Application of NtGS2 gene in increasing amino acid content and chlorophyll content of tobacco leaves

Abstract

The invention discloses an application of an NtGS2 gene in increasing amino acid and chlorophyll content of tobacco leaves. According to the research, two GS2 family genes NtGS2-1 and NtGS2-2 are cloned from a tobacco cultivated variety K326; the glutamine synthetase activities of NtGS2-1 and NtGS2-2 overexpressed plant leaves are respectively increased by 41% and 13% compared with those of a control group, and the total amino acid contents are respectively 1.46 times and 1.93 times of those of a wild type. NtGS2-1 and NtGS2-2 play an important role in amino acid metabolism of tobacco, and besides glutamine, the NtGS2-1 and NtGS2-2 can promote synthesis of various other amino acids, which shows that the NtGS2-1 and NtGS2-2 have great application potential in tobacco quality improvement and production.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, and in particular to application of NtGS2 gene in improving the amino acid and chlorophyll contents of tobacco leaves. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an important leaf-type economic crop in my country, with the largest planting area and yield in the world. Amino acids and chlorophyll are the main nitrogen-containing compounds in tobacco leaves, which not only have important physiological functions, but are also closely related to tobacco leaf quality. The Maillard reaction between amino acids and reducing sugars is one of the main ways to form tobacco leaf aroma substances. Increasing the amino acid content of the Maillard reaction substrate has an important role in promoting tobacco leaf quality, especially in aroma. Glutamine, aromatic amino acids, alanine, etc. are not only high in tobacco leaves, but also have a high conversion rate in the Maillard reaction. The threshold of small molecules such as acetic acid, 2-acetylfuran, 2,6-dimethylpyrazine, 2,5-pyrrolidinedione, furfural, styrene, benzaldehyde, etc. produced by the cracking of the Maillard reaction products is low, and the contribution rate to the aroma of tobacco leaves is large. In addition, aromatic amino acids themselves can also be directly cracked to produce aroma substances, such as phenylalanine, which can be cracked into benzyl alcohol, phenylethanol and other aroma substances that can cover up the impurities. Chlorophyll can be converted into neophytadiene during the baking process. Neophytadiene has the highest content in tobacco neutral volatiles and is an important aroma substance in tobacco leaves. Therefore, increasing the content of glutamine, aromatic amino acids, alanine, etc. in tobacco leaves plays an important role in improving the quality of tobacco leaves.

[0003] 95% of NH4+ in higher plants is assimilated into amino acids through the glutamine synthetase / glutamate synthetase (GS / GOGAT) cycle. Glutamine synthetase (GS) catalyzes glutamate and ammonium to produce glutamine, playing a very important role in nitrogen metabolism. GS exists in plants as a group of isozymes, located in the cytosol (GS1) or plastids (GS2), and participates in various physiological processes in the plant life cycle. It plays a very important regulatory role in plant growth and development, nitrogen assimilation, and many other aspects.

[0004] Glutamine synthetase exists in animals, plants and microorganisms. At present, GS in organisms can be divided into three categories: GSⅠ mainly exists in prokaryotes, GSⅡ mainly exists in eukaryotes, and GSⅢ mainly exists in Bacteroides, Butyrivibrio and some cyanobacteria. GS in higher plants belongs to GSⅡ, which is encoded by a small nuclear gene family and is divided into two forms, namely GS1 and GS2, with different subcellular localization and molecular weight. The molecular weight of cytoplasmic glutamine synthetase (GS1) isozyme is 38-40kDa, encoded by 3-5 genes, and varies from species to species. Glutamine synthetase isozyme (GS2) is distributed in plastids, with a molecular weight of 44-45kDa, usually encoded by only one gene, and rarely by two genes. It is currently recognized that the structure of GS active isozyme is a decamer, composed of two face-to-face pentamer rings of identical subunits, with a total of ten active sites, each of which is formed between every two adjacent subunits in each ring.

[0005] The metabolic process of nitrogen in tobacco leaves is very complex. Tobacco has two glutamine synthetase genes, and their regulatory effects on tobacco amino acids and chlorophyll are still unclear. Genetic engineering may be an effective way to improve the nitrogen flow process in tobacco leaves and make the content of specific amino acids more in line with our expectations. Therefore, it is urgent to study the genes that regulate the distribution and flow of nitrogen in tobacco leaves in order to improve the quality of tobacco. Summary of the invention

[0006] In view of this, the object of the present invention is to provide an application of NtGS2 gene in increasing the amino acid and chlorophyll contents of tobacco leaves.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. Application of NtGS2 gene in increasing the amino acid and chlorophyll content of tobacco leaves.

[0009] In some embodiments of the present invention, the coding region sequence of the NtGS2 gene is shown as Nitab4.5_0000059g0010.1 or Nitab4.5_0003070g0010.1.

[0010] In some embodiments of the present invention, the method for expressing the NtGS2 gene is to construct the NtGS2 gene into a plant binary expression vector, then transform tobacco under the mediation of Agrobacterium tumefaciens, and screen positive transformed plants with increased expression of the NtGS2 gene.

[0011] In some embodiments of the present invention, the amino acid is glutamine, phenylalanine, alanine, arginine, asparagine, leucine, isoleucine, threonine or methionine.

[0012] In some embodiments of the present invention, the chlorophyll is chlorophyll a or chlorophyll b.

[0013] In some embodiments of the present invention, the tobacco is cultivated tobacco.

[0014] The beneficial effects of the present invention are as follows: in this study, two GS2 family genes NtGS2-1 and NtGS2-2 were cloned from the tobacco cultivar K326; by constructing a GFP fusion expression vector and transiently expressing it in Nicotiana benthamiana, it was confirmed that the subcellular localization of NtGS2-1 and NtGS2-2 proteins were both in chloroplasts; the tissue expression patterns of NtGS2-1 and NtGS2-2 were similar, mainly expressed in leaves, with a small amount of expression in stems and buds, and basically no expression in roots, and the expression level of NtGS2-2 in various tissue parts was significantly higher than that of NtGS2-1.

[0015] By constructing an overexpression vector and genetically transforming tobacco K326, the phenotype of transgenic positive plants was identified, glutamine synthetase activity was determined, chlorophyll content was determined, and total amino acids and 19 amino acids were determined. The results showed that overexpression of NtGS2-1 and NtGS2-2 in tobacco had no significant effect on plant growth, the chlorophyll b content of NtGS2-1 overexpression plants was significantly increased compared with the wild type, and the chlorophyll a and total chlorophyll of NtGS2-2 overexpression plants were significantly increased compared with the wild type. The glutamine synthetase activity of leaves of NtGS2-1 and NtGS2-2 overexpression plants increased by 41% and 13% respectively compared with the control, and the total amino acid content was 1.46 times and 1.93 times that of the wild type, respectively. The contents of 11 amino acids in NtGS2-1 overexpressing plants were significantly increased compared with the wild type, including glutamine, asparagine, serine, glycine, arginine, phenylalanine, leucine, isoleucine, alanine, threonine and methionine. Among them, glutamine, phenylalanine, alanine and arginine had higher activity in Maillard reaction, which were increased by 18%, 19%, 34% and 21% respectively compared with the wild type, and the contents of other amino acids were not significantly different from those of wild type. In NtGS2-2 overexpressing plants, the contents of 16 amino acids were significantly increased compared with the wild type, among which the contents of glutamine, phenylalanine, alanine and tyrosine were increased by 50%, 36%, 40% and 28% respectively compared with the wild type, and the contents of other amino acids were not significantly different from those of wild type. The results showed that NtGS2-1 and NtGS2-2 play an important role in the amino acid metabolism of tobacco, and in addition to glutamine, they can also promote the synthesis of many other amino acids.

[0016] The mutation of NtGS2 gene also caused the changes of multiple amino acid contents in tobacco leaves. These results show that NtGS2 gene has great application potential in tobacco leaf quality improvement and production. By using these materials, we can further study the molecular mechanism of coordinating different amino acid contents in tobacco leaves to improve tobacco leaf quality, and provide theoretical and material support for the breeding of high-quality tobacco varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0018] Figure 1 The NtGS2 gene was amplified by PCR; lane 1 and lane 2 were the amplified bands of NtGS2-1 and NtGS2-2, respectively.

[0019] Figure 2 Subcellular localization of GS2-1 and GS2-2.

[0020] Figure 3 Expression pattern analysis of NtGS2-1 and NtGS2-2; A: Expression pattern analysis of NtGS2-1; B: Expression pattern analysis of NtGS2-2; C: Comparison of the expression of NtGS2-1 and NtGS2-2 in tissues and organs.

[0021] Figure 4 Identification of NtGS2-1 and NtGS2-2 overexpressing transgenic plants; A: qRT-qPCR detection of the expression level of NtGS2-1 in NtGS2-1 overexpressing plants; B: qRT-PCR detection of the expression level of NtGS2-2 in NtGS2-2 overexpressing plants, asterisks indicate statistical significance, ** represents p<0.01, *** represents p<0.001 (analyzed by Ordinary one-way ANOVA).

[0022] Figure 5 The aboveground phenotypes of NtGS2-1 and NtGS2-2 overexpressing plants; A: 30 days after plant transplantation; B: 80 days after plant transplantation; CH: agronomic trait statistics of plant height, stem girth, internode length, maximum leaf length, maximum leaf width, and number of effective leaves.

[0023] Figure 6 BE represents the underground phenotype of NtGS2-1 and NtGS2-2 overexpressing plants; BE represents the total length of root system, total surface area of ​​root system, total volume of root system and number of root tips, respectively.

[0024] Figure 7 Glutamine synthetase activity and total amino acid content in NtGS2-1 and NtGS2-2 overexpressing plants were determined.

[0025] Figure 8Amino acid determination of NtGS2-1 and NtGS2-2 overexpressing plants; the overexpression amino acid content was determined about 30 days after the plants were transplanted.

[0026] Fig. 9 This is a cluster heat map of amino acid content in NtGS2-1 and NtGS2-2 overexpressing plants.

[0027] Fig.10 To determine the chlorophyll content in NtGS2-1 and NtGS2-2 overexpressing plants.

[0028] Fig.11 Schematic diagram of the NtGS2 gene editing vector.

[0029] Fig.12 qRT-PCR was used to detect the expression of NtGS2-1 and NtGS2-2 in gene-edited strains.

[0030] Fig.13 Phenotypes of homozygous edited plants of NtGS2-1 and NtGS2-2; A: 10 days after plant transplantation; B: Observation of aboveground phenotypes of plants 30 days after plant transplantation; C: Observation of underground phenotypes of plants 30 days after plant transplantation; DG: Statistics of underground traits including total root length, total root surface area, total root volume, and number of root tips 30 days after plant transplantation.

[0031] Fig.14 Phenotypic observation and agronomic trait statistics of gene-edited plants 80 days after transplanting.

[0032] Fig.15 Glutamine synthetase activity and total amino acid content were determined in gene-edited plants.

[0033] Fig.16 Determination of amino acid content in gene-edited plants.

[0034] Fig.17 Cluster analysis of amino acid content in gene-edited plants.

[0035] Fig.18 NH4 for NtGS2 gene editing plants + Content determination.

[0036] Fig.19 Determination of chlorophyll content in gene-edited plants.

[0037] Fig. 20 To detect the expression level of NtGS1 in the leaves and roots of gene-edited plants.

[0038] Fig.21 To detect the expression level of NtGDH1 in the leaves and roots of gene-edited plants. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] Example 1. Cloning of NtGS2-1 and NtGS2-2

[0041] Two full-length coding region sequences of glutamine synthetase 2 (Nitab4.5_0000059g0010.1 and Nitab4.5_0003070g0010.1) were obtained from the tobacco database, and primers GS2-TF and GS2-TR were designed based on the sequences using Primer Premier 5. Total RNA was extracted from K326 tobacco leaves and reverse transcribed into cDNA. The cDNA was used as a template and the high-fidelity enzyme MegaFi was used to amplify the RNA. TM Fidelity 2X PCR Master Mix was used to perform PCR amplification of the target gene fragment. The specific reaction system is shown in Table 1, the reaction procedure is shown in Table 2, and the primers used in the present invention are shown in Table 3 and Table 2.

[0042] Table 1 Gene cloning reaction system

[0043]

[0044] Table 2 PCR reaction program

[0045]

[0046]

[0047] Table 3 Primers used in this study

[0048]

[0049]

[0050] The PCR products were subjected to electrophoresis, and the results were as follows: Figure 1As shown, the fragment size has a single band in the upper region of 1000bp. The fragment gel is recovered, connected to the T vector and transformed into Escherichia coli. After bacterial inspection, the positive clones are sequenced. The sequencing results show that 2 NtGS2s are amplified, and they are completely consistent with the reference sequence, without deletions and mismatches. The length of the 2 NtGS2 sequences is 1299bp, and both encode 432 amino acids. The CDS sequences of NtGS2-1 and NtGS2-2 are highly similar, with a difference of 29bp, and the encoded proteins have a difference of 1 amino acid. The gene sequence obtained by amplifying the Nitab4.5_0000059g0010.1 sequence is named NtGS2-1, and the Nitab4.5_0003070g0010.1 sequence is named NtGS2-2.

[0051] Example 2. Subcellular localization of NtGS2-1 and NtGS2-2

[0052] In order to clarify the subcellular localization of two glutamine synthetase 2 in tobacco, this study constructed the coding sequence of GS2-1 and GS2-2 proteins into the pCAMBIA1300-eGFP vector and transformed Agrobacterium GV3101 for transient expression in Nicotiana benthamiana. After 2 days of dark culture, fluorescence was observed using a confocal microscope. The specific steps are as follows:

[0053] (1) Streak the positive Agrobacterium onto YEB solid medium and culture at 28°C for 2 days.

[0054] (2) Pick a single spot and inoculate it into 10 mL YEB liquid culture of the corresponding resistance, and culture it at 28°C, 200 rpm, and shake overnight.

[0055] (3) Pipette 2 mL of overnight cultured bacterial solution into 40 mL YEB liquid culture of corresponding resistance, incubate at 28°C, 200 rpm, and shake for 4-6 h. Take 2 mL of bacterial solution and measure OD600 = 0.5.

[0056] (4) Centrifuge at 5000 rpm for 10 min at room temperature, discard the supernatant, resuspend the bacterial solution in MES (2.132 g of 10 mM MES powder, 0.9521 g of 10 mM MgCl2, 2.031 g of MgCl2.H20, add ddH2O to make up to 1 L, pH 5.6), and add 50 mg / mL AS (1000:1).

[0057] (5) Place the bacterial solution in a 28°C incubator for 2 h-4 h, and use a 1 mL sterile syringe to inject the bacterial solution into the back of the tobacco leaf so that the entire leaf is soaked. Keep it in the dark for 24 h and in the light for 24 h.

[0058] (6) Two days after the injection, use a handheld fluorescence microscope to observe whether there is fluorescence. If there is a signal, use a confocal microscope to observe and take pictures.

[0059] The results are as follows Figure 2 As shown, NtGS2-1-GFP and NtGS2-2-GFP both showed dot-like green fluorescence, which completely overlapped with the chloroplast autofluorescence, so GS2-1 and GS2-2 proteins were localized in chloroplasts. A careful observation revealed that the green fluorescence distribution of NtGS2-1-GFP and NtGS2-2-GFP was different, and some of the dot-like fluorescence of NtGS2-2-GFP showed a ring distribution. By observing the bright field, it was found that the ring existed between epidermal cells, so the NtGS2-2 protein existed in guard cells in addition to chloroplasts in epidermal cells.

[0060] Example 3. Expression pattern analysis

[0061] In order to clarify the expression of NtGS2 in various tissues and organs of tobacco, RNA was extracted from roots, stems, leaves, and buds of wild-type tobacco K326 30 days after transplantation and reverse transcribed into cDNA. The relative expression levels of NtGS2-1 and NtGS2-2 were detected by real-time fluorescence quantitative PCR. Green qPCR SuperMix was used for qRT-PCR experiments, mainly for detecting the relative expression of genes. The reaction system and amplification procedures are shown in Tables 4 and 5. The quantitative results were analyzed using the corresponding software Bio-Rad CFX 3.1 / Bio Rad CFXManager.

[0062] Table 4 qRT-PCR reaction system

[0063]

[0064] Table 5 qRT-PCR amplification program

[0065]

[0066] The results showed that the expression patterns of NtGS2-1 and NtGS2-2 were similar, with the highest expression in leaves, a small amount of expression in stems and buds, and almost no expression in roots. The results showed that the expression of NtGS2-1 and NtGS2-2 was tissue-specific ( Figure 3 , A, B). An independent sample t-test was performed on the relative expression levels of NtGS2-1 and NtGS2-2 in various parts. The results showed that the expression levels of NtGS2-2 in roots, stems, leaves, and buds were significantly higher than those of NtGS2-1, indicating that the expression level of NtGS2-2 in tobacco was higher than that of NtGS2-1 ( Figure 3 , C).

[0067] Example 4. Construction of overexpression vector and detection of transgenic plant traits

[0068] 1. Construction of overexpression vector and screening of positive transgenic plants

[0069] In order to further study the functions of NtGS2-1 and NtGS2-2 in tobacco and create tobacco germplasm materials with more coordinated amino acid content and higher quality, the overexpression vectors of NtGS2-1 and NtGS2-2 were constructed respectively, and genetic transformation was carried out with K326 as the background. The pCAMBIA1300-35S-EGFP vector was used to construct the p35S::NtGS2-1-eGFP vector. The overexpression vector was linearized by double restriction endonucleases BamHⅠ and XbaⅠ. Amplification primers GS2-1-1300-F and GS2-1-1300-R containing BamHⅠ and XbaⅠ restriction sites were designed. The positive plasmid with correct sequencing was used as a template to amplify the full-length CDS sequence of NtGS2-1. The sequence was connected to the vector backbone using the homologous recombination method, and then transformed into Escherichia coli. The vector universal primers 35S-F+GS2-1-1300-R were used for bacterial inspection. The amplified size was consistent with the expectation (1383bp). The bacterial solution with positive bacterial inspection was sent for sequencing. After correct sequencing, the plasmid was extracted and named pCAMBIA1300-NtGS2-1.

[0070] The pCAMBIA1302 vector was used to construct the p35S::NtGS2-2-eGFP vector. The pCAMBIA1302 vector was linearized by restriction endonucleases NcoⅠ and SpeⅠ, and primers GS2-2-1302-F+GS2-2-1302-R containing NcoⅠ and SpeⅠ restriction sites were designed. The coding region sequence of NtGS2-2 was amplified using the positive plasmid with correct sequencing as a template, and the sequence was connected to the vector backbone by homologous recombination. Escherichia coli was transformed and bacterial testing was performed using the vector universal primers 1302-F+GS2-2-1302-R. The test results showed that the size of the amplified fragment was in line with expectations (1732bp). The bacterial solution with positive bacterial testing was sent for sequencing. After sequencing was correct, the plasmid was extracted and named pCAMBIA1302-NtGS2-2.

[0071] Plasmids pCAMBIA1300-NtGS2-1 and pCAMBIA1302-NtGS2-2 were used for genetic transformation with the excellent tobacco cultivar K326 as the genetic background. 14 NtGS2-1 overexpressing transgenic plants and 12 NtGS2-2 overexpressing transgenic plants were obtained. In order to further determine the overexpression effect of transgenic plants, the gene expression of NtGS2-1 and NtGS2-2 was detected by qRT-PCR. NtGS2-1 and NtGS2-2 specific quantitative primers and internal reference primers were designed at NCBI, and total RNA from NtGS2-1, NtGS2-2 overexpressing plants and wild-type plants was extracted and reverse transcribed into cDNA. The quantitative results showed that in the NtGS2-1 overexpressing plants, the expression levels of NtGS2-1 in L1, L6, L7, L11, and L14 were significantly different from those in the wild-type K326 ( Figure 4 , A), which were 6.8, 4.9, 6.1, 5.7 and 12.5 times the expression of the wild type, respectively. The expression of other plants NtGS2-1 had no significant difference compared with the wild type. The gene expression of NtGS2-2 in NtGS2-2 overexpressing plants L2, L5, L8, L9 and L12 was significantly different from that of wild type K326 ( Figure 4 , B), which were 2.7 times, 3.2 times, 2.0 times, 1.5 times and 2.7 times the expression levels of the wild type, respectively. There was no significant difference in the expression levels of other plants NtGS2-2 compared with the wild type.

[0072] After harvesting the seeds of the T0 generation NtGS2-1 and NtGS2-2 overexpression transgenic lines, the T1 generation NtGS2-1 overexpression lines L1, L6, L7, L14 plants and NtGS2-2 overexpression lines L2, L5, L9, L12 plants were planted in the laboratory according to the identification results of the T0 generation overexpression plants. About 10 days after the T0 generation transgenic plant seeds germinated, the T1 generation overexpression plants were positively identified, and the specific detection primer combination GS2-1-1300-JC-F and GS2-1-1300-JC-R for the NtGS2-1 overexpression recombinant plasmid was designed, and the amplified fragment size was expected to be 719 bp. The specific detection primer combination 1302-F+GS2-2-1302-JC-R for the NtGS2-2 overexpression recombinant plasmid was designed, and the amplified fragment size was expected to be 940 bp. The CTAB method was used to extract plant DNA and used as a template for PCR amplification. ddH2O was used as a template and a negative control. The amplification size was consistent with expectations, and the positive rate of transgenic plants was high. The plants identified as positive were transplanted and the phenotype was observed.

[0073] II. Phenotypic Identification of NtGS2-1 and NtGS2-2 Overexpressing Plants

[0074] In order to clarify the effect of overexpression of NtGS2-1 and NtGS2-2 on plant growth, this study observed the growth of overexpressed plants. It was found that there was no significant difference in the growth of NtGS2-1 and NtGS2-2 overexpressed plants compared with the wild type ( Figure 5 , A, B). Agronomic traits were measured about 80 days after plant transplantation, including plant height, stem girth, internode length, maximum leaf length, maximum leaf width, and number of effective leaves of overexpressing plants and wild-type controls. Figure 5 As shown in Figure , CH, the analysis found that there were no significant differences in the agronomic traits of NtGS2-1 and NtGS2-2 overexpressing plants compared with the wild-type control.

[0075] The growth of the underground parts of NtGS2-1 and NtGS2-2 overexpressing plants was observed and statistically analyzed. Figure 6 The analysis results showed that the total root length, total surface area, total volume and number of root tips of NtGS2-1 overexpressing plants were not significantly different from those of the wild type; the total root length of NtGS2-2 overexpressing plants was significantly higher than that of the wild type, but the total root surface area, total volume and number of root tips were not significantly different from those of the wild type.

[0076] 3. Glutamine synthetase activity assay

[0077] In order to clarify the effect of overexpression of NtGS2 on the glutamine synthetase activity of plants, the glutamine synthetase activity of plants was measured about 30 days after transplantation of NtGS2-1 and NtGS2-2 overexpression plants. The glutamine synthetase activity was determined using the glutamine synthetase kit produced by Suzhou Grace Biotechnology Co., Ltd. Figure 7 As shown in A, the glutamine synthetase activity of NtGS2-1 and NtGS2-2 overexpressing plants was significantly higher than that of the wild type, which were 1.41 times and 1.13 times of the glutamine synthetase activity in the leaves of the wild type, respectively. This indicates that overexpression of NtGS2-1 and NtGS2-2 increased the glutamine synthetase activity in the leaves.

[0078] 4. Determination of free amino acid and total amino acid content

[0079] In order to analyze the effect of overexpression of NtGS2-1 and NtGS2-2 on the amino acid content of plants, the contents of 19 amino acids and total amino acids in NtGS2-1 and NtGS2-2 overexpressing plants were determined about 30 days after transplantation. The total amino acid content was determined using the total amino acid determination kit of Nanjing Jiancheng Bioengineering Research Institute and adjusted according to the results of the preliminary experiment. The total amino acid determination results are shown in Figure 7As shown in Figure , B, the total amino acid contents of NtGS2-1 and NtGS2-2 overexpressing plants were significantly higher than that of the wild type, which were 1.46 times and 1.93 times that of the wild type plants, respectively.

[0080] The contents of 19 amino acids in NtGS2-1 and NtGS2-2 overexpressing plants were compared with those in wild-type control plants by one-way ANOVA. The middle tobacco leaves of transgenic plants and wild-type control plants of the same period were selected as samples for amino acid determination. Suzhou Panomik Biopharmaceutical Technology Co., Ltd. performed targeted detection of free amino acids, specifically using mass spectrometry chromatography. The results are shown in the figure. Figure 8 As shown in the results, the contents of 11 amino acids in NtGS2-1 overexpressing plants were significantly increased compared with the wild type, including glutamine, asparagine, serine, glycine, arginine, phenylalanine, leucine, isoleucine, alanine, threonine and methionine. Among them, glutamine, phenylalanine, alanine and arginine had higher Maillard reaction activity, which were increased by 18%, 19%, 34% and 21% respectively compared with the wild type. The contents of other amino acids were not significantly different from those of wild type. In NtGS2-2 overexpressing plants, the contents of 16 amino acids were significantly increased compared with the wild type, among which the contents of glutamine, phenylalanine, alanine and tyrosine were increased by 50%, 36%, 40% and 28% respectively compared with the wild type. The contents of other amino acids were not significantly different from those of wild type. The results showed that NtGS2-1 and NtGS2-2 played an important role in the amino acid metabolism of tobacco. In addition to glutamine, they could also promote the synthesis of many other amino acids.

[0081] Agglomerate hierarchical clustering was used to analyze the relative quantitative values ​​of amino acid content in NtGS2-1, NtGS2-2 overexpressing transgenic plants and wild type. Fig. 9 As shown, the contents of most amino acids in NtGS2-1 and NtGS2-2 overexpressing plants were increased compared with the wild type, among which the contents of arginine, threonine, phenylalanine, serine, glutamine and other amino acids were increased compared with the wild type. Compared with NtGS2-1 overexpressing plants, NtGS2-2 overexpressing plants had greater differences compared with the control, and more types of upregulated amino acids were found.

[0082] Amino acids are important indirect precursors of tobacco aroma components and are closely related to aroma and taste. Properly increasing the amino acid content helps to improve the strength and richness of tobacco leaves (Deng Guobin et al., 2011; Geng Z et al., 2023). Amino acids produce Amadori compounds through the Maillard reaction. This intermediate product is then heated, rearranged, dehydrated and cracked to produce a large amount of flavor substances, giving tobacco a unique aroma (Kanzler et al., 2017). The decomposition of phenylalanine can directly form aroma precursors such as benzyl alcohol and phenylethanol; tyrosine can generate p-methylphenol, which can react with sugar to generate glycosides; tryptophan can generate methyl indole, hydroxy indole and indole (Cheng Changhe et al., 2014). The synthesis of nicotine, an important quality substance in tobacco, begins with the decarboxylation reaction of ornithine and arginine (Duan Shengzhi et al., 2015; Li Wangjie et al., 2016; Li Jinping et al., 2010). In addition, the increase in aromatic amino acids in tobacco will increase the content of phenylpropanoid metabolites and improve tobacco's tolerance to stress (Oliva et al., 2020). These results show that multiple amino acids in tobacco are significantly correlated with the intrinsic quality of tobacco leaves. In this study, multiple materials with changed amino acid content and composition of tobacco leaves were created by overexpression and gene editing of the NtGS2 gene. Among them, the overexpression of NtGS2 in tobacco significantly increased the content of various free amino acids with high Maillard reaction activity in tobacco leaves, such as Gln, Phe, and Ala, which had a positive effect on the formation of tobacco leaf quality during late baking and processing of tobacco. NtGS2 gene editing also caused changes in the content of multiple amino acids in tobacco leaves. These results show that the NtGS2 gene has great application potential in tobacco leaf quality improvement and production. These materials can be used in subsequent studies to further study the molecular mechanism of coordinating the content of different amino acids in tobacco leaves to improve tobacco leaf quality, and provide theoretical and material support for the breeding of high-quality tobacco varieties.

[0083] 5. Determination of chlorophyll content

[0084] Chlorophyll is the main pigment for photosynthesis in plants. The chlorophyll content in leaves is an indicator that reflects the ability of plants to photosynthesize. In addition, chlorophyll can be converted into neophytadiene during the baking process. Neophytadiene has the highest content in tobacco neutral volatiles and is an important aroma substance in tobacco leaves. We selected the NtGS2-1 overexpression strain L7 and the NtGS2-2 overexpression strain L12 to determine the content of chlorophyll a, chlorophyll b and total chlorophyll. The test results are shown in the figure. Fig.10As shown, the chlorophyll b content of the L7 plant of the NtGS2-1 overexpression line was significantly higher than that of the wild-type control, which was 1.31 times that of the wild-type, while chlorophyll a and total chlorophyll were not significantly different from those of the wild-type. In the NtGS2-2 overexpression line L12, chlorophyll a and total chlorophyll were significantly increased by 13% relative to the wild-type, while chlorophyll b was not significantly different from the wild-type. The results show that overexpression of NtGS2-1 and NtGS2-2 is beneficial to the increase of chlorophyll content.

[0085] Example 5. Knockout of NtGS2-1 and NtGS2-2 to verify the function and difference of regulating amino acid content

[0086] 1. Obtaining edited plants, identifying editing effects, and screening homozygous edited plants

[0087] In order to clearly study the functions and differences of the two glutamine synthetases 2 in tobacco, we constructed NtGS2-1 and NtGS2-2 gene editing vectors and performed genetic transformation based on the Boyuan Bio CRISPR / Cas gene editing vector pHSbdcas9i (K5). The enhanced CaMV35S promoter was used to efficiently express the Cas9 gene, and the CaMV35S promoter was used to efficiently express the hygromycin resistance gene. Target sites were designed on the website (http: / / crispr.hzau.edu.cn / CRISPR / ). Since the two NtGS2 sequences are highly similar, three targets were designed to simultaneously knock out NtGS2-1 and NtGS2-2. These three targets (Target 1, Target2, Target 3) were designed in the first three exon regions of NtGS2-1 and NtGS2-2, respectively. The plasmids of the recombinant vectors are as follows: Fig.11 shown.

[0088] After genetic transformation of the gene editing vector plasmid, we obtained 11 NtGS2 gene-edited plants, which were numbered CRISPR-L1, L2, L12, L14, L15, L18, L20, L21, L24, L25, and L28. First, we identified whether the gene-edited plants were positive, designed primers to detect cas9 protein fragments, and used the CTAB method to extract plant DNA for amplification. The expected amplified fragment size was 1153bp, and all 11 plants were able to successfully amplify the expected size fragment, indicating that all 11 plants were transgenic positive plants.

[0089] In order to further identify the gene editing effect of gene-edited plants NtGS2-1 and NtGS2-2, specific amplification primers GS2-1-BJJC-F and GS2-1-BJJC-917R were designed for the genomic sequences on both sides of the three target sites of the NtGS2-1 gene sequence, and PCR amplification and electrophoresis detection were performed using the gene-edited plant DNA as a template, and the amplified PCR products that met the expectations were sequenced. Finally, the sequencing results were compared with the genome sequence of the wild-type K326 using the software snapgene, and the specific editing forms of NtGS2-1 and NtGS2-2 in each edited plant were analyzed. Among the 11 positive gene-edited plants, CRISPR-L1 and CRISPR-L28 were double allele homozygous mutant plants in which both NtGS2-1 and NtGS2-2 were edited, and the rest of the plants were heterozygous mutant plants.

[0090] After bagging the T0-generation gene-edited plants and harvesting the seeds, the T1-generation gene-edited plants of various strains were planted in the laboratory. About 10 days after the plants were transplanted, the plant DNA was extracted, and the sequences of the three target regions were amplified with specific primers to identify the specific editing effects of the NtGS2-1 and NtGS2-2 genes of each gene-edited strain.

[0091] We screened out three biallelic homozygous mutant plants with single editing of NtGS2-1 in the T1 generation of CRISPR-L21. The editing forms were consistent. In the mutants, NtGS2-1 mutated at Target 1 and Target 3, and the base ACAGA was deleted at 185-189bp (Target 1), resulting in a frameshift mutation, which led to the early generation of the stop codon TGA and the early termination of protein synthesis. In the T1 generation of transgenic plants of strain CRISPR-L24, three biallelic homozygous mutant plants with single editing of NtGS2-2 were screened out. In the mutants, NtGS2-2 mutated at both Target 1 and Target 3, and 13 bases were deleted at 176-188bp (Target 1), resulting in a frameshift mutation, which led to the early generation of the stop codon TAA and the early termination of protein synthesis. In the mutant strain CRISPR-L28, NtGS2-1 lost the base A at 187bp, resulting in a frameshift mutation, which led to the premature generation of the stop codon TAA and the premature termination of protein synthesis. In the mutant strain CRISPR-L28, NtGS2-2 lost 84 bases at 160-242bp, resulting in the loss of 28 amino acids, and lost the base GCA at 367-369bp, resulting in the loss of alanine.

[0092] We named the gs2-1 / gs2-2 double mutant plants screened from strain CRISPR-L28, the gs2-1 single mutant plants screened from strain CRISPR-L21, and the gs2-2 single mutant plants screened from strain CRISPR-L24 as gs2-1 / gs2-2-L28, gs2-1-L21, and gs2-2-L24, respectively, for subsequent experiments (Table 6).

[0093] Table 6 Detection results of editing forms of T1 generation positive gene-edited plants NtGS2-1 and NtGS2-2

[0094]

[0095] About 20 days after the gene-edited transgenic plants were transplanted, RNA was extracted from gs2-1-L21, gs2-2-L24, and gs2-1 / gs2-2-L28 plants and reverse transcribed into cDNA. qRT-PCR was used to detect the expression of NtGS2-1 and NtGS2-2 in each strain. The test results are shown in Fig.12 In the gs2-1 / gs2-2-L28 strain, NtGS2-1 was hardly expressed, while the relative expression of NtGS2-2 was significantly lower than that of the wild type, which was 0.2 times that of the wild type; in the gs2-1-L21 strain, NtGS2-1 was hardly expressed, but NtGS2-2 was upregulated relative to the wild type; in the gs2-2-L24 strain, NtGS2-2 was hardly expressed, and NtGS2-1 was slightly downregulated relative to the wild type. The expression of NtGS2-1 and NtGS2-2 in the gene-edited strains was consistent with the sequencing results of NtGS2-1 and NtGS2-2 in each strain.

[0096] 2. Phenotypic identification of gene-edited plants

[0097] In order to clarify the effects of knocking out NtGS2-1 or NtGS2-2 alone and knocking out both NtGS2s at the same time on plant growth, this study observed the growth of each gene-edited strain. It was found that the germination rate of the gs2-1 / gs2-2-L28 double mutant plants was slower than that of the wild type, and the growth was slow and the leaves were yellow; while the gs2-1-L21 single mutant plants and the gs2-2-L24 single mutant plants could germinate and grow normally, with no significant difference in growth rate from the wild type, and the leaf color was normal ( Fig.13 , A).

[0098] About 30 days after the plant was transplanted, the growth status of the aboveground and underground parts of the plant is as follows: Fig.13 , B and Fig.13As shown in Figure , C, the aboveground parts of gs2-1-L21 and gs2-2-L24 single mutant plants were not much different from the wild type; while the leaves of gs2-1 / gs2-2-L28 double mutant plants lost green leaves, and the difference from the wild type plants gradually increased. In the statistical analysis of the underground parts, it was found that the root system of gs2-2-L21 single mutant plants had no significant difference from that of the wild type; the total root volume and root tip number of gs2-2-L24 single mutant plants were significantly reduced compared with the wild type; the various agronomic indicators of the root system of gs2-1 / gs2-2-L28 double mutant plants were significantly lower than those of the wild type ( Fig.13 , DG).

[0099] The growth status of the plant about 80 days after transplanting is as follows Fig.14 As shown, the gs2-1 / gs2-2-L28 double mutant plants grew slower, and the leaves became darker green than in the early stage. The aboveground agronomic traits of the gene-edited plants were measured and counted, including plant height, stem girth, internode, maximum leaf length, maximum leaf width and number of effective leaves. Statistical analysis showed that the agronomic traits of the mutant gs2-1-L21 were not significantly different from those of the wild type, indicating that knocking out NtGS2-1 alone did not affect plant growth; while the plant height, number of effective leaves, internode and stem girth of the gs2-2-L24 single mutant plants were significantly lower than those of the wild type, indicating that knocking out NtGS2-2 had a certain effect on the growth of tobacco. The plant height, stem girth, internode, maximum leaf length, maximum leaf width and number of effective leaves of the gs2-1 / gs2-2-L28 double mutant were significantly lower than those of the wild type, indicating that knocking out NtGS2-1 and NtGS2-2 at the same time had a great effect on the growth of tobacco.

[0100] 3. Determination of glutamine synthetase activity in edited plants

[0101] To clarify the effect of knocking out NtGS2 on the activity of glutamine synthetase in plants, we measured the activity of glutamine synthetase in the leaves of edited plants about 30 days after transplanting. Fig.15 As shown in Figure A, the glutamine synthetase activity in the leaves of gs2-1-L21 and gs2-2-L24 single mutant plants decreased slightly, but there was no significant difference compared with the control; while the glutamine synthetase activity in the leaves of the gs2-1 / gs2-2-L28 double mutant was significantly lower than that of the wild type, decreasing by 23%. This indicates that knocking out NtGS2-1 or NtGS2-2 alone has little effect on glutamine synthetase activity, while knocking out two NtGS2s at the same time will significantly reduce glutamine synthetase activity.

[0102] 4. Determination of amino acid content in edited plants

[0103] In order to clarify the effects of knocking out NtGS2-1 or NtGS2-2 alone and knocking out both NtGS2s at the same time on the amino acid content of plants, this study measured the total amino acid content of gene-edited plants and wild-type controls. About 30 days after the gene-edited plants were transplanted, the total amino acid content in the leaves was measured. Fig.15 As shown in Figure B, the total amino acid content in the leaves of gs2-1-L21, gs2-2-L24 single mutant plants and gs2-1 / gs2-2-L28 double mutant plants was significantly lower than that of the wild type, decreasing by 23%, 43%, and 54%, respectively. The results showed that knocking out NtGS2-1 or NtGS2-2 alone or knocking out both NtGS2 genes simultaneously would lead to a significant decrease in the total amino acid content in the leaves, and the decrease caused by knocking out both NtGS2 genes simultaneously was the most significant.

[0104] Since the double mutant plant gs2-1 / gs2-2-L28 grows very slowly, it is not suitable to measure the amino acid content in the early stage of plant growth. Therefore, about 70 days after the T1 generation plants of the NtGS2 gene-edited line were transplanted, the content of 19 amino acids in the leaves of the gene-edited plants was measured. A one-way ANOVA was performed on the amino acid content of the gene-edited plants and the wild-type control. The results are as follows Fig.16 As shown in the figure, the gs2-1-L21 single mutant plants had 4 kinds of amino acids (phenylalanine, alanine, glycine, and threonine) significantly decreased compared with the wild type, which were 0.74 times, 0.7 times, 0.82 times, and 0.66 times of the wild type amino acid content, respectively. The tryptophan content was significantly increased compared with the wild type, which was 1.33 times of the wild type. There were no significant differences in the other 14 amino acids compared with the wild type.

[0105] The gs2-2-L24 single mutant plants had 7 amino acids that were significantly decreased compared to the wild type, including asparagine, serine, histidine, arginine, tyrosine, lysine, and threonine. Among them, the decrease in aspartic acid, serine, and histidine was the largest, which were 0.45 times, 0.48 times, and 0.65 times the corresponding amino acid content of the wild type, respectively. In addition, 4 amino acids (aspartic acid, glycine, glutamic acid, and tryptophan) were significantly increased relative to the wild type, which were 1.32 times, 1.37 times, 1.46 times, and 1.41 times the corresponding amino acids of the wild type, respectively. There was no significant difference in the content of 8 amino acids compared to the wild type.

[0106] The gs2-1 / gs2-2-L28 double mutant plants had 8 amino acid contents significantly increased compared with the wild type, including glutamine, asparagine, aspartic acid, proline, glutamic acid, serine, alanine, and glycine. Among them, the contents of glutamine, asparagine, and aspartic acid increased the most, which were 9.73 times, 7.47 times, and 3.01 times of the corresponding amino acid contents of the wild type, respectively. In addition, 7 amino acids were significantly decreased compared with the wild type, including tryptophan, tyrosine, phenylalanine, isoleucine, histidine, serine, and valine. Among them, tryptophan, histidine, and isoleucine decreased the most, which were 0.15 times, 0.35 times, and 0.44 times of the corresponding amino acid contents of the wild type, respectively. In addition, the contents of 4 amino acids were not significantly different from those of the wild type.

[0107] Agglomerative hierarchical clustering was used to analyze the relative quantitative values ​​of amino acid content in gene-edited plants. The results are as follows: Fig.17 As shown, the analysis found that the amino acid content of the gs2-1-L21 single mutant plant was the least different from the wild type, followed by the gs2-2-L24 single mutant plant. The amino acid content of the gs2-1 / gs2-2-L28 double mutant plant was the most different from the wild type, and the content of multiple amino acids such as glutamine was increased, which was not in line with expectations. After the two NtGS2 knockouts in tobacco, the double mutant plants still contained a high amino acid content in the leaves 80 days after transplantation. It is speculated that this may be due to the large difference in plant growth conditions, or the existence of other genes that compensate for the function of NtGS2.

[0108] 5. Determination of ammonium ion content in plants

[0109] Glutamine synthetase is a key catalytic enzyme in the assimilation and utilization of nitrogen in plants. + The response of the gene-edited plants to the NH4 + The content was determined ( Fig.18 Error! Reference source not found.). The results showed that NH4 + The content was not significantly different from that of the wild type, indicating that knocking out NtGS2-1 or NtGS2-2 alone had little effect on the NH4 + The gs2-1 / gs2-2-L28 double mutant has NH4 + The content was significantly increased compared with the wild type, up to 40.47 times that of the wild type amino acids, which indicates that the simultaneous knockout of NtGS2-1 and NtGS2-2 will seriously hinder the NH4 +assimilation, which may lead to serious impact on plant growth and development.

[0110] VI. Determination of chlorophyll content in NtGS2 gene-edited plants

[0111] It was observed that knocking out both NtGS2-1 and NtGS2-2 had a significant impact on tobacco growth, causing yellowing of leaves and uneven leaf color in the later stages. About 40 days after the gene-edited plants were transplanted, the contents of chlorophyll a, chlorophyll b, and total chlorophyll were measured. Fig.19 As shown in the figure, the chlorophyll a, chlorophyll b and total chlorophyll contents of gs2-1-L21 and gs2-2-L24 single mutant plants were not significantly different from those of the wild type. However, the chlorophyll a, chlorophyll b and total chlorophyll contents of gs2-1 / gs2-1-L28 double mutant plants were significantly lower than those of the wild type, decreasing by 76%, 51% and 66%, respectively. This indicates that knocking out NtGS2-1 or NtGS2-2 alone does not affect chlorophyll synthesis, but knocking out NtGS2-1 and NtGS2-2 simultaneously will hinder the synthesis of chlorophyll in plants.

[0112] VII. Compensatory mechanisms after NtGS2 functional loss

[0113] In the phenotypic observation of gene-edited plants, we found that the phenotypic changes of gs2-1 single mutant plants and gs2-2 single mutant plants were small during transplantation, but the growth and development of gs2-1 / gs2-2 double mutant plants were extremely slow during the growth period, and the leaves showed yellowing symptoms. However, 60 days after transplantation, the yellowing gradually eased, the leaf color began to turn green, and the growth was slightly restored, and the seeds could be harvested. The content of 19 amino acids in double mutant plants was measured, and the results showed that the content of 8 amino acids, including glutamine, glutamic acid, asparagine, and aspartic acid, increased, which contradicted the results of the upregulation of amino acid content in overexpression plants. Therefore, we speculate that there are certain genes in mutant plants that can compensate for the loss of NtGS2 function and reduce the impact of NtGS2 mutation on plants. Studies have shown that the defect of Arabidopsis Gln2;0 function can be compensated by increasing the expression of AtGln1;1 and glutamate dehydrogenase (GDH1) (Ferreira et al., 2019). We compared the tobacco sequences with the highest homology to Arabidopsis AtGln1;1 and AtGDH1 through the NCBI website and the Solanaceae database: NtGS1 and GDH1. We also tested the expression levels of NtGS1 and GDH1 in gene-edited plants and wild-type plants. The results are as follows: Fig. 20 , Fig.21 As shown:

[0114] NtGS1 is expressed in both roots and leaves, with a higher expression level in roots. After NtGS2-1 mutation, the expression level of NtGS1 in leaves did not change significantly, but the expression level in roots decreased significantly; after NtGS2-2 mutation, the expression level of NtGS1 in leaves did not change significantly, but the expression level in roots increased significantly; in the double mutant, NtGS1 was significantly upregulated in both leaves and roots, with the expression level in leaves being 7.3 times that of wild-type plants and the expression level in roots being 4.4 times that of wild-type plants.

[0115] NtGDH1 is highly expressed in roots but hardly expressed in leaves. After the NtGS2-1 mutation, the expression of NtGDH1 in leaves did not change significantly, but the expression in roots decreased significantly; after the NtGS2-2 mutation, the expression of NtGDH1 in leaves and roots did not change significantly; in the double mutant, NtGDH1 was significantly upregulated in both leaves and roots, with the expression in leaves being 1.5 times that of wild-type plants and the expression in roots being 4.4 times that of wild-type plants.

[0116] The above results show that NtGS1 and NtGDH1 have no obvious compensatory effect in gs2-1 single mutant plants and gs2-2 single mutant plants. After NtGS2-1 and NtGS2-2 double mutations, glutamine synthesis is blocked, resulting in the inhibition of glutamate metabolism, thus affecting various biological metabolic processes involved in glutamate. NtGS1, as a cytoplasmic GS isozyme, synthesizes glutamine in roots and leaves, compensating for the functional loss of NtGS2; while NtGDH1 catalyzes the reaction of α-ketoglutarate with NH4 + Combined to form glutamate, which assimilates NH4 + , and effectively supplemented the source of glutamate and reduced NH4 + In addition, we found that in the double mutant, NtGS1-1 and NtGDH1 were expressed at higher levels in the roots, suggesting that tobacco can transport glutamine and glutamate synthesized in the roots to the leaves for use. This is consistent with the results of Ferreira et al. (2019), and tobacco also has a GS2 functional defect compensation mechanism similar to that reported in Arabidopsis.

[0117] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of NtGS2 gene in increasing the amino acid and chlorophyll content of tobacco leaves.

2. The application according to claim 1, characterized in that: The coding region sequence of the NtGS2 gene is shown in Nitab4.5_0000059g0010.1 or Nitab4.5_0003070g0010.

1.

3. The application according to claim 1, characterized in that: The method for overexpressing the NtGS2 gene is to construct the NtGS2 gene into a plant binary expression vector, then transform tobacco under the mediation of Agrobacterium tumefaciens, and screen positive transformed plants with increased expression of the NtGS2 gene.

4. The application according to claim 3, characterized in that: The amino acid is glutamine, phenylalanine, alanine, arginine, asparagine, leucine, isoleucine, threonine or methionine.

5. The use according to claim 3, characterized in that: The chlorophyll is chlorophyll a or chlorophyll b.

6. The use according to claim 3, characterized in that: The tobacco is cultivated tobacco.

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