CN116240225A - Series tobacco NtHQT gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of tobacco genetic engineering, and in particular relates to a series of tobacco NtHQT genes and applications thereof. The series of tobacco NtHQT genes include tobacco hydroxycinnamoyl-CoA quinyl hydroxycinnamon transferase NtHQT2 gene and NtHQT3 gene. In this application, two new NtHQT2 and NtHQT3 genes were excavated based on the existing research on tobacco NtHQT genes. By using gene editing technology to silence these two genes and through overexpression experiments on these two genes, the results show that the expression of these two genes is positively correlated with the content of chlorogenic acid in tobacco leaves. Based on these results, it can not only lay a certain theoretical foundation for the research on the regulation pathway of secondary metabolites in tobacco, but also lay a certain technical foundation for the cultivation of new varieties of related plants.

Description

A Series of Tobacco NtHQT Genes and Their Applications

technical field

The invention belongs to the technical field of tobacco genetic engineering, and specifically relates to a series of tobacco NtHQT genes and applications thereof.

Background technique

Chlorogenic acid (CGA), also known as coffee tannin, is produced by plants through the phenylalanine pathway in the process of aerobic respiration in the natural synthesis pathway. Esters of acids such as caffeic acid, p-coumaric acid and ferulic acid.

As a phenolic antioxidant, chlorogenic acid can quickly eliminate hydroxyl radicals. In actual development and application, in addition to utilizing the antioxidant properties of chlorogenic acid, studies have also found that chlorogenic acid can inhibit and kill a variety of pathogenic bacteria and viruses, anti-tumor, inhibit mutation, protect liver and gallbladder, lower blood pressure, lower Blood lipids and many other functions. For example: studies have found that chlorogenic acid can inhibit gene mutations, the formation of carcinogenic N-nitroso compounds and DNA damage in vitro; it can significantly inhibit the proliferation and infiltration of tumor cells; Pharmacological effects such as viruses. Chlorogenic acid can also reduce triglycerides and cholesterol, prevent cardiovascular diseases such as hypertension and coronary heart disease, and resist obesity and diabetes by regulating glucose metabolism. It can also inhibit inflammation and reduce pain. Studies have shown that the wide range of biological functions of chlorogenic acid is positively related to its significant antioxidant activity. Some studies have found that the ability of chlorogenic acid and its derivatives to scavenge free radicals is significantly higher than that of classic antioxidants ascorbic acid and tocopherol. Effectively scavenge hydroxyl radicals and superoxide anion radicals. Chlorogenic acid can be directly absorbed by the small intestine in the body, or it can be converted into caffeic acid after being hydrolyzed by coliform bacteria. Caffeic acid, like chlorogenic acid, also has strong antioxidant capacity.

As a secondary metabolite, chlorogenic acid is expressed and distributed in a variety of plants. Measurements and surveys show that the content of chlorogenic acid in tobacco leaves is 3%-5%, which is one of the plant species with better content. Moreover, chlorogenic acid is the polyphenol compound with the highest content in tobacco leaves, accounting for about 70-90% of the total polyphenol content. Therefore, it is of great technical significance to study the regulation pathway of chlorogenic acid synthesis in tobacco leaves for the cultivation of tobacco leaf varieties and the development of tobacco resources.

Studies on the relevant synthesis mechanism have shown that hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) is a key enzyme in the synthetic and metabolic pathway of plant chlorogenic acid, which can catalyze caffeoyl-CoA and quinic acid Produces chlorogenic acid. Using genetic engineering technology, in some plants, through overexpression or gene silencing, the content of chlorogenic acid in plants can be increased or decreased accordingly. However, as a member of the BAHD (acyl-CoA-dependent acyltransferase) family, HQT itself has a relatively complex gene structure and includes multiple gene types. The in-depth study of pathways and the cultivation of related plant varieties are of great technical significance.

Contents of the invention

On the basis of the applicant's existing research work on tobacco NtHQT genes, the purpose of this application is to provide two new tobacco hydroxycinnamoyl-CoA quinyl hydroxycinnamon transferase NtHQT genes (NtHQT2, NtHQT3), so as to provide tobacco (tobacco) The regulation mechanism of secondary metabolites and the cultivation of new tobacco varieties have laid a certain technical foundation.

The technical solution adopted by this application is described in detail as follows.

A series of tobacco NtHQT genes, including tobacco hydroxycinnamoyl-CoA quinyl hydroxycinnamon transferase NtHQT2 gene and NtHQT3 gene;

The NtHQT2 gene is related to the synthesis of chlorogenic acid in tobacco (tobacco leaves) (or related to the regulation of chlorogenic acid content in tobacco leaves), and its clone is obtained from common tobacco K326, and its base sequence is shown in SEQ ID No.1; details as follows:

NtHQT2 gene sequence (1311bp)

ATGGGAAGTGAAAAGATGATGAAGATTAACATCAAAGAATCAACATTAGTAAAACCATCAAAACCAACACCAACAAAGAGACTTTGGAATTCTAACTTAGATTTAATAGTTGGAAGAATTCATCTTTTAACAGTATATTCTATAAACCAAATGGATCTTCAAATTTCTTTGATTCTAAAGTAATGAAAGAAGCATTAAGTAATGTTCTTGTTTCATTTTATCCAATG GCTGGGAGATTTAGCTAGAGATGAACAAGGAAGAATTGAGATAAATTGTAATGGAGAAGGAGTTTTGTTTGTTGAAGCTGAAAGTGATGCTTTTGTTGATGATTTTGGTGATTTTACTCCAAGTTTGGAACTTAGGAAACTTATTCCTACTGTTGATACTTCTGGTGATATTTCTACTTCCCCCTCATCTTTCAGGTTACTCGTTTCAAATGTGGTGGAGT TTCACTTGGTGGAGGAGTATTCCACACTCTATCAGATGGTCTCTCATCCATTCACTTCATCAACACATGGTCCGATATTGCCCGAGGCCTCTCCGTCGTCATCCCGCCGTTCATTGACCGGACCCCTCCTCCGTGCACGGGACCCACCGACATCGTCGTTCGAACACGTCGAGTATCATCCTCCTCCATCTCTTATTTCATCATCAAAAACCAAAGAATCCACTAGCCC AAAGCCTAGTACCACAACCATGTTAAAATTCTCTAGTGACCAACTTGGGCTTCTAAAGTCCAAGTCCAAACATGATGGTAGCACTTACGAAATCCTCGCGGCCCATATTTGGCGTTGCACGTGCAAGGCACGTGCACTGTCCGACGATCAATTGACCAAATTACATGTGGCCACTGATGGTAGGTCTAGGCTTTGCCCTCCTTTGCCACCAGGTTACTTAGGAA ATGTTGTGTTCACAGGCACACCTATGGCAAAATCAAGTGAACTTTTACAAAGAACCATTGACAAATTCAGCCAAGAGAATTCATAGTGCATTATCAAAAATGGATGACAATTACCTAAGATCAGCTCTCGATTACCTCGAATTACTGCCCGATTTATCGGCTTTAATCCGTGGACCGACGTACTTTGCTAGCCCTAATCTTAATATTAATAGTTGGACTAGATTGCCTGTTCATGA TTCAGATTTTGGATGGGGAAGGCCAATTCATATGGGACCAGCTTGCATTTTATATGAAGGGACAGTTTATATTGCCAAGTCCAAATAGTAAAGATAGGAACTTGCGTTTGGCTGTTTGTTTAGATGCTGATCACATGCCACTATTTGAGAAGTATTTGTATGAATTTTGA;

Correspondingly, the amino acid sequence (436AA) of the NtHQT2 protein encoded by the NtHQT2 gene is as follows:

MGSEKMMKINIKESTLVKPSKPTPTKRLWNSNLDLIVGRIHLLTVYFYKPNGSNFFDSKVMKEALSNVLVSFYPMAGRLARDEQGRIEINCNGEGVLFVEAESDAFVDDFGDFPSLERKLIPTVDTSGDISTFPLIIFQVTRFKCGGVSLGGGVFHTLSDGLSSIHFINTWSDIARGLSV VIPPFIDRTLLRARDPDPTSSFEHVEYHPPPSLISSSKTKESTSPKPSTTTMMLKFSSDQLGLLKSKSKHDGSTYEILAAHIWRCTCKARALSDDQLTKLHVATDGRRLLCPPLPPGYLGNVVFTGTPMAKSSELLQEPLTNSAKRIHSALSKMDDNYLRSALDYLELLPDLSALIRGPTYFASPNLNINSWTRLPVHDSDFGW GRPIHMGPACILYEGTVYILPSPNSKDRNLRLAVCLDADHMPLFEKYLYEF;

The NtHQT3 gene is a homologous gene of the NtHQT2 gene, which is related to the synthesis of chlorogenic acid in tobacco (tobacco leaves) (or related to the regulation of chlorogenic acid content in tobacco leaves), and its clone is obtained from common tobacco K326. The base sequence is as follows: Shown in SEQ ID No.2; details are as follows (1311bp):

ATGAAGATCGACATTAAGGAATCTATAATGGTGAAACCATCAAAGCCAACTCCTTCAAAAAGGCTATGGAACTCAAATTTGGATTTAATAGTGGGAAGAATTCATCTTTTGACAGTATATTTCTACAAAACCAAATGGAATTTCTCCAAATTTCTTTGATTTTAGAATTATGAAGGAGGCTTTAAGCAATATTTTAGTTTCATTTTATCCAATGGCTGGGAGATT GGCTAAGGATAATAATGAAAAAGGAAGAATTGAGATAAATTGTAATGGAGAAGGGGTTCTTTTTATTGAAGCTGAAATTGATGAATTTATTGATGATTTTGGTGATGATTTTACTCCAAGTTTGGAAATAAAGAAGATGTTTAATTCCTAATGTTGTGATCATCTTCTTTTCCACTTGTCATATTTCAGGTAACTCGTTTCAAGTGTGGTGGA GTCTCTTTAGGGTGTGGCGTTTTCCATACATTATCTGACGGCATTTCATCCCTTCATTTCATCAACACATGGTCGGAAGTTGCTCGAGGCCTCTCCGTCGCCGTCTCGCCGTTCATCGACCGGTCCCTCCTTCGTGCACGGGACCCCACCAACCCCCGCTTATAAACACGTCGAGTATCACCCCCCTCCGACCCTAAAATCATCGTCAGAATACGTTCATGATCAA GTTAACGGTCCAAAATCCAGCATCACGGCCATGTTTAAAGATCACAAGTGACCAGCTAACCCTACTCAAGACCAAGTCAAAAAAAGAGGGTAGTACTTATGAAGTACTCGCAGCCCATATTTGGCGTTGCACGTGCAAAGCACGTGCCCTAGACAATGACCAATTGACAAAGTTACATGTTGCTACAGATGGTAGGTCAAAGACTTTTTTCCTCCTTTACCACCAGGTTACC TAGGAAATGTTGTTTTTCACAGCTACACCAATTGCCAAATCTGGTGAAATTTTATCAGAGCCATTGATGAACACAGCTCAAAGAATTCATAGCGCGTTAGCAAGAATGGACGACGAATACTTAAGATCAGCGATAGATTATCTCGAATTACAGACAGATTTATCTAAATTAATCCGAGGACCGACGTATTTTGCTAGTCCTAATCTTAATATTAACAGTTGGACTAGGT TGCCTGTTCATGATTCAGATTTTGGATGGGGAAAACCAATTCATATGGGACCTGCATGTATTTTGTATGAAGGGACAGTTTATATTGCCAAGTCCAAGCAATGACAAGAATTTGAGGTTGGCTGTGTGTTTAGATGCTGATCATATGCCACTTTTTGAGAAGTACCTGTATGACTTTTGA;

Correspondingly, the amino acid sequence (436AA) of the NtHQT3 protein encoded by the NtHQT3 gene is as follows:

MKIDIKESIMVKPSKPTPSKRLWNSNLDLIVGRIHLLTVYFYKPNGISPNFFDFRIMKEALSNILVSFYPMAGRLAKDNNEKGRIEINCNGEGVLFIEAEIDEFIDDFGDDFTPSLEIKKMLIPNVDTSGDISSFPLVIFQVTRFKCGGVSLGCGVFHTLSDGISSLHFINTWSEVARGLSVAVSPFID RSLLRARDPPTPAYKHVEYHPPPTLKSSSEYVHDQVNGPKSSITAMFKITSDQLTLLKTKSKKEGSTYEVLAAHIWRCTCKARALDNDQLTKLHVATDGRRLFPPLPPGYLGNVVFTATPIAKSGEILSEPLMNTAQRIHSALARMDDEYLRSAIDYLELQTDLSKLIRGPTYFASPPNLNINSWTRLPVHD SDFGWGKPIHMGPACILYEGTVYILPSPSNDKNLRLAVCLDADHMPLFEKYLYDF;

In specific applications, after overexpressing the NtHQT2 gene or NtHQT3 gene, it can significantly increase (upregulate) the content of chlorogenic acid in tobacco leaves; after down-regulating the expression of NtHQT2 gene or NtHQT3 gene through gene editing or gene silencing, it can significantly reduce ( Down-regulated) the content of chlorogenic acid in tobacco leaves;

At the same time, overexpression (overexpression) of NtHQT2 gene or NtHQT3 gene can significantly increase the plant height, promote early flowering, and increase the leaf area of the middle leaf;

Using the method for cultivating new plant varieties of the series of tobacco NtHQT genes, using genetic engineering technology, overexpressing the NtHQT2 gene (or NtHQT3 gene) or underexpressing the NtHQT2 gene (or NtHQT3 gene) in the new variety to obtain high expression of chlorogenic acid content Or new plant varieties with low expression;

By expressing the NtHQT2 gene (or NtHQT3 gene), a new tobacco variety with increased plant height, early flowering, and increased leaf area at the middle leaf position can be obtained.

In this application, on the basis of existing studies on tobacco NtHQT genes, two new NtHQT2 and NtHQT3 genes were excavated. By using gene editing technology to silence these two genes and through overexpression experiments on these two genes, the results show that the expression of these two genes is positively correlated with the content of chlorogenic acid in tobacco leaves. At the same time, the statistical results of related growth phenotypes showed that when these two genes were overexpressed, the plant height could be significantly increased, the leaf area of the middle leaf position could be increased, and early flowering could be promoted. Based on these results, it can not only lay a certain theoretical foundation for the research on the regulation pathway of secondary metabolites in tobacco, but also lay a certain technical foundation for the cultivation of new varieties of related plants.

Description of drawings

Figure 1 is the expression characteristics of NtHQT2 gene in different tissues;

Figure 2 is the expression characteristics of NtHQT3 gene in different tissues;

Figure 3 is a schematic diagram of the target site of NtHQT2 gene knockout (the 20 bp target site is followed by the PAM region, + indicates the positive-sense strand, and the asterisk indicates the relative position of the sgRNA);

Figure 4 is a schematic diagram of the target site of NtHQT3 gene knockout (the 20 bp target site is followed by the PAM region, + indicates the sense strand, and the asterisk indicates the relative position of the sgRNA);

Figure 5 shows the sequencing results of the knockout target site for the T0 transgenic line of the NtHQT2 gene;

Fig. 6 is the sequencing result of knockout target site for NtHQT3 gene T0 generation transgenic line;

Figure 7 is a T2 generation overexpression and gene editing NtHQT2 gene phenotype diagram for the NtHQT2 gene;

Figure 8 is a T2 generation overexpression and gene editing NtHQT3 gene phenotype diagram for the NtHQT3 gene;

Figure 9 is the NtHQT2 gene expression detection of NtHQT2 overexpression plants;

Figure 10 is the determination of chlorogenic acid content in the leaves of T2 generation overexpression and gene editing transgenic lines for NtHQT2 gene;

Figure 11 is the detection of NtHQT3 gene expression in overexpressed plants;

Fig. 12 is the measurement of chlorogenic acid content in leaves of T2 generation overexpression and gene editing transgenic lines targeting NtHQT3 gene.

Detailed ways

The present application will be further explained below in conjunction with the embodiments. Before introducing specific embodiments, a brief introduction to some experimental backgrounds in the following embodiments is as follows.

biomaterials:

Tobacco K326, a tetraploid tobacco variety commonly used in tobacco cultivation and research; the applicant, as a professional tobacco research institution, has cultivated and preserved this material for a long time; this biological material can also be obtained from public channels;

Super pCAMBIA1300 plasmid, which is a commonly used and common plasmid vector in molecular biology experimental research, can be obtained from public channels;

CRISPER/Cas9 plasmid, a common and commonly used plasmid vector for gene editing in existing molecular biology research experiments, can be obtained from public sources. Provided by the laboratory as a gift;

DH5α competent cells were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.;

LBA4404 Agrobacterium strain, commonly used and common strains in genetic engineering technology, can be obtained from public channels;

The synthesis and sequencing of relevant primer sequences were provided by Beijing Liuhe Huada Bioengineering Co., Ltd.;

Other experimental reagents and main equipment:

DNA/RNA extraction kit, product of Gene Answer;

Gel Recovery Kit/Reverse Transcription Kit, product of Bao Bioengineering (Dalian) Co., Ltd.;

Gel electrophoresis instrument (Bio-Rad), PCR instrument (Biometra), UVP gel imaging system (GelDoc-It310), etc. are common and commonly used instruments and equipment in existing molecular biology experiments, and will not be repeated here.

Example 1

On the basis of the previous research work on tobacco NtHQT genes, combined with the existing tobacco genome work, the inventor speculated that there are similar HQT homologous genes (family genes) in tobacco, in order to further understand the homologous genes (family genes) related to HQT in tobacco ) situation, the inventor carried out further gene cloning, and the related work is briefly introduced as follows.

First, based on the existing tobacco genome data and existing tobacco NtHQT gene research, the inventors designed the primer sequences for PCR amplification as follows:

NtHQT2-F: 5'-ATGGGAAGTGAAAAGATGAT-3',

NtHQT2-R: 5'-TCAAAATTCATACAAATACTT-3';

NtHQT3-F: 5'-ATGAAGATCGACATTAAGG-3',

NtHQT3-R: 5'-TCAAAAGTCATACAGGTACT-3'.

Subsequently, the leaves of tobacco at different growth and development stages were used as samples to extract RNA (refer to the instructions of the Gene Answer RNA extraction kit for extraction), and refer to the instructions of the reverse transcription kit (Takara) to reverse transcribe the extracted RNA into cDNA.

Then, using the cDNA prepared above as a template, PCR amplification was performed using the aforementioned NtHQT2-F/R and NtHQT3-F/R primer pairs respectively;

During PCR amplification, the 25 μL reaction system was designed as follows:

cDNA template, 2 μL;

Upstream primer, 0.4 μL;

Downstream primer, 0.4 μL;

PremixTaq, 12.5 μL;

_ddH2O , 9.7 μL;

Reaction program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 60-90 s, 30 cycles; extension at 72 °C for 10 min, the amplified product was directly detected by subsequent electrophoresis or 4 °C Keep warm.

Then, the PCR amplification products were detected by 1.0% agarose gel electrophoresis (DNA Maker selected DL2000, the electrophoresis condition was 120V/20 min), and observed under the ultraviolet scanner, and the DNA gel recovery kit was used for the target DNA fragment ( Takara) recovered and measured the concentration and stored at -20°C for later use or directly for subsequent experimental operations.

Finally, after linking the recovered and purified target fragment with the pMD19-T vector, the competent cell DH5α was transformed. After screening, shaking table expansion and identification, the recombinant plasmid containing the target fragment was selected and sent to Beijing Liuhe Huada Biotechnology Co., Ltd. The company sequences the gene of interest.

Sequencing results showed that the tobacco hydroxycinnamoyl-CoA quiny hydroxycinnamon transferase NtHQT2 gene amplified by PCR included 1311 bases and encoded a protein with a length of 436 amino acids. The specific nucleotide sequence is shown in SEQ ID No.1 , the NtHQT3 gene is a homologous gene of the NtHQT2 gene, which includes 1311 bases and encodes a protein with a length of 436 amino acids. The specific nucleotide sequence is shown in SEQ ID No.2.

Based on the above sequencing results, after comparison with the existing NtHQT sequence, combined with related structural analysis, it can be determined that the NtHQT2 gene and NtHQT3 gene obtained in this example belong to two new genes of the HQT family.

Example 2

On the basis of Example 1, with the help of a real-time fluorescent quantitative PCR (BIO-RAD, USA) instrument, the inventors conducted a preliminary analysis of the tissue expression patterns of the NtHQT2 gene and NtHQT3 gene involved in this application, so as to provide a basis for interpreting these two genes To lay a certain foundation for the parts of the organizational structure that play a role, the relevant test process is briefly introduced as follows.

(1) Design primers

On the basis of the gene sequence obtained in Example 1, the primer sequences for fluorescent quantitative PCR detection were designed as follows:

NtHQT2-Q-F: 5'-AGTATCATCCTCCTCCATCT-3';

NtHQT2-Q-R: 5'-TTCGTAAGTGCTACCATCAT-3';

NtHQT3-Q-F: 5'-TACTTCTGGTGACATCTCTTC-3';

NtHQT3-Q-R: 5'-GCCGTCAGATAATGTATGGA-3';

In the process of fluorescent quantitative PCR measurement, L25 was used as the internal reference gene, and correspondingly, the primer pair was designed as follows:

L25-F: 5'-CCCCTCACCACAGAGTCTGC-3';

L25-R: 5'-AAGGGTGTTGTTGTCCTCAATCTT-3'.

(2) Sampling

The stems, stem nodes, leaves (5 leaves, 10 leaves, 15 leaves), flowers, flower buds, axillary buds, lateral roots, fibrous roots and other samples of K326 in full flowering stage were used to extract RNA and reverse transcribe into cDNA for future use.

(3) Fluorescent quantitative PCR detection

For fluorescent quantitative PCR, the 20 μL reaction system was designed as follows:

cDNA template, 4 μL (40 ng/μL);

Upstream and downstream primers, 1 μL each;

SYBR Green, 10 μL;

_ddH2O , 4 μL.

The results of fluorescence quantitative detection are shown in Figure 1 and Figure 2. Analysis shows that:

The NtHQT2 gene was expressed in the roots, leaves, flowers and other tissues of the common tobacco K326 at the full flowering stage, but the expression level was the highest in the leaves, and the expression level of the leaves at the 5th leaf position was higher; while the tissue expression pattern of the NtHQT3 gene Similar to the NtHQT2 gene, it is also expressed in multiple tissues and organs, and the expression level is also the highest in leaves, but it is obviously different from the expression site of the NtHQT2 gene. For example, the NtHQT3 gene seems to be more concentrated in the leaves, and in the The expression level was higher in leaves at the 5th leaf position. This result also indicates that it is precisely because of the detailed differences in the expression sites of different NtHQT genes that they play a comprehensive role in regulating the content of related secondary metabolites in leaves.

In general, only in terms of tissue expression patterns, the tissue expression patterns of these two genes are similar, that is, they are mainly expressed in leaf tissues, but the expression levels of these two genes and their expression levels in tissues and organs such as China and roots are similar. There were also significant differences in expression.

Example 3

On the basis of the work in Examples 1 and 2, in order to more intuitively clarify the relationship between the NtHQT2 gene and NtHQT3 gene and the chlorogenic acid content phenotype in tobacco leaves, the inventors used the CRISPER/Cas9 gene editing technology to genetically modify the NtHQT2 and NtHQT3 genes respectively. Editing (gene silencing) down-regulated the expression of NtHQT2 and NtHQT3 genes. On this basis, the changes of chlorogenic acid content in tobacco leaves caused by different gene expression were detected and analyzed. The main technical operation process is as follows: first construct gene editing vectors targeting NtHQT2 and NtHQT3 genes respectively; then transform the gene editing vectors into Agrobacterium; use Agrobacterium-mediated genetic transformation technology to transform tobacco, and carry out kana gene resistance on transformed tobacco After screening, the positive transgenic tobacco was obtained, and continued to grow until it set seeds; the obtained seeds were planted again (T1 generation tobacco plants), and the metabolites of chlorogenic acid in the transgenic plants were analyzed. The specific experimental conditions are as follows.

(1) Construction of gene editing vector

According to the genome and coding region sequences of NtHQT2 and NtHQT3 genes sequenced in Example 1, and referring to the design principles of gene editing target sites, target sites of about 20 bp were designed on the first exon sequences of NtHQT2 and NtHQT3 genes ( The schematic diagrams are shown in Figure 3 and Figure 4), and the knockout primer sequences were designed as follows:

NtHQT2-C-F: GATTGTTGAGGTCTGATTTGAGGAG;

NtHQT2-C-R:AAACCTCCTCAAATCAGACCTCAAC;

NtHQT3-C-F:GATTGTCAAATTTGGATTTAATAGT;

NtHQT3-C-R: AAACACTATTAAATCCAAATTTGAC;

Subsequently, PCR amplification was used to obtain the DNA double strands of the target site; the reference design of the 20 μL reaction system is as follows:

Upstream and downstream primers, 4 μL each (50 μmol/L);

Annealing Buffer for DNA Oligos (5×), 4 μL;

Make up to 20 µL with ddH ₂ O;

Reaction program: 95°C for 5 min, drop by 0.1°C every 8s until 25°C; store the reaction product at 4°C for future use.

Then, ligate the annealed product obtained from the above PCR reaction with the plasmid pORE-Cas9/gRNA (pre-digested with BsaI); the reference for 10 µL ligation system is:

Digested vector, 3 µL;

Annealed product, 2 µL;

Solution I, 5 µL;

Incubate at 16°C for 30 min.

Then, the above ligation product was transformed into DH5α competent cells and cultured for resistance selection (37°C for about 12 hours).

Finally, after positive clones were selected for colony PCR identification, the correctly identified plasmids were further sent to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing identification to ensure correct plasmid recombination.

It should be noted that in the colony PCR identification, for the NtHQT2 editing vector, the primer pair for identification was designed as follows:

U26-jiance-F: 5'-TTAGGTTTACCCGCCAATA-3',

NtHQT2-C-R: 5'-AAACCTCCTCAAATCAGACCTCAAC-3';

For the NtHQT3 editing vector, the primer pair for identification is designed as follows:

U26-jiance-F: 5'-TTAGGTTTACCCGCCAATA-3',

NtHQT3-C-R: 5'-AAACATCTCCCAGCCATTGGATAAC-3'.

(2) Preparation of transfection solution

The editing vector prepared in step (1) was transformed into Agrobacterium LBA4404, and the transfection solution was further prepared. The specific operation is as follows.

First, take 1 μl of the gene editing vector, add it to a centrifuge tube containing 100 μl of Agrobacterium competent cells, place it on ice for 30 minutes, transfer to liquid nitrogen for quick freezing for 1 minute, and then incubate at 37°C for 5 minutes;

Then, add 1 ml LB liquid medium, shake and culture at 28°C for 3 hours; after the culture, centrifuge at 5000 rpm for 1 minute, discard the supernatant (medium), add 200 μl LB liquid medium, and resuspend the pellet;

Finally, take 200 μl of the resuspended bacteria and evenly spread it on the LB solid plate containing 20 mg/L Rif and 50 mg/L Kanamycin (Kan). After culturing at 28°C for 2-3 days, select positive plasmids for amplification Afterwards, carry out colony PCR identification to ensure that the plasmid is transformed correctly, and save the transformed recombinant strain for future use. The correct transformed strains were further shaken to amplify, centrifuged to collect the bacteria, and then resuspended with MSO to prepare as a transfection solution for later use. (For related operations, please refer to conventional operations in the prior art, and details will not be repeated).

It should be noted that Agrobacterium competent cells can be prepared by referring to the following operations:

First, pick a single colony of Agrobacterium LBA4404 and culture it overnight at 28°C in 2ml LB (containing 20 mg/mL Rif);

Then, inoculate 2 ml of well-grown bacteria solution (containing 25 mg/L Rif) into 50 ml LB liquid medium, and culture with shaking at 28°C until OD600=0.5;

Then, transfer the bacterial solution to a 50 ml centrifuge tube, put it on ice for 30 minutes, and then centrifuge at 5000 rpm for 5 minutes at 4°C to collect the bacterial cells;

Then, after lightly suspending the bacteria in 10ml, 0.15M pre-cooled sodium chloride solution, centrifuge at 5000 rpm for 5 minutes at 4°C to collect the bacteria;

Finally, 20 ml of pre-cooled 20 mM calcium chloride solution was added to suspend the cells to complete the preparation of competent cells; the prepared competent cells were divided into 100 μl/tube and stored at -80°C for later use.

(3) Transformed Tobacco

The tissue culture regeneration method is used to transform tobacco (the specific process is entrusted to a relevant company to operate), and the specific operation is as follows.

First, take vigorously growing tobacco leaves, cut them into small pieces of about 1 cm ² after disinfection and sterilization, place them in the medium for pre-cultivation for 2 days, and then place them in the infection solution prepared in step (2) to fully infect 10- About 15 minutes;

Subsequently, the above-mentioned infected leaf pieces were placed in the medium for dark culture for 4 days, and then transferred to the medium to induce differentiation to induce the formation of clustered adventitious bud cuts (during this period, the medium was changed every 10 days as needed, and at the same time Antibiotics were added to the culture medium for resistance screening);

When the adventitious buds grow to 1-2cm, the clustered adventitious buds are divided into individual small buds, and further transferred to the medium to induce rooting;

Finally, after the development and growth of the root system is completed, the tissue cultured seedlings are taken out, transferred to flowerpots filled with loose sterile soil, and routinely managed and cultivated.

For the transformed transgenic tissue-cultured regenerated seedlings, an appropriate amount of leaves of regenerated tobacco plants were collected as samples, and their genomic DNA was extracted, and PCR method was used to detect whether the exogenous DNA fragments were inserted into the plant genome.

For PCR amplification verification, the primers were designed as follows:

Cas9-F: 5'-GGGACCCTAAGAAGTACGGC-3',

Cas9-R:5'-TATTCTCGGCCTGCTCTCTG-3';

The 25 μL reaction system reference design is:

Gene upstream and downstream primers, 1 µL each (10 µmol/L);

DNA template, 1 µL (100 ng/µL);

10×Taq Buffer (Mg ²⁺ ), 2.5 µL;

dNTPs, 2 µL (2.5 mmol/L);

Taq DNA polymerase, 0.25 µL;

Make up to 25 µL with ddH ₂ O;

PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 20 s, 30 cycles; extension at 72°C for 10 min; storage at 4°C.

After the PCR is completed, the PCR amplification product is detected by 1% agarose gel electrophoresis to determine whether the exogenous DNA fragment (as9 protein) is inserted into the plant genome.

In order to further determine whether the target gene was successfully edited and mutated for the transgenic positive plants identified by the above preliminary PCR screening, and to confirm the type of mutation, the inventor conducted further detection and identification, and the specific situation is as follows.

First, according to the NtHQT2 gene sequence and the position of the target site, the primer pair for detection was designed as follows:

NtHQT2-BJ-F:5'-ggagtgagtacggtgtgcAAACCAACACCAACAAAGAG-3',

NtHQT2-BJ-R:5'-gagttggatgctggatggTCTTCCTTGTTCATCTCTAGC-3';

According to the NtHQT2 gene sequence and the position of the target site, the primer pair for detection was designed as follows:

NtHQT3-BJ-F:5'-ggagtgagtacggtgtgcAACCATCAAAGCCAACTC-3',

NtHQT3-BJ-R:5'-gagttggatgctggatggATTATCCTTAGCCAATCTCC-3';

Subsequently, using the above-mentioned Cas9 positive transgenic plant DNA as a template, carry out PCR amplification;

The 50 μL amplification system reference design is:

Upstream and downstream primers, 2 µL each (10 µmol/L);

DNA template, 2 µL (100 ng/µL);

10 × Taq Buffer (Mg ^{2 +} ), 5 µL;

dNTPs, 4 µL (2.5 mmol/L);

Taq DNA polymerase, 0.5 µL;

Make up to 50 µL with ddH ₂ O;

PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 20 s, 35 cycles; extension at 72°C for 10 min, storage at 4°C;

After the PCR amplification products were detected by electrophoresis, the PCR products were recovered and purified, and sent to Xi'an Qingxue Biotechnology Co., Ltd. for Hi-TOM sequencing, and the mutation rate of the gene was compared and analyzed according to the sequencing results.

Part of the sequencing results are shown in Figure 5 and Figure 6. The results show that:

For the NtHQT2 transgenic plants, there were 3 types of gene mutations in the 11 T0 generation plants, which were deletions of 1 and 8 bases, and insertions of 1 base. These base mutations all occurred at the knockout target site, but no mutation was detected in the NtHQT2 gene of wild-type plants. This shows that the plants of the T0 generation have mutated the NtHQT2 gene in the form of base deletion or insertion.

For NtHQT3 transgenic plants, there were 4 types of gene mutations in 9 T0 generation plants, which were 1 base insertion, 1, 2 and 3 base deletions. These base mutations all occurred at the knockout target site, but no mutation was detected in the NtHQT3 gene of wild-type plants. This shows that the plants of the T0 generation have mutated the NtHQT3 gene in the form of base deletion or insertion.

In order to verify whether the relevant phenotype can be stably inherited to the next generation, the seeds of the positive plants of the T0 generation were collected, and the T2 generation plants were obtained after continued planting, and the detection and analysis were carried out. The results showed that: T0 generation plants can be stably inherited to T2 generation.

Example 4

Based on the work in the above examples, in order to further verify the relationship between the NtHQT2 gene and NtHQT3 gene and the chlorogenic acid content phenotype in tobacco leaves, the inventor constructed a related overexpression vector to up-regulate the expression of NtHQT2 and NtHQT3 genes, The changes of chlorogenic acid content in tobacco leaves caused by different gene expression levels were further detected and analyzed. The specific experimental situation is briefly introduced as follows.

(1) Construction of overexpression vector

First, design the primer pair for PCR amplification as follows:

For the NtHQT2 gene,

Upstream primer: 5'- GGATCCATGGGAAGTGAAAAGATGAT-3',

Downstream primer: 5'-CCATGGTCAAAAATTCATACAAATACTT-3';

For the NtHQT3 gene,

Upstream primer: 5'-TCTAGAATGAAGATCGACATTAAGG-3',

Downstream primer: 5'-GGTACCTCAAAAAGTCATACAGGTACT-3';

Subsequently, PCR amplification was performed using the T plasmid correctly sequenced in Example 1 containing the NtHQT2 (or NtHQT3) gene as a template, and the amplified product was recovered;

Then, the PCR product was digested (for the NtHQT2 gene, BamH I and Nco I double enzyme digestion was performed, and for the NtHQT3 gene, Xba 1 and Kpn 1 double enzyme digestion was performed), and the digested fragment and pCAMBIA1300 empty vector vector connect;

Finally, the ligated product was transformed into Escherichia coli DH5α. And carry out resistance screening and identification to ensure correct plasmid recombination.

Relevant operations can refer to the foregoing or existing technologies, and will not be repeated here.

(2) Preparation of transfection solution

For related operations, refer to Embodiment 3, and details are not repeated here.

(3) Transformed Tobacco

Similar to Example 3, the tissue culture regeneration method was used to transform tobacco, and the specific operation will not be repeated.

It should be noted that when performing PCR identification on transgenic positive plants:

For the NtHQT2 gene, the primer pair was designed as (the upstream primer specifically binds to the NtHQT2 gene, and the downstream primer specifically binds to the GFP tag):

Upstream primer: 5'-TTGCCACCAGGTTACTTAG-3';

Downstream primer: 5'-CGTCGTCCTTGAAGAAGAT-3';

For the NtHQT3 gene, the primer pair is designed as (the upstream primer specifically binds to the NtHQT3 gene, and the downstream primer specifically binds to the GFP tag):

Upstream primer: 5'-AAGAATGGACGACGAATACT-3'.

Downstream primer: 5'-ATGGCGGACTTGAAGAAG-3';

The genomic DNA of the control, K326 and T0 generation seedlings were used as templates for PCR amplification, and the positive seedlings of the T0 generation were amplified with specific bands.

Further, using the tobacco L25 gene as an internal reference gene, the expression level of NtHQT2 (NtHQT3) gene in positive transgenic seedlings was detected by qPCR method; when detecting, the relevant primers were designed as follows:

Targeting the NtHQT2 gene:

Upstream primer: 5'-AGTATCATCCTCCTCCATCT-3',

Downstream primer: 5'-TTCGTAAGTGCTACCATCAT-3';

Targeting the NtHQT3 gene:

Upstream primer: 5'-TACTTCTGGTGACATCTCTTC-3'

Downstream primer: 5'-GCCGTCAGATAATGTATGGA-3';

For L25 internal reference gene:

Upstream primer: 5'-CCCCTCACCACAGAGTCTGC-3',

Downstream primer: 5'- AAGGGTGTTGTTGTCCTCAATCTT-3';

Finally, according to the CT values obtained by qPCR, the relative expression levels of NtHQT2 and NtHQT3 genes were calculated using the ^2-△△CT method.

Example 5

For the transgenic lines obtained in Example 3 and Example 4, the inventors conducted further phenotype observation and detection of chlorogenic acid content, and the relevant results are as follows.

(1) Growth phenotype

The comparison results of some typical phenotypes are shown in Figure 7 and Figure 8. It can be seen from the comparison:

For the NtHQT2 gene, the statistical results of related growth phenotypes showed that after overexpressing the NtHQT2 gene, the plant height increased by an average of 12.5 cm compared with the control K326, the total number of effective leaves decreased by 3-4 pieces, and the flowering time was about 10 days earlier. The leaf length of the largest leaf increased by 8.2 cm, and the leaf width increased by 13.7 cm; while the phenotype of the NtHQT2 gene-edited plants did not change much;

That is: editing (gene silencing) targeting the NtHQT2 gene does not lead to significant differences in related growth phenotypes, but overexpression of the gene can significantly increase plant height, promote early flowering, and increase the number of middle leaves while reducing total effective leaves. bit leaf area;

For the NtHQT3 gene, its growth phenotype is similar to that of the NtHQT2 gene, but there are also significant differences in the specific phenotypic difference data. Specifically, the plant height increased by an average of 12.0 cm compared with the control K326, and the total number of leaves decreased by about 3 , the flowering time was about 7 days earlier, the leaf length of the largest leaf in the middle increased by 4.6 cm, and the leaf width increased by 12.8 cm; while the phenotype of NtHQT3 gene-edited plants and control K326 did not change much;

That is, editing (gene silencing) targeting the NtHQT3 gene does not lead to significant differences in related growth phenotypes, but overexpression of the gene can significantly increase plant height, promote early flowering, and increase the number of middle leaves while reducing total effective leaves. Leaf area; (however, the phenotypic effect data such as the increase in plant height, the early flowering time, and the degree of leaf area increase are smaller than those of the NtHQT2 gene)

(2) Gene expression level

The gene expressions of NtHQT2 and NtHQT3 in some transgenic lines are shown in Fig. 9 and Fig. 11 . It can be seen that the expression levels of NtHQT2 and NtHQT3 genes in the overexpression lines have been significantly improved.

(3) Chlorogenic acid content

Using the GC-MS analysis method, the inventor detected the content of chlorogenic acid in the transgenic lines. During the detection, the HPLC-UV absolute quantitative analysis conditions: chromatographic column Symmetry C18 (4.6×250 mm, 5 µm), detection wavelength 340 nm , injection volume 5 µL, flow rate 1.0 mL/min;

Mobile phase A is water/methanol/acetic acid (44/5/1, v/v/v), mobile phase B is methanol/water/acetic acid (44/5/1, v/v/v);

Gradient elution:

0~15.0 min, 10%B-30%B;

15.0~26.0 min, 30%B-90%B;

26.0~28.0 min, 90% B;

28.1-35.0 min, 10% B.

The results showed that for the NtHQT2 gene strains (as shown in Figure 10):

Compared with the control (K326) gene-edited plants of the T2 generation (BJ1, BJ2, BJ3), the content of chlorogenic acid in the middle leaf of the middle leaf mature stage decreased by 34.6%, 29.1% and 21.5%, and the overexpressed tobacco plants of the T2 generation (OE- 1, OE-2, OE-3) Compared with the control (K326), the content of chlorogenic acid in the middle leaf maturity stage increased by 71.2%, 82.4% and 101.7%, respectively. This result indicated that NtHQT2 gene plays an important role in the synthesis of tobacco chlorogenic acid.

For the NtHQT3 gene line: Compared with the control (K326), the content of chlorogenic acid in the leaves of the 10th leaf position in the middle leaf maturity stage of the T2 transgenic plants (OE-1, OE-2, OE-3) overexpressing NtHQT4 An increase of 98.1%, 81.2% and 101.7%. The content of chlorogenic acid in the 10th leaf position of the T2 generation gene-edited plants (BJ1, BJ2, BJ3) decreased by 15.4%, 22.0% and 28.2% at the middle leaf maturity stage. This result indicated that the use of NtHQT3 gene plays an important role in tobacco chlorogenic acid synthesis.

Claims (7)

1. The series of tobacco NtHQT genes are characterized by comprising two tobacco hydroxycinnamoyl coenzyme A quinihydroxycinnamoyl transferase NtHQT2 genes and NtHQT3 genes;

the NtHQT2 gene is related to chlorogenic acid synthesis in tobacco, and the base sequence is shown in SEQ ID No. 1;

the NtHQT3 gene is related to chlorogenic acid synthesis in tobacco, and the base sequence is shown in SEQ ID No. 2.

2. The primer pair for PCR amplification of the series tobacco NtHQT genes as claimed in claim 1, wherein when the series tobacco NtHQT genes are prepared by PCR amplification, specific primer pairs are designed as follows:

NtHQT2-F：5 '-ATGGGAAGTGAAAAGATGAT-3 '，

NtHQT2-R：5 '-TCAAAATTCATACAAATACTT-3 '；

NtHQT3-F：5 '-ATGAAGATCGACATTAAGG-3 '，

NtHQT3-R：5 '-TCAAAAGTCATACAGGTACT-3 '。

3. the super-expression plasmid prepared by using the series of tobacco NtHQT genes as claimed in claim 1, wherein the pCAMBIA1300 plasmid is used as a vector, and the NtHQT2 genes or the NtHQT3 genes are recombined.

4. Use of the series of tobacco NtHQT genes according to claim 1 in tobacco for increasing the content of chlorogenic acid in the tobacco leaves by over-expression of the NtHQT2 gene or the NtHQT3 gene;

the method is used for reducing the content of chlorogenic acid in tobacco leaves by utilizing a gene editing or gene silencing technology to down-regulate the expression level of the NtHQT2 gene or the NtHQT3 gene.

5. Use of the series of tobacco NtHQT genes according to claim 1 in tobacco for increasing plant height, promoting early flowering, increasing leaf area in the middle leaf position by over-expression of the NtHQT2 gene or the NtHQT3 gene.

6. The method for cultivating new tobacco varieties by using the series of tobacco NtHQT genes as claimed in claim 1, which is characterized in that the new tobacco varieties with high or low chlorogenic acid content are obtained by using a genetic engineering technology to overexpress the NtHQT2 gene or the NtHQT3 gene or to underexpress the NtHQT2 gene or the NtHQT3 gene.

7. The method for cultivating new tobacco varieties by using the series of tobacco NtHQT genes as claimed in claim 1, which is characterized in that the new tobacco varieties with increased plant height, early flowering and increased leaf area in the middle leaf position are obtained by over-expressing the NtHQT2 gene or the NtHQT3 gene by using a genetic engineering technology.

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