CN115725640A - Application of tobacco transcription factor NtHB6 - Google Patents

Abstract

The invention discloses an application of a tobacco transcription factor NtHB6, wherein the nucleotide sequence of a coding region of the NtHB6 is shown as SEQ ID No.3, the tobacco transcription factor NtHB6 increases the content of nitrides in tobacco leaves, such as phenylalanine, glutamine, spermidine, isoleucine, glutamic acid, valine and the like, through positive regulation and control, more high-activity reaction substrate amino acids are provided for Maillard reaction, and thus the quality of the tobacco leaves is improved; meanwhile, the growth period of tobacco plants is shortened, and the method has important significance for quality breeding of tobacco leaves.

Description

Application of tobacco transcription factor NtHB6

Technical Field

The invention relates to the technical field of biology, in particular to application of a tobacco transcription factor NtHB6.

Background

Tobacco (Nicotiana tabacum L.) is a solanaceous crop with tobacco leaves as a main economic part, and nitrogen is a key factor influencing the yield and quality of the tobacco leaves. The nitrogen flow direction in the tobacco leaves determines the composition of nitrogen-containing compounds in the tobacco leaves, and the nitrogen-containing compounds in the tobacco leaves are closely related to the quality of flue-cured tobacco. The nitrogen transported to tobacco leaves in tobacco is stored in different cell structures of leaf cells and some free nitrogen-containing compounds, and the formula determines the strength of leaf photosynthesis, influences the toughness of leaves and the chemical defense strength. Amino acid, as one of the main nitrogen-containing compounds, has important physiological functions and is closely related to the quality of tobacco leaves. The activity of the glutamine and the aromatic amino acid participating in the Maillard reaction is higher, the content of the corresponding Amadori compound in the flue-cured tobacco leaves is also high, and the contribution to the Maillard flavor production in the baking process is larger.

A number of transcription factors are also involved in regulating the nitrogen flux in plants. Studies have shown that overexpression of the heterodimeric transcription factor E2Fa-DPa in arabidopsis results in the division of ectopic cells, leading to increased intracellular replication and arrest of early development in arabidopsis plants. After transcriptome analysis of E2Fa-DPa transgenic plants, the expression level of AtHB6 and other nitrogen metabolism related genes in the HD-Zip transcription factor family is obviously up-regulated, and the secondarily induced genes code proteins involved in the biosynthesis of Arabidopsis thaliana cell walls, and the functions of the transcription and signal transduction proteins are not clear. From the data presented by them, it was shown that many of the up-regulated expressed genes are involved in nitrate assimilation, nitrogen in transgenic plants is more available for nucleotide synthesis, while the reduction of synthesis of other nitrogen-containing compounds results in a reduced nitrate assimilation.

The homeodomain-Leucine Zipper (HD-Zip) protein is a transcription factor specific to higher plants, and plays an important role in aspects of vegetative growth, organ development, stress resistance and the like of the plants. The HD-Zip family transcription factor is composed of a Homeodomain (HD) domain encoded by a Homeobox gene (Homeobox, hox) and A Leucine Zipper (HALZ) element associated with the homeodomain, the HD domain being capable of specifically binding to DNA and the HALZ element being capable of mediating the formation of protein dimers. The HD-Zip transcription factor family comprises 4 subfamilies (HD-Zip I, II, III and IV) which are involved in the regulation and control of hormone level and interact with other proteins so as to regulate and control a plurality of biological processes of vegetative growth, reproductive growth, photomorphogenesis, leaf nitrogen flow regulation, fruit development, plant adversity response and the like of plants.

Therefore, it is necessary to research the genes for controlling the distribution and flow direction of tobacco leaf nitrogen in tobacco to improve the quality of tobacco.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of a tobacco transcription factor NtHB6 in improving tobacco leaf quality; the second purpose of the invention is to provide the application of the tobacco transcription factor NtHB6 in regulating and controlling the content of nitrogenous compounds in tobacco leaves; the invention also aims to provide the application of the tobacco transcription factor NtHB6 in shortening the growth period of tobacco plants.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the application of the tobacco transcription factor NtHB6 in improving the quality of tobacco leaves, wherein the nucleotide sequence of a coding region of the tobacco transcription factor NtHB6 is shown as SEQ ID NO. 3; the quality of the tobacco leaves is aroma quality, aroma quantity, taste or miscellaneous gas.

2. The application of the tobacco transcription factor NtHB6 in regulating and controlling the content of nitrogenous substances in tobacco leaves.

In a preferred embodiment of the present invention, the nitride-containing compound is at least one of phenylalanine, glutamine, spermidine, isoleucine, glutamic acid and valine.

3. The application of the tobacco transcription factor NtHB6 in shortening the growth period of tobacco strains, wherein the nucleotide sequence of the coding region of the tobacco transcription factor NtHB6 is shown as SEQ ID NO. 3.

The invention has the beneficial effects that: the invention discloses an application of a tobacco transcription factor NtHB6, wherein the NtHB6 gene is cloned from tobacco, and expression analysis shows that the NtHB6 has higher-level expression in tobacco leaves and flowers and the expression level in stems is relatively low; subcellular localization showed that NtHB6 localizes to the nucleus.

High-throughput transcriptome sequencing shows that the total number of genes up-regulated and expressed in the NtHB6 overexpression strain is 747, the total number of genes down-regulated and expressed in the NtHB6 overexpression strain is 276, and the total number of genes with expression quantity difference of more than 4 times is 44, so that the NtHB6 transcription factor is involved in regulating and controlling amino acid transport in the co-expression network and regulating related genes of nitrogen metabolites, and has a positive regulation function on the nitrogen metabolites in tobacco leaves.

The field difference phenotype shows that the plant leaf shape of the NtHB6 overexpression plant is similar to that of the K326, the plant leaf shape of the NtHB6 interference plant is obviously different from that of the K326, the leaves are short, the included angle of stem veins is small, and the number of the leaves is reduced. The plant height of the NtHB6 interference plant is higher than that of an overexpression plant and K326 in the early stage, the plant height of the NtHB6 interference plant is not obviously different from that of a control K326 in the later stage, and the leaf number, the length and the width of the top-cut waist leaves are obviously different from that of the K326; the over-expression strain height and the leaf number are obviously lower than K326. The NtHB6 gene expression quantity is increased or reduced, so that the tobacco plant can bloom in advance. The NtHB6 transcription factor can be presumed to regulate the growth and development process of the common cultivated tobacco, and shows that the transgenic plants enter the vigorous growth period earlier, so that the utilization efficiency of nitrogen is improved, and the synthesis, the morphogenesis (plant height), the reproductive growth and the like of nitrogen flowing to nitrogen-containing compounds (such as amino acid) in the tobacco leaves are changed.

The transcriptome results show that 2575 differentially expressed genes are identified in total in the NtHB6 overexpression strain compared with the control, wherein 186 genes up-regulated and expressed in the NtHB6 overexpression strain are included, 2389 genes down-regulated and expressed in the NtHB6 overexpression strain are included, and 62 genes with more than 4-fold difference in expression amount are included, including 18 up-regulated and 44 down-regulated genes. Differentially expressed genes up-regulated in transcriptome analysis of tobacco cultivated in a culture room are mainly enriched in cellular nitrogen compound metabolism (GO: 034641), N-terminal protein amino acid modification (GO: 031365), peptide or protein amino-terminal blocking (GO: 018409), N-terminal peptidyl-glycine N-myristoylation (GO: 018008), and protein amino acid phosphorylation (GO: 006468). The differential expression gene for down-regulating expression is mainly enriched in RNA silencing (GO: 031047), negative regulation of translation caused by miRNA silencing (GO: 035278), reproductive development process (GO: 003006) and phase transition of meristem from nutrition to reproduction (GO: 010228). The quantity of the DEGs which are down-regulated and expressed in the transcriptome sequencing data of the indoor cultivated tobacco is obviously more than that of the DEGs which are up-regulated and expressed. The overexpression of the NtHB6 transcription factor is presumed to reduce the expression of related genes in the processes of RNA silencing in the transcription process, translation negative regulation caused by miRNA silencing, ncRNA-mediated translation negative regulation and the like.

According to the difference analysis of nitrogen-containing compounds in NtHB6 transgenic tobacco, the content of spermidine in tobacco leaves of NtHB6 over-expression plants is remarkably higher than that of a control, and the content of isoleucine, glutamic acid, glutamine, valine and phenylalanine is remarkably higher than that of the control; the amount of spermine in tobacco leaves of an interference plant of NtHB6 is remarkably higher than that of a control, the amount of glutamine is remarkably lower than that of the control, and the content of glutamic acid is remarkably lower than that of the control.

The flue-cured tobacco quality analysis on the aspects of aroma quality, aroma quantity, taste, miscellaneous gas and the like of the tobacco leaves shows that the tobacco leaf quality of the NtHB6 overexpression transgenic plant is superior to that of the control K326.

Therefore, the NtHB6 can be used for regulating and controlling the content of the nitride in the tobacco leaves, improving the quality of the tobacco leaves and shortening the growing period of tobacco plants.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the gene structure of NtHB6, which includes 3 exons and 2 introns;

FIG. 2 shows the structural features and domain positions of the NtHB6 protein;

FIG. 3 shows the transcriptional activation activity of NtHB6 (A: full length, N-terminal, and C-terminal segmented fusion vector of the coding region of NtHB6 (NtHB 6: 342 amino acids in full length of the protein encoded by the coding region of NtHB 6; ntHB6-N: 270 amino acids encoded by the N-terminal of NtHB 6; ntHB6-C: 72 amino acids encoded by the C-terminal of NtHB 6); B: growth of vector-transformed AH109 yeast on SD/-Trp medium, growth of C: SD/-Trp/-His/-Ade medium);

FIG. 4 shows the expression level of NtHB6 in different tissues and organs of tobacco;

FIG. 5 shows phenotypic identification of K326 and NtHB6 transgenic lines (5-1: control K326 plant (A), ntHB6 overexpression (B) and interfering plant (C) field plant growth status; 5-2: ntHB6 overexpression (OV 1-1, OV1-2, OV3-1, OV3-2, OV8-1, OV 8-2) before growth (R1-1, R1-2, R4-1, R4-2, R5-1, R5-2) plant height, leaf number, leaf length and leaf width (A-D)), and 5-3: ntHB6 overexpression (OV 1-1, OV1-2, OV3-1, OV3-2, OV8-1, OV 8-2) after growth (R1-2, R4-1, R4-2, R5-1, R5-2) plant height, leaf length and leaf width (A-D)).

FIG. 6 shows the bud emergence rates 45 days and 48 days after the transplantation of K326 and NtHB6 overexpression (OV 1-1, OV1-2, OV3-1, OV3-2, OV8-1, OV 8-2), interference (R1-1, R1-2, R4-1, R4-2, R5-1, R5-2) strains.

FIG. 7 shows the subcellular localization of the NtHB6-pEarleyGate101 expression vector (A: taYFP fluorescence; B: marker nuclear localization fluorescence; C: ESID-T1 bright field; D: merged synthetic field).

FIG. 8 is a schematic diagram of PCA analysis of nitrogen-containing compounds in tobacco leaves (A: ntHB6 overexpression; B: interference with NtHB6 expression).

FIG. 9 is a NtHB6 overexpression strain PLS-DA analysis VIP scatterplot.

FIG. 10 is a NtHB6 interference strain PLS-DA analysis VIP scattergram.

FIG. 11 shows the content of differential nitrogen-containing compounds in the NtHB6 transgenic lines and K326 tobacco leaves (over-expression (A-E), interference (F-O) of NtHB6 and K326 tobacco leaves).

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 obtaining and cloning of NtHB6 Gene

Through high-throughput metabonomics analysis and transcriptome sequencing result analysis of tobacco leaves at different fertilization levels, a transcription factor NtHB6 which can possibly regulate and control the nitrogen flow direction of the tobacco leaves is screened out. According to NCBI: (http:// www.ncbi.nlm.nih.gov/) The Coding region sequence (CDS) of the Nicotiana benthamiana NtHB6 disclosed above is subjected to PCR amplification by using Geneius software to design a specific amplification primer NtHB6-F/NtHB6-R, taking tobacco cDNA as a template and taking NtHB6-F and/NtHB 6-R as primers:

NtHB6-F：5’-caccATGAAGAGAGTTCGTAGCTCTGATTC-3’(SEQ ID NO.1)；

NtHB6-R：5’-GGAATCTTTCCAATTATTCCAGTCCTCAG-3’(SEQ ID NO.2)；

PCR cloning reaction was performed using SEQ ID NO.1 and SEQ ID NO.2 as primers and 2 XPCR Precision Master Mix with dye kit (ABM) to obtain the coding region CDS full length (SEQ ID NO. 3) of the NtHB6 transcription factor. And carrying out electrophoresis detection on the obtained PCR product by using 1% agarose gel, recovering the PCR product gel, constructing an NtHB6-T5_ Zero cloning vector for sequencing, carrying out blast comparison on a sequencing result in an NCBI website, and finding that the size of the sequencing result is consistent with that of an expected gene coding region. Indicating that the cloned gene fragment can be used as a coding region fragment required by subsequent experiments.

Analysis of the full-length sequence of the NtHB6 genome revealed that the full-length sequence of the NtHB6 genome was 2135bp in length (SEQ ID NO. 4), comprised 3 exons and 2 introns, the Coding sequence (CDS) was 1029bp in length and 39.4% in GC content, as shown in FIG. 1. The number of amino acids of the tobacco NtHB6 protein is 342, the NtHB6 protein has the mass of about 35kD, the core region consists of an HD structural domain and a HALZ element, the composition and the structural characteristics are shown in figure 2, the HD structural domain is positioned between 53 and 106 amino acids, the HALZ structure is connected with the HD structural domain through threonine (Thr), and the HALZ element is positioned between 108 and 148 amino acids.

Example 2 analysis of NtHB6 transcriptional activation Activity

In order to confirm the transcription activation activity of NtHB6, yeast expression vectors pGBKT7-NtHB6, pGBKT7-NtHB6N and pGBKT7-NtHB6C are constructed, an unloaded pGBKT7 is taken as a negative control (FIG. 3, A), yeast strains AH109 are respectively transferred, the transformed yeast strains are screened in SD/-Trp, SD/-Trp/-His/-Ade solid culture media, and are cultured for 3-5d at 30 ℃, observed and photographed. The results show that AH109 yeast strains transformed with pGBKT7-NtHB6 and PGBKT7 can normally grow on SD/-Trp solid plates, which indicates that the vector construction is successful, and the transformed vectors have no toxicity to the yeast strains. And (3) selecting a single spot on the SD/-Trp, placing the single spot in a proper amount of sterilized water, gently blowing and mixing the single spot with a pipette, placing the single spot on the SD/-Trp/-His/-Ade solid medium, and allowing the yeast strain transformed into pGBKT7-NtHB6 to grow normally, wherein the yeast strain transformed into PGBKT7 cannot grow, which indicates that the NtHB6 has the transcription activation activity in the full length.

To further refine the region in which the transcriptional activation active site of NtHB6 is located, the N-terminal (SEQ ID NO. 5) and C-terminal (SEQ ID NO. 6) segmented fusion vectors pGBKT7-NtHB6-N, pGBKT7-NtHB6-C, and the control pGBKT7 were transformed into AH109, respectively. The results show that the yeast strains AH109 which are transferred into pGBKT7-NtHB6-N, pGBKT7-NtHB6-C and pGBKT7 can normally grow on SD/-Trp, and show that the construction of the segmented fusion vector is successful and the segmented fusion vector has no toxicity to the yeast strains. Further, growth conditions of AH109 transformed into pGBKT7-NtHB6-N, pGBKT7-NtHB6-C and pGBKT7 on SD/-Trp/-His/-Ade solid medium were analyzed, and only the strains transformed into pGBKT7-NtHB6-N and pGBKT7-NtHB6-C were found to grow normally, indicating that both the N-terminal and the C-terminal of NtHB6 have transcription activation activity (FIG. 3, B).

Example 3 analysis of NtHB6 expression Pattern

In order to clarify the expression pattern of NtHB6 in K326 major tissues and organs, RNA was extracted at the same time after K326 plants blossomed (125 d after transplantation), and reverse-transcribed into cDNA, and then the expression pattern of NtHB6 in K326 stems, leaves and flowers was analyzed by qRT-PCR. The results are shown in fig. 4, the NtHB6 has higher expression level in K326 tobacco leaves and flowers, and the expression level in stems is relatively low, which indicates that the NtHB6 is differentially expressed in different tissues and organs.

Example 4 construction of NtHB6 overexpression vector and interference vector

Construction of overexpression vector: the method comprises the steps of carrying out electrophoresis on an NtHB6 coding region sequence obtained by amplification, purifying the sequence by using a gel recovery kit, using a gel recovery product of an amplified fragment for TOPO cloning, using the TOPO kit to construct an intermediate vector of the NtHB6-PENTR according to the kit steps, carrying out Enzyme digestion reaction on the vector NtHB6-PENTR by using MluI restriction endonuclease, then recovering and purifying a linearized fragment gel, then putting a target fragment LR on a target vector pEarleyGate101, using a Gateway LR clone II Enzyme Mix of Yingweiji (Shanghai) trade company Limited, carrying out reaction according to the instruction steps by using an operation method, then carrying out Enzyme digestion recovery product of an entry clone positive plasmid (pENTR-HB 6) and an over-expression vector of the YileyGate LR 101 according to a Gateway LR reaction kit program, carrying out reaction for one hour at 37 ℃, transforming an Escherichia coli competent cell TOP10, taking a single-clone of a white clone, identifying the single colony, and carrying out correct detection by using PCR and then using the NtHB6-pEarlE as a bacterial liquid to obtain a NyHB 6-pEarleR-pE clone.

Constructing an interference vector: construction of RNA interference (RNAi) vector for the NtHB6 transcription factor based on the cloned nicotiana benthamiana NtHB6 transcription factor, a region corresponding to the full-length cDNA of the NtHB6 gene was used as an interference fragment for a specific conserved region of the gene family, and primers were designed:

to facilitate the directed cloning, a BamH I and an AatII site were introduced 5 'to the upstream primer and an XbaI and an NcoI site were introduced 5' to the downstream primer. The total cDNA of cultivated tobacco K326 is taken as a template, a primer combination RNAi-HB6-F + RNAi-HB6-R is used for amplifying an interference fragment of a transcription factor NtHB6, a 50 mu L standard system contains 0.5 mu L template and 1.5U Taq DNA polymerase, and other components have conventional contents. The PCR instrument setting program is as follows: denaturation at 94 deg.C for 5min, and circulating; denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 30s, extension at 72 ℃ for 1min,35 cycles; final extension at 72 ℃ for 10min. The PCR product was subjected to 1% agarose gel electrophoresis, and the band of interest (1026bp NtHB6 gene fragment) was excised after electrophoresis and recovered according to the instructions of the full-scale gold colloid recovery kit. Connecting the recovered HB6 fragment with a T5-ZERO vector according to a 10 mu L standard system according to the instruction of a connecting kit to obtain T5-HB6, then transforming DH5 alpha escherichia coli competent cells, screening through Kan (100 mg/L) resistance, identifying the bacterium liquid positive detection of white colony clones by adopting PCR, using RNAi-HB6-F + M13R and M13F + RNAi-HB6-R as detection primers, and carrying out sample sending and sequencing on the positive clones by the same procedure as above.

The DH5 alpha single clone containing T5-HB6 is cultured to the late logarithmic phase, and plasmid is extracted by adopting a plasmid extraction kit. T5-HB6 was completely digested simultaneously with NcoI + Aat II, and a 50. Mu.L system contained 20. Mu.g of plasmid, 10U of each of the two enzymes, and 1 XTango buffer. pFGC5941M is also subjected to the same complete double enzyme digestion, enzyme digestion is carried out for 1h at 37 ℃, electrophoresis is respectively carried out after the enzyme digestion is complete, then the vector skeletons of purified antisense fragments RNAi-HB6 and pFGC5941M are recovered, the two recovered fragments are connected by a 10 mu L standard system through a connecting kit, and the antisense fragment is subcloned between a CaMV 35S promoter and a spacer region in the pFGC 5941M. The ligation product is transformed into escherichia coli DH5 alpha competence, kan (100 mg/mL) resistant monoclonal colonies are selected for culture, bacterium liquid PCR detection is carried out on the clones by using primer combinations F35S3N + HB6-F and HB6-R + RBnPAP212, and full-positive clones are selected to extract plasmids which are named as pFGC5941M-HB6-1. The plasmid T5-HB6 and pFGC5941M-HB6-1 were completely digested with BamHI + XbaI in a double digestion system as described above. Carrying out enzyme digestion for 1h at 37 ℃, carrying out electrophoresis respectively after the enzyme digestion is completed, recovering a sense fragment HB6 and an open-loop pFGC5941M-HB6 skeleton after the electrophoresis, carrying out 10 mu L standard system recombination connection on the two according to the previous method, converting DH5 alpha competence after the reaction is completed, selecting a monoclonal colony, carrying out PCR detection on various bacteria liquid of a recombinant vector plasmid PFGC5941M-HB6-2, using primers F35S3N + RBnPAP212 and FBnPAP212+ ROCST5N, and using a full-positive person as a recombinant plasmid pFGC5941M-HB6-RNAi, namely an RNAi vector of NtHB6.

Example 5 transgenic plant acquisition and detection

The constructed vector is used for transforming the recombinant vector into tobacco K326 through an agrobacterium-mediated leaf disc method, a transgenic strain is screened, then plant phenotype and agronomic characters are observed, and the result is shown in the figure, the plant leaf shape of the NtHB6 overexpression plant is similar to that of the K326, the plant leaf shape of the NtHB6 interference plant is obviously different from that of the K326, the leaves are short, the included angle of stem veins is small, and the number of leaves is reduced (figure 5-1). The plant height of the NtHB6 interference plant is higher than that of an over-expression plant and K326 (figure 5-2) in the early stage, the plant height of the NtHB6 interference plant is not obviously different from that of a control K326 in the later stage, and the leaf number, the length and the width of the waist leaf after topping are obviously different from that of the K326; the over-expression strain height and leaf number are significantly lower than K326 (FIG. 5-3). Both increased and decreased expression levels of the NtHB6 gene can lead to premature flowering of tobacco plants (FIG. 6). The NtHB6 can shorten the growth period of the tobacco plant, so that the tobacco plant can enter reproductive growth in advance, and the agronomic character index of the tobacco plant is influenced.

Example 6 subcellular localization of NtHB6 transcription factors

In the subcellular localization experiment of the NtHB6 transcription factor, the constructed expression vector NtHB6-pEarleyGate101-EYFP with Enhanced yellow-green fluorescent protein tag (EYFP) is transformed into escherichia coli, and a full-scale gold plasmid macroextraction kit is used for extracting over-expression vector plasmids with high concentration to enable the concentration to reach 0.1mg/ml. And (3) transforming the overexpression vector into the prepared Nicotiana benthamiana protoplast, taking the protoplast after 16h to observe bright field, nuclear Marker fluorescence and EYFP fluorescence of the overexpression vector tag fusion protein, simultaneously collecting photos, and processing, wherein the result is shown in figure 7. The laser confocal results of the subcellular localization experiments for NtHB6 transcription factors were consistent with those expected from the SMART (http:// SMART. Embl. De /) website, with NtHB6 localized to the nucleus and completely overlapping with the control signal.

Example 7 Effect of NtHB6 on tobacco leaf quality of tobacco plants

(1) Differential analysis of nitrogen-containing compounds in NtHB6 transgenic tobacco

In order to further understand the difference between the nitrogen-containing compounds in the tobacco leaves of the NtHB6 transgenic plants planted in the field and the control, 3 strains of the NtHB6 which is over-expressed and interfered are respectively selected to measure the nitrogen-containing compounds, and the nitrogen-containing compounds are compared with the control. A supervised mode PAC-partial least squares discriminant analysis method (PLS-DA) model is established. As shown in FIG. 8, ntHB6 overexpression and interference strains can be distinguished from the control K326, which indicates that the nitrogenous compounds have certain differences compared with the control K326.

According to the VIP value (threshold value is 1) of the PLS-DA model, 8 nitrogen-containing compounds which greatly contribute to tobacco leaves in the Ov-NtHB6 vs K326 are screened, wherein the nitrogen-containing compounds are spermidine, Y-aminobutyric acid, asparagine, isoleucine, glutamic acid, glutamine, valine and phenylalanine respectively (figure 9); there are 13 nitrogen-containing compounds contributing greatly to tobacco leaves in RNAi-NtHB6 vs K326, which are spermine, methionine, glutamine, arginine, proline, glycine, asparagine, ammonium ion, aspartic acid, glutamic acid, leucine, isoleucine, and lysine, respectively (fig. 10).

Independent sample t-tests were performed for nitrogen-containing species with VIP >1.0 as shown in table 1. Compared with a control K326 and an NtHB6 overexpression strain and a control K326, compared with the screened VIP is more than 1.0, and 5 and 10 nitrogen-containing compounds with significant differences in t-test are respectively selected.

TABLE 1 test of nitrogen-containing Compounds in NtHB6 transgenic plants and K326 tobacco leaves

The spermidine in the tobacco leaves of the NtHB6 overexpression plants is remarkably higher than a control, and the contents of isoleucine, glutamic acid, glutamine, valine and phenylalanine are all remarkably higher than the control; the amount of the NtHB6 interfering plant in tobacco leaf is significantly higher than that of the control, the amount of glutamine is significantly lower than that of the control, and the content of glutamic acid is significantly lower than that of the control (figure 11). These results indicate that NtHB6 may have an effect on tobacco leaf quality mainly by regulating the contents of glutamine and phenylalanine.

(3) NtHB6 overexpression, interference expression and K326 middle leaf primary roasting sensory quality analysis

The sensory quality of the primary flue-cured tobacco leaves of NtHB6 over-expression, interference and contrast K326 plants, such as aroma quality, aroma quantity, taste, miscellaneous gas and the like, is evaluated. The result of the product suction is shown in table 2, the fragrance quality, fragrance amount, taste and miscellaneous gas scores of the primary flue-cured tobacco of the over-expressed NtHB6 transgenic plant are all higher than those of the control K326, the strength, combustibility and gray of the primary flue-cured tobacco are equivalent to those of the control K326, the fragrance quality, fragrance amount, taste and miscellaneous gas scores of the primary flue-cured tobacco of the interference strain NtHB6 are all lower than those of the control K326, and therefore, the tobacco leaf quality of the over-expressed NtHB6 transgenic plant is better than that of the control K326.

TABLE 2 sensory quality analysis of NtHB6 overexpression plants and K326 flue-cured tobacco leaves

In conclusion, the tobacco transcription factor NtHB6 can increase the content of glutamine, phenylalanine and the like in tobacco leaves through positive regulation, provide more high-activity reaction substrate amino acids for the Maillard reaction and further improve the quality of the tobacco leaves; meanwhile, the growing period of the tobacco plants is shortened.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (4)

1. The application of the tobacco transcription factor NtHB6 in improving the tobacco leaf quality is characterized in that: the nucleotide sequence of the coding region of the tobacco transcription factor NtHB6 is shown in SEQ ID NO. 3; the quality of the tobacco leaves is aroma quality, aroma quantity, taste or miscellaneous gas.

2. Application of the tobacco transcription factor NtHB6 in regulating and controlling the content of nitrogenous substances in tobacco leaves.

3. Use according to claim 2, characterized in that: the nitride-containing compound is at least one of phenylalanine, glutamine, spermidine, isoleucine, glutamic acid and valine.

4. The application of the tobacco transcription factor NtHB6 in shortening the growth period of tobacco plants is characterized in that: the nucleotide sequence of the coding region of the tobacco transcription factor NtHB6 is shown in SEQ ID NO. 3.

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