CN116751787B - Tobacco nicotine metabolism-related transcription factor NtbHLH66 and its encoding gene, RNAi interference vector and application - Google Patents

Abstract

The present invention discloses a tobacco nicotine metabolism-related transcription factor NtbHLH66, a coding gene thereof, an RNAi interference vector and an application thereof. The tobacco nicotine metabolism-related transcription factor coding gene NtbHLH66 gene of the present invention has a nucleotide sequence as follows: (1) a nucleotide sequence shown in SEQ ID NO.1; (2) a nucleotide sequence in which the nucleotide sequence shown in SEQ ID NO.1 is replaced and/or deleted and/or one or more nucleotides are added and the nucleotide sequence expressing the same functional protein. By interfering with the expression of the tobacco NtbHLH66 gene, it is found that after silencing the NtbHLH66 gene, the nicotine content in the leaves of the NtbHLH66 gene interference strain at the seedling stage is significantly reduced compared with the control strain. The present invention provides a new strategy and path for reducing the nicotine content in tobacco, and has broad application prospects and huge economic benefit potential in the field of low-nicotine tobacco variety breeding.

Description

Tobacco nicotine metabolism related transcription factor NtbHLH and coding gene, RNAi interference vector and application thereof

Technical Field

The invention relates to a tobacco nicotine metabolism related transcription factor NtbHLH and a coding gene, an RNAi interference vector and application thereof, belonging to the technical field of bioengineering.

Background

Alkaloids, commonly referred to as phytoalkaloids, are the most widely occurring class of secondary metabolites in plants, except terpenes, and are generally basic. Only a small part of plants in the plant kingdom contain alkaloids, which are mainly present in the Solanaceae plants, and the biological functions of the alkaloids are mainly insect resistance. Among the tobacco (Nicotiana tabacum l.), there are mainly 4 alkaloids, nicotine (nicotine), nornicotine, neonicotinoid and pseudoscouring. The nicotine is the most main alkaloid of tobacco, accounting for 90-95% of the total alkaloid content in cultivated tobacco, and the tobacco is an important cash crop, is one of plants with the largest biomass in nature, has great excavation and utilization potential, however, the high nicotine content in the tobacco limits the multipurpose development and utilization of the tobacco to a great extent. Thus, studies on nicotine biosynthesis and regulation have long been one of the focus of research in the field of secondary metabolism.

The nicotine biosynthesis pathway has been clearly known through many years of research: namely ornithine decarboxylase (ornithine decarboxylase, ODC) catalyzes ornithine or arginine decarboxylase (arginine decarboxylase, ADC) catalyzes arginine to produce putrescine, the putrescine is catalyzed by putrescine-N-methyltransferase (Putrescine N-METHYLTRANSFERASE, PMT) to produce N-methyl putrescine, and the N-methyl putrescine is catalyzed by N-methyl putrescine oxidase (N-methylputrescine oxidase, MPO) to produce 4-methyl butyraldehyde; nicotinic acid or derivatives formed via the aspartate pathway may form a pyridine ring; the pyridine ring is condensed with pyrrolidine ring formed by natural cyclization of 4-methyl butyraldehyde to form nicotine. However, there has been no breakthrough in the molecular regulation of nicotine biosynthesis, and the role of the site of nicotine synthesis in the tobacco itself, other than the plant hormone, has yet to be further studied.

Up to now, more than 10 transcription factor genes involved in nicotine synthesis are found, and the transcription factor genes belong to different families, but in order to further understand the molecular regulation of nicotine synthesis, more accurate tobacco breeding is realized, and the problem to be solved is that the mining and screening of transcription factor regulation genes involved in nicotine metabolism is urgent.

Disclosure of Invention

It is a first object of the present invention to provide a gene NtbHLH which encodes a tobacco nicotine metabolism-related transcription factor gene NtbHLH which is capable of encoding a tobacco nicotine metabolism-related transcription factor NtbHLH.

It is a second object of the present invention to provide a tobacco nicotine metabolism-related transcription factor NtbHLH, 66, which is closely related to tobacco nicotine anabolism.

The third object of the present invention is to provide an RNAi interference vector, which can realize efficient silencing of NtbHLH66 genes, successfully obtain NtbHLH66 gene-silenced tobacco plants, and further reduce the content of nicotine in the silenced plants.

The fourth object of the invention is to provide the application of the coding gene NtbHLH of the transcription factor related to the tobacco nicotine metabolism or the RNAi interference vector in the cultivation of low-nicotine tobacco varieties, and after the NtbHLH gene in the tobacco plants is silenced by RNAi interference technology, the nicotine content in the silenced plant is found to be obviously reduced, so that new gene resources are provided for the breeding of the low-nicotine tobacco varieties.

In order to achieve the above purpose, the technical scheme adopted by the gene NtbHLH for coding the transcription factor related to the tobacco nicotine metabolism is as follows:

the nucleotide sequence of the coding gene NtbHLH of the tobacco nicotine metabolism related transcription factor is as follows:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

(2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein.

The beneficial effects of the technical scheme are that: according to the invention, a specific primer is designed, the coding gene of the tobacco nicotine metabolism related transcription factor NtbHLH is cloned and obtained, and the NtbHLH gene is mainly expressed in roots, stems, leaves and flower buds, especially the highest expression amount in the roots in normal tobacco plants through fluorescent quantitative PCR analysis.

In order to achieve the above purpose, the technical scheme adopted by the tobacco nicotine metabolism related transcription factor NtbHLH and 66 of the invention is as follows:

a tobacco nicotine metabolism-related transcription factor NtbHLH, having the amino acid sequence:

(1) An amino acid sequence shown in SEQ ID NO. 2;

(2) The amino acid sequence shown in SEQ ID NO.2 is a derivative protein with identical functions and with one or more amino acid residues replaced and/or deleted and/or added.

The beneficial effects of the technical scheme are that: it is verified that the transcription factor NtbHLH related to the nicotine metabolism in tobacco is closely related to the anabolism of nicotine in tobacco.

In order to achieve the above purpose, the technical scheme adopted by the RNAi interference vector of the invention is as follows:

An RNAi-interfering vector comprising as a targeting sequence a nucleotide fragment specific for NtbHLH genes.

The beneficial effects of the technical scheme are that: the interference strain obtained by RNAi has better gene silencing effect and stability compared with the VIGS strain, and can be transferred to the next generation.

As a further improvement, the nucleotide sequence of the guide sequence is shown in SEQ ID NO. 3.

As a further improvement, the guide sequence is inserted into a PBWA (V) HS empty vector, and screening, sequencing and identification are carried out.

In order to achieve the above purpose, the technical scheme adopted by the application of the coding gene NtbHLH gene of the transcription factor related to the tobacco nicotine metabolism or the RNAi interference vector in the cultivation of low-nicotine tobacco varieties is as follows:

The application of a tobacco nicotine metabolism related transcription factor coding gene NtbHLH gene or RNAi interference vector in the cultivation of low-nicotine tobacco varieties.

The beneficial effects of the technical scheme are that: according to the invention, through an RNA interference silencing technology, ntbHLH gene expression is disturbed in tobacco to obtain a NtbHLH66 gene silencing transgenic plant, and detection shows that the nicotine content in leaves of the NtbHLH gene silencing plant in a seedling stage is obviously reduced, so that a new strategy and path are provided for the nicotine content regulation molecular breeding of tobacco and other plants, and a foundation is laid for the accurate breeding of low-nicotine-content tobacco.

As a further improvement, the nicotine content of the tobacco leaves was significantly reduced after NtbHLH a gene silencing.

As a further improvement, the reduced nicotine tobacco variety is obtained by the following method: and (3) transforming agrobacterium with the RNAi vector to serve as an invader solution, transforming tobacco, and screening and identifying to obtain the tobacco variety with obviously reduced leaf nicotine content.

The beneficial effects of the technical scheme are that: the interference strain obtained by the agrobacterium transformation method has better gene silencing effect and the next generation gene silencing is more stable.

The invention discovers that the gene NtbHLH of the transcription factor coding gene related to the nicotine metabolism of the tobacco is expressed in each tissue of the tobacco through real-time PCR, and the expression in tobacco leaves, flowers and roots is relatively higher. To further confirm the function of the NtbHLH gene, RNAi vectors for silencing NtbHLH66 gene were constructed by RNAi technology, and a silencing strain for inhibiting NtbHLH66 gene expression was successfully obtained after transformation. The detection result shows that compared with a control plant, the leaf nicotine, nornicotine and neonicotine content in the transgenic silent plant are obviously reduced, which indicates that NtbHLH gene is highly related to the biosynthesis metabolism of tobacco alkaloid, thus providing an important reference for the regulation and control research of plant secondary metabolism and laying a foundation for cultivating new varieties of new plants with reduced nicotine.

Drawings

FIG. 1 is an analysis of the expression pattern of NtbHLH gene in experimental example 1 of the present invention;

FIG. 2 is an electrophoresis chart of PCR products of NtbHLH gene in experimental example 1 of the present invention (M is marker, lane 1 is forward sequence PCR product, lane 2 is reverse sequence PCR product);

FIG. 3 is a diagram showing the RNAi interference vector pBWA (V) HS-RNAi pattern in Experimental example 2 of the present invention;

FIG. 4 shows the cleavage map of pBWA (V) HS-NtbHLH-RNAi in Experimental example 2 (M is marker, lane 1 is cleavage product);

FIG. 5 is a diagram showing the gene expression analysis of NtbHLH-RNAi transgenic line NtbHLH66 in Experimental example 3 of the present invention;

FIG. 6 shows the alkaloid content analysis of NtbHLH-RNAi transgenic line and control K326 in Experimental example 4 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples, experimental examples and comparative examples were all commercially available, except for the specific descriptions.

The tobacco NtbHLH gene codes a tobacco nicotine metabolism related transcription factor NtbHLH66, and the transcription factor comprises an amino acid sequence shown in SEQ ID: no. 2. The transcription factor may also be a gene consisting of SEQ ID No:2 via substitution and/or deletion and/or addition of one or more amino acid residues, and has a derivative polypeptide which influences the accumulation of tobacco nicotine. The substitution and/or deletion and/or addition of one or several amino acid residues refers to substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The nucleotide sequence contained in the coding gene of the tobacco nicotine metabolism related transcription factor NtbHLH is shown as SEQ ID: no. 1. Or can be matched with SEQ ID No in a sequence table under high-stringency conditions: 1, a nucleotide sequence which hybridizes to the DNA sequence defined in 1; or with SEQ ID No:1, and the DNA sequence which has more than 90 percent of homology and codes the same functional protein.

The application of the gene NtbHLH to the gene coding the transcription factor related to the tobacco nicotine metabolism is to inhibit the expression of the NtbHLH gene in tobacco plants, so that the content of tobacco leaf nicotine in the seedling stage can be changed. Expression of NtbHLH gene can be inhibited by a variety of methods mediated by RNA, such as: a method for mediating gene silencing by a plant virus vector, a method for mediating and transforming RNAi interference vector by agrobacterium, a method for optimizing and modifying a gene coding frame, a method for optimizing a gene promoter and the like. The method of inhibiting gene expression according to the present invention is not limited to the above-mentioned methods, as long as NtbHLH a 66 expression can be inhibited.

All plant tissue material in the examples below was taken from K326 transgenic plants of flue-cured tobacco (Nicotiania tabacum l.) variety K326 and RNAi interference NtbHLH 66. Tobacco material was planted in a climatic chamber at a growth temperature of 25℃with a photoperiod of 12 hours light/12 hours darkness. Tobacco in seedling stage is collected, leaves are quickly frozen by liquid nitrogen and used for subsequent molecular experiments; and collecting leaves, and freeze-drying for subsequent metabolism detection.

Example 1 of the Gene NtbHLH A66 encoding a transcription factor related to Nicotiana tabacum nicotine metabolism

The nucleotide sequence of the tobacco nicotine metabolism related transcription factor encoding gene NtbHLH and 66 gene in this example is:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

(2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein.

Example 1 of tobacco Nicotine metabolism-related transcription factor NtbHLH66

The amino acid sequence of the tobacco nicotine metabolism-related transcription factor NtbHLH in this example is:

(1) An amino acid sequence shown in SEQ ID NO. 2;

(2) The amino acid sequence shown in SEQ ID NO.2 is a derivative protein with identical functions and with one or more amino acid residues replaced and/or deleted and/or added.

EXAMPLE 1 RNAi interference vector

The RNAi interference vector of this example contains a specific nucleotide fragment of NtbHLH gene as a leader sequence, the nucleotide sequence of which is shown in SEQ ID NO. 3.

Example 1 application of tobacco Nicotine metabolism-related transcription factor encoding Gene NtbHLH Gene or RNAi interference vector in cultivation of Low Nicotine variety

In the embodiment, after the RNAi interference vector containing the specific nucleotide fragment of NtbHLH-66 gene is transformed into a tobacco plant, the tobacco plant with NtbHLH-66 gene silencing is constructed, the contents of nicotine, nornicotine and neonicotinoid in leaves of the plant are obviously reduced, and the tobacco variety with low nicotine content is obtained.

Experimental example 1NtbHLH expression pattern analysis of Gene and cloning of NtbHLH Gene fragment

1. Extraction of tobacco RNA

Taking the root, stem, leaf, flower and other tissues of cultivated tobacco K326 grown to a seedling stage as samples, fully grinding the samples into powder by liquid nitrogen, then placing about 100mg of the powder material into a 1.5ml centrifuge tube containing 1.0ml TRIZOL reagent, adding 200 μl of chloroform, shaking and mixing uniformly, centrifuging, carefully taking out an upper aqueous phase, transferring the upper aqueous phase into another centrifuge tube, adding 500 μl of isopropanol, precipitating, centrifuging to separate RNA, washing by 75% alcohol, slightly drying at room temperature, and then adding RNASE FREE volumes of water for full dissolution. The total RNA extracted is treated by DNase I, and the digestion reaction system is shown in Table 1.

TABLE 1 reaction System for DNase I digestion

Composition of components	Dosage of
		RNA	1μg
10×reaction buffer with MgCl2	1μl
		DNase I,RNase-free	1μl(1U)
DEPC-treated water	to 10μl

The reaction conditions were set in a water bath at 37℃for 30min.

2. CDNA Synthesis

The template RNA/primer mixture of Table 2 was prepared in a sterile 0.2ml centrifuge tube, incubated at 70℃for 10min, rapidly quenched on ice for more than 2min, and centrifuged for several seconds to allow the denatured solution of the template RNA/primer to accumulate at the bottom of the centrifuge tube.

TABLE 2 preparation of RNA/primer mixture

Name of the name	Dosage of
		RN A(100ng/μl)	1μl
Oligo(dT)Primer(50μM)	1μl
		RNase free dH2O	5μl
Total Volume	7μl

Preparing reverse transcription reaction liquid in table 3 in the centrifuge tube, and then preserving heat for 1h at 42 ℃; the cDNA obtained was used for PCR amplification after incubation at 70℃for 15min and cooling on ice.

TABLE 3 preparation of reverse transcription reaction solution

Reagent name	Dosage of
		The template RNA/primer denaturing solution	7μl
5×M-MLV buffer	2μl
		DNTP mix (10 mM each)	0.5μl
RNase Inhibitor(40U/μl)	0.25μl
		RTase M-MLV(RNase H-)(200U/μl)	0.25μl
Total Volume	10μl

3. Analysis of expression patterns of NtbHLH gene in different tissues and organs of tobacco

The expression level of NtbHLH gene in each tissue and organ was detected by real-time quantitative PCR using K326 cDNA as a template. Taking tobacco 26S gene as an internal reference, carrying out fluorescent quantitative PCR detection, wherein the primer sequences are as follows:

the detection primers and internal reference primers are shown below.

The qRT-PCR primers are as follows:

q NtbHLH66-F:5'-CAATAAGACGGACAAGGC-3' (shown as SEQ ID NO. 4);

q NtbHLH66-R:5'-CCACCCAATCTACTCATACTC-3' (shown as SEQ ID NO. 5).

The internal reference gene primer is as follows:

26s-F:5'-GAAGAAGGTCCCAAGGGTTC-3' (shown as SEQ ID NO. 6);

26s-R:5'-TCTCCCTTTAACACCAACGG-3' (shown as SEQ ID NO. 7).

As shown in FIG. 1, it can be seen that NtbHLH.sup.66 gene is mainly expressed in roots, stems, leaves and flower buds, especially the highest expression amount in roots, in normal tobacco plants.

4. Cloning of NtbHLH66 Gene

And (3) taking the K326 cDNA as a template, designing a primer according to the information of the tobacco genome database, and carrying out PCR amplification of NtbHLH genes to obtain a PCR amplification product.

Primers for amplifying forward NtbHLH gene fragment:

NtbHLH66-F:5'-cagtGGTCTCacaacgccatgaaccagtacgatcc-3' (shown as SEQ ID NO. 8);

NtbHLH66-R:5'-cgatGGTCTCacctgcaggtgctgaagaggattag-3' (shown as SEQ ID NO. 9).

LOOP primer:

f:5'-cgatGGTCTCacaggtctagtttttctcctt-3' (shown as SEQ ID NO. 10);

R:5'-cgatGGTCTCagcccgggctctgtaactatc-3' (shown as SEQ ID NO. 11).

Primers for amplifying reverse NtbHLH gene fragment:

NtbHLH66-F:5'-cagtGGTCTCagggctgctgaagaggattagaagg-3' (as shown in SEQ ID NO. 12);

NtbHLH66-R:5'-cagtGGTCTCatacagccatgaaccagtacgatcc-3' (shown as SEQ ID NO. 13).

The PCR amplification system is shown in Table 4, and the reaction procedure is shown in Table 5.

TABLE 4PCR amplification System

Reagent name	Dosage of
		GXL polymerase	1μl
5×GXL buffer	10μl
		dNTP Mixture(10mM)	4μl
Primer-F/R	8μl
		ddH2O	26μl
cDNA	1μl
		Total Volume	50μl

TABLE 5PCR reaction procedure

The amplified PCR product was subjected to 1% agarose gel electrophoresis, and the result of the gel electrophoresis is shown in FIG. 2. After electrophoresis, adopting TAKARA PCR product purification kit, recovering and purifying the PCR product by 200bp according to the product specification, and sending to Shanghai worker for sequencing, and verifying the sequence result.

Experimental example 2 construction of RNAi vector

1. Connection

After purification of the PCR product containing the information linker sequence obtained in example 1, the fragment of interest was ligated into pBWA (V) HS-RNAi vector using an information ligase, the pBWA (V) HS-RNAi vector diagram is shown in FIG. 3. The information connection system is shown in Table 6.

Table 6 information connection system

Name of the name	Dosage of
		5 X Information ligase	2μl
PBWA (V) HS-RNAi after cleavage	2μl
		Gene fragment	6μl
Total Volume	10μl

The fragment mixture was reacted at 50℃for 15 minutes, and then placed on ice for 2 to 3 minutes.

2. Conversion by heat shock

10 Μl of the ligation product was added to E.coli competent cells under sterile conditions, gently mixed and then ice-bathed for 30min. And (3) heat-shocking at 42 ℃ for 90s, and rapidly transferring the centrifuge tube into an ice bath and placing for 2-3min. 800 μl of LB medium without antibiotics was added, and the mixture was gently shaken for about 1h at 37℃in a shaker (100-160 rpm). 200 μl of the culture solution is smeared on LB solid medium containing 50 μg/ml of antibiotics, X-Gal and IPTG are added to be smeared before the bacteria smearing solution, and the culture is inverted at 37 ℃ for 12-16h.

3. Positive clone screening and identification

A plurality of blue and white bacterial plaques grow in the culture medium, when the bacterial plaques grow to a proper size, a plurality of white plaques are picked up by a sterilized gun head, and the bacterial plaques are respectively cultured for 12-16 hours in an LB liquid culture medium containing 50 mug/ml kanamycin in a shaking way. The construction result of the EcoRV digestion identification vector of the extracted plasmid is shown in figure 4, 3 strips are clear after digestion, the size of the target sequence is 1100bp, and the RNA vector is successfully constructed. The correct RNAi vector was identified by retention of cleavage and designated pBWA (V) HS-NtbHLH-RNAi.

Experimental example 3 Agrobacterium-mediated transformation of tobacco and identification of transgenic plants

1. Freeze thawing process of transforming agrobacterium

Adding 1 mu g pBWA (V) HS-NtbHLH-RNAi vector into 100 mu EHA105 agrobacterium L competence, standing on ice for 30min after uniform mixing, putting into liquid nitrogen for freezing for 5min, taking out from the liquid nitrogen, putting into a water bath at 37 ℃ for water bath for 5min, standing on ice for 5min, adding 500 mu L LB solution, recovering to culture for 4h under the condition of full shaking at 28 ℃, and finally uniformly smearing bacterial liquid on a selective plate culture medium, and culturing for 24-48h at 28 ℃.

2. Leaf disc method for transforming tobacco plants

The invention uses a leaf disc method to transform the RNAi vector successfully constructed in the embodiment 2 into K326 tobacco, and the specific implementation operation is as follows:

(a) Under aseptic condition, putting tobacco K326 seeds into an EP pipe, and flushing with aseptic water for 2-3 times; soaking in 75% alcohol for 30-60s, treating with 0.1% mercuric chloride for 5min, washing with sterile water for 5 times, seeding on MS culture medium, and placing culture flask in artificial climatic chamber to ensure normal germination and growth of tobacco seedling.

(B) When the tobacco seedlings grow to 3-5cm (about 20-30 d), taking terminal buds, placing the terminal buds on a culture medium of MS+BA 0.2mg/L (strong buds, enabling the strong buds to grow rapidly), and carrying out secondary culture. After 14 days of subculture (only with small leaves), taking leaves with the size of 1cm multiplied by 1cm, cutting off leaf stems, scratching the surfaces and edges of the leaves, placing the leaves on a preculture medium with the pH of 6.0-6.5 and MS+BA of 1.0mg/L, placing the leaves with the front face facing downwards tightly against the culture medium, and preculturing the leaves for 2-3 days under dark conditions.

(C) Taking out the pre-cultured leaf or stem segment, and putting the leaf or stem segment into an agrobacterium infection solution for infection. At night the day before infection, 2 bottles of agrobacteria shake. The 2mL centrifuge tube is filled with bacterial liquid, centrifuged at 4000r/min for 5min, and washed twice with the bacterial suspension. Placing 1 pipe 1.5mL of bacterial suspension into the bacterial suspension according to the ratio of 1:10 (10 mL of bacterial suspension is placed into 1 pipe), adding 25mg/L acetosyringone (As), continuously shaking the dyeing liquid to enable the dyeing liquid to be fully contacted with the cut parts of the leaves and the stems, taking out the bacterial suspension after 15min, and placing the bacterial suspension on sterilized dry filter paper to suck the bacterial suspension.

The preparation method of the agrobacterium infection solution comprises the following steps: culturing transformed Agrobacterium in a refrigerator at-80deg.C, and adding 50mg/L Kan and 50mg/L Rif into LB solid plate; picking up single bacterial plaque into 5mL LB liquid medium containing 50mg/L Kan and 50mg/L Rif, placing into a shaking table, and culturing at 28deg.C and 200r/min overnight (12-16 h); when the concentration of the bacterial liquid reaches about OD600 = 1.5, 2mL of bacterial liquid is taken and added into a centrifuge tube, and the centrifugation is carried out for 5min at 4000 r/min; removing supernatant, sucking 1mL of new MS liquid culture medium, re-suspending agrobacterium, and centrifuging at 4000r/min for 5min; repeating the step (6) for 1 time; after re-suspending the bacteria with 1mL of MS liquid medium, the bacteria are added into 40mL of MS liquid medium (containing 40 mu L of 25mg/L As), and the bacteria are the invader solution. After 2h of standing, the infection is carried out.

200ML of the suspension was prepared as follows:

100 x macroelement mother liquor 2mL(NH₄NO₃ 165g/L,KNO₃ 190g/L,MgSO₄·7H₂O 37g/L,KH₂PO417g/L,CaCl₂·2H₂O 44g/L)

100X 2ml of organic element mother liquor (inositol 10g/L, nicotinic acid 0.05g/L, pyridoxine hydrochloride 0.05g/L, thiamine nicotinate 0.01g/L, glycine 0.2 g/L)

100×Ferric salt mother liquor 2mL (EDTA disodium, 37.3mg/L, feSO ₄·7H₂ O27.8 mg/L)

100 X trace element mother liquor 2mL(MnSO₄·4H₂O 22.3mg/L,ZnSO₄·7H₂O 8.6mg/L,CoCl₂·6H₂O0.025mg/L,CuSO₄·5H₂O 0.025mg/L,Na₂MoO₄·2H₂O 0.25mg/L,KI 0.83mg/L,H₃BO₃6.2mg/L)

Sucrose 5.6g

Deionized water was added to a constant volume of 200mL.

(D) Putting the leaves and the stem segments back on the preculture medium, and co-culturing for 2-3 days under the dark condition at 28 ℃ until micro-bacterial plaques are formed around the cut of the leaves; taking out co-cultured tobacco leaves and stem segments, washing with sterile water added with 500mg/L Cef for 6 times, placing on a shaking table for shaking for 30min for the first time, and 5min each time to wash out agrobacterium on the surface of the explant;

(e) Sucking with filter paper, transferring to tobacco bud induction culture medium with pH of MS+BA 1.0 mg/L+Hyg25mg/L+Cef500mg/L and pH of 5.8; after 2 weeks observation, if no growth of bacteria was found, the Cef concentration was decreased. If bacteria grow, the Cef concentration continues to be maintained.

(F) The medium was changed 1 time every 2 weeks until adventitious buds grew (typically 2 weeks). Cutting off regenerated plantlets (about 1 cm), and transferring into a secondary culture medium MS+BA0.2-0.1 mg/L+Hyg25mg/L+Cef500mg/L pH 5.8; growing until the young seedling grows to 2cm long (the young seedling can grow), transferring into rooting culture medium MS+NAA 0.2-0.1mg/L, culturing at 25deg.C for 12 hr, and growing into thick root system.

(G) And (5) growing until the roots grow to 2-3cm. When the seedling is about 7-10cm high, the triangular flask is removed to wash off the root culture medium, and the seedling is transplanted into a flowerpot for greenhouse culture.

3. Identification of transgenic plants

Extracting genome DNA of transgenic tobacco seedlings by using a Takara company DNA extraction kit, designing Kan resistance gene primers for PCR amplification, screening positive plants, and detecting 10 positive plants.

The Kan resistance gene primer is as follows:

Kan-F:5'-TCTGGACGAAGAGCATCAGG-3' (as shown in SEQ ID NO. 14);

Kan-R:5'-ATGAATCCAGAAAAGCGGCC-3' (shown as SEQ ID NO. 15).

RNA was extracted from wild-type K326 tobacco and 3 transgenic NtbHLH-RNAi transgenic tobacco lines and cDNA was synthesized as described in example 1. The expression of NtbHLH66 in the different transgenic lines was detected by fluorescent quantitative PCR, and the detection primers and internal reference primers were as described in example 1.

As shown in FIG. 5, the RNAi interference vector constructed by the present invention can effectively interfere NtbHLH with the expression of NtbHLH gene. And selecting a transgenic line (RNAi 2) with the lowest expression level as a research object, detecting the nicotine content of leaves, and performing function verification of NtbHLH genes.

Experimental example 4 determination of alkaloid content in tobacco leaves

Uniformly sowing cultivated tobacco K326 plants and RNAi interference transgenic plant line transgenic tobacco seeds into small basins containing nutrient soil, and culturing in an illumination culture room. In the seedling stage, taking the leaf liquid nitrogen for quick freezing, freeze-drying by a freeze-dryer. Weighing 0.3g of freeze-dried fresh leaf powder, putting the freeze-dried fresh leaf powder into a 50mL screw pressure-resistant test tube, adding 2.5mL 5% sodium hydroxide solution, wetting a sample, standing for 15min, adding 20mL 0.01% triethylamine/methyl tertiary butyl ether solution, sealing by a cover, putting into an ultrasonic generator, performing ultrasonic extraction for 15min at room temperature, and centrifuging for 5min at 6000 r/min. Taking 2mL of organic phase for GC/MS analysis, and detecting the nicotine content; accurately transferring 10mL of organic phase, concentrating to 1mL, performing GC/MS analysis, and detecting the content of other 10 alkaloids. Detection of nicotine and other 10 alkaloids was done in two injections, using retention time and Selective Ion (SIM) mode characterization, double internal standard amounts. As shown in FIG. 6, after NtbHLH66 gene silencing, the contents of nicotine, nornicotine and neonicotinoid in leaves of NtbHLH-RNAi transgenic tobacco plants were significantly reduced.

The last explanation is: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (4)

1. Application of silencing the tobacco nicotine metabolism-related transcription factor encoding gene NtbHLH66 in the breeding of low-nicotine tobacco varieties, characterized in that: after silencing the NtbHLH66 gene, the nicotine content of tobacco leaves is significantly reduced; the nucleotide sequence of the NtbHLH66 gene is shown in SEQ ID NO.1. 2. Application of an RNAi interference vector for silencing the tobacco nicotine metabolism-related transcription factor encoding gene NtbHLH66 in the breeding of low-nicotine tobacco varieties, characterized in that: the nucleotide sequence of the NtbHLH66 gene is as shown in SEQ ID NO.1; the RNAi interference vector contains a specific nucleotide fragment of the NtbHLH66 gene as a guide sequence. 3. Use of the RNAi interference vector for silencing the tobacco nicotine metabolism-related transcription factor encoding gene NtbHLH66 gene in the breeding of low-nicotine tobacco varieties according to claim 2, characterized in that the nucleotide sequence of the guide sequence is shown in SEQ ID NO.3. 4. Use of the RNAi interference vector for silencing the tobacco nicotine metabolism-related transcription factor encoding gene NtbHLH66 according to claim 2 or 3 in the breeding of low-nicotine tobacco varieties, characterized in that the low-nicotine tobacco variety is obtained by the following method: the RNAi interference vector is transformed into Agrobacterium as an infection liquid, and then the tobacco is transformed, and the tobacco variety with significantly reduced nicotine content in the leaves is obtained by screening and identification.