B. Target site primer design
Synthesizing a target site primer according to the target site designed by A:
P1：5’-ATTGGAATTGGTTGCGATGAATCG-3’，
P2：5’-AAACCGATTCATCGCAACCAATTC-3’；
C. designing detection primers of the editing material at two sides of the target site,
NtCOI1-1-SdF:5’-AGCAACACTACTCTGCCACT-3’，
NtCOI1-1-SdR 5'-TACTATAGCTCAGTAGCAAGC-3', and the amplification length is 819bp;
NtCOI1-2-SdF:5’-ACTAACATCCGCTGTTACGC-3’，
NtCOI1-2-SdR 5'-TACAGCTCACTAGTAAGTTGC-3', the amplification length is 1046bp.
D. Preparation of dsDNA
And (C) annealing the primer designed and synthesized in the step (B) to form a complementary DNA oligo, which comprises the following specific steps:
the reaction system is 50 μ L, including P1 20 μ L, P2 20 μ L,10 × analizing buffer 5 μ L, and sterilized double distilled water 5 μ L. The annealing procedure is as follows: 95 ℃ for 5min; at 90 ℃ for 1min; at 80 ℃ for 1min; 1min at 70 ℃;60 ℃ for 1min;50 ℃ for 1min; at 40 ℃ for 1min; at 30 ℃ for 1min; at 20 deg.C for 1min;10 ℃ for 1min.
E. The pHSE401 vector was cleaved and ligated with the dsDNA prepared in step D.
The pHSE401 vector is cut by Bsa I enzyme, and the cutting system is 50 mu L, and comprises the following components: plasmid 5. Mu.L, 10 XBuffer 5. Mu.L, bsa I2. Mu.L, sterile double distilled water 38. Mu.L. The enzyme was cleaved at 37 ℃ for 1h.
After enzyme digestion, carrying out electrophoresis detection analysis on the enzyme digestion product, wherein two bands of 1200bp and 11520bp can be seen, and recycling the enzyme digestion product of 11520bp for later use;
and D, utilizing T4 DNA ligase to connect the recovered large fragment enzyme digestion product with the dsDNA prepared in the step D, wherein a connection system is 20 mu L: 3 mu L of recovered vector enzyme digestion product, 10 mu L of annealed dsDNA product, 2 mu L of T4 DNA buffer, 1 mu L of T4 DNA ligase and 4 mu L of sterilized double distilled water. Ligation was performed overnight at 16 ℃;
F. sequencing verification
Transforming the ligation product in the step E into Escherichia coli, screening positive clones (resistance of the pHSE401 vector is kan) and carrying out colony PCR detection,
when colony PCR detection is carried out, the primers are designed as follows:
U6-26p F：5’-TGTCCCAGGATTAGAATGATTAGGC-3’；
P2：5’-AAACCGATTCATCGCAACCAATTC-3’；
and (3) carrying out sequencing analysis on the cultured and amplified positive clone strains which are correctly detected and verified by colony PCR, wherein primers used in sequencing are as follows:
U6-26p-F:5’-TGTCCCAGGATTAGAATGATTAGGC-3’。
the sequencing results were analyzed and the correct clone (pHSE 401-COI 1/2) was selected for storage.
(2) Agrobacterium transformation
Taking out Agrobacterium tumefaciens competent cells (C58C 1) from a-80 ℃ refrigerator, placing on ice for dissolving, and adding a vector pHSE401-COI1/2 mu L; quickly freezing for 1 minute by using liquid nitrogen, transferring into a water bath at 37 ℃ for 5 minutes, then performing ice bath for 2 minutes, adding 1mL of LB liquid culture medium into the mixture, and culturing for 3-4 hours at 28 ℃ and 220 rpm; the culture was spread on LB solid medium containing 100mg/L kanamycin and 25mg/L rifampicin, and inverted cultured at 28 ℃ for 2-3 days, whereby Agrobacterium clones containing the target vector were observed.
(3) Tobacco transformation
A. Selecting agrobacterium clone containing target vector, streaking on LB plate containing kanamycin and rifampicin, and culturing at 28 deg.c for 2-3 days; scraping streak plaque and inoculating bacteria into an LB culture medium containing kanamycin and rifampicin, carrying out shake culture at 28 ℃ and 220rpm, and infecting when the concentration of the bacteria liquid reaches OD = 0.5-0.8;
B. putting the tobacco leaves into a 500mL wide-mouth bottle, adding a proper amount of 75% ethanol, and rinsing for 1min; removing the ethanol, adding 0.1 percent of HgCl2 solution, and placing the mixture on a shaking table to oscillate for 15 to 30 minutes at room temperature; discarding the solution, and washing with sterile water for 6 times;
C. taking out the leaves, washing off surface liquid by using sterile absorbent paper, cutting the sterile leaves into small pieces of 1cm multiplied by 1cm by using scissors, putting the cut tobacco leaves into sterile LB liquid culture medium suspension bacteria liquid containing a target carrier, and standing for 15-20 min; taking out tobacco leaves, removing redundant bacteria liquid with sterile filter paper, and performing dark culture in MS culture medium containing 6-BA (0.02 mg/L) and NAA (2 mg/L) at 25 deg.C for two days; transferring tobacco leaves into a differentiation culture medium, contacting the cut with the culture medium, wherein the differentiation culture medium is an MS culture medium containing 6-BA (0.5 mg/L), NAA (0.1 mg/L), hygromycin (20 mg/L) and cefamycin (500 mg/L), subculturing once every 2-3 weeks, gradually forming callus at the cut, and finally differentiating to bud;
D. cutting off buds growing to 3-5 cm, transferring the buds into an MS culture medium to induce rooting, taking out the transgenic plants after rooting from the rooting culture medium, washing the culture medium with tap water, and transplanting the transgenic plants into sterilized nutrient soil.
(4) Sequencing screening editing material
And (2) growing the T0 generation transgenic seedlings for about 1 week, selecting 20 tobacco seedlings, taking leaves, extracting DNA by using DNeasy Plant Mini Kit (QIAGEN), amplifying by using the primer SdF/SdR designed in the step (1) C, purifying the amplified product, and sequencing by using a forward primer. Analyzing the sequencing result, an editing material (figure 3) that NtCOI1-1 gene lacks 1 base A and TCGGG in the NtCOI1-2 gene is converted into 1C is obtained. Planting the editing material T1 generation plant, screening double-gene homozygous mutant single plant by sequencing and harvesting to obtain T2 generation seeds.
(5) Editing material nicotine content
Homozygous T2 generation material ntcoi1-1/2 and controls were edited by greenhouse pot planting. The application of the tobacco composite fertilizer (N: P: nicotine = 10: 15) is carried out for 5g per plant by using pure nitrogen for 5 g. And in the bud blooming period, the whole tobacco leaf is taken to be de-enzymed and then the nicotine content is detected. The results are shown in fig. 4, the nicotine content of the edited material is obviously reduced compared with that of the control, and the NtCOI1-1 and NtCOI 2 genes are knocked out to influence the accumulation of nicotine in tobacco leaves.
According to the invention, a tobacco receptor protein gene NtCOI1-1 and NtCOI 2 is obtained from tobacco by using a homologous cloning technology, and the function verification is carried out on the NtCOI1-1 and NtCOI 2 by using a CRISPR/CAS9 editing technology, and the result shows that the NtCOI1-1 and NtCOI 2 genes have the function of regulating the accumulation of nicotine in tobacco leaves, so that a target gene is provided for regulating the nicotine content of the tobacco.
Drawings
FIG. 1 is an agarose gel electrophoresis of the CDS products of NtCOI1-1 and NtCOI 2 genes amplified by primer pairs NtCOI1-1, 2F/NtCOI1-1 and 2R, the amplification products being about 2000bp in size: 1kb DNA ladder,1: ntCOI1-1,2: ntCOI1-2 CDS PCR product;
FIG. 2 shows the result of tissue-specific expression analysis of NtCOI1-1 and NtCOI 2 genes;
FIG. 3 shows the sequencing result of the CRISPR/CAS9 editing NtCOI1-1 and NtCOI 2 gene material editing target sites, wherein NtCOI1-1 and NtCOI1-2 are wild type sequences, and NtCOI1-1 and NtCOI1-2 are editing material sequences;
FIG. 4 shows comparison of nicotine content in NtCOI1-1 and NtCOI 2 gene editing material and control tobacco leaves. ntcoi1-1/2 is editing material and WT is control.
Detailed Description
The present invention is further described with reference to the following examples and drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The tobacco receptor protein gene comprises NtCOI1-1 and NtCOI1-2, and the nucleotide sequences are shown in SEQ ID No.1 and SEQ ID No. 2.
The amino acid sequences coded by the tobacco receptor protein gene NtCOI1-1 and the tobacco receptor protein gene NtCOI1-2 are shown as SEQ ID No.3 and SEQ ID No. 4.
The cloning method of the tobacco receptor protein gene comprises the following steps:
A. extracting RNA of tobacco root tissue, and performing reverse transcription to obtain first-chain cDNA;
B. using cDNA as a template, designing primers according to the sequence of a tobacco receptor protein gene NtCOI1-1 and a tobacco receptor protein gene NtCOI1-2, carrying out PCR amplification, and recovering and purifying PCR amplification products;
C. linking the purified amplification product with a vector, and specifically carrying out the following processes: 4 μ L of purified product, 1 μ L of salt solution, 1 μ L
-Blunt II-TOPO (Invitrogen) and water bath at 25 deg.C for 30min; the connected vector is transformed into Escherichia coli DH5 alpha by heat shock, added with liquid culture medium for shaking culture and coated to contain 100mg/L kanamycinOvernight culturing on an LB plate, selecting colonies for bacterial liquid culture, extracting plasmids, detecting PCR, screening positive clones, and sequencing the positive clones.
The primer in the step B is as follows:
NtCOI1-1F：5’-ATGGAGGAGCGTAGCTCCACTC-3’
NtCOI1-1R：5’-CTATTCAGCGAGAAGGAAATTTGG-3’；
NtCOI1-2F：5’-ATGGAGGAGCGTAGCTCCACG-3’
NtCOI1-2R：5’-CTATTCAGCGAGAAGGGAATTTG-3’。
the PCR reaction system in the step B is as follows: total volume 50 μ L, including: 200ng cDNA,5 XPPhusion HF reaction buffer 10. Mu.L, 1. Mu.L of 10mM dNTP, 2U
High-Fidelity DNA Polymerase, 1. Mu.L each of 10. Mu.M forward and reverse primers, and water was added to 50. Mu.L.
The reaction procedure of PCR amplification in step B is as follows: 30 seconds at 98 ℃;7 seconds at 98 ℃;30 seconds at 66 ℃;60 seconds at 72 ℃;35 cycles; extension at 72 ℃ for 7 minutes.
The application of the tobacco receptor protein gene is the application of the tobacco receptor protein gene in obtaining a transgenic tobacco plant for regulating and controlling the nicotine content of tobacco.
The invention is further illustrated by the following specific examples:
example 1
Cloning of tobacco nicotine synthesis regulatory gene NtCOI1-1,2
A. Searching an NCBI database according to a partial nucleotide sequence (AB 433899) of the tobacco NtCOI1 gene to obtain NtCOI1-1 and NtCOI 2 sequences, and designing a gene cloning primer by utilizing the sequences:
NtCOI1-1F：5’-ATGGAGGAGCGTAGCTCCACTC-3’
NtCOI1-1R：5’-CTATTCAGCGAGAAGGAAATTTGG-3’。
NtCOI1-2F：5’-ATGGAGGAGCGTAGCTCCACG-3’
NtCOI1-2R：5’-CTATTCAGCGAGAAGGGAATTTG-3’。
B. extracting RNA of tobacco root tissues, and performing reverse transcription to obtain first-strand cDNA;
C. performing PCR amplification by using a primer NtCOI1-1F/R, ntCOI1-2F/R and taking the first strand cDNA obtained by reverse transcription as a template, and recovering and purifying a PCR product after agarose gel electrophoresis separation (figure 1);
D. the purified product is connected with a carrier, and the connection system and the process are as follows: 4 μ L of purified product, 1 μ L of salt solution, 1 μ L
-Blunt II-TOPO (Invitrogen) and water bath at 25 deg.C for 30min; transforming the connected vector into escherichia coli DH5 alpha by heat shock, adding a liquid culture medium for shake culture, coating the cultured product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting bacterial colonies for bacterial liquid culture, extracting plasmids and detecting PCR (polymerase chain reaction), screening positive clones, and sequencing the positive clones.
And C, selecting a Phusion high-fidelity amplification enzyme reaction system as a PCR amplification reaction system in the step C, wherein the total volume of the system is 50 mu L, and the method comprises the following steps: 200ng cDNA,5 XPPhusion HF reaction buffer 10. Mu.L, 1. Mu.L of 10mM dNTP, 2U
High-Fidelity DNA Polymerase, 1. Mu.L each of 10. Mu.M forward and reverse primers, and water was added to 50. Mu.L.
The reaction conditions for PCR amplification in step C are
The pro amplification instrument is used for carrying out the following reaction procedures: 30 seconds at 98 ℃;7 seconds at 98 ℃; at 66 ℃;30 seconds, 72 ℃;60 seconds, 35 cycles; extension at 72 ℃ for 7 min;
example 2
Tobacco NtCOI1-1,2 gene tissue specific expression analysis
A. Planting and cultivating the tobacco Yunyan 87, extracting root, stem, leaf and flower RNA in flowering, and carrying out reverse transcription to obtain first-strand cDNA.
B. qRT-PCR primer designed according to NtCOI1-1 and NtCOI 2 gene sequences
NtCOI1-1QF：AAGGTGCAGTTACGCACAGA，
NtCOI1-1QR：TCTCTCCTCTCGGTCAAGCA。
NtCOI1-2QF：AGCGGTGAACAACCCAGTTC，
NtCOI1-2QR：TGAGCCACCACCAAACTCCT。
Taking tobacco Actin gene as an internal reference, wherein the expression of Actin-F: CTGAGGTCCTTTTCCAACCA and Actin-R: TACCCGGGAACATGGTAGAG. The fluorescent quantitative PCR was carried out using the root, stem, leaf and flower cDNAs as templates, and the reaction was carried out on a Roche LightCycler 480 with a LightCycler 480SYBR Green I master, and the 20. Mu.L system contained 10. Mu.L of LightCycler 480SYBR Green I master (2X), 1. Mu.L (10. Mu. Mol/L) of each of the forward and reverse primers, 1. Mu.L of cDNA (diluted 4 times with the reverse transcription product), and 7. Mu.L of sterile distilled water. The reaction procedure was as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 20s,58 ℃ (NtCOI 1-1) or 60 ℃ (NtCOI 1-2) annealing for 15s, and extension at 72 ℃ for 15s;40 cycles. Fluorescent quantitative PCR results
The method calculates the relative expression quantity of the NtCOI1-1 and NtCOI 2 genes. Each treatment was set to 3 biological replicates and histograms were drawn by Excel software (figure 2). The results showed that NtCOI1-1 and NtCOI 2 were expressed in various organs of tobacco.
Example 3
Functional identification of tobacco NtCOI1-1 and 2 genes by using CRSIPR/CAS9 editing technology
(1) Construction of CRISPR/CAS9 vectors
A. The CRISPR/CAS9 target site (PAM), namely GAATTGGTTGCGATGAATCGGGG, is designed according to NtCOI1-1 and NtCOI 2 genome sequences.
B. Target site primer design
Synthesizing a target site primer according to the target site designed by A:
P1：5’-ATTGGAATTGGTTGCGATGAATCG-3’，
P2：5’-AAACCGATTCATCGCAACCAATTC-3’；
C. designing detection primers of the editing material at two sides of the target site,
NtCOI1-1-SdF:5’-AGCAACACTACTCTGCCACT-3’，
NtCOI1-1-SdR 5'-TACTATAGCTCAGTAGCAAGC-3', and the amplification length is 819bp;
NtCOI1-2-SdF:5’-ACTAACATCCGCTGTTACGC-3’，
NtCOI1-2-SdR 5'-TACAGCTCACTAGTAAGTTGC-3', the amplification length is 1046bp.
D. Preparation of dsDNA
And B, annealing the primer designed and synthesized in the step B to form a complementary DNA oligo, which comprises the following specific steps:
the reaction system is 50 μ L, including P1 20 μ L, P2 20 μ L,10 × analizing buffer 5 μ L, and sterilized double distilled water 5 μ L. The annealing procedure is as follows: 95 ℃ for 5min; at 90 ℃ for 1min; at 80 ℃ for 1min; 1min at 70 ℃; at 60 ℃ for 1min;50 ℃ for 1min; at 40 ℃ for 1min; at 30 ℃ for 1min; at 20 ℃ for 1min;10 ℃ for 1min.
E. The pHSE401 vector was cleaved and ligated with the dsDNA prepared in step D.
The pHSE401 vector is cut by Bsa I enzyme, and the cutting system is 50 mu L, and comprises the following components: plasmid 5. Mu.L, 10 XBuffer 5. Mu.L, bsa I2. Mu.L, sterile double distilled water 38. Mu.L. The enzyme was cleaved at 37 ℃ for 1h.
Carrying out electrophoresis detection analysis on the enzyme digestion product after enzyme digestion to obtain two bands of 1200bp and 11520bp, and recovering the enzyme digestion product of 11520bp for later use;
and D, utilizing T4 DNA ligase to connect the recovered large fragment enzyme digestion product with the dsDNA prepared in the step D, wherein the connection system is 20 mu L: 3 mu L of recovered vector enzyme digestion product, 10 mu L of dsDNA product formed by annealing, 2 mu L of T4 DNA buffer, 1 mu L of T4 DNA ligase and 4 mu L of sterilized double distilled water. Ligation was performed overnight at 16 ℃;
F. sequencing validation
E, transforming the ligation product in the step E into escherichia coli, screening positive clones (the resistance of the pHSE401 vector is kan) and carrying out colony PCR detection,
when colony PCR detection is carried out, the primers are as follows:
U6-26p F：5’-TGTCCCAGGATTAGAATGATTAGGC-3’；
P2：5’-AAACCGATTCATCGCAACCAATTC-3’
and (3) carrying out sequencing analysis on the cultured and amplified positive clone strains which are correctly detected and verified by colony PCR, wherein primers used in sequencing are as follows:
U6-26p-F:5’-TGTCCCAGGATTAGAATGATTAGGC-3’。
the sequencing results were analyzed and the correct clone (pHSE 401-COI 1/2) was selected for storage.
(2) Agrobacterium transformation
Taking out Agrobacterium-infected competent cells (C58C 1) from-80 deg.C refrigerator, placing on ice for dissolving, and adding vector pHSE401-COI1/2 μ L; quickly freezing for 1 minute by using liquid nitrogen, transferring into a water bath at 37 ℃ for 5 minutes, then performing ice bath for 2 minutes, adding 1mL of LB liquid culture medium into the mixture, and culturing for 3-4 hours at 28 ℃ and 220 rpm; the culture was spread on LB solid medium containing 100mg/L kanamycin and 25mg/L rifampicin, and inverted cultured at 28 ℃ for 2 to 3 days, whereby Agrobacterium clones containing the target vector were observed.
(3) Tobacco transformation
A. Selecting agrobacterium clone containing a target vector, streaking on an LB (Langmycin) plate containing natamycin and rifampicin, and culturing for 2-3 days at 28 ℃; scraping streak plaque and inoculating bacteria into an LB culture medium containing natamycin and rifampicin, carrying out shake culture at 28 ℃ and 220rpm, and infecting when the concentration of the bacteria liquid reaches OD = 0.5-0.8;
B. putting the tobacco leaves into a 500mL wide-mouth bottle, adding a proper amount of 75% ethanol, and rinsing for 1min; removing the ethanol, adding 0.1 percent of HgCl2 solution, and placing the mixture on a shaking table to oscillate for 15 to 30 minutes at room temperature; discarding the solution, and washing with sterile water for 6 times;
C. taking out the leaves, washing off surface liquid by using sterile absorbent paper, cutting the sterile leaves into small pieces of 1cm multiplied by 1cm by using scissors, putting the cut tobacco leaves into sterile LB liquid culture medium suspension bacteria liquid containing a target carrier, and standing for 15-20 min; taking out tobacco leaf, removing excess bacterial liquid with sterile filter paper, and performing dark culture in MS culture medium containing 6-BA (0.02 mg/L) and NAA (2 mg/L) at 25 deg.C for two days; transferring tobacco leaves into a differentiation culture medium, contacting the cut with the culture medium, wherein the differentiation culture medium is an MS culture medium containing 6-BA (0.5 mg/L), NAA (0.1 mg/L), hygromycin (20 mg/L) and cefamycin (500 mg/L), subculturing once every 2-3 weeks, gradually forming callus at the cut, and finally differentiating to bud;
D. cutting off buds growing to 3-5 cm, transferring the buds into an MS culture medium to induce rooting, taking out the transgenic plants after rooting from the rooting culture medium, washing the culture medium with tap water, and transplanting the transgenic plants into sterilized nutrient soil.
(4) Sequencing screening editing material
And (3) growing the T0 generation transgenic seedlings for about 1 week, selecting 20 tobacco seedlings, taking leaves, extracting DNA by using DNeasy Plant Mini Kit (QIAGEN), amplifying by using the primer SdF/SdR designed in the step (1) C, purifying the amplified product, and sequencing by using a forward primer. Analyzing the sequencing result, an editing material (figure 3) that NtCOI1-1 gene lacks 1 base A and TCGGG in the NtCOI1-2 gene is converted into 1C is obtained. Planting the editing material T1 generation plant, screening double-gene homozygous mutant single plant by sequencing and harvesting to obtain T2 generation seeds.
(5) Editing material nicotine content
Homozygous T2 generation material ntcoi1-1/2 and controls were edited by greenhouse pot planting. The application of the tobacco composite fertilizer (N: P: nicotine = 10: 15) is carried out for 5g per plant by using pure nitrogen for 5 g. And in the bud emergence and flowering period, the nicotine content is detected after the whole tobacco leaves are subjected to enzyme deactivation. The results are shown in fig. 4, the nicotine content of the edited material is obviously reduced compared with that of the control, and the NtCOI1-1 and NtCOI 2 gene knockout is shown to influence the accumulation of nicotine in the tobacco leaves.
SEQUENCE LISTING
<110> research institute of tobacco agricultural science in Yunnan province
<120> tobacco receptor protein gene and cloning method and application thereof
<130> 2020
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 1821
<212> DNA
<213> nucleotide sequence of tobacco receptor protein gene NtCOI1-1
<400> 1
atggaggagc gtagctccac tcgattacca acggggagct acacgaacga caataccgta 60
tgggagtgcg tgataccgta cataacggaa tcgcgtgaca gggacgcggt gtcgacggtg 120
tgcaagaggt ggtggcagat cgatgcaata acacgaaagc atataaccat ggctttgtgc 180
tatacggcga aaccggaaca actttctaga aggttcccgc acctcgaatc gcttaaactt 240
aaaggaaaac cacgcgctgc tatgtttaat ttgatacctg aggattgggg agggtatgtt 300
acaccttggg tagtggaaat tactaagtcg tttagtaaat tgaaggcgct tcattttcgg 360
agaatgattg ttagggattc ggatctagaa ttggttgcga tgaatcgggg taaagtgctt 420
caggttttga agctggataa gtgttctgga ttctctaccg atggccttct gcatatttgt 480
cgttcctgca ggagcttaag aactttgttt ctggaagaga gttctatagt tgagaatgat 540
ggagaatggg tacatgaact agcggtgaac aacactgttc ttgagaattt gaatttttac 600
atgacggatc ttgtgcaagt tagagctgaa gatcttgaat tgatggcaag aaattgtaaa 660
tctcttgtct caatgaaaat tagcgagtgc gaacttgcca atcttcttgg cttctttaga 720
gctgctgttg cattggagga gtttggtggt ggctcgttta atgaccagcc agaacctgtt 780
gctgaaaatg gctataacga gcaattggaa aaatatgctg ctgtagtttc ccctccaaga 840
ttgtgccaat tgggcttgac gtacttgggg aaatatgaga tgcccattct ctttcctatt 900
gcttctcgtc tgacgaaatt ggatcttctt tatgcacttc ttgacacagc agcccattgt 960
ttcttgctgc aaaggtgccc caacttggta attcttgaga caaggaatgt tgttggggat 1020
agaggattgg aagtacttgg ccagtactgt aagaggttaa agcggctcag gattgagcga 1080
ggagctgatg atcaggagat ggaggatgaa caaggtgcag ttacgcacag aggattgact 1140
gatttggcga agggatgcct tgaactagaa tacatggctg tttatgtgtc agatattact 1200
aatgaagctt ttgaaaatat tggcacatat ttgaaaaatc tgtgtgattt tcggctggtt 1260
ttgcttgacc gagaggagag aataacagat ctaccacttg ataatggtgt ccgtgcttta 1320
ctaagaggtt gctataagct tagaaggttt gccctctatg tccggcctgg gggccttact 1380
gatgtaggtc tcagttatgt cgggcgatac agtccaaatg tgagatggat gcttctggga 1440
tatgttgggg aatccgatga aggccttctg gagttctcta aaggatgtcc tagcctgcaa 1500
aagctagaag tgagaggctg ctgttttagt gaacgtgcat tagctttggc tgccatgcag 1560
ctaaaatcat taaggtactt gtgggtacaa ggatacaggg catcttcaac tggtcgggat 1620
ctcttggcca tggctcggcc attctggaat attgagttga ttcctgcaag acgagttgtt 1680
gccagtgagg gaaataatgg agaaactatc gttgctgagc atcccgccca tatactttcc 1740
tactattctc ttgctgggcg aagaaccgat tttccagaca cagtcaggcc tttggaccca 1800
aatttccttc tcgctgaata g 1821
<210> 2
<211> 1821
<212> DNA
<213> nucleotide sequence of tobacco receptor protein gene NtCOI1-2
<400> 2
atggaggagc gtagctccac gcgattacca acggggagct acacgaacga caataccgta 60
tgggagtgcg tgataccgta cataacggaa tcgcgtgaca gggacgcggt gtcgacggtg 120
tgcaagaggt ggtggcagat cgatgcgata acacgaaagc atataaccat ggctttgtgc 180
tatacggcga aaccggagca actttctaga aggttcccgc acctcgaatc gcttaagctt 240
aaaggaaaac cacgcgcggc tatgtttaat ttgatacctg aagattgggg agggtatgtt 300
acaccttggg tagtggaaat tactaagtcg tttagtaaat tgaaggcgct tcattttcgg 360
agaatgattg ttagggattc ggatctagaa ttggttgcga tgaatcgggg taaagtgctt 420
caggttttga agctggataa gtgttctggg ttctctaccg atggtctgct gcatatttgt 480
cgttcctgca ggaacttaag aactttgttt ctggaagaga gttctatagt tgagaatgat 540
ggagaatggg tgcatgaact agcggtgaac aacccagttc ttgagaattt gaatttttac 600
atgacggatc ttgtgcaagt tagagctgaa gatcttgaat tgatagcaag aaattgtaaa 660
tctctcgtct caatgaaaat tagcgagtgc gaacttgcca atcttcttgg cttctttaga 720
gctgctgttg cattggagga gtttggtggt ggctcattta atgaccagcc agaacctgtt 780
gccgaaaatg gctataacga gcaattggaa aaatatgctg cagttgtttc ccctccaaga 840
ttgtgccagt tgggcttgac gtacttgggg aaatatgaga tgcccattct ctttcctatt 900
gcttctcgtc tgacgaaatt ggatcttctt tatgcacttc ttgacacagc agcccattgt 960
ttcttactgc aaaggtgccc caacttggaa attcttgaga caaggaatgt tgttggggat 1020
agaggattgg aagtgcttgg ccagtactgt aagaggttaa agcatctcag gattgagcga 1080
ggagctgatg atcaggagat ggaggatgaa caaggtgcag ttacccacag aggattgact 1140
gatttggcga agggatgcct tgaactagaa tacatggctg tttatgtgtc agatattacc 1200
aatgaagctt ttgaaaatat tggcacatat ttgaaaaatc tgtgtgattt tcggctggtt 1260
ttgctcgacc gagaagagag aataacagat ctacctcttg ataatggtgt ccgtgcttta 1320
ctaagaggtt gttataagct tagaaggttt gccctctatg tccggccagg gggccttact 1380
gatgtaggtc tcagttatgt cgggcaatac agcccaaatg tgagatggat gcttctggga 1440
tacgttgggg aatccgatga aggccttctg gagttctcta aaggatgccc taacctgcaa 1500
aagctagaag tgagaggctg ctgttttagt gagcgtgcat tagctttggc tgccatgcag 1560
ctaaaatcat taaggtactt gtgggtacaa ggatacaggg catcttcaac tggtcgggat 1620
ctcttggcga tggctcggcc attctggaat attgagttga ttcctgcaag acgagttgtt 1680
gccagtgagg gaaataatgg agaaactgta gttgctgagc atccagccca tatacttgcc 1740
tactattctc ttgctggaca aagaaccgat tttccacaca cagtcaggcc tttggaccca 1800
aattcccttc tcgctgaata g 1821
<210> 3
<211> 606
<212> PRT
<213> tobacco receptor protein gene NtCOI1-1 amino acid sequence
<400> 3
Met Glu Glu Arg Ser Ser Thr Arg Leu Pro Thr Gly Ser Tyr Thr Asn
1 5 10 15
Asp Asn Thr Val Trp Glu Cys Val Ile Pro Tyr Ile Thr Glu Ser Arg
20 25 30
Asp Arg Asp Ala Val Ser Thr Val Cys Lys Arg Trp Trp Gln Ile Asp
35 40 45
Ala Ile Thr Arg Lys His Ile Thr Met Ala Leu Cys Tyr Thr Ala Lys
50 55 60
Pro Glu Gln Leu Ser Arg Arg Phe Pro His Leu Glu Ser Leu Lys Leu
65 70 75 80
Lys Gly Lys Pro Arg Ala Ala Met Phe Asn Leu Ile Pro Glu Asp Trp
85 90 95
Gly Gly Tyr Val Thr Pro Trp Val Val Glu Ile Thr Lys Ser Phe Ser
100 105 110
Lys Leu Lys Ala Leu His Phe Arg Arg Met Ile Val Arg Asp Ser Asp
115 120 125
Leu Glu Leu Val Ala Met Asn Arg Gly Lys Val Leu Gln Val Leu Lys
130 135 140
Leu Asp Lys Cys Ser Gly Phe Ser Thr Asp Gly Leu Leu His Ile Cys
145 150 155 160
Arg Ser Cys Arg Ser Leu Arg Thr Leu Phe Leu Glu Glu Ser Ser Ile
165 170 175
Val Glu Asn Asp Gly Glu Trp Val His Glu Leu Ala Val Asn Asn Thr
180 185 190
Val Leu Glu Asn Leu Asn Phe Tyr Met Thr Asp Leu Val Gln Val Arg
195 200 205
Ala Glu Asp Leu Glu Leu Met Ala Arg Asn Cys Lys Ser Leu Val Ser
210 215 220
Met Lys Ile Ser Glu Cys Glu Leu Ala Asn Leu Leu Gly Phe Phe Arg
225 230 235 240
Ala Ala Val Ala Leu Glu Glu Phe Gly Gly Gly Ser Phe Asn Asp Gln
245 250 255
Pro Glu Pro Val Ala Glu Asn Gly Tyr Asn Glu Gln Leu Glu Lys Tyr
260 265 270
Ala Ala Val Val Ser Pro Pro Arg Leu Cys Gln Leu Gly Leu Thr Tyr
275 280 285
Leu Gly Lys Tyr Glu Met Pro Ile Leu Phe Pro Ile Ala Ser Arg Leu
290 295 300
Thr Lys Leu Asp Leu Leu Tyr Ala Leu Leu Asp Thr Ala Ala His Cys
305 310 315 320
Phe Leu Leu Gln Arg Cys Pro Asn Leu Val Ile Leu Glu Thr Arg Asn
325 330 335
Val Val Gly Asp Arg Gly Leu Glu Val Leu Gly Gln Tyr Cys Lys Arg
340 345 350
Leu Lys Arg Leu Arg Ile Glu Arg Gly Ala Asp Asp Gln Glu Met Glu
355 360 365
Asp Glu Gln Gly Ala Val Thr His Arg Gly Leu Thr Asp Leu Ala Lys
370 375 380
Gly Cys Leu Glu Leu Glu Tyr Met Ala Val Tyr Val Ser Asp Ile Thr
385 390 395 400
Asn Glu Ala Phe Glu Asn Ile Gly Thr Tyr Leu Lys Asn Leu Cys Asp
405 410 415
Phe Arg Leu Val Leu Leu Asp Arg Glu Glu Arg Ile Thr Asp Leu Pro
420 425 430
Leu Asp Asn Gly Val Arg Ala Leu Leu Arg Gly Cys Tyr Lys Leu Arg
435 440 445
Arg Phe Ala Leu Tyr Val Arg Pro Gly Gly Leu Thr Asp Val Gly Leu
450 455 460
Ser Tyr Val Gly Arg Tyr Ser Pro Asn Val Arg Trp Met Leu Leu Gly
465 470 475 480
Tyr Val Gly Glu Ser Asp Glu Gly Leu Leu Glu Phe Ser Lys Gly Cys
485 490 495
Pro Ser Leu Gln Lys Leu Glu Val Arg Gly Cys Cys Phe Ser Glu Arg
500 505 510
Ala Leu Ala Leu Ala Ala Met Gln Leu Lys Ser Leu Arg Tyr Leu Trp
515 520 525
Val Gln Gly Tyr Arg Ala Ser Ser Thr Gly Arg Asp Leu Leu Ala Met
530 535 540
Ala Arg Pro Phe Trp Asn Ile Glu Leu Ile Pro Ala Arg Arg Val Val
545 550 555 560
Ala Ser Glu Gly Asn Asn Gly Glu Thr Ile Val Ala Glu His Pro Ala
565 570 575
His Ile Leu Ser Tyr Tyr Ser Leu Ala Gly Arg Arg Thr Asp Phe Pro
580 585 590
Asp Thr Val Arg Pro Leu Asp Pro Asn Phe Leu Leu Ala Glu
595 600 605
<210> 4
<211> 606
<212> PRT
<213> tobacco receptor protein gene NtCOI1-2 amino acid sequence
<400> 4
Met Glu Glu Arg Ser Ser Thr Arg Leu Pro Thr Gly Ser Tyr Thr Asn
1 5 10 15
Asp Asn Thr Val Trp Glu Cys Val Ile Pro Tyr Ile Thr Glu Ser Arg
20 25 30
Asp Arg Asp Ala Val Ser Thr Val Cys Lys Arg Trp Trp Gln Ile Asp
35 40 45
Ala Ile Thr Arg Lys His Ile Thr Met Ala Leu Cys Tyr Thr Ala Lys
50 55 60
Pro Glu Gln Leu Ser Arg Arg Phe Pro His Leu Glu Ser Leu Lys Leu
65 70 75 80
Lys Gly Lys Pro Arg Ala Ala Met Phe Asn Leu Ile Pro Glu Asp Trp
85 90 95
Gly Gly Tyr Val Thr Pro Trp Val Val Glu Ile Thr Lys Ser Phe Ser
100 105 110
Lys Leu Lys Ala Leu His Phe Arg Arg Met Ile Val Arg Asp Ser Asp
115 120 125
Leu Glu Leu Val Ala Met Asn Arg Gly Lys Val Leu Gln Val Leu Lys
130 135 140
Leu Asp Lys Cys Ser Gly Phe Ser Thr Asp Gly Leu Leu His Ile Cys
145 150 155 160
Arg Ser Cys Arg Asn Leu Arg Thr Leu Phe Leu Glu Glu Ser Ser Ile
165 170 175
Val Glu Asn Asp Gly Glu Trp Val His Glu Leu Ala Val Asn Asn Pro
180 185 190
Val Leu Glu Asn Leu Asn Phe Tyr Met Thr Asp Leu Val Gln Val Arg
195 200 205
Ala Glu Asp Leu Glu Leu Ile Ala Arg Asn Cys Lys Ser Leu Val Ser
210 215 220
Met Lys Ile Ser Glu Cys Glu Leu Ala Asn Leu Leu Gly Phe Phe Arg
225 230 235 240
Ala Ala Val Ala Leu Glu Glu Phe Gly Gly Gly Ser Phe Asn Asp Gln
245 250 255
Pro Glu Pro Val Ala Glu Asn Gly Tyr Asn Glu Gln Leu Glu Lys Tyr
260 265 270
Ala Ala Val Val Ser Pro Pro Arg Leu Cys Gln Leu Gly Leu Thr Tyr
275 280 285
Leu Gly Lys Tyr Glu Met Pro Ile Leu Phe Pro Ile Ala Ser Arg Leu
290 295 300
Thr Lys Leu Asp Leu Leu Tyr Ala Leu Leu Asp Thr Ala Ala His Cys
305 310 315 320
Phe Leu Leu Gln Arg Cys Pro Asn Leu Glu Ile Leu Glu Thr Arg Asn
325 330 335
Val Val Gly Asp Arg Gly Leu Glu Val Leu Gly Gln Tyr Cys Lys Arg
340 345 350
Leu Lys His Leu Arg Ile Glu Arg Gly Ala Asp Asp Gln Glu Met Glu
355 360 365
Asp Glu Gln Gly Ala Val Thr His Arg Gly Leu Thr Asp Leu Ala Lys
370 375 380
Gly Cys Leu Glu Leu Glu Tyr Met Ala Val Tyr Val Ser Asp Ile Thr
385 390 395 400
Asn Glu Ala Phe Glu Asn Ile Gly Thr Tyr Leu Lys Asn Leu Cys Asp
405 410 415
Phe Arg Leu Val Leu Leu Asp Arg Glu Glu Arg Ile Thr Asp Leu Pro
420 425 430
Leu Asp Asn Gly Val Arg Ala Leu Leu Arg Gly Cys Tyr Lys Leu Arg
435 440 445
Arg Phe Ala Leu Tyr Val Arg Pro Gly Gly Leu Thr Asp Val Gly Leu
450 455 460
Ser Tyr Val Gly Gln Tyr Ser Pro Asn Val Arg Trp Met Leu Leu Gly
465 470 475 480
Tyr Val Gly Glu Ser Asp Glu Gly Leu Leu Glu Phe Ser Lys Gly Cys
485 490 495
Pro Asn Leu Gln Lys Leu Glu Val Arg Gly Cys Cys Phe Ser Glu Arg
500 505 510
Ala Leu Ala Leu Ala Ala Met Gln Leu Lys Ser Leu Arg Tyr Leu Trp
515 520 525
Val Gln Gly Tyr Arg Ala Ser Ser Thr Gly Arg Asp Leu Leu Ala Met
530 535 540
Ala Arg Pro Phe Trp Asn Ile Glu Leu Ile Pro Ala Arg Arg Val Val
545 550 555 560
Ala Ser Glu Gly Asn Asn Gly Glu Thr Val Val Ala Glu His Pro Ala
565 570 575
His Ile Leu Ala Tyr Tyr Ser Leu Ala Gly Gln Arg Thr Asp Phe Pro
580 585 590
His Thr Val Arg Pro Leu Asp Pro Asn Ser Leu Leu Ala Glu
595 600 605
<210> 5
<211> 22
<212> DNA
<213> NtCOI1-1F
<400> 5
atggaggagc gtagctccac tc 22
<210> 6
<211> 24
<212> DNA
<213> NtCOI1-1R
<400> 6
ctattcagcg agaaggaaat ttgg 24
<210> 7
<211> 21
<212> DNA
<213> NtCOI1-2F
<400> 7
atggaggagc gtagctccac g 21
<210> 8
<211> 23
<212> DNA
<213> NtCOI1-2R
<400> 8
ctattcagcg agaagggaat ttg 23
Claims (4)
1. Tobacco receptor protein geneNtCOI1-1AndNtCOI1-2application in obtaining transgenic tobacco plants for regulating and controlling the nicotine content of tobacco,NtCOI1-1andNtCOI1-2the nucleotide sequences of (A) are shown as SEQ ID No.1 and SEQ ID No.2, and the corresponding coded amino acid sequences are shown as SEQ ID No.3 and SEQ ID No. 4; it is characterized in that CRSIPR/CAS9 editing technology is used for knocking out tobacco plantsNtCOI1-1AndNtCOI1-2the nicotine content in the tobacco leaves of the obtained transgenic tobacco plants is obviously reduced compared with that of wild plants.
2. Root of herbaceous plantUse according to claim 1, characterized in that the tobacco receptor protein geneNtCOI1-1AndNtCOI1-2the cloning method comprises the following steps:
A. extracting RNA of tobacco root tissue, and performing reverse transcription to obtain first-chain cDNA;
B. using cDNA as template, according to tobacco receptor protein geneNtCOI1-1And tobacco receptor protein geneNtCOI1-2Designing forward and reverse primers according to the sequence, carrying out PCR amplification, and recovering and purifying PCR amplification products; the forward and reverse primers are as follows:
NtCOI1-1F：5’- ATGGAGGAGCGTAGCTCCACTC-3’
NtCOI1-1R：5’- CTATTCAGCGAGAAGGAAATTTGG-3’；
NtCOI1-2F：5’- ATGGAGGAGCGTAGCTCCACG -3’
NtCOI1-2R：5’- CTATTCAGCGAGAAGGGAATTTG-3’；
C. connecting the purified amplification product with a carrier, and specifically carrying out the following processes: uniformly mixing 4 mu L of the purified product, 1 mu L of salt solution and 1 mu L of PCR (polymerase chain reaction) -Blunt II-TOPO, and carrying out water bath for 30min at the temperature of 25 ℃; and transforming the connected vector into escherichia coli DH5 alpha through heat shock, adding a liquid culture medium, performing shake culture, coating the obtained product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting a bacterial colony, performing bacterial liquid culture, performing plasmid extraction and PCR (polymerase chain reaction) detection, screening positive clones, and sequencing the positive clones.
3. The use of claim 2, wherein the PCR reaction system in step B is: total volume 50 μ L, including: 200ng cDNA, 10 uL of 5 XPHUSION HF reaction buffer, 1 uL of 10mM dNTP, 2U of Phusion High-Fidelity DNA Polymerase,1 uL of 10 uM forward and reverse primers respectively, and water is supplemented to 50 uL.
4. The use of claim 2, wherein the reaction procedure of PCR amplification in step B is: 30 seconds at 98 ℃;7 seconds at 98 ℃;30 seconds at 66 ℃;60 seconds at 72 ℃;35 cycles; extension at 72 ℃ for 7 min.
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| CN110684779A (en) * | 2019-10-22 | 2020-01-14 | 云南省烟草农业科学研究院 | Cloning and application of tobacco nicotine synthesis regulatory gene NtERF91 |
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| CN111118031A (en) * | 2020-02-17 | 2020-05-08 | 云南省烟草农业科学研究院 | Cloning and application of negative regulation gene NtARF6 for synthesizing tobacco alkaloid |
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