阅读说明：本技术 一种原花青素物质调控因子NtMYB330及其表达载体、转化体、试剂盒与方法 (Proanthocyanidins substance regulatory factor NtMYB330, and expression vector, transformant, kit and method thereof ) 是由 赵璐 宋中邦 王丙武 高玉龙 隋学艺 师君丽 张谊寒 李永平 于 2021-08-19 设计创作，主要内容包括：本发明“一种原花青素物质调控因子NtMYB330及其表达载体、转化体、试剂盒与方法”属于遗传工程技术领域。所述一种原花青素物质调控因子NtMYB330的氨基酸序列中包含：R2重复序列、R3重复序列、[D/E]Lx-(2)[R/K]x-(3)Lx-(6)Lx-(3)R结构域、VI[R/P]TKAx-(1)RC[S/T]结构域。所述一种原花青素物质调控因子NtMYB330的氨基酸序列如SEQ ID NO：2所示。基于过表达原花青素物质调控因子NtMYB330获得的转基因植株花中的儿茶酸含量平均提高431.5％；表儿茶酸含量平均提高406.5％。说明烟草NtMYB330基因在提高烟草花中原花青素含量方面具有较大的应用前景。(The invention discloses a proanthocyanidin substance regulatory factor NtMYB330, an expression vector, a transformant, a kit and a method thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the procyanidine substance regulatory factor NtMYB330 comprises: r2 repetitive sequence, R3 repetitive sequence, [ D/E]Lx 2 [R/K]x 3 Lx 6 Lx 3 R domain, VI [ R/P]TKAx 1 RC[S/T]A domain. The procyanidinThe amino acid sequence of the substance regulatory factor NtMYB330 is shown as SEQ ID NO: 2, respectively. The catechin content in the transgenic plant flowers obtained on the basis of the over-expression procyanidine substance regulatory factor NtMYB330 is averagely improved by 431.5%; the epicatechin content increased by 406.5% on average. The tobacco NtMYB330 gene has a wide application prospect in the aspect of improving the content of procyanidine in tobacco flowers.)

1. A proanthocyanidin substance regulatory factor NtMYB330, which has an amino acid sequence comprising: r2 repetitive sequence, R3 repetitive sequence, [ D/E]Lx₂[R/K]x₃Lx₆Lx₃R domain, VI [ R/P]TKAx₁RC[S/T]A domain.

2. The procyanidin substance regulatory factor NtMYB330 of claim 1, wherein the amino acid sequence thereof comprises an R2 repetitive sequence, an R3 repetitive sequence, [ D/E ] in order from amino terminus to carboxy terminus]Lx₂[R/K]x₃Lx₆Lx₃R domain, VI [ R/P]TKAx₁RC[S/T]A domain;

preferably, said [ D/E]Lx₂[R/K]x₃Lx₆Lx₃The R domain is located within the R3 repeat sequence;

preferably, said [ D/E]Lx₂[R/K]x₃Lx₆Lx₃The R domain is located after amino acid 12 of the R3 repeat;

preferably, the amino acid sequence of the procyanidine substance regulatory factor NtMYB330 at the 11 th-63 th amino acid positions is an R2 repetitive sequence;

preferably, the amino acid sequence of the procyanidine substance regulatory factor NtMYB330 at the 64 th-115 th amino acid position is an R3 repetitive sequence;

preferably, the amino acid sequence 188-197 of the procyanidine substance regulatory factor NtMYB330 is VI [ R/P ]]TKAx₁RC[S/T]A domain;

preferably, the amino acid sequence of the procyanidine substance regulatory factor NtMYB330 at the 76 th-95 th position is [ D/E ]]Lx₂[R/K]x₃Lx₆Lx₃An R domain.

3. The procyanidin substance modulating factor NtMYB330 of claim 1 or 2, which has an amino acid sequence as set forth in SEQ ID NO: 2, respectively.

4. The procyanidin substance regulatory factor NtMYB330 of claim 3, which has a gene sequence as set forth in SEQ ID NO: 1 is shown in the specification;

preferably, the procyanidin substance regulatory factor NtMYB330 is tobacco Nicotiana tabacum l.

5. An expression vector capable of regulating the synthesis of procyanidine, which is characterized in that a gene sequence of the procyanidine substance regulating factor NtMYB330 as claimed in any one of claims 1 to 4 is connected.

Preferably, the expression vector for regulating the synthesis of the procyanidine substance refers to a gene sequence of the procyanidine substance regulating factor NtMYB330 connected to the expression vector;

preferably, the expression vector is selected from the group consisting of: pK2GW7 vector;

preferably, the regulation is positive regulation;

preferably, the procyanidin material comprises catechin, epicatechin;

preferably, the positive control means that the content of procyanidin substances is preferably increased by more than 3 times.

6. A transformant capable of regulating the synthesis of a proanthocyanidin substance, which is transformed with the expression vector of claim 5.

Preferably, the transformant capable of regulating the synthesis of the procyanidine substance refers to an expression vector which is transformed in a host cell and is capable of regulating the synthesis of the procyanidine substance;

preferably, the host cell is selected from the group consisting of: plant cells or microbial cells;

preferably, the plant is tobacco;

preferably, the microorganism is selected from: escherichia coli and Agrobacterium.

7. A kit for regulating and controlling the synthesis of procyanidine substances is characterized by comprising: the procyanidin production regulatory factor NtMYB330 of any one of claims 1 to 4, and/or the expression vector for regulating procyanidin production of claim 5, and/or the transformant for regulating procyanidin production of claim 6.

8. The kit for regulating synthesis of procyanidin of claim 7, further comprising: an amplification primer of a gene sequence of the proanthocyanidin substance regulatory factor NtMYB330, a PCR (polymerase chain reaction) common reagent, a connection transformation common reagent and a transgene common reagent;

preferably, the amplification primer of the gene sequence of the procyanidine substance regulatory factor NtMYB330 comprises:

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’；

Preferably, the common reagents for PCR include: DNA polymerase, dNTP, PCR buffer solution and double distilled water;

the DNA polymerase is preferablyHigh-Fidelity DNA Polymerase, wherein the PCR buffer solution is preferably 5 XPHUSION HF reaction buffer solution;

preferably, the common reagents for ligation transformation include: restriction enzyme, connection buffer solution, expression vector, protease, competent cell and culture medium;

preferably, the restriction enzyme is preferably BamH I, Xho I; the restriction enzyme is preferably Clonease^TMII, enzyme Mix; the connection Buffer solution is preferably TE Buffer; the protease is preferably protease K;

preferably, the expression vector is selected from the group consisting of: pK2GW7 vector;

preferably, the competent cell is selected from the group consisting of: competent cells of Escherichia coli and competent cells of Agrobacterium;

preferably, the medium is selected from LB medium.

9. A method for regulating the synthesis of procyanidin substances, which comprises overexpressing the procyanidin substance regulatory factor NtMYB330 of any one of claims 1 to 4 in a plant, and/or transforming the expression vector for regulating the synthesis of procyanidin substances of claim 5 in a plant, and/or infecting the plant with the transformant for regulating the synthesis of procyanidin substances of claim 6.

10. The method of claim 9, wherein the controllable synthesis of procyanidins comprises: PCR amplifying the gene sequence of the proanthocyanidin substance regulatory factor NtMYB330 to obtain a PCR amplification product;

preferably, the PCR amplification system comprises: 4 ng/. mu.L plant cDNA, 5 XPPhusion HF reaction buffer 0.2. mu.L/. mu.L, 0.2mM dNTP, 0.04U/. mu.LHigh-Fidelity DNA Polymerase, 0.2 mu M forward and reverse primers, and the balance of water;

preferably, the forward and reverse primers comprise:

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’；

Preferably, the PCR amplification procedure comprises: 30 seconds at 98 ℃; at 98 ℃ for 7 seconds; 30 seconds at 62 ℃; 1 cycle at 72 ℃ for 45 seconds, and 35 cycles in total; extension at 72 ℃ for 7 min;

preferably, the method further comprises: connecting the PCR amplification product with an expression vector to obtain an expression vector for synthesizing the adjustable procyanidin substance;

preferably, the connection refers to an expression vector pENTR connected with a gene sequence of the proanthocyanidin substance regulatory factor NtMYB330^TMThe NtMYB330 is further connected through LR reaction to obtain an expression vector pK2GW7-NtMYB330 capable of regulating procyanidine synthesis;

preferably, the system for LR reaction comprises: pENTR of 6.25 ng/. mu.L-18.75 ng/. mu.L^TMNtMYB330, 0.0625 μ L/μ L of a connecting vector, and the balance TE Buffer;

preferably, the LR reaction step comprises: connecting the reaction system, mixing, performing ice bath for 2min, and mixing; add 0.2. mu.L/. mu.L LR Clonease^TMII, mixing uniformly, centrifuging, and carrying out water bath at 25 ℃ for 1 h; then adding 0.9 mu L/mu L of protease K, uniformly mixing, and carrying out water bath at 37 ℃ for 10 min; the blending mode is preferably light and elastic;

preferably, the method further comprises: transforming an expression vector pK2GW7-NtMYB330 capable of regulating the synthesis of the procyanidine substances into competent cells to obtain a transformant capable of regulating the synthesis of the procyanidine substances;

preferably, the method further comprises: infecting plant cells with a transformant capable of regulating the synthesis of procyanidine substances;

preferably, the competent cell is an agrobacterium competent cell; the transformant for regulating the synthesis of the procyanidine substances refers to agrobacterium clone containing an expression vector pK2GW7-NtMYB330 for regulating the synthesis of the procyanidine substances;

preferably, the plant is preferably tobacco;

preferably, the regulation is positive regulation;

preferably, the procyanidin substance: including catechin, epicatechin;

preferably, the positive control means that the content of procyanidin substances is preferably increased by more than 3 times.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a proanthocyanidin substance regulatory factor NtMYB330, an expression vector, a transformant, a kit and a method thereof.

Background

Tobacco (scientific name: Nicotiana tabacum L.) is an annual herb of the genus Nicotiana of the family Solanaceae. Native to south america. China is widely cultivated in the north and south provinces. The plant can be used as raw material of tobacco industry; the whole plant can also be used as pesticide; it can also be used as anesthetic, sweating, sedative and emetic.

The procyanidine is an important phenol secondary metabolite in plants, and has strong oxidation resistance. The over-expression of the procyanidine pathway related gene (CsDFR, CsANR) in the tea in the tobacco plant can obviously improve the procyanidine substance content of the transgenic tobacco, simultaneously improve the capability of the transgenic tobacco in removing active oxygen free radicals and resist the infection resistance of Spodoptera litura (Spodoptera litura) of the tobacco leaf. In view of the influence of procyanidine on plant stress resistance and the like, more and more researches are focused on the regulation of metabolic pathways of procyanidine, particularly the regulation of transcription level, and the regulation function of a heterologous gene on procyanidine synthesis is verified in flowers of tobacco plants by taking tobacco as a model plant.

Transcription regulation and control of procyanidine approaches in higher plants such as arabidopsis thaliana, tomatoes, grapes and the like are widely researched at home and abroad, but related reports in tobacco are rare, and cloning of procyanidine substance regulation and control factors and related genes in related tobacco in the field is blank.

Disclosure of Invention

Aiming at the blank existing in the prior art in the field, the invention provides a positive control gene NtMYB330 for synthesizing procyanidine in tobacco flower, a cloning method of the positive control gene NtMYB330 for synthesizing procyanidine in tobacco flower, and application of the positive control gene NtMYB330 for synthesizing procyanidine in tobacco flower.

The technical scheme of the invention is as follows:

a proanthocyanidin substance regulatory factor NtMYB330, which has an amino acid sequence comprising: r2 repetitive sequence, R3 repetitive sequence, [ D/E]Lx₂[R/K]x₃Lx₆Lx₃R domain, VI [ R/P]TKAx₁RC[S/T]A domain.

The amino acid sequence of the procyanidine substance regulatory factor NtMYB330 sequentially comprises an R2 repetitive sequence, an R3 repetitive sequence and a [ D/E ] sequence from an amino terminal to a carboxyl terminal]Lx₂[R/K]x₃Lx₆Lx₃R domain, VI [ R/P]TKAx₁RC[S/T]A domain;

preferably, said [ D/E]Lx₂[R/K]x₃Lx₆Lx₃The R domain is located within the R3 repeat sequence;

preferably, said [ D/E]Lx₂[R/K]x₃Lx₆Lx₃The R domain is located after amino acid 12 of the R3 repeat;

preferably, the amino acid sequence of the procyanidine substance regulatory factor NtMYB330 at the 11 th-63 th amino acid positions is an R2 repetitive sequence;

preferably, the amino acid sequence of the procyanidine substance regulatory factor NtMYB330 at the 64 th-115 th amino acid position is an R3 repetitive sequence;

preferably, the amino acid sequence 188-197 of the procyanidine substance regulatory factor NtMYB330 is VI [ R/P ]]TKAx₁RC[S/T]A domain;

preferably, the amino acid sequence of the procyanidine substance regulatory factor NtMYB330 at the 76 th-95 th position is [ D/E ]]Lx₂[R/K]x₃Lx₆Lx₃An R domain.

The amino acid sequence of the procyanidine substance regulatory factor NtMYB330 is shown as SEQ ID NO: 2, respectively.

The gene sequence of the proanthocyanidin substance regulatory factor NtMYB330 is shown in SEQ ID NO: 1 is shown in the specification;

preferably, the procyanidin substance regulatory factor NtMYB330 is tobacco Nicotiana tabacum l.

An expression vector capable of regulating and controlling the synthesis of procyanidine substances is characterized in that the expression vector is connected with a gene sequence of a procyanidine substance regulating factor NtMYB 330.

Preferably, the expression vector for regulating the synthesis of the procyanidine substance refers to a gene sequence of the procyanidine substance regulating factor NtMYB330 connected to the expression vector;

preferably, the expression vector is selected from the group consisting of: pK2GW7 vector;

preferably, the regulation is positive regulation;

preferably, the procyanidin material comprises catechin, epicatechin;

preferably, the positive control means that the content of procyanidin substances is preferably increased by more than 3 times.

A transformant capable of regulating the synthesis of procyanidine substances is transformed with the expression vector capable of regulating the synthesis of procyanidine substances.

Preferably, the transformant capable of regulating the synthesis of the procyanidine substance refers to an expression vector which is transformed in a host cell and is capable of regulating the synthesis of the procyanidine substance;

preferably, the host cell is selected from the group consisting of: plant cells or microbial cells;

preferably, the plant is tobacco;

preferably, the microorganism is selected from: escherichia coli and Agrobacterium.

A kit for regulating and controlling the synthesis of procyanidine substances is characterized by comprising: the proanthocyanidin substance regulating factor NtMYB330 and/or the expression vector capable of regulating the proanthocyanidin substance synthesis and/or the transformant capable of regulating the proanthocyanidin substance synthesis.

The kit for regulating and controlling the synthesis of the procyanidine substance further comprises: an amplification primer of a gene sequence of the proanthocyanidin substance regulatory factor NtMYB330, a PCR (polymerase chain reaction) common reagent, a connection transformation common reagent and a transgene common reagent;

preferably, the amplification primer of the gene sequence of the procyanidine substance regulatory factor NtMYB330 comprises:

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’；

Preferably, the common reagents for PCR include: DNA polymerase, dNTP, PCR buffer solution and double distilled water;

the DNA polymerase is preferablyHigh-Fidelity DNA Polymerase, wherein the PCR buffer solution is preferably 5 XPHUSION HF reaction buffer solution;

preferably, the common reagents for ligation transformation include: restriction enzyme, connection buffer solution, expression vector, protease, competent cell and culture medium;

preferably, the restriction enzyme is preferably BamH I, Xho I; the restriction enzyme is preferably Clonease^TMII, enzyme Mix; the connection Buffer solution is preferably TE Buffer; the protease is preferably protease K;

preferably, the expression vector is selected from the group consisting of: pK2GW7 vector;

preferably, the competent cell is selected from the group consisting of: competent cells of Escherichia coli and competent cells of Agrobacterium;

preferably, the medium is selected from LB medium.

A method for regulating and controlling the synthesis of procyanidine substances is characterized in that a procyanidine substance regulating factor NtMYB330 is overexpressed in a plant body, and/or an expression vector for regulating and controlling the synthesis of procyanidine substances is transformed in the plant body, and/or the plant is infected by a transformant for regulating and controlling the synthesis of procyanidine substances.

The method for synthesizing the adjustable procyanidin substance comprises the following steps: PCR amplifying the gene sequence of the proanthocyanidin substance regulatory factor NtMYB330 to obtain a PCR amplification product;

preferably, the PCR amplification system comprises: 4 ng/. mu.L plant cDNA, 5 XPPhusion HF reaction buffer 0.2. mu.L/. mu.L, 0.2mM dNTP, 0.04U/. mu.LHigh-Fidelity DNA Polymerase, 0.2 mu M forward and reverse primers, and the balance of water;

preferably, the forward and reverse primers comprise:

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’；

Preferably, the PCR amplification procedure comprises: 30 seconds at 98 ℃; at 98 ℃ for 7 seconds; 30 seconds at 62 ℃; 1 cycle at 72 ℃ for 45 seconds, and 35 cycles in total; extension at 72 ℃ for 7 min;

preferably, the method further comprises: connecting the PCR amplification product with an expression vector to obtain an expression vector for synthesizing the adjustable procyanidin substance;

preferably, the connection refers to an expression vector pENTR connected with a gene sequence of the proanthocyanidin substance regulatory factor NtMYB330^TMThe NtMYB330 is further connected through LR reaction to obtain an expression vector pK2GW7-NtMYB330 capable of regulating procyanidine synthesis;

preferably, the system for LR reaction comprises: pENTR of 6.25 ng/. mu.L-18.75 ng/. mu.L^TMNtMYB330, 0.0625 μ L/μ L of a connecting vector, and the balance TE Buffer;

preferably, the LR reaction step comprises: connecting the reaction system, mixing, performing ice bath for 2min, and mixing; add 0.2. mu.L/. mu.L LR Clonease^TMII, mixing uniformly, centrifuging, and carrying out water bath at 25 ℃ for 1 h; then adding 0.9 mu L/mu L of protease K, uniformly mixing, and carrying out water bath at 37 ℃ for 10 min; the blending mode is preferably light and elastic;

preferably, the method further comprises: transforming an expression vector pK2GW7-NtMYB330 capable of regulating the synthesis of the procyanidine substances into competent cells to obtain a transformant capable of regulating the synthesis of the procyanidine substances;

preferably, the method further comprises: infecting plant cells with a transformant capable of regulating the synthesis of procyanidine substances;

preferably, the competent cell is an agrobacterium competent cell; the transformant for regulating the synthesis of the procyanidine substances refers to agrobacterium clone containing an expression vector pK2GW7-NtMYB330 for regulating the synthesis of the procyanidine substances;

preferably, the plant is preferably tobacco;

preferably, the regulation is positive regulation;

preferably, the procyanidin substance: including catechin, epicatechin;

preferably, the positive control means that the content of procyanidin substances is preferably increased by more than 3 times.

The invention provides a positive regulatory gene NtMYB330 for synthesizing procyanidine in tobacco flower, which is characterized in that the nucleotide sequence of the positive regulatory gene NtMYB330 for synthesizing procyanidine in tobacco flower is shown as SEQ ID NO: 1 is shown.

The amino acid sequence coded by the procyanidine substance synthesis positive control gene NtMYB330 in the tobacco flower is shown as SEQ ID NO: 2, respectively.

The cloning method of the positive regulatory gene NtMYB330 for synthesizing procyanidine substances in the tobacco flowers is characterized by comprising the following steps of:

A. extracting tobacco flower tissue RNA, and performing reverse transcription to obtain first-strand cDNA;

B. designing and synthesizing a specific primer according to the NtMYB330 gene sequence, performing PCR amplification by using a first strand cDNA obtained by reverse transcription as a template, and recovering and purifying a PCR product;

C. connecting the purified amplification product with a TOPO carrier, wherein the connection system and the process are as follows: 4 μ L of purified product, 1 μ L of salt solution, 1 μ LMixing with Blunt II-TOPO, and water bathing at 25 deg.C for 30 min; and (3) carrying out heat shock transformation on the connected vector to enter escherichia coli DH5 alpha, adding a liquid culture medium for shaking culture, then coating the obtained product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting a bacterial colony for bacterial liquid culture, carrying out plasmid extraction and carrying out PCR (polymerase chain reaction) detection. Screening positive clones, and sequencing the positive clones.

The primer in the step B is as follows:

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’(SEQ ID NO.3)，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’(SEQ ID NO.4)。

And B, selecting a Phusion high-fidelity amplification enzyme reaction system as a PCR amplification reaction system in the step B, wherein the total volume of the system is 50 mu L, and the method comprises the following steps: 200ng cDNA, 10. mu.L of 5 XPPhusion HF reaction buffer, 1. mu.L of 10mM dNTP, 2UHigh-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 of the PCR amplification in the step B areThe pro amplification instrument is used for carrying out the following reaction procedures: 30 seconds at 98 ℃; 7 seconds at 98 ℃; 30 seconds at 62 ℃; 72 ℃ for 45 seconds; 35 cycles; extension at 72 ℃ for 7 minutes.

The overexpression vector of the gene NtMYB330 for positively regulating and controlling the content of procyanidine in the tobacco flower is characterized in that the overexpression vector of the gene NtMYB330 for positively regulating and controlling the content of procyanidine in the tobacco flower is pK2GW7-NtMYB 330.

The application of the positive control gene NtMYB330 for procyanidine synthesis in tobacco flowers is characterized in that the positive control gene NtMYB330 for procyanidine synthesis in tobacco flowers is applied to obtaining transgenic plants with the content of procyanidine substances in tobacco flowers remarkably improved.

The method for obtaining the NtMYB330 transgenic tobacco comprises the following steps:

A. construction of overexpression cloning vectors:

1. cloned NtMYB330 gene linked TOPO vector

(1) Taking cDNA of flower tissue of a tobacco variety Yunyan 87 as a template, utilizing NtMYB330 gene specific primers to amplify to obtain a gene fragment with the size of about 1.2kb, and purifying and recycling the gene fragment;

(2) the recovered NtMYB330 gene fragment is subjected to TOPO cloning and connected toTransforming Escherichia coli DH5 alpha competent cells after Blunt II-TOPO (3.5kb) vector, extracting plasmid for PCR detection, selecting plasmid with amplification product size of about 1.2kb to extract DNA, and naming the constructed vector as pTOPO-NtMYB 330;

(3) because the upstream and downstream primers of the gene respectively have recognition sites of BamH I and Xho I, the two enzymes are selected to carry out double enzyme digestion detection on a plasmid sample with correct PCR detection, and the enzyme digestion results generate two fragments with the sizes of about 3.5kb and 1.2kb respectively, which indicates that the target fragment is inserted into a TOPO vector, and the target gene fragment (1.2kb) is recovered by glue.

2. Construction of plant overexpression vectors

a. Entry clone pENTR^TMConstruction of 2B-NtMYB330

(1) BamH I/Xho I digestion of pTOPO-NtMYB330 and pENTR^TM2B, obtaining a target gene fragment NtMYB330 and a carrier pENTR^TM2B linearization segment, gel recovery, connection and transformation of competent cell DH5 alpha;

(2) the clone transformed into DH5 alpha is picked up, plasmid DNA is extracted, enzyme digestion is carried out by BamH I/Xho I, the enzyme digestion result is 3.5kb carrier fragment and about 1.2kb fragment are positiveThe exact clone, the correct clone was designated pENTR^TM2B-NtNtMYB330；

b. Obtaining plant expression vectors by LR reaction

(1) Entry clone pENTR^TMAfter the reaction of NtMYB330 and expression vector pK2GW7 LR, E.coli DH5 alpha competent cells are transformed, and plant expression vector pK2GW7-NtMYB330 is obtained. Using the BamH I/Xho I cleavage to identify pK2GW7-NtMYB330, the correct clone cut out two fragments of approximately 1.2kb and 11 kb.

The above LR reaction system: successfully constructed entry vector pENTR^TM-NtMYB330(50-150ng) 1-7. mu.L, 0.5. mu.L Destination Vector, TE Buffer to a total volume of 8. mu.L; mixing, ice-cooling for 2min, and flicking for 2 times; add 2. mu.L LR Clonease^TMII, flicking, uniformly mixing, centrifuging and carrying out water bath at 25 ℃ for 1 h; then 1. mu.L of protease K is added for flicking, mixed evenly and bathed in water at 37 ℃ for 10 min.

B. Genetic transformation of tobacco:

(1) expression vector transformation of agrobacterium

Taking out the agrobacterium-infected cells from a refrigerator at the temperature of-80 ℃, placing the cells on ice for dissolving, and adding 4 mu L of recombinant expression vector pK2GW7-NtMYB 330; quickly freezing for 1min with liquid nitrogen, transferring into 37 deg.C water bath for 5min, ice-cooling for 2min, adding 1mL LB liquid culture medium into the mixture, culturing at 28 deg.C and 220rpm for 3-4 hr; the culture is coated on LB solid culture medium containing 100mg/L spectinomycin and 25mg/L rifampicin, inverted culture is carried out for 2-3 days at 28 ℃, and agrobacterium clone containing a target vector can be seen;

(2) tobacco transformation

a. Selecting agrobacterium clone containing a target vector, streaking on an LB (Langerhans) plate containing spectinomycin and rifampicin, and culturing for 2-3 days at 28 ℃; scraping streak plaque, inoculating bacteria in an MS culture medium containing spectinomycin and rifampicin, carrying out shake culture at 28 ℃ and 220rpm, centrifuging at 6,000rpm for 5 minutes when the bacterial liquid concentration reaches OD (0.5-0.8) to enrich the bacteria, removing supernatant, and suspending the bacteria by using 20mL liquid MS culture medium to obtain agrobacterium suspension bacterial liquid containing a target carrier;

b. putting the tobacco leaves into a 500mL wide-mouth bottle, adding a proper amount of 75% ethanol, and rinsing for 1 min; discarding ethanol, adding0.1% HgCl₂Placing the solution on a shaking table, and oscillating for 15-30 minutes at room temperature; discard HgCl₂The solution is washed with sterile water for 6 times;

c. taking out the tobacco leaves, absorbing surface liquid by using sterile absorbent paper, cutting the sterile leaves into small pieces of about 1cm multiplied by 1cm by using scissors, putting the cut tobacco leaves into sterile MS liquid culture medium suspension bacteria liquid containing a target carrier, and standing for 15-20 min; taking out tobacco leaf, removing excess bacteria solution with sterile filter paper, and dark culturing in MS culture medium containing 6-BA (0.02mg/L) and NAA (2mg/L) at 25 deg.C for two days; then, the tobacco leaves are transferred into a differentiation culture medium, and the cut contacts the culture medium to perform differentiation culture under the greenhouse condition; the differentiation culture medium is MS culture medium containing 6-BA (0.5mg/L), NAA (0.1mg/L), kanamycin (100mg/L) and cefamycin (500mg/L), subcultured for 1 time every 2-3 weeks, callus gradually grows from incision, and finally differentiation buds;

d. cutting off buds growing to 3-5cm, transferring the buds into an MS culture medium to induce rooting, taking out the transgenic plants after rooting, washing the culture medium with tap water, and transplanting the transgenic plants into sterilized nutrient soil;

e. the transgenic plant is subjected to NPTII gene specific primers:

NPTII-F:5’-TCGGCTATGACTGGGCACAACAGA-3’(SEQ ID NO.5)，

NPTII-R: 5'-AAGAAGGCGATAGAAGGCGATGCG-3' (SEQ ID NO.6) PCR-verified amplification, identifying transgenic positive plants.

The first purpose of the invention is realized by that the nucleotide sequence of the positive regulatory gene NtMYB330 for synthesizing procyanidine substances in the tobacco flower is shown in a sequence table SEQ ID NO. 1.

The second object of the present invention is achieved by comprising the steps of:

A. extracting tobacco flower tissue RNA, and performing reverse transcription to obtain first-strand cDNA;

B. designing and synthesizing a specific primer according to the NtMYB330 gene sequence, performing PCR amplification by using a first strand cDNA obtained by reverse transcription as a template, and recovering and purifying a PCR product;

C. the purified amplification product is connected with the TOPO carrier through a kit reactionThe connection system and the process are as follows: 4 μ L of purified product, 1 μ L of salt solution, 1 μ LMixing with Blunt II-TOPO, and water bathing at 25 deg.C for 30 min; and (3) carrying out heat shock transformation on the connected vector to enter escherichia coli DH5 alpha, adding a liquid culture medium for shaking culture, then coating the obtained product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting a bacterial colony for bacterial liquid culture, carrying out plasmid extraction and carrying out 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 positive control gene NtMYB330 for synthesizing the procyanidine substances in the tobacco flowers to obtain transgenic plants with the content of the procyanidine substances in the tobacco flowers remarkably improved; namely, the positive control gene NtMYB330 for synthesizing the procyanidine substance in the tobacco flower is used for improving the content of the procyanidine substance in the tobacco flower.

The invention discloses a proanthocyanidin substance regulatory factor NtMYB330, an expression vector, a transformant, a kit and a method thereof, wherein the proanthocyanidin substance synthetic forward regulatory gene NtMYB330 nucleotide sequence is shown as SEQ ID: no.1, the coded amino acid sequence is shown as SEQ ID: no. 2. The invention discloses a cloning method of a positive regulatory factor NtMYB330 gene synthesized by procyanidine substances in tobacco flowers, which comprises the following specific steps: A. determining the sequence of the NtMYB330 gene; B. extracting tobacco flower tissue RNA, and performing reverse transcription to obtain first-strand cDNA; C. designing and synthesizing a specific primer according to the NtMYB330 gene sequence, and carrying out PCR amplification by taking cDNA as a template; D. recovering and purifying PCR products, and sequencing; E. constructing an overexpression vector containing the NtMYB330 gene. And (3) carrying out overexpression on the overexpression vector in tobacco through agrobacterium-mediated transformation to prepare a transgenic plant. Compared with an empty vector control, the content of the catechin in the obtained transgenic plant flowers is averagely increased by 431.5%; the epicatechin content increased by 406.5% on average. The tobacco NtMYB330 gene has a wide application prospect in the aspect of improving the content of procyanidine in tobacco flowers.

The invention obtains the structural gene co-expression with the flavonoid substance synthetic pathway by transcriptome sequencingSeveral MYB transcription factors are expressed, wherein the NtMYB330 gene encodes a protein sequence which, except for the amino-terminal R2R3 repeat sequence and the highly conserved [ D/E ] binding to bHLH protein]Lx₂[R/K]x₃Lx₆Lx₃Outside the R domain, VI [ R/P ] is present in the sequence at the carboxy terminus]TKAx₁RC[S/T]The structural domain, and the R2R3-MYB type transcription factor containing the structural domain is thought to be involved in regulating the synthesis of procyanidine, and belongs to the second evolutionary branch of procyanidine regulating factors. Homology analysis is carried out on the protein sequence of the NtMYB330 and the protein sequence of other plant R2R3-MYB transcription factors with known regulation functions, and the NtMYB330 is found to belong to the second evolutionary branch of the procyanidine regulation factor.

The transcription factor complex is involved in regulating the transcription activity of structural genes of different branch pathways of the flavonoid (such as anthocyanin and procyanidine branch pathways), and is a main mechanism for regulating and controlling the synthesis of the flavonoid substance. The transcription factor complex includes R2R 3-type MYB transcription factors responsible for binding to DNA, as well as bHLH transcription factors and WD40 regulatory proteins, where the R2R3-MYB transcription factors determine which target genes the complex is able to regulate. The tobacco NtMYB330 cloned by the invention is an R2R3-MYB type transcription factor for regulating and controlling flavonoid ways, and directly participates in the synthesis of transcription regulation procyanidine substances. The invention shows that the over-expression NtMYB330 gene can obviously improve the content of catechin and epicatechin in the tobacco flower through related experimental results, and the result provides a target gene for improving the content of procyanidine substances in the tobacco flower by utilizing a plant genetic engineering technology.

The tobacco procyanidine regulating factor NtMYB330 cloned by the invention discloses a regulating mechanism of a metabolic pathway of the procyanidine of the tobacco to a certain extent, enriches the research of higher plants in the field, and is also beneficial to the functional research of the procyanidine related genes of other plants by taking the tobacco as a model plant and a research platform.

The invention obtains a positive control gene NtMYB330 synthesized by procyanidine substances in tobacco flowers from tobacco, and the method comprises the following specific steps: a plurality of MYB transcription factors co-expressed with structural genes of flavonoid substance synthesis pathways are obtained through transcriptome sequencing, wherein homology analysis of protein sequences coded by NtMYB330 genes and protein sequences of other plant R2R3-MYB transcription factors with known regulation functions suggests that NtMYB330 belongs to procyanidine regulation factor clade two. Designing a gene specific primer according to the gene sequence information to carry out PCR reaction to obtain a target product; sequencing a target product to obtain a NtMYB330 gene sequence; an Overexpression (OE) strain of the NtMYB330 gene is obtained by utilizing an agrobacterium-mediated genetic transformation method, and the function identification is carried out on the NtMYB330, so that the result shows that the overexpression of the NtMYB330 gene can obviously improve the accumulation amount of procyanidine substances in tobacco flowers, and compared with an empty vector control, the content of catechin in the obtained transgenic plant flowers is averagely improved by 431.5%; the epicatechin content increased by 406.5% on average. The tobacco NtMYB330 gene has a wide application prospect in the aspect of improving the content of procyanidine in tobacco flowers.

Drawings

FIG. 1 is an agarose gel electrophoresis chart of PCR amplification product of the NtMYB330 gene in Experimental example 1, wherein the size of the NtMYB330 is 1164bp, and L is DL 5000DNA Marker;

FIG. 2 shows the Agrobacterium transformation effect of the recombinant over-expression vector pK2GW7-NtMYB330 detected by colony PCR in Experimental example 3, wherein L is DL 2000DNA Ladder, and 1, 2 and 3 are PCR products;

FIG. 3 shows the result of tissue-specific expression analysis of the NtMYB330 gene in Experimental example 3 of the present invention. Because the relative expression amount of the gene is greatly different among different tissues. The histogram of fig. 3 represents the treatment of Y-axis breaks as a common graphical treatment method in prior art articles in the field, and the purpose of this treatment is that when the difference between different treatments/tissues is large, the columns for "flowers" and "seeds" are high, while the columns for "stems" and "leaves" are substantially invisible. To express a tissue with a low expression level, the Y-axis is fractured, and the difference is visually reduced in the vacant part.

FIG. 4 shows T in Experimental example 4 of the present invention₂Analyzing the expression level of a procyanidine substance biosynthesis pathway gene in a transgenic plant flower of the NtMYB330 gene; VC is an empty vector control, OE1 and OE2 are transgenic lines, A is NtDFR1, B is NtDFR2, C is NtANS1, D is NtANS2, E is NtLAR1, F is NtLAR2, G is NtANR1,h is NtANR 2. "x" and "x" indicate that the relative expression of the gene of the transgenic plant was significantly different from the empty vector control VC at the 0.05 and 0.01 levels, respectively;

FIG. 5 is T₂Analyzing the content of procyanidine substances in the transgenic plant flowers of the NtMYB330 gene; a is catechin content analysis, B is epicatechin content analysis, VC is empty vector control, and OE1 and OE2 are transgenic strains. "x" and "x" indicate that the procyanidin content of the transgenic plants differed significantly from the empty vector control VC at 0.05 and 0.01 levels, respectively.

Detailed Description

The present invention is further illustrated by the following examples and the accompanying 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.

Group 1 examples of proanthocyanidin substance regulatory factor NtMYB330 of the present invention

The present group of embodiments provides a procyanidin substance modulating factor NtMYB 330. All embodiments of this group share the following common features: the amino acid sequence of the proanthocyanidin substance regulatory factor NtMYB330 comprises: R2R3 repetitive sequence, [ D/E]Lx₂[R/K]x₃Lx₆Lx₃R domain and VI [ R/P]TKAx₁RC[S/T]A domain.

[D/E]Lx₂[R/K]x₃Lx₆Lx₃The R domain is described in "A single amino acid mutation in the R3 domain of GLABRA1 leads to inhibition of chromosome formation in Arabidopsis with out interaction with GLABRA 3";

VI[R/P]TKAx₁RC[S/T]the domain is described in "The Arabidopsis TT2 gene codes an R2R3 MYB domain protein that acts as a key reagent for a protease amplification in a purification seed";

the R2R3 repeat sequence is described in "Ectopic expression of the coleus R2R3 MYB-Type proanthocyanidin regulator gene SsMYB3 alcohols the flow color in transgenic tobaca".

In some embodiments, the amino acid sequence of the procyanidin substance regulatory factor NtMYB330 comprises R2R3 repetitive sequence and [ D/E ] from amino terminal to carboxyl terminal in sequence]Lx₂[R/K]x₃Lx₆Lx₃R domain, VI [ R/P]TKAx₁RC[S/T]A domain.

In some embodiments, the amino acid sequence of the procyanidin modulator NtMYB330 is as set forth in SEQ ID NO: 2, respectively.

In other embodiments, the nucleotide sequence of the procyanidine substance regulatory factor NtMYB330 gene is as set forth in SEQ ID NO: 1 is shown in the specification;

preferably, the procyanidin substance regulatory factor NtMYB330 is tobacco Nicotiana tabacum l.

Group 2 example, expression vectors of the present invention for regulating the Synthesis of procyanidin substances

The embodiments of this group provide an expression vector that can regulate the synthesis of procyanidine substances. All embodiments of this group share the following common features: linked with the gene sequence of the procyanidine substance regulatory factor NtMYB330 provided in any one of group 1.

Preferably, the expression vector for regulating the synthesis of the procyanidine substance refers to a gene sequence of the procyanidine substance regulating factor NtMYB330 connected to the expression vector;

preferably, the expression vector is selected from the group consisting of: pK2GW7 vector;

preferably, the regulation of procyanidin synthesis is positive regulation;

preferably, the procyanidin substance: including catechin, epicatechin;

preferably, the positive control means that the content of procyanidin substances is preferably increased by more than 3 times.

Group 3 example, transformants capable of regulating the Synthesis of procyanidin substance of the invention

The present group of embodiments provides a transformant capable of regulating the synthesis of a procyanidin substance. All embodiments of this group share the following common features: an expression vector for regulating the synthesis of procyanidine provided in any one of the embodiments of group 2 is transformed in the transformant for regulating the synthesis of procyanidine.

Preferably, the transformant capable of regulating the synthesis of the procyanidine substance refers to an expression vector which is transformed in a host cell and is capable of regulating the synthesis of the procyanidine substance;

preferably, the host cell is selected from the group consisting of: plant cells or microbial cells;

preferably, the plant is selected from the group consisting of: arabidopsis thaliana and tobacco;

preferably, the microorganism is selected from: escherichia coli and Agrobacterium.

According to the teaching of the present invention, those skilled in the art can try to use the proanthocyanidin substance regulatory factor NtMYB330, the expression vector for regulating the synthesis of the proanthocyanidin substance, and the transformant for regulating the synthesis of the proanthocyanidin substance of the present invention to perform transgenosis on plants other than arabidopsis thaliana and tobacco through conventional transgenic operations in the art, that is, to use the gene overexpression vector for transferring the proanthocyanidin substance regulatory factor NtMYB330 into plants other than arabidopsis thaliana and tobacco, so that the effect of positively regulating proanthocyanidin substances (catechin and epicatechin) similar to that of the transgenic tobacco of the present invention can be theoretically obtained. Group 4 example, kits for controlled Synthesis of procyanidins of the invention

The group of embodiments provides a kit capable of regulating and controlling procyanidine substance synthesis. All embodiments of this group share the following common features: the kit comprises: the procyanidin substance modulating factor NtMYB330 provided in any one of group 1 embodiments, and/or an expression vector capable of regulating and controlling procyanidin substance synthesis provided in any one of group 2 embodiments, and/or a transformant capable of regulating and controlling procyanidin substance synthesis provided in any one of group 3 embodiments.

In a further embodiment, the kit for regulating the synthesis of procyanidin substances further comprises: an amplification primer of a gene sequence of the proanthocyanidin substance regulatory factor NtMYB330, a PCR (polymerase chain reaction) common reagent, a connection transformation common reagent and a transgene common reagent;

preferably, the amplification primer of the gene sequence of the procyanidine substance regulatory factor NtMYB330 comprises:

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’；

Preferably, the common reagents for PCR include: DNA polymerase, dNTP, PCR buffer solution and double distilled water;

in some embodiments, the DNA polymerase is preferablyHigh-Fidelity DNA Polymerase, wherein the PCR buffer solution is preferably 5 XPHUSION HF reaction buffer solution;

preferably, the common reagents for ligation transformation include: restriction enzyme, connection buffer solution, expression vector, protease, competent cell and culture medium;

preferably, the restriction enzyme is preferably BamH I, Xho I; the LR reaction system is preferably Clonease^TMII, enzyme Mix; the connection Buffer solution is preferably TE Buffer; the protease is preferably protease K;

preferably, the expression vector is selected from the group consisting of: pK2GW7 vector; based on the common technical knowledge in the field, the skilled person can choose to use other expression vectors commonly used in the field according to the actual production requirement by combining the teaching of the present invention.

Preferably, the competent cell is selected from the group consisting of: competent cells of Escherichia coli and competent cells of Agrobacterium; based on the common technical knowledge in the field, the skilled person can choose to use other competent cells commonly used in the field according to the actual production requirement by combining the teaching of the present invention. Competent cells are also host cells for expression vectors in biological nature.

Preferably, the medium is selected from LB medium.

Example 5, a method of regulating procyanidin Synthesis according to the invention

The present group of embodiments provide a method for regulating the synthesis of a procyanidin substance. All embodiments of this group share the following common features: overexpresses the procyanidin substance regulatory factor NtMYB330 provided in any one of group 1 examples in a plant, and/or transforms an expression vector that regulates procyanidin substance synthesis provided in any one of group 2 examples in a plant, and/or infects a plant with a transformant that regulates procyanidin substance synthesis provided in any one of group 3 examples.

In a specific embodiment, the method for regulating the synthesis of the procyanidin substance comprises the following steps: PCR amplifying the gene sequence of the proanthocyanidin substance regulatory factor NtMYB330 to obtain a PCR amplification product;

preferably, the PCR amplification system comprises: 4 ng/. mu.L plant cDNA, 5 XPPhusion HF reaction buffer 0.2. mu.L/. mu.L, 0.2mM dNTP, 0.04U/. mu.LHigh-Fidelity DNA Polymerase, 0.2 mu M forward and reverse primers, and the balance of water;

preferably, the forward and reverse primers comprise:

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’；

Preferably, the PCR amplification procedure comprises: 30 seconds at 98 ℃; at 98 ℃ for 7 seconds; 30 seconds at 62 ℃; 1 cycle at 72 ℃ for 45 seconds, and 35 cycles in total; extension at 72 ℃ for 7 min;

preferably, the method further comprises: connecting the PCR amplification product with an expression vector to obtain an expression vector for synthesizing the adjustable procyanidin substance;

preferably, the connection refers to an expression vector pENTR connected with a gene sequence of the proanthocyanidin substance regulatory factor NtMYB330^TMThe NtMYB330 is further connected through LR reaction to obtain an expression vector pK2GW7-NtMYB330 capable of regulating procyanidine synthesis;

preferably, the system for LR reaction comprises: pENTR of 6.25 ng/. mu.L-18.75 ng/. mu.L^TMNtMYB330, 0.0625 μ L/μ L of a connecting vector, and the balance TE Buffer;

preferably, the LR reaction step comprises: connecting the reaction system, mixing, performing ice bath for 2min, and mixing; add 0.2. mu.L/. mu.L LR Clonease^TMII, mixing uniformly, centrifuging, and carrying out water bath at 25 ℃ for 1 h; then adding 0.9 mu L/mu L of protease K, uniformly mixing, and carrying out water bath at 37 ℃ for 10 min; the blending mode is preferably light and elastic;

preferably, the method further comprises: transforming an expression vector pK2GW7-NtMYB330 capable of regulating the synthesis of the procyanidine substances into competent cells to obtain a transformant capable of regulating the synthesis of the procyanidine substances;

preferably, the method further comprises: infecting plant cells with a transformant capable of regulating the synthesis of procyanidine substances;

preferably, the competent cell is an agrobacterium competent cell; the transformant for regulating the synthesis of the procyanidine substances refers to agrobacterium clone containing an expression vector pK2GW7-NtMYB330 for regulating the synthesis of the procyanidine substances;

preferably, the plant is preferably tobacco;

preferably, the regulation of procyanidin synthesis is positive regulation;

preferably, the procyanidin substance: including catechin, epicatechin;

preferably, the positive control means that the content of procyanidin substances is preferably increased by more than 3 times.

The most specific embodiments of the present invention are described below:

the nucleotide sequence of the procyanidine synthesis positive control gene NtMYB330 in the tobacco flower is shown in a sequence table SEQ ID NO: 1 is shown.

The amino acid sequence coded by the procyanidine synthesis positive control gene NtMYB330 in the tobacco flower is shown as SEQ ID NO: 2, respectively.

The invention relates to a cloning method of a procyanidine synthesis positive control gene NtMYB330 in tobacco flowers, which is characterized by comprising the following steps of:

A. extracting tobacco flower tissue RNA, and performing reverse transcription to obtain first-strand cDNA;

B. designing and synthesizing a specific primer according to the NtMYB330 gene sequence, performing PCR amplification by using a first strand cDNA obtained by reverse transcription as a template, and recovering and purifying a PCR product;

C. the purified amplification product is connected with a TOPO carrier through a kit reaction, and the connection system and the process are as follows: 4 μ L of purified product, 1 μ L of salt solution, 1 μ LMixing with Blunt II-TOPO, and water bathing at 25 deg.C for 30 min; and (3) carrying out heat shock transformation on the connected vector to enter escherichia coli DH5 alpha, adding a liquid culture medium for shaking culture, then coating the obtained product on an LB (lysogeny broth) plate containing 100mg/L kanamycin for overnight culture, selecting a bacterial colony for bacterial liquid culture, carrying out plasmid extraction and carrying out PCR (polymerase chain reaction) detection. Screening positive clones, and sequencing the positive clones.

And B, selecting a Phusion high-fidelity amplification enzyme reaction system as a PCR amplification reaction system in the step B, wherein the total volume of the system is 50 mu L, and the method comprises the following steps: 200ng cDNA, 10. mu.L of 5 XPPhusion HF reaction buffer, 1. mu.L of 10mM dNTP, 2UHigh-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 of the PCR amplification in the step B areThe pro amplification instrument is used for carrying out the following reaction procedures: 30 seconds at 98 ℃; 7 seconds at 98 ℃; 30 seconds at 62 ℃; 72 ℃ for 45 seconds; 35 cycles; extension at 72 ℃ for 7 minutes.

The application of the positive regulation gene NtMYB330 for synthesizing the procyanidine substances in the tobacco flowers is the application of the positive regulation gene NtMYB330 for synthesizing the procyanidine substances in the tobacco flowers in obtaining transgenic plants with the content of the procyanidine substances in the tobacco flowers remarkably improved.

The method for obtaining the transgenic plant with the remarkably improved content of the procyanidine substances in the tobacco flowers comprises the following steps:

A. construction of overexpression cloning vectors:

1. cloned NtMYB330 ligation TOPO vector

(1) Taking cDNA of flower tissue of a tobacco variety Yunyan 87 as a template, utilizing NtMYB330 gene specific primers to amplify to obtain a gene fragment with the size of about 1.2kb, and purifying and recycling the gene fragment;

(2) the recovered NtMYB330 gene fragment is subjected to TOPO cloning and connected toTransforming Escherichia coli DH5 alpha competent cells after Blunt II-TOPO (3.5kb) vector, extracting plasmid for PCR detection, selecting plasmid with amplification product size of about 1.2kb to extract DNA, and naming the constructed vector as pTOPO-NtMYB 330;

(3) because the upstream and downstream primers of the gene respectively have recognition sites of BamH I and Xho I, the two enzymes are selected to carry out double enzyme digestion detection on a plasmid sample with correct PCR detection, and the enzyme digestion results generate two fragments with the sizes of about 3.5kb and 1.2kb respectively, which indicates that the target fragment is inserted into a TOPO vector, and the target gene fragment (1.2kb) is recovered by glue.

2. Construction of plant overexpression vectors

a. Entry clone pENTR^TMConstruction of 2B-NtMYB330

(1) BamH I/Xho I digestion of pTOPO-NtMYB330 and pENTR^TM2B, obtaining a target gene fragment NtMYB330 and a carrier pENTR^TM2B linearization segment, gel recovery, connection and transformation of competent cell DH5 alpha;

(2) selecting clone transformed into DH5 alpha, extracting plasmid DNA, carrying out enzyme digestion by BamH I/Xho I, wherein the enzyme digestion result is that a carrier fragment of 3.5kb and a fragment of about 1.2kb are correct clones, and the correct clones are named as pENTR^TM2B-NtNtMYB330；

b. Obtaining plant expression vectors by LR reaction

(1) Entry clone pENTR^TMNtMYB330 and expression vector pK2GW7 LR are reacted and transformed into Escherichia coli DH5 alphaCompetent cells, resulting in the plant expression vector pK2GW7-NtMYB 330. Using the BamH I/Xho I cleavage to identify pK2GW7-NtMYB330, the correct clone cut out two fragments of approximately 1.2kb and 11 kb.

The above LR reaction system: successfully constructed entry vector pENTR^TMNtMYB330(50-150ng) 1-7. mu.L, 0.5. mu.L Destination Vector, TE Buffer to a total volume of 8. mu.L; mixing, ice-cooling for 2min, and flicking for 2 times; add 2. mu.L LR Clonease^TMII, flicking, uniformly mixing, centrifuging and carrying out water bath at 25 ℃ for 1 h; then 1. mu.L of protease K is added for flicking, mixed evenly and bathed in water at 37 ℃ for 10 min.

B. Genetic transformation of tobacco:

(1) expression vector transformation of agrobacterium

Taking out the agrobacterium-infected cells from a refrigerator at the temperature of-80 ℃, placing the cells on ice for dissolving, and adding 4 mu L of recombinant expression vector pK2GW7-NtMYB 330; quickly freezing for 1min with liquid nitrogen, transferring into 37 deg.C water bath for 5min, ice-cooling for 2min, adding 1mL LB liquid culture medium into the mixture, culturing at 28 deg.C and 220rpm for 3-4 hr; the culture is coated on LB solid culture medium containing 100mg/L spectinomycin and 25mg/L rifampicin, inverted culture is carried out for 2-3 days at 28 ℃, and agrobacterium clone containing a target vector can be seen;

(2) tobacco transformation

a. Selecting agrobacterium clone containing a target vector, streaking on an LB (Langerhans) plate containing spectinomycin and rifampicin, and culturing for 2-3 days at 28 ℃; scraping streak plaque, inoculating bacteria in an MS culture medium containing spectinomycin and rifampicin, carrying out shake culture at 28 ℃ and 220rpm, centrifuging at 6,000rpm for 5 minutes when the bacterial liquid concentration reaches OD (0.5-0.8) to enrich the bacteria, removing supernatant, and suspending the bacteria by using 20mL liquid MS culture medium to obtain agrobacterium suspension bacterial liquid containing a target carrier;

b. putting the tobacco leaves into a 500mL wide-mouth bottle, adding a proper amount of 75% ethanol, and rinsing for 1 min; ethanol was removed and 0.1% HgCl was added₂Placing the solution on a shaking table, and oscillating for 15-30 minutes at room temperature; discard HgCl₂The solution is washed with sterile water for 6 times;

c. taking out the tobacco leaves, absorbing surface liquid by using sterile absorbent paper, cutting the sterile leaves into small pieces of about 1cm multiplied by 1cm by using scissors, putting the cut tobacco leaves into sterile MS liquid culture medium suspension bacteria liquid containing a target carrier, and standing for 15-20 min; taking out tobacco leaf, removing excess bacteria solution with sterile filter paper, and dark culturing in MS culture medium containing 6-BA (0.02mg/L) and NAA (2mg/L) at 25 deg.C for two days; then, the tobacco leaves are transferred into a differentiation culture medium, and the cut contacts the culture medium to perform differentiation culture under the greenhouse condition; the differentiation culture medium is MS culture medium containing 6-BA (0.5mg/L), NAA (0.1mg/L), kanamycin (100mg/L) and cefamycin (500mg/L), subcultured for 1 time every 2-3 weeks, callus gradually grows from incision, and finally differentiation buds;

d. cutting off buds growing to 3-5cm, transferring the buds into an MS culture medium to induce rooting, taking out the transgenic plants after rooting, washing the culture medium with tap water, and transplanting the transgenic plants into sterilized nutrient soil;

e. the transgenic plant is subjected to NPTII gene specific primers:

NPTII-F:5’-TCGGCTATGACTGGGCACAACAGA-3’(SEQ ID NO.5)，

NPTII-R: 5'-AAGAAGGCGATAGAAGGCGATGCG-3' (SEQ ID NO.6) PCR-verified amplification, identifying transgenic positive plants.

The invention is further illustrated by the following specific experimental examples:

experimental example 1, isolation and cloning of NtMYB330 Gene, comprising the following steps:

1. determination of NtMYB330 gene sequence:

a plurality of MYB transcription factors co-expressed with structural genes of flavonoid substance synthesis pathways are obtained through transcriptome sequencing, wherein homology analysis of protein sequences coded by NtMYB330 genes and protein sequences of other plant R2R3-MYB transcription factors with known regulation functions suggests that NtMYB330 belongs to procyanidine regulation factor clade two. Designing a gene cloning primer according to the gene sequence to carry out PCR reaction to obtain a target product;

forward primer NtMYB330-BamH I: 5-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’(SEQ ID NO.3)，

Reverse primer NtMYB330-Xho I: 5' -CTCGAGTCAAGAGGAGAACCCATTAATCC-3’(SEQ ID NO.4)。

To construct an overexpression vector, BamH I and Xho I sites (underlined) were primed in the above primers;

2. the RNA of floral tissue of tobacco variety yunyan 87 was extracted using Trizol kit (Invitrogen) and the procedure was performed according to the instructions provided in the kit.

3. Taking the first strand cDNA obtained by reverse transcription as a template, carrying out PCR amplification by using a primer NtMYB330-BamH I F/NtMYB330-Xho I R pair, and selecting a Phusion high fidelity amplification enzyme reaction system with the total volume of 50 mu L, wherein the system comprises: 200ng cDNA, 10. mu.L of 5 XPPhusion HF reaction buffer, 1. mu.L of 10mM dNTP, 2UHigh-Fidelity DNA Polymerase, 1. mu.L each of 10. mu.M forward and reverse primers, and water was added to 50. mu.L. The PCR reaction is carried out inThe pro amplification instrument is used for carrying out the following reaction procedures: 30 seconds at 98 ℃; 7 seconds at 98 ℃; 30 seconds at 62 ℃; 72 ℃ for 45 seconds; 35 cycles; extension at 72 ℃ for 7 minutes. The PCR product was recovered and purified. The electrophoretogram of the PCR amplified product is shown in FIG. 1.

4. Connecting the purified amplification product with a carrier: 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 30 min; and transforming the connected vector into escherichia coli DH5 alpha by heat shock, adding a liquid culture medium for shaking culture, then coating the liquid culture medium on an LB (Langmuir-Blodgett) 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.

The formula and preparation method of the culture medium in the step 4 are that 8-12g of tryptone, 5g of yeast extract and 10g of NaCl are weighed and dissolved in 1L of distilled water, and the culture medium is obtained by sterilizing for 25min at 121 ℃.

Experimental example 2 construction of overexpression vector

1. Cloned NtMYB330 ligation TOPO vector

(1) Taking cDNA of flower tissue of a tobacco variety Yunyan 87 as a template, utilizing NtMYB330 gene specific primers to amplify to obtain a gene fragment with the size of about 1.2kb, and purifying and recycling the gene fragment;

(2) the recovered NtMYB330 gene fragment is subjected to TOPO cloning and connected toTransforming Escherichia coli DH5 alpha competent cells after Blunt II-TOPO (3.5kb) vector, extracting plasmid for PCR detection, selecting plasmid with amplification product size of about 1.2kb to extract DNA, and naming the constructed vector as pTOPO-NtMYB 330;

(3) because the forward and reverse primers of the gene are respectively provided with recognition sites of BamH I and Xho I, the two enzymes are selected to carry out double enzyme digestion detection on a plasmid DNA sample, and the enzyme digestion result generates two fragments with the sizes of about 3.5kb and 1.2kb respectively, which indicates that a target fragment is inserted into a TOPO vector;

2. construction of plant overexpression vectors

a. Entry clone pENTR^TMConstruction of 2B-NtMYB330

(1) BamH I/Xho I digestion of pTOPO-NtMYB330 and pENTR^TM2B, obtaining a target gene fragment NtMYB330 and a carrier pENTR^TM2B linearization segment, gel recovery, connection and transformation of competent cell DH5 alpha;

(2) selecting clone transformed into DH5 alpha, extracting plasmid DNA, carrying out enzyme digestion by BamH I/Xho I, wherein the enzyme digestion result is that a carrier fragment of 3.8kb and a fragment of about 1.2kb are correct clones, and the correct clones are named as pENTR^TM2B-NtNtMYB330；

b. Obtaining plant expression vectors by LR reaction

(1) Entry clone pENTR^TMAfter the reaction of NtMYB330 and expression vector pK2GW7 LR, E.coli DH5 alpha competent cells are transformed, and plant expression vector pK2GW7-NtMYB330 is obtained. Using BamH I/Xho I digestion to identify pK2GW7-NtMYB330, two fragments, approximately 1.2kb and 11kb, were excised from the correct clone.

The above LR reaction system: successfully constructed entry loadBody pENTR^TMNtMYB330(50-150ng) 1-7. mu.L, 0.5. mu.L Destination Vector (i.e., pK2GW7 Vector), TE Buffer to a total volume of 8. mu.L; mixing, ice-cooling for 2min, and flicking for 2 times; add 2. mu.L LR Clonease^TMII, flicking, uniformly mixing, centrifuging and carrying out water bath at 25 ℃ for 1 h; then 1. mu.L of protease K is added for flicking, mixed evenly and bathed in water at 37 ℃ for 10 min.

Experimental example 3 genetic transformation of tobacco

(1) Expression vector transformation of agrobacterium

Taking out the agrobacterium-infected cells from a refrigerator at the temperature of-80 ℃, placing the cells on ice for dissolving, and adding 4 mu L of recombinant expression vector pK2GW7-NtMYB 330; quickly freezing for 1min with liquid nitrogen, transferring into 37 deg.C water bath for 5min, ice-cooling for 2min, adding 1mL LB liquid culture medium into the mixture, culturing at 28 deg.C and 220rpm for 3-4 hr; the culture is coated on LB solid culture medium containing 100mg/L spectinomycin and 25mg/L rifampicin, inverted culture is carried out for 2-3 days at 28 ℃, and agrobacterium clone containing a target vector can be seen;

(2) tobacco transformation

a. Selecting agrobacterium clone containing a target vector, streaking on an LB (Langerhans) plate containing spectinomycin and rifampicin, and culturing for 2-3 days at 28 ℃; scraping streak plaque, inoculating bacteria in an MS culture medium containing spectinomycin and rifampicin, carrying out shake culture at 28 ℃ and 220rpm, centrifuging at 6,000rpm for 5 minutes when the bacterial liquid concentration reaches OD (0.5-0.8) to enrich the bacteria, removing supernatant, and suspending the bacteria by using 20mL liquid MS culture medium to obtain agrobacterium suspension bacterial liquid containing a target carrier;

b. putting the tobacco leaves into a 500mL wide-mouth bottle, adding a proper amount of 75% ethanol, and rinsing for 1 min; ethanol was removed and 0.1% HgCl was added₂Placing the solution on a shaking table, and oscillating for 15-30 minutes at room temperature; discard HgCl₂The solution is washed with sterile water for 6 times;

c. taking out tobacco leaf, removing surface liquid with sterile absorbent paper, cutting the tobacco leaf into small pieces of about 1cm × 1cm with scissors, placing the cut tobacco leaf into sterile MS liquid culture medium suspension bacteria solution containing target carrier, and standing for 15-20 min; taking out tobacco leaf, removing excess bacteria solution with sterile filter paper, and dark culturing in MS culture medium containing 6-BA (0.02mg/L) and NAA (2mg/L) at 25 deg.C for two days; then, the tobacco leaves are transferred into a differentiation culture medium, and the cut contacts the culture medium to perform differentiation culture under the greenhouse condition; the differentiation culture medium is MS culture medium containing 6-BA (0.5mg/L), NAA (0.1mg/L), kanamycin (100mg/L) and cefamycin (500mg/L), subcultured for 1 time every 2-3 weeks, callus gradually grows from incision, and finally differentiation buds;

d. cutting off buds growing to 3-5cm, transferring the buds into an MS culture medium to induce rooting, taking out the transgenic plants after rooting, washing the culture medium with tap water, and transplanting the transgenic plants into sterilized nutrient soil;

e. the transgenic plant is subjected to NPTII gene specific primers:

NPTII-F:5’-TCGGCTATGACTGGGCACAACAGA-3’(SEQ ID NO.5)，

NPTII-R:5’-AAGAAGGCGATAGAAGGCGATGCG-3’(SEQ ID NO.6)

PCR verification and amplification are carried out to identify the transgenic positive plants. The results of colony PCR are shown in FIG. 2.

(3) Gene tissue specific expression analysis experiment

Planting and cultivating the tobacco Yunyan 87, extracting root, stem, leaf, flower and seed RNA in flowering, and carrying out reverse transcription to obtain first-strand cDNA. Designing qRT-PCR primers according to the NtMYB330 gene sequence:

NtMYB330-F:5’-GGATCCATGGGAAGAAAGCCTTGTTGTTC-3’(SEQ ID NO.3)，

NtMYB330-R:5’-CTCGAGTCAAGAGGAGAACCCATTAATCC-3’(SEQ ID NO.4)。

taking tobacco Actin gene as an internal reference, wherein the expression of Actin-F: CTGAGGTCCTTTTCCAACCA and Actin-R: TACCCGGGAACATGGTAGAG are provided. Fluorescent quantitative PCR was performed using seed cDNA as template, and the reaction was performed on Roche LightCycler 480SYBR Green I master, 20. mu.L system containing 10. mu.L LightCycler 480SYBR Green I master (2X), 1. mu.L each of forward and reverse primers (10. mu. mol/L), 1. mu.L of cDNA (reverse transcription product diluted 4 times), and 7. mu.L of sterile distilled water. The reaction procedure was as follows: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 7s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 45 s; 35 cycles. Fluorescent quantitative PCR results Using 2^-△△CtThe method calculates the relative expression of the NtMYB330 gene. Each processing device3 biological replicates, histograms were drawn by Excel software (figure 3). The result of the tissue-specific expression analysis of the NtMYB330 gene is shown in FIG. 3, and the gene has higher expression level in tissues such as roots, flowers and seeds of tobacco.

Experimental example 4 analysis of transgenic plants

T₂Total RNA was extracted from the transgenic lines, and PrimeScript was obtained by TaKaRa^TMThe RT reagent Kit reverse transcription Kit synthesizes cDNA as a template to perform real-time fluorescent quantitative PCR analysis, and the obtained over-expressed (OE) strain is subjected to phenotype analysis (figure 4). T is₂The experimental results of the generation transgenic strains show that compared with the empty vector control, the content of the catechin in the obtained transgenic plant flowers is averagely increased by 431.5 percent by the NtMYB330 gene overexpression strains; the epicatechin content increased on average 406.5% (fig. 5).

SEQUENCE LISTING

<110> research institute of tobacco agricultural science in Yunnan province

<120> proanthocyanidin substance regulatory factor NtMYB330, and expression vector, transformant, kit and method thereof

<130> P210400/YCN

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1164

<212> DNA

<213> Artificial Sequence

<220>

<223> Nicotiana tabacum L. procyanidine substance regulatory factor gene NtMYB330

<400> 1

atgggaagaa agccttgttg ttctaaagaa ggattaaaca aaggggcatg gactcctatg 60

gaggataaaa ttctaataga ttatatcaaa gtaaatggtg aagggaaatg gagaaatctt 120

cccaaaagag ctggtcttaa aagatgtgga aagagttgca gactaaggtg gctgaattat 180

ctaaggccag acattaagag gggaaatata actccagatg aagaagatct cattatcaga 240

cttcataaac ttcttggaaa tagatggtct ctgatagctg gaaggttacc agaacgaaca 300

gacaatgaaa tcaagaatta ttggaacaca aacatcggca aaaaactaca acaaggagtt 360

gctcctggtc agccaaaccg cataatatct tccattaatc gtcagcgccc tcgttctagt 420

catgccaaat cttccaagtc cgacccagtt acccaaccaa acaaaaataa tcaagaacac 480

acagttccta atcaggattc acagtatttg ctaacagacg ttggattcgg aggatcatcg 540

tcttcttcat ccccgtgttt ggttatccgc acaaaggcaa ttaggtgcac taaagttttt 600

attactcctc ctcctactag tagttcggtt gctgagccac agaatgttga tcagtctcac 660

aatgagattg ctcaaagggc tagtaattct cactcagtct tcccaccttg caccaggaat 720

cccgttgagt tcttacgctt tcatgttgac aactcaattc ttgataatga taacgatgac 780

aaggtaatgg cggaggattt gacaatagaa aatgcaaata ctattgtagc atcgtcctca 840

tcatcgtcat cattatcagt gtcatctttg tccgagcagc aacaaccaat atcaggatca 900

acaccaactt tctctggaga attggaaaat tataacttta attttatgtt tggttttgat 960

atggacgatc cttttctttc tgagcttcta aatgcacctg atatatgtga aaacttggag 1020

aatacaacta ctgttggaga tagttgcagc aaaaacgaaa aggaaaggag ctatttccct 1080

tcgaattata gtcaaacaac attgttcgca gaagatacgc aacacaacga tttggaactt 1140

tggattaatg ggttctcctc ttga 1164

<210> 2

<211> 387

<212> PRT

<213> Artificial Sequence

<220>

<223> Nicotiana tabacum L. procyanidin substance regulatory factor NtMYB330

<220>

<221> REPEAT

<222> (11)..(63)

<223> R2 repetitive sequence

<220>

<221> REPEAT

<222> (64)..(115)

<223> R3 repetitive sequence

<220>

<221> misc_feature

<222> (76)..(95)

<223> [ DE ] Lx2[ RK ] x3Lx6Lx3R Domain

<220>

<221> misc_feature

<222> (188)..(197)

<223> VI [ R/P ] TKAx1RC [ S/T ] Domain

<400> 2

Met Gly Arg Lys Pro Cys Cys Ser Lys Glu Gly Leu Asn Lys Gly Ala

1 5 10 15

Trp Thr Pro Met Glu Asp Lys Ile Leu Ile Asp Tyr Ile Lys Val Asn

20 25 30

Gly Glu Gly Lys Trp Arg Asn Leu Pro Lys Arg Ala Gly Leu Lys Arg

35 40 45

Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp

50 55 60

Ile Lys Arg Gly Asn Ile Thr Pro Asp Glu Glu Asp Leu Ile Ile Arg

65 70 75 80

Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu

85 90 95

Pro Glu Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Asn Ile

100 105 110

Gly Lys Lys Leu Gln Gln Gly Val Ala Pro Gly Gln Pro Asn Arg Ile

115 120 125

Ile Ser Ser Ile Asn Arg Gln Arg Pro Arg Ser Ser His Ala Lys Ser

130 135 140

Ser Lys Ser Asp Pro Val Thr Gln Pro Asn Lys Asn Asn Gln Glu His

145 150 155 160

Thr Val Pro Asn Gln Asp Ser Gln Tyr Leu Leu Thr Asp Val Gly Phe

165 170 175

Gly Gly Ser Ser Ser Ser Ser Ser Pro Cys Leu Val Ile Arg Thr Lys

180 185 190

Ala Ile Arg Cys Thr Lys Val Phe Ile Thr Pro Pro Pro Thr Ser Ser

195 200 205

Ser Val Ala Glu Pro Gln Asn Val Asp Gln Ser His Asn Glu Ile Ala

210 215 220

Gln Arg Ala Ser Asn Ser His Ser Val Phe Pro Pro Cys Thr Arg Asn

225 230 235 240

Pro Val Glu Phe Leu Arg Phe His Val Asp Asn Ser Ile Leu Asp Asn

245 250 255

Asp Asn Asp Asp Lys Val Met Ala Glu Asp Leu Thr Ile Glu Asn Ala

260 265 270

Asn Thr Ile Val Ala Ser Ser Ser Ser Ser Ser Ser Leu Ser Val Ser

275 280 285

Ser Leu Ser Glu Gln Gln Gln Pro Ile Ser Gly Ser Thr Pro Thr Phe

290 295 300

Ser Gly Glu Leu Glu Asn Tyr Asn Phe Asn Phe Met Phe Gly Phe Asp

305 310 315 320

Met Asp Asp Pro Phe Leu Ser Glu Leu Leu Asn Ala Pro Asp Ile Cys

325 330 335

Glu Asn Leu Glu Asn Thr Thr Thr Val Gly Asp Ser Cys Ser Lys Asn

340 345 350

Glu Lys Glu Arg Ser Tyr Phe Pro Ser Asn Tyr Ser Gln Thr Thr Leu

355 360 365

Phe Ala Glu Asp Thr Gln His Asn Asp Leu Glu Leu Trp Ile Asn Gly

370 375 380

Phe Ser Ser

385

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> forward primer NtMYB330-BamH I

<400> 3

ggatccatgg gaagaaagcc ttgttgttc 29

<210> 4

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer NtMYB330-Xho I

<400> 4

ctcgagtcaa gaggagaacc cattaatcc 29

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer NPTII-F

<400> 5

tcggctatga ctgggcacaa caga 24

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> primer NPTII-R

<400> 6

aagaaggcga tagaaggcga tgcg 24