Series of tobacco NtHQT genes and their applications

Abstract

The present invention belongs to the technical field of tobacco genetic engineering, and specifically relates to a series of tobacco NtHQT genes and applications thereof. The series of tobacco NtHQT genes include two tobacco hydroxycinnamoyl-CoA quinoline hydroxycinnamoyltransferase NtHQT2 genes and NtHQT3 genes. In the present application, two new NtHQT2 and NtHQT3 genes were excavated based on the existing research on tobacco NtHQT genes. By silencing these two genes using gene editing technology and overexpressing these two genes, the results showed that the expression levels of these two genes were positively correlated with the chlorogenic acid content in tobacco leaves. Based on these results, it can not only lay a certain theoretical foundation for the study of the regulatory pathways of secondary metabolites in tobacco, but also lay a certain technical foundation for the cultivation of new varieties of related plants.

Description

Technical Field

[0001] 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. Background Art

[0002] Chlorogenic acid (CGA), also known as coffee tannin, is produced naturally by plants through the phenylalanine pathway during aerobic respiration. It is an ester compound formed by quinic acid and certain trans-cinnamic acids such as caffeic acid, p-coumaric acid and ferulic acid.

[0003] Chlorogenic acid, a phenolic antioxidant, can rapidly eliminate hydroxyl free radicals. In practical applications, in addition to leveraging its antioxidant properties, research has also shown that chlorogenic acid exhibits a variety of functions, including inhibiting and killing various pathogenic bacteria and viruses, fighting tumors, suppressing mutations, protecting the liver and promoting bile secretion, lowering blood pressure, and lowering blood lipids. For example, studies have shown that chlorogenic acid can inhibit gene mutations, the formation of carcinogenic N-nitroso compounds, and DNA damage in vitro; significantly inhibit tumor cell proliferation and infiltration; and possess pharmacological effects against various pathogenic bacteria and viruses in vitro. Chlorogenic acid can also lower triglycerides and cholesterol, prevent cardiovascular diseases such as hypertension and coronary heart disease, and protect against obesity and diabetes by regulating glucose metabolism. It can also inhibit inflammation and reduce pain. Research suggests that the broad range of chlorogenic acid's biological functions is positively correlated with its significant antioxidant activity. Studies have shown that chlorogenic acid and its derivatives have significantly higher free radical scavenging abilities than classic antioxidants such as ascorbic acid and tocopherol, effectively scavenging hydroxyl and superoxide anion radicals. Chlorogenic acid can be directly absorbed by the small intestine in the body, or it can be hydrolyzed by the large intestinal flora into caffeic acid. Like chlorogenic acid, caffeic acid also has strong antioxidant capacity.

[0004] Chlorogenic acid, a secondary metabolite, is expressed and distributed in a wide variety of plants. Measurements and surveys have shown that tobacco leaves contain 3%-5% chlorogenic acid, one of the highest concentrations found in plant species. Furthermore, chlorogenic acid is the most abundant polyphenolic compound in tobacco leaves, accounting for approximately 70-90% of the total polyphenol content. Therefore, understanding the regulatory pathways for chlorogenic acid synthesis in tobacco leaves is of great technological significance for tobacco variety breeding and tobacco resource development.

[0005] Studies on the biosynthesis mechanism have shown that hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) is a key enzyme in the chlorogenic acid biosynthesis pathway in plants, catalyzing the conversion of caffeoyl-CoA and quinic acid to chlorogenic acid. Using genetic engineering techniques, overexpression or gene silencing in some plants can increase or decrease chlorogenic acid content accordingly. However, as a member of the BAHD (acyl-CoA-dependent acyltransferase) family, HQT has a complex genetic structure and comprises multiple gene types. Therefore, in-depth analysis of the structural differences among different HQT genes is of great technical significance for further research on the chlorogenic acid biosynthesis pathway and the breeding of related plant varieties. Summary of the Invention

[0006] Based on the applicant's existing research work on tobacco NtHQT genes, the purpose of this application is to provide two new tobacco hydroxycinnamoyl-CoA quinoline hydroxycinnamoyltransferase NtHQT genes (NtHQT2 and NtHQT3), thereby laying a certain technical foundation for the regulation mechanism of secondary metabolites in tobacco (tobacco leaves) and the breeding of new tobacco varieties.

[0007] The technical solutions adopted in this application are described in detail as follows.

[0008] A series of tobacco NtHQT genes, including tobacco hydroxycinnamoyl-CoA quinoline hydroxycinnamoyltransferase NtHQT2 and NtHQT3 genes;

[0009] 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). It was cloned from common tobacco K326, and its base sequence is shown in SEQ ID No. 1; specifically, it is as follows:

[0010] NtHQT2 gene sequence (1311 bp)

[0011]

[0012] Correspondingly, the amino acid sequence (436AA) of the NtHQT2 protein encoded by the NtHQT2 gene is as follows:

[0013] MGSEKMMKINIKESTLVKPSKPTPTKRLWNSNLDLIVGRIHLLTVYFYKPNGSSNFFDSKVMKEALSNVLVSFYPMAGRLARDEQGRIEINCNGEGVLFVEAESDAFVD DFGDFTPSLELRKLIPTVDTSGDISTFPLIIFQVTRFKCGGVSLGGGVFHTLSDGLSSIHFINTWSDIARGLSVVIPPFIDRTLLRARDPPTSSFEHVEYHPPPSLISS SKTKESTSPPKPSTTTMLKFSSDQLGLLKSKSKHDGSTYEILAAHIWRCTCKARALSDDQLTKLHVATDGRSRLCPPLPPGYLGNVVFTGTPMAKSSELLQEPLTNSAKR IHSALSKMDDNYLRSALDYLELLPDLSALIRGPTYFASPNLNINSWTRLPVHDSDFGWGRPIHMGPACILYEGTVYILPSPNSKDRNLRLAVCLDADHMPLFEKYLYEF;

[0014] The NtHQT3 gene is a homologous gene of the NtHQT2 gene and is related to the synthesis of chlorogenic acid in tobacco (or the regulation of chlorogenic acid content in tobacco leaves). It was cloned from common tobacco K326 and its base sequence is shown in SEQ ID No. 2; specifically, it is as follows (1311 bp):

[0015]

[0016] Correspondingly, the amino acid sequence (436AA) of the NtHQT3 protein encoded by the NtHQT3 gene is as follows:

[0017] MKIDIKESIMVKPSKPTPSKRLWNSNLDLIVGRIHLLTVYFYKPNGISPNFFDFRIMKEALSNILVSFYPMAGRLAKDNNEKGRIEINCNGEGVLFIEAEIDEFIDDFGDDFTPSLEIKKMLIPNVDTSGDISSFPLVIFQVTRFKCGGVSLGCGVFHTLSDGISSLHFINTWSEVARGLSVAVSPFIDRSLLRARDPPTPAYKHVEYHPPPTLKSSS EYVHDQVNGPKSSITAMFKITSDQLTLLKTKSKKEGSTYEVLAAHIWRCTCKARALDNDQLTKLHVATDGRSRLFPPLPPPGYLGNVVFTATPIAKSGEILSEPLMNTAQ RIHSALARMDDEYLRSAIDYLELQTDLSKLIRGPTYFASPNLNINSWTRLPVHDSDFGWGKPIHMGPACILYEGTVYILPSPSNDKNLRLAVCLDADHMPLFEKYLYDF;

[0018] In specific applications, overexpression of the NtHQT2 gene or the NtHQT3 gene can significantly increase (upregulate) the content of chlorogenic acid in tobacco leaves; downregulation of the NtHQT2 gene or the NtHQT3 gene expression through gene editing or gene silencing techniques can significantly reduce (downregulate) the content of chlorogenic acid in tobacco leaves;

[0019] At the same time, overexpression of NtHQT2 or NtHQT3 can significantly increase plant height, promote early flowering, and increase the area of ​​middle leaf leaves;

[0020] A method for breeding new plant varieties using 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 varieties to obtain new plant varieties with high or low expression of chlorogenic acid content;

[0021] Among them, expressing the NtHQT2 gene (or NtHQT3 gene) can obtain new tobacco varieties with increased plant height, early flowering, and increased leaf area in the middle leaf position.

[0022] In this application, based on the existing research on tobacco NtHQT genes, two new NtHQT2 and NtHQT3 genes were discovered. By silencing these two genes using gene editing technology and conducting overexpression experiments on these two genes, the results showed that the expression levels of these two genes were positively correlated with the chlorogenic acid content 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, the leaf area of ​​the middle leaf position, and early flowering could be significantly increased. Based on these results, it can not only lay a certain theoretical foundation for the study of the regulatory pathways of secondary metabolites in tobacco, but also lay a certain technical foundation for the cultivation of new varieties of related plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the expression characteristics of NtHQT2 gene in different tissues;

[0024] Figure 2 is the expression characteristics of NtHQT3 gene in different tissues;

[0025] Figure 3 Schematic diagram of the target site for NtHQT2 gene knockout (the 20 bp target site is followed by the PAM region, + indicates the positive strand, and the asterisk indicates the relative position of the SgRNA);

[0026] Figure 4 Schematic diagram of the target site for NtHQT3 gene knockout (the 20 bp target site is followed by the PAM region, + indicates the positive strand, and the asterisk indicates the relative position of the SgRNA);

[0027] Figure 5 This is the sequencing result of the target site knockout of the T0 transgenic line of the NtHQT2 gene;

[0028] Figure 6 This is the sequencing result of the target site knockout of the NtHQT3 gene T0 transgenic line;

[0029] Figure 7 This is a phenotype diagram of the T2 generation of NtHQT2 gene overexpression and gene editing;

[0030] Figure 8 This is a phenotype diagram of the T2 generation of NtHQT3 gene overexpression and gene editing;

[0031] Figure 9 To detect NtHQT2 gene expression in NtHQT2 overexpressing plants;

[0032] Figure 10 To determine the chlorogenic acid content in leaves of T2 generation overexpression and gene-edited transgenic lines of NtHQT2 gene;

[0033] Figure 11 To detect the expression of NtHQT3 gene in overexpressing plants;

[0034] Figure 12 To determine the chlorogenic acid content in the leaves of T2 generation overexpression and gene-edited transgenic lines of the NtHQT3 gene. DETAILED DESCRIPTION

[0035] The present application will be further explained below with reference to the following examples. Before introducing the specific examples, a brief description of some experimental backgrounds in the following examples is given below.

[0036] Biomaterials:

[0037] Nicotiana tabacum K326 is a common tetraploid tobacco variety commonly used in tobacco cultivation and research. The applicant, as a professional tobacco research institution, has long-term cultivated and preserved this material. This biological material is also available through public channels.

[0038] Super pCAMBIA1300 plasmid is a commonly used plasmid vector in molecular biology experimental research and is available through public channels;

[0039] CRISPER / Cas9 plasmids are common and frequently used gene editing plasmid vectors in current molecular biology research experiments and are publicly available. The plasmids (pORE-Cas9 / gRNA) used in the examples were kindly provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University.

[0040] DH5α competent cells were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.;

[0041] LBA4404 Agrobacterium strain, a commonly used and common strain in genetic engineering technology, is available from public channels;

[0042] The relevant primer sequence synthesis and sequencing work were provided by Beijing Liuhe BGI Biotechnology Co., Ltd.

[0043] Other experimental reagents and main equipment:

[0044] DNA / RNA extraction kit, product of Gene Answer;

[0045] Gel recovery kit / reverse transcription kit, products of Takara Biotechnology (Dalian) Co., Ltd.

[0046] Gel electrophoresis apparatus (Bio-Rad), PCR apparatus (Biometra), UVP gel imaging system (GelDoc-It310), etc. are all common and commonly used instruments and equipment in existing molecular biology experiments and will not be described in detail.

[0047] Example 1

[0048] Based on previous research on tobacco NtHQT genes and combined with existing tobacco genome work, the inventors speculated that similar HQT homologous genes (family genes) exist in tobacco. To further understand the situation of HQT-related homologous genes (family genes) in tobacco, the inventors conducted further gene cloning. The relevant work is briefly described below.

[0049] First, based on existing tobacco genome data and previous tobacco NtHQT gene research, the inventors designed the following primer sequences for PCR amplification:

[0050] NtHQT2-F:5'-ATGGGAAGTGAAAAGATGAT-3',

[0051] NtHQT2-R:5'-TCAAAATTCATAAAATACTT-3';

[0052] NtHQT3-F: 5'-ATGAAGATCGACATTAAGG-3',

[0053] NtHQT3-R:5'-TCAAAAGTCATACAGGTACT-3'.

[0054] Subsequently, tobacco leaves at different growth and development stages were used as samples to extract RNA (refer to the instructions for the Gene Answer RNA extraction kit for extraction), and the extracted RNA was reverse transcribed into cDNA according to the instructions for the reverse transcription kit (Takara).

[0055] Then, the cDNA prepared above was used as a template to perform PCR amplification using the aforementioned NtHQT2-F / R and NtHQT3-F / R primer pairs;

[0056] For PCR amplification, the 25 μL reaction system was designed as follows:

[0057] cDNA template, 2 μL;

[0058] upstream primer, 0.4 μL;

[0059] downstream primer, 0.4 μL;

[0060] PremixTaq, 12.5 μL;

[0061] ddH2O, 9.7 μL;

[0062] Reaction procedure: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 60-90 s, for 30 cycles; extension at 72°C for 10 min. The amplified product was directly subjected to subsequent electrophoresis detection or kept warm at 4°C for later use.

[0063] Then, the PCR amplification products were detected by 1.0% agarose gel electrophoresis (DNA Maker DL2000, electrophoresis conditions 120V / 20 min) and observed under a UV scanner. The target DNA fragments were recovered using a DNA gel recovery kit (Takara) and the concentration was measured and stored at -20°C for future use or directly used for subsequent experimental operations.

[0064] Finally, the recovered purified target fragment was connected to the pMD19-T vector and transformed into competent cells DH5α. After screening, shaking expansion and identification, the recombinant plasmid containing the target fragment was selected and sent to Beijing Liuhe BGI Biotechnology Co., Ltd. for sequencing of the target gene.

[0065] Sequencing results showed that the tobacco hydroxycinnamoyl-CoA quinoline-hydroxycinnamoyltransferase NtHQT2 gene amplified by PCR consists of 1311 bases and encodes a protein with a length of 436 amino acids. The specific nucleotide sequence is shown in SEQ ID No. 1. The NtHQT3 gene, as a homologous gene of the NtHQT2 gene, consists of 1311 bases and encodes a protein with a length of 436 amino acids. The specific nucleotide sequence is shown in SEQ ID No. 2.

[0066] Based on the above sequencing results, after comparison with the existing NtHQT sequence and combined with related structure analysis, it can be determined that the NtHQT2 gene and NtHQT3 gene obtained in this example belong to two new genes in the HQT family.

[0067] Example 2

[0068] Based on Example 1, the inventors conducted a preliminary analysis of the tissue expression patterns of the NtHQT2 and NtHQT3 genes involved in this application using a real-time fluorescence quantitative PCR instrument (BIO-RAD, USA), thereby laying a certain foundation for interpreting the tissue structural locations where these two genes play a role. The relevant experimental process is briefly described below.

[0069] (1) Primer design

[0070] Based on the gene sequence obtained in Example 1, the primer sequences for fluorescence quantitative PCR detection were designed as follows:

[0071] NtHQT2-QF:5'-AGTATCATCCTCCTCCATCT-3';

[0072] NtHQT2-QR:5'-TTCGTAAGTGCTACCATCAT-3';

[0073] NtHQT3-QF:5'-TACTTCTGGTGACATCTCTTC-3';

[0074] NtHQT3-QR:5'-GCCGTCAGATAATGTATGGA-3';

[0075] During the fluorescence quantitative PCR assay, L25 was used as the internal reference gene, and the corresponding primer pairs were designed as follows:

[0076] L25-F: 5'-CCCCTCACCACAGAGTCTGC-3';

[0077] L25-R: 5'-AAGGTGTTGTTGTCCTCAATCTT-3'.

[0078] (2) Sampling

[0079] RNA was extracted from the stems, nodes, leaves (5 leaves, 10 leaves, 15 leaves), flowers, buds, axillary buds, lateral roots, and fibrous roots of K326 during its peak flowering period and reverse transcribed into cDNA for later use.

[0080] (III) Fluorescence quantitative PCR detection

[0081] For fluorescence quantitative PCR, the 20 μL reaction system was designed as follows:

[0082] cDNA template, 4 μL (40 ng / μL);

[0083] Upstream and downstream primers, 1 μL each;

[0084] SYBR Green, 10 μL;

[0085] ddHO, 4 μL.

[0086] Fluorescence quantitative detection results Figure 1 、 Figure 2 As shown. The analysis shows that:

[0087] The NtHQT2 gene is expressed in roots, leaves, and flowers of tobacco K326 during the peak flowering period, but its expression is highest in leaves, with the highest expression in leaves at the fifth leaf position. The tissue expression pattern of the NtHQT3 gene is similar to that of NtHQT2, with expression in multiple tissues and organs, and also highest expression in leaves. However, the expression sites of the NtHQT3 gene differ from those of NtHQT2. For example, the NtHQT3 gene appears to be more concentrated in leaves, with the highest expression levels in leaves at the fifth leaf position. This result indicates that it is precisely the detailed differences in the expression sites of different NtHQT genes that play a comprehensive regulatory role in the content of related secondary metabolites in leaves.

[0088] Overall, in terms of tissue expression patterns alone, the tissue expression patterns of the two genes are similar, that is, they are mainly expressed in leaf tissues, but the expression levels of the two genes and their expression in tissues and organs such as flowers and roots also show obvious differences.

[0089] Example 3

[0090] Based on the work of Examples 1 and 2, to more clearly clarify the relationship between the NtHQT2 and NtHQT3 genes and the chlorogenic acid content phenotype in tobacco leaves, the inventors used CRISPER / Cas9 gene editing technology to gene-edit (gene silencing) the NtHQT2 and NtHQT3 genes, respectively, downregulating their expression levels. Based on this, the changes in chlorogenic acid content in tobacco leaves caused by different gene expression levels were detected and analyzed. The main technical operation process is as follows: first, gene editing vectors targeting the NtHQT2 and NtHQT3 genes were constructed; then, these gene editing vectors were transformed into Agrobacterium; tobacco was transformed using Agrobacterium-mediated genetic transformation technology, and the transformed tobacco was screened for kanamycin resistance to obtain positive transgenic tobacco plants, which were then cultivated to seed production; the obtained seeds were then planted (T1 generation tobacco plants), and the chlorogenic acid metabolites in the transgenic plants were analyzed. The specific experimental details are briefly described below.

[0091] (I) Construction of gene editing vector

[0092] According to the genome and coding region sequences of NtHQT2 and NtHQT3 genes obtained by sequencing in Example 1, and referring to the principles of gene editing target site design, target sites of about 20 bp were designed on the first exon sequences of NtHQT2 and NtHQT3 genes, respectively (schematic diagram shown in FIG. Figure 3 、 Figure 4 The knockout primer sequences were designed as follows:

[0093] NtHQT2-CF:GATTGTTGAGGTCTGATTTGAGGAG;

[0094] NtHQT2-CR: AAACCTCCTCAAATCAGACCTCAAC;

[0095] NtHQT3-CF:GATTGTCAAATTTGGATTTAATAGT;

[0096] NtHQT3-CR:AAACACTATTAAATCCAAATTTGAC;

[0097] Subsequently, PCR amplification was performed to obtain the double-stranded DNA at the target site. The reference design of the 20 μL reaction system is as follows:

[0098] Upstream and downstream primers, 4 μL each (50 μmol / L);

[0099] Annealing Buffer for DNA Oligos (5×), 4 μL;

[0100] Add ddH2O to 20 µL;

[0101] Reaction procedure: 95°C, 5 min, decrease 0.1°C every 8 s until 25°C; the reaction product is stored at 4°C until use.

[0102] Next, ligate the annealed product from the PCR reaction with the plasmid pORE-Cas9 / gRNA (pre-digested with BsaI). The 10 µL ligation system is as follows:

[0103] Enzyme-digested vector, 3 µL;

[0104] annealed product, 2 μL;

[0105] Solution I, 5 μL;

[0106] Ligate at 16°C for 30 min.

[0107] Then, the ligation product was transformed into DH5α competent cells and cultured for resistance screening (cultured at 37°C for about 12 hours).

[0108] Finally, after selecting positive clones for colony PCR identification, the correctly identified plasmids were further sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing identification to ensure the correct plasmid recombination.

[0109] It should be noted that in the colony PCR identification, the primer pair for the NtHQT2 editing vector was designed as follows:

[0110] U26-jiance-F:5'-TTAGGTTTACCGCCAATA-3',

[0111] NtHQT2-CR: 5'-AAACCTCCTCAAATCAGACCTCAAC-3';

[0112] For the NtHQT3 editing vector, the primer pair for identification was designed as follows:

[0113] U26-jiance-F:5'-TTAGGTTTACCGCCAATA-3',

[0114] NtHQT3-CR: 5'-AAACATCTCCCAGCCATTGGATAAC-3'.

[0115] (II) Preparation of transfection solution

[0116] Transform the editing vector prepared in step (1) into Agrobacterium tumefaciens LBA4404 and prepare the transfection solution. Detailed instructions are as follows.

[0117] First, take 1 μl of gene editing vector and add it to a centrifuge tube containing 100 μl of Agrobacterium competent cells. After placing it on ice for 30 minutes, transfer it to liquid nitrogen for quick freezing for 1 minute, and then incubate it at 37°C for 5 minutes.

[0118] Subsequently, 1 ml of LB liquid medium was added and cultured at 28°C with shaking for 3 hours. After the culture was completed, the cells were centrifuged at 5000 rpm for 1 minute, the supernatant (culture medium) was discarded, and 200 μl of LB liquid medium was added to resuspend the pellet.

[0119] Finally, evenly spread 200 μl of the resuspended bacterial solution onto a solid LB plate containing 20 mg / L Rif and 50 mg / L kanamycin (Kan). After incubation at 28°C for 2-3 days, select positive plasmids for amplification and perform colony PCR to confirm correct plasmid transformation. The correctly transformed recombinant strains are then stored for future use. Correctly transformed strains are further amplified by shaking, harvested by centrifugation, and resuspended in MSO to prepare the transfection solution for future use. (Related procedures can be followed by reference to conventional techniques and are not detailed here.)

[0120] It should be noted that Agrobacterium competent cells can be prepared by referring to the following steps:

[0121] First, a single colony of Agrobacterium LBA4404 was picked and cultured in 2 ml of LB (containing 20 mg / mL Rif) at 28°C overnight;

[0122] Subsequently, 2 ml of the well-grown bacterial solution (containing 25 mg / L Rif) was inoculated into 50 ml of LB liquid medium and cultured with shaking at 28°C until OD600 = 0.5.

[0123] Then, transfer the bacterial solution to a 50 ml centrifuge tube, place on ice for 30 minutes, and centrifuge at 4°C and 5000 rpm for 5 minutes to collect the bacteria.

[0124] Then, the cells were lightly suspended in 10 ml of 0.15 M pre-cooled sodium chloride solution and centrifuged at 5000 rpm for 5 min at 4°C to collect the cells.

[0125] Finally, add 20 ml of pre-cooled 20 mM calcium chloride solution to suspend the bacteria to complete the preparation of competent cells; after the prepared competent cells are packaged into 100 µl / tube, store them at -80℃ for later use.

[0126] (3) Transforming tobacco

[0127] The tissue culture regeneration method was used to transform tobacco (the specific process was entrusted to a related company). The specific operation is as follows.

[0128] First, take the vigorously growing tobacco leaves, sterilize them and cut them into 1 cm 2 Place the small pieces in the culture medium for pre-culture for 2 days, then place them in the infection solution prepared in step (2) and fully infect for about 10-15 minutes;

[0129] Subsequently, the infected leaf pieces were placed in a culture medium for dark culture for 4 days, and then transferred to a culture medium for differentiation induction to induce the formation of clustered adventitious buds (during this period, the culture medium was changed every 10 days as needed, and antibiotics were added to the culture medium for resistance screening);

[0130] When the adventitious buds grow to 1-2 cm, the clustered adventitious buds are cut into individual buds and further transferred to the culture medium for rooting induction;

[0131] Finally, after the root system has completed its development and growth, the tissue culture seedlings are taken out and transferred to a pot filled with loose sterile soil for routine management and cultivation.

[0132] For the transformed transgenic plantlets, an appropriate amount of leaves of the regenerated tobacco plants were collected as samples, and their genomic DNA was extracted. The PCR method was used to detect and confirm whether the exogenous DNA fragments were inserted into the plant genome.

[0133] For PCR amplification verification, the primers were designed as follows:

[0134] Cas9-F:5'-GGGACCCTAAGAAGTACGGC-3',

[0135] Cas9-R:5'-TATTCTCGGCCTGCTCTCTG-3';

[0136] The reference design for a 25 μL reaction system is:

[0137] Upstream and downstream primers of the gene, 1 μL each (10 μmol / L);

[0138] DNA template, 1 µL (100 ng / µL);

[0139] 10× Taq Buffer (Mg 2+ ), 2.5 μL;

[0140] dNTPs, 2 µL (2.5 mmol / L);

[0141] Taq DNA polymerase, 0.25 µL;

[0142] Add ddH2O to 25 µL;

[0143] PCR reaction program: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 20 s; extension at 72°C for 10 min; and storage at 4°C.

[0144] After the PCR was completed, the PCR amplification products were subjected to 1% agarose gel electrophoresis to determine whether the exogenous DNA fragment (as9 protein) was inserted into the plant genome.

[0145] For the transgenic positive plants identified by the preliminary PCR screening, the inventors conducted further testing and identification to further determine whether the target gene was successfully edited and mutated, and to specifically confirm the type of mutation. The details are briefly described below.

[0146] First, based on the NtHQT2 gene sequence and target site location, the following primer pairs were designed for detection:

[0147] NtHQT2-BJ-F:5'-ggagtgagtacggtgtgcAAACCAACACCAACAAAGAG-3',

[0148] NtHQT2-BJ-R:5'-gagttggatgctggatggTCTTCCTTGTTCATCTCTAGC-3';

[0149] Based on the NtHQT2 gene sequence and target site location, the following primer pairs were designed for detection:

[0150] NtHQT3-BJ-F:5'-ggagtgagtacggtgtgcAACCATCAAAGCCAACTC-3',

[0151] NtHQT3-BJ-R:5'-gagttggatgctggatggATTATCCTAGCCAATCTCC-3';

[0152] Subsequently, PCR amplification was performed using the above-mentioned Cas9-positive transgenic plant DNA as a template;

[0153] The reference design of the 50 μL amplification system is:

[0154] Upstream and downstream primers, 2 µL each (10 µmol / L);

[0155] DNA template, 2 µL (100 ng / µL);

[0156] 10× Taq Buffer (Mg 2 + ), 5 μL;

[0157] dNTPs, 4 µL (2.5 mmol / L);

[0158] Taq DNA polymerase, 0.5 µL;

[0159] Add ddH2O to 50 µL;

[0160] PCR reaction program: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 59°C annealing for 30 s, 72°C extension for 20 s, 35 cycles; 72°C extension for 10 min, 4°C storage;

[0161] After electrophoresis detection of the PCR amplification products, the PCR products were recovered and purified, and sent to Xi'an Qingxue Biotechnology Co., Ltd. for Hi-TOM sequencing. The mutation rate of the gene was analyzed based on the sequencing results.

[0162] Some sequencing results are as follows Figure 5 、 Figure 6 The analysis results show that:

[0163] Eleven T0-generation NtHQT2 transgenic plants showed three types of mutations: deletions of one and eight bases, and an insertion of one base. These mutations occurred at the target site of the knockout, while no mutations were detected in the NtHQT2 gene of wild-type plants. This suggests that the T0-generation plants have achieved mutations in the NtHQT2 gene, either through deletions or insertions.

[0164] In the nine T0-generation NtHQT3 transgenic plants, four types of gene mutations were observed: one-base insertions, and one-, two-, and three-base deletions. These base mutations all occurred at the knockout target site, while no mutations were detected in the NtHQT3 gene of wild-type plants. This indicates that the T0-generation plants have achieved mutations in the NtHQT3 gene in the form of base deletions or insertions.

[0165] To verify whether the relevant phenotypes can be stably inherited to the next generation, seeds from positive plants in the T0 generation were collected and planted to obtain T2 generation plants, which were then tested and analyzed. The results showed that the T0 generation plants can be stably inherited to the T2 generation.

[0166] Example 4

[0167] Building on the work in the aforementioned examples, to further validate the relationship between the NtHQT2 and NtHQT3 genes and the chlorogenic acid content phenotype in tobacco leaves, the inventors constructed overexpression vectors to upregulate the expression of the NtHQT2 and NtHQT3 genes. The authors then further analyzed the changes in chlorogenic acid content in tobacco leaves caused by varying gene expression levels. The specific experimental details are summarized below.

[0168] (1) Construction of overexpression vector

[0169] First, design the primer pairs for PCR amplification as follows:

[0170] Targeting the NtHQT2 gene,

[0171] Upstream primer: 5'-GGATCCATGGGAAGTGAAAAGATGAT-3',

[0172] Downstream primer: 5′-CCATGGTCAAAATTCATACAAATACTT-3′;

[0173] Targeting the NtHQT3 gene,

[0174] Upstream primer: 5'-TCTAGAATGAAGATCGACATTAAGG-3',

[0175] Downstream primer: 5′-GGTACCTCAAAAGTCATACAGGTACT-3′;

[0176] Subsequently, PCR amplification is performed using the T plasmid containing the NtHQT2 (or NtHQT3) gene sequenced correctly in Example 1 as a template, and the amplified product is recovered;

[0177] Then, the PCR products were digested with enzymes (for NtHQT2 gene, double digestion with BamH I and Nco I; for NtHQT3 gene, double digestion with Xba 1 and Kpn 1), and the digested fragments were ligated with the pCAMBIA1300 empty vector.

[0178] Finally, the ligation product was transformed into E. coli DH5α and resistance screening and identification were performed to ensure that the plasmid was correctly recombined.

[0179] For related operations, please refer to the above or existing technologies and will not be described in detail.

[0180] (II) Preparation of transfection solution

[0181] The related operations can be referred to Example 3 and will not be described in detail.

[0182] (3) Transforming tobacco

[0183] Similar to Example 3, the tissue culture regeneration method was used to transform tobacco, and the specific operations are not repeated here.

[0184] It should be noted that when PCR identification is performed on transgenic positive plants:

[0185] For the NtHQT2 gene, the primer pair was designed as follows (the upstream primer specifically binds to the NtHQT2 gene, and the downstream primer specifically binds to the GFP tag):

[0186] Upstream primer: 5′-TTGCCACCAGGTTACTTAG-3′;

[0187] Downstream primer: 5′-CGTCGTCCTTGAAGAAGAT-3′;

[0188] For the NtHQT3 gene, the primer pair was designed as follows (the upstream primer specifically binds to the NtHQT3 gene, and the downstream primer specifically binds to the GFP tag):

[0189] Upstream primer: 5′-AAGAATGGACGACGAATACT-3′.

[0190] Downstream primer: 5′-ATGGCGGACTTGAAGAAG-3′;

[0191] PCR amplification was performed using the genomic DNA of the control, K326 and T0 generation seedlings as templates, and the T0 generation positive seedlings were those that amplified specific bands.

[0192] Furthermore, the expression level of the NtHQT2 (NtHQT3) gene in the positive transgenic seedlings was detected by qPCR using the tobacco L25 gene as an internal reference gene. The relevant primers were designed as follows:

[0193] Targeting the NtHQT2 gene:

[0194] Upstream primer: 5'-AGTATCATCCTCCTCCATCT-3',

[0195] Downstream primer: 5′- TTCGTAAGTGCTACCATCAT-3′;

[0196] Targeting the NtHQT3 gene:

[0197] Upstream primer: 5'-TACTTCTGGTGACATCTCTTC-3'

[0198] Downstream primer: 5′- GCCGTCAGATAATGTATGGA-3′;

[0199] For L25 internal reference gene:

[0200] Upstream primer: 5'-CCCCTCACCACAGAGTCTGC-3',

[0201] Downstream primer: 5′- AAGGGTGTTGTTGTCCTCAATCTT-3′;

[0202] Finally, according to the CT value obtained by qPCR, 2 -△△CT Methods The relative expression levels of NtHQT2 and NtHQT3 genes were calculated.

[0203] Example 5

[0204] For the transgenic lines obtained in Example 3 and Example 4, the inventors conducted further phenotypic observations and chlorogenic acid content detection, and the relevant results are briefly described below.

[0205] (1) Growth phenotype

[0206] Some typical phenotypic comparison results are as follows Figure 7 、 Figure 8 As shown. By comparison, we can see that:

[0207] For the NtHQT2 gene, statistical results of related growth phenotypes showed that after overexpressing the NtHQT2 gene, plant height increased by an average of 12.5 cm compared to the control K326, the total number of effective leaves decreased by 3-4, flowering time was advanced by about 10 days, the length of the largest leaf in the middle increased by 8.2 cm, and the leaf width increased by 13.7 cm. However, the phenotypic changes of the NtHQT2 gene-edited plants were minimal.

[0208] That is, editing (gene silencing) the NtHQT2 gene does not lead to significant differences in related growth phenotypes, but overexpressing the gene can significantly increase plant height, promote early flowering, and increase the area of ​​mid-leaf leaves while reducing the total number of effective leaves.

[0209] The growth phenotype of the NtHQT3 gene was similar to that of the NtHQT2 gene, but there were also significant differences in specific phenotypic data. Specifically, plant height increased by an average of 12.0 cm compared to the control K326, the total number of leaves decreased by approximately 3, flowering time was advanced by approximately 7 days, the length of the largest leaf in the middle increased by 4.6 cm, and the leaf width increased by 12.8 cm. However, the phenotypes of the NtHQT3 gene-edited plants and the control K326 did not change much.

[0210] That is, editing (silencing) the NtHQT3 gene did not result in significant differences in related growth phenotypes. However, overexpressing the gene significantly increased plant height, promoted early flowering, and increased the area of ​​mid-leaf leaves while reducing the total number of effective leaves. (However, the phenotypic effects of the NtHQT2 gene, such as the increase in plant height, early flowering time, and increased leaf area, were smaller than those of the NtHQT2 gene.)

[0211] (II) Gene expression

[0212] The expression of NtHQT2 and NtHQT3 genes in some transgenic lines is as follows Figure 9 、 Figure 11 As shown in Figure 2, it can be seen that the expression levels of NtHQT2 and NtHQT3 genes in the overexpression lines were significantly increased.

[0213] (III) Chlorogenic acid content

[0214] The inventors used GC-MS analysis to detect the chlorogenic acid content in the transgenic lines. The HPLC-UV absolute quantitative analysis conditions were as follows: Symmetry C18 column (4.6×250 mm, 5 µm), detection wavelength 340 nm, injection volume 5 µL, flow rate 1.0 mL / min;

[0215] Mobile phase A was water / methanol / acetic acid (44 / 5 / 1, v / v / v), and mobile phase B was methanol / water / acetic acid (44 / 5 / 1, v / v / v);

[0216] Gradient elution:

[0217] 0-15.0 min, 10% B-30% B;

[0218] 15.0~26.0 min, 30%B-90%B;

[0219] 26.0~28.0 min, 90% B;

[0220] 28.1-35.0 min, 10% B.

[0221] The results showed that for NtHQT2 gene strains (such as Figure 10 ):

[0222] Compared to the control (K326), the chlorogenic acid content in the middle leaves of the T2-generation gene-edited plants (BJ1, BJ2, and BJ3) decreased by 34.6%, 29.1%, and 21.5% at the mid-leaf maturity stage. However, the chlorogenic acid content in the middle leaves of the T2-generation overexpressing tobacco plants (OE-1, OE-2, and OE-3) increased by 71.2%, 82.4%, and 101.7%, respectively, at the mid-leaf maturity stage compared to the control (K326). This result indicates that the NtHQT2 gene plays an important role in chlorogenic acid biosynthesis in tobacco.

[0223] Regarding NtHQT3 transgenic lines: Compared to the control (K326), the chlorogenic acid content in leaves at the tenth leaf position (mid-leaf maturity) of T2-generation transgenic plants overexpressing NtHQT4 (OE-1, OE-2, and OE-3) increased by 98.1%, 81.2%, and 101.7%, respectively. Chlorogenic acid content in leaves at the tenth leaf position (mid-leaf maturity) of T2-generation gene-edited plants (BJ1, BJ2, and BJ3) decreased by 15.4%, 22.0%, and 28.2%, respectively. These results demonstrate that the NtHQT3 gene plays a significant role in chlorogenic acid biosynthesis in tobacco.

Claims

1. Application of a series of tobacco NtHQT genes in tobacco, characterized in that: Overexpression of NtHQT2 gene or NtHQT3 gene is used to increase plant height, promote early flowering, and increase the leaf area of ​​the middle leaf; The NtHQT2 gene, the base sequence is shown in SEQ ID No. 1; The base sequence of the NtHQT3 gene is shown in SEQ ID No.

2.

2. The use of the tobacco NtHQT gene series in tobacco according to claim 1, characterized in that: When the NtHQT2 gene or the NtHQT3 gene is overexpressed, a recombinant plasmid expression vector with the pCAMBIA1300 plasmid as a vector and recombinant NtHQT2 gene or NtHQT3 gene is used.

3. The use of the tobacco NtHQT gene series in tobacco according to claim 2, characterized in that: When the pCAMBIA1300 plasmid is used as a vector and the NtHQT2 gene or the NtHQT3 gene is recombined, the specific primer pairs are designed as follows when the series of tobacco NtHQT genes are prepared by PCR amplification: NtHQT2-F:5'-ATGGGAAGTGAAAAGATGAT-3', NtHQT2-R:5'-TCAAAATTCATAAAATACTT-3'; NtHQT3-F: 5'-ATGAAGATCGACATTAAGG-3', NtHQT3-R:5'-TCAAAAGTCATACAGGTACT-3'.

4. A method for breeding new tobacco varieties with a series of tobacco NtHQT genes, characterized in that: By using genetic engineering technology, by overexpressing the NtHQT2 gene or the NtHQT3 gene, tobacco varieties with increased plant height, early flowering, and increased leaf area in the middle leaf position are obtained; The NtHQT2 gene, the base sequence is shown in SEQ ID No. 1; The base sequence of the NtHQT3 gene is shown in SEQ ID No.

2.

5. The method for breeding new tobacco varieties with a series of tobacco NtHQT genes as claimed in claim 4, characterized in that: When the NtHQT2 gene or the NtHQT3 gene is overexpressed, a recombinant plasmid expression vector with the pCAMBIA1300 plasmid as a vector and recombinant NtHQT2 gene or NtHQT3 gene is used.

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