Plant NtBES1 protein and application thereof in regulating and controlling 6-phosphate-trehalose level in tobacco body

Abstract

The invention provides a plant NtBES1 protein and application of the plant NtBES1 protein in regulation and control of 6-phosphoric acid-trehalose level in tobacco bodies, and belongs to the technical field of tobacco regulation and control. The invention provides a complete tobacco-derived gene coding sequence, the regulation effect of the plant NtBES1 protein on the T6P level is defined in the tobacco body through a genetic transformation means, and due to the fact that the T6P in the plant body cannot be regulated through direct exogenous application of the T6P, the discovery of the regulation effect of the plant NtBES1 protein on the T6P level has the advantages that the regulation effect of the plant NtBES1 protein on the T6P level is obviously better than that of the plant NtBES1 protein on the T6P level; the method can be applied to tobacco endogenous T6P level regulation in the later period, so that the yield / quality of tobacco is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco regulation, and in particular relates to a plant NtBES1 protein and application thereof in regulating the level of 6-phosphate-trehalose in tobacco. Background Art

[0002] In plants, sugar is the direct product of plant photosynthesis and the common basis of material metabolism and energy metabolism. It participates in a series of basic physiological and biochemical processes such as carbon accumulation and energy supply. It is also the material basis of macromolecular compounds such as polysaccharides, proteins, and fats in plants. It plays an extremely important role in improving the yield and quality of tobacco leaves. In plants, photosynthesis, nutrient transport and distribution are all regulated by sugar. In the past, the effects of sugar on gene expression and plant growth and development were often considered to be secondary effects of material metabolism and energy metabolism, but with the deepening of research, more and more evidence shows that sugar is also a signal molecule. In particular, trehalose and its precursor trehalose-6-phosphate (T6P) are important signal molecules that regulate plant growth and development, and participate in plant metabolic regulation and gene expression regulation (O'Hara et al. 2013; Schluepmann et al. 2004; van Dijken et al. 2004). T6P can be used as a regulator of carbon metabolism and an enhancer of photosynthetic capacity (Paul et al. 2001; Eastmond and Graham 2003). Studies in wheat have shown that an increase in T6P can promote grain filling, thereby increasing yield. In addition, exogenous application of T6P analogs can significantly promote development and improve crop yield (Chemical intervention in plant sugar signaling increases yield and resilience).

[0003] Trehalose (α-D-pyranose glucose, α-D-pyranose glucoside) is a non-reducing disaccharide composed of two glucose molecules. T6P is the metabolic precursor of trehalose. It is the product of the condensation reaction of UDP-glucose and glucose-6-phosphate catalyzed by trehalose-6-phosphate synthase (TPS), which can be further dephosphorylated by trehalose-6-phosphate phosphatase (TPP) to produce trehalose (Cabib and Leloir 1958). At present, there are at least five different pathways known for trehalose synthesis, namely the OtsA-OtsB pathway, the TreP pathway, the TreS pathway, the TreY-TreZ pathway and the TreT pathway (Avonce et al. 2006; Paul et al. 2008). In plants, only the OtsA-OtsB trehalose synthesis pathway exists, which includes two enzymatic reactions. First, TPS catalyzes the reaction of uridine diphosphate-glucose with glucose-6-phosphate to form T6P, and then TPP catalyzes the dephosphorylation of T6P to form trehalose, which is further catalyzed by trehalase to generate two molecules of glucose (Avonce et al. 2006). TPS is the rate-limiting step in the synthesis of T6P. Its activity has a direct determining effect on the content of T6P. TPP determines the degradation of T6P. Therefore, the expression of TPS and TPP will affect the level of T6P in plants (chluepmann, H., Pellny, T., van Dijken, A., et al. (2003). "Trehalose6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsisthaliana." Proceedings of the National Academy of Sciences, 100 (11), 6849-6854).

[0004] In Arabidopsis, the TPS family contains 11 members and the TPP family contains 10 members, among which TPS1 is considered to be the main gene controlling T6P synthesis. In addition, these two enzymes are not only crucial in metabolic regulation, but also participate in signal transduction, regulating carbon metabolism and energy balance. The transcription of TPS and TPP genes is subject to complex transcriptional regulation. Studies have shown that these genes have differential expression patterns in different developmental stages and different tissues, and are regulated by factors such as sugar status, hormone levels, and environmental signals. The level of T6P in plants can regulate the transcription of TPS and TPP genes through feedback. Some regulatory factors (such as bZIP11 transcription factor) regulate the transcriptional activities of these genes by binding to the promoters of TPS and TPP genes.

[0005] Brassinosteroids (BRs) are an important class of plant hormones that regulate plant growth, development, and responses to stress through their signal transduction pathways. BZR / BES is a key transcription factor in the BR signaling pathway, which is directly regulated by BR signals and regulates the expression of downstream genes, thereby affecting plant growth and development. Currently, this gene has been analyzed, isolated, cloned, and functionally identified in model plants such as Arabidopsis thaliana, rice (Oryza sativa), corn (Zea mays L.), and potato (Solanumtuberosum L.).

[0006] With the development of genome sequencing technology, although the functions of homologous genes are inferred through the functions identified in other species, due to the possible differences between different species and the particularity of tobacco as a tetraploid plant, its function in tobacco still needs to be verified by scientific and reliable technology. So far, there has been no functional identification of BZR1 / BES1 in tobacco, let alone the analysis of its mechanism of regulation of sugar signal T6P. Summary of the invention

[0007] The present invention provides a plant NtBES1 protein and its use in regulating the level of 6-phosphate-trehalose in tobacco. Experiments have shown that the plant NtBES1 protein can effectively regulate the level of sugar signal T6P in tobacco leaves, so that it can be used to regulate the endogenous T6P level of tobacco to improve the yield / quality of tobacco.

[0008] In order to achieve the above object, the present invention provides a plant NtBES1 protein, including NtBES1S protein and NtBES1T protein, the amino acid sequence of the former is shown in SEQ ID NO: 1, and the amino acid sequence of the latter is shown in SEQ ID NO: 2.

[0009] The present invention also provides a gene encoding the plant NtBES1 protein according to the above technical solution.

[0010] Preferably, the encoding gene is selected from NtBES1S gene and / or NtBES1T gene, wherein the nucleotide sequence of NtBES1S gene is shown in SEQ ID NO: 3, and the nucleotide sequence of NtBES1T gene is shown in SEQ ID NO: 4.

[0011] The present invention also provides an overexpression material or a deletion material containing the coding gene described in the above technical solution.

[0012] The present invention also provides a use of the plant NtBES1 protein according to the above technical solution or the encoding gene according to the above technical solution in regulating the level of 6-phosphate-trehalose in tobacco leaves.

[0013] Preferably, the NtBES1S gene and the NtBES1T gene negatively regulate to inhibit the synthesis of T6P before the leaves mature; and the NtBES1S gene and the NtBES1T gene positively regulate to promote the synthesis of T6P after the leaves mature.

[0014] Preferably, under the action of exogenous sucrose, the regulation of T6P by NtBES1T gene is induced by sucrose, and the regulation of T6P by NtBES1S gene is inhibited by sucrose.

[0015] The present invention also provides a method for improving tobacco yield / quality, which improves tobacco yield / quality by introducing the coding gene described in the above technical solution into tobacco.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are:

[0017] The present invention provides a complete tobacco-derived gene coding sequence, and through genetic transformation means, clarifies the regulatory effect of plant NtBES1 protein on T6P level in tobacco. Since T6P in plants cannot be regulated by direct exogenous application of T6P, the discovery of the regulatory effect of plant NtBES1 protein on T6P level can be later applied to the regulation of endogenous T6P level in tobacco, thereby improving tobacco yield / quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The phylogenetic relationship of BES1 in tobacco, Arabidopsis, potato, and tomato provided in the embodiments of the present invention, wherein the red box represents common tobacco (N.tabacum); the blue triangle represents forest tobacco (N.sylvestris); the gray triangle represents hairy tobacco (N.tomentosiformis); the purple box represents the species potato (S.tuberosum); the blue circle represents tomato (S.lycopersicum); the green circle represents Arabidopsis thaliana;

[0019] Figure 2 The transcriptional activation activity analysis of tobacco NtBES1 and NtBZR1 provided in the embodiment of the present invention, wherein, in the transcriptional activation test in yeast: empty pGBKT7 is used as a negative control; AtDREBA-pGBKT7 is used as a positive control, pGBKT7 is an empty vector, which is a negative control, 100, 10 -1 , 10 -2 , 10-3 represents the dilution factor of yeast;

[0020] Figure 3 The subcellular localization of tobacco NtBES1 and NtBZR1 provided in the embodiments of the present invention, wherein GFP: the fluorescence signal of GFP fluorescent protein observed under the excitation light of 488nm wavelength; DAPI: the fluorescence signal observed under the excitation light of 405nm wavelength; Merge: the image after the bright field and dark field are merged; Bright: the image observed under the bright field;

[0021] Figure 4 The invention provides the creation of NtBES1 genetic material, wherein A. NtBES1 gene editing target sequence design; B. Gene editing sequencing results; C. Amino acid sequence alignment after editing; D. Regeneration stage of transgenic plants during tobacco genetic transformation; E. Analysis of relative expression of NtBES1T gene in overexpressed plants; F. Analysis of relative expression of NtBES1S gene in overexpressed plants;

[0022] Figure 5 Analysis of the synthesis and metabolic pathways of T6P provided in the embodiments of the present invention;

[0023] Figure 6 The contents of T6P and trehalose (AB), the contents of TPS and TPP enzymes (CD), and the expression levels of NtTPS and NtTPP (EF) in the NtBES1 genetic material provided in the embodiments of the present invention;

[0024] Figure 7 The T6P content of the NtBES1 genetic material after sucrose treatment provided by the embodiment of the present invention;

[0025] Figure 8 The TPS content of the NtBES1 genetic material after sucrose treatment provided by the embodiment of the present invention;

[0026] Fig. 9 The TPP content of the NtBES1 genetic material after sucrose treatment provided by the embodiment of the present invention;

[0027] Fig.10 The gene expression levels of NtTPS (A) and NtTPP (B) after sucrose treatment of the NtBES1 genetic material provided in the embodiment of the present invention;

[0028] Fig.11 This is the NtTPP gene expression level after sucrose treatment of the NtBES1 genetic material provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] Phylogenetic tree of plant NtBES1 proteins

[0032] In order to study the orthologous relationship between BES1 members, a phylogenetic tree was constructed using the full gene sequence length of BES1 proteins from Solanaceae and Arabidopsis thaliana to better discover potential orthologs. Based on the neighbor-joining method, phylogenetic analysis was performed, and each homolog was named with a different prefix to distinguish different species (common tobacco (Nt, N. tabacum, K326), forest tobacco (Nsyl, N. sylvestris), hairy tobacco (Ntom, N. tomentosiformis), potato (St, S. tuberosum), tomato (Si, S. lycopersicum)). Figure 1 As shown in Figure 2, homology analysis classified 57 BES1 proteins into five major categories, named I to V. Based on the clustering and sequence analysis in their phylogenetic trees, most of the BES genes in NsylBES1s and NtomBES1s are homologous. The above studies prove that most BES genes were preserved through interspecific hybridization of ancestors.

[0033] Considering the evolution of Solanaceae, the genes NsylBEH7, NsylBEH7.1, NsylBEH7.2 and NsylBEH7.3 in tobacco lack orthologous genes, the common tobacco genes NtBEH2.1, NtBEH6.1, NtBEH6.2, NtBEH6.4S and NtBEH6.5S are clustered into a separate category, and the potato StBEH5 is clustered into a separate category. No orthologous genes were found in Arabidopsis and tobacco. This shows that the common tobacco NtBES1 gene family is divided into three groups. The first group includes 7 genes, the second group includes 6 genes, and the third group includes 5 genes. All three groups can find orthologous genes in the closely related species of tobacco, tobacco, and forest tobacco. At the same time, NsylBEH4.1, NsylBEH4.2, NtomBEH4, NtBEH4.1S and NtBEH4.2S are grouped together. This shows that common tobacco is a tetraploid, and gene replication still occurs after the formation of allotetraploidy. The genome size is much larger than that of species such as Arabidopsis, rice, and corn that have been studied. The functional division of genes and redundancy are more complex. This also requires scientific means to isolate, clone, and identify the genes obtained from the tobacco genome database in order to confirm their functions in tobacco.

[0034] Basic information of tobacco NtBES1 gene family members

[0035] The latest genome annotation information of the above-mentioned common tobacco (N. tabacum, K326), forest tobacco (N. sylvestris), hairy tobacco (N. tomentosiformis), potato (S. tuberosum), and tomato (S. lycopersicum) was downloaded from the SGN (Sol Genomics Network, http: / / solgenomics.net / ) database. The previously reported AtBES1 full-length and homeodomain amino acid sequences were retrieved from the Arabidopsis Information Resource (TAIR: http: / / www.arabidopsis.org / ), aligned with MAFFTv5.3 to generate Stockholm files, and then HMMERv3.0 was used to construct HMM (Hidden Markov Model) profiles. The HMM profile was applied to perform HMM searches on the annotated Solanaceae protein database with an E value cutoff of 1.0. In addition, using the full-length and homeodomain amino acid sequences of AtBESs, BLASTP searches were performed to identify other potential WOX proteins with an E value cutoff of 0.01. The protein sequences obtained from the above two methods were combined and redundant entries were manually removed. The hit sequences were then analyzed with Pfam (https: / / pfam.xfam.org / ) and SMART (http: / / smart.embl.de / ) to ensure the presence of conserved functional domains.

[0036] The relative molecular mass (Mr) and isoelectric point (pI) of proteins were analyzed using the online tool ProtParam, whose website is http: / / web.expasy.org / protparam / .

[0037] The tool used to predict the subcellular localization of BES1 family genes is ProtComp 9.0, the website of which is http: / / www.softberry.com / berry.phtml?topic=protcomppl&group=programs&subgroup=proloc.

[0038] ClustalW software was used to align the amino acid sequences of BES1 transcription factors of selected species, including S. lycopersicum, S. tuberosum, N. tabacum, N. tomentosiformis, N. sylvestri and Arabidopsis; then the evolutionary tree was constructed using the Neighbor-Joining method (NJ) and MEGA 6.0 software. The parameters were set as Poisson correction, pairwise deletion and bootstrap value (1000 replicates).

[0039] In summary, a total of 18 NtBES1 homologous sequences were searched in the common tobacco database. The tobacco NtBES1 gene family information is shown in Table 1. The protein amino acid sequences of each member of the tobacco BES1 transcription factor family contain 179-415 amino acids, with an average of 289; the molecular weight is 2.0388104-4.5121104 Daltons (Da), with an average of 3.1433104; the theoretical isoelectric point (PI) is in the range of 5.12 to 9.44. Overall, the isoelectric point of most tobacco BES1 family proteins is greater than 7, the isoelectric point is in the alkaline range, and the protein molecules are rich in basic amino acids; the results of protein hydrophobicity analysis show that the hydrophobicity of the 18 proteins is less than 0, indicating that tobacco BES1 proteins are all hydrophilic proteins, but there are differences between different proteins. Considering that the BZR1 family and BES1 family have been studied more in various species, especially their functions in the BR signaling pathway, the four genes NtBES1S, NtBES1T, NtBZR1S and NtBZR1T were further screened for subsequent experiments.

[0040] Table 1 Information on tobacco NtBES1 gene family

[0041]

[0042]

[0043] Example 2

[0044] 2.1 Both NtBES1 and NtBZR1 have transcriptional activation activity

[0045] Firstly, the tobacco NtBES1S, NtBES1T, NtBZR1S and NtBZR1TCDS were amplified using primers NtBES1S1098F / R, NtBES1T 1227F / R, NtBZR1S1186F / R and NtBZR1T1170F / R, respectively. In order to detect whether NtBES1S, NtBES1T, NtBZR1S and NtBZR1T have transcriptional activation activity, the CDS of NtBES1S, NtBES1T, NtBZR1S and NtBZR1T transcripts were constructed into the pGBKT7 vector (containing the DNA-BD domain), respectively; the empty pGBKT7 was used as a negative control; and AtDREBA-pGBKT7 was used as a positive control. These plasmids were mixed with the empty AD vector plasmid, respectively, and transformed into yeast (Y2HGold) containing the reporter gene. The yeast was cultured at 30°C for 3 days, and the growth of the yeast on the SD / -Trp-His selective medium was checked and verified by β-gal color development.

[0046] The results are as follows Figure 2 As shown, the four yeast strains can grow on Trp-deficient medium, and only the negative control pGBKT7 cannot grow on Trp-His-Ade-deficient medium, indicating that NtBES1S, NtBES1T, NtBZR1S and NtBZR1T all have transcriptional activation activity.

[0047] 2.2 NtBES1 and NtBZR1 are localized in the nucleus

[0048] The subcellular localization of NtBES1S, NtBES1T, NtBZR1S and NtBZR1T was analyzed. The stop codons of NtBES1S, NtBES1T, NtBZR1S and NtBZR1T were removed, and the fragments of NtBES1S, NtBES1T, NtBZR1S and NtBZR1T were amplified using primers BES1S / TP57F and BES1SP57R, BES1S / TP57F and BES1TP57R, BZR1S / TP57F and BZR1SP57R, BZR1S / TP57F and BZR1TP57R, and inserted between the 35S promoter and GFP coding sequence of the P35S::GFP vector by restriction digestion and ligation to construct an expression vector P 35S ::NtBES1S:GFP and P 35S ::NtBES1T:GFP,P 35S ::NtBZR1S:GFP and P 35S ::NtBZR1T:GFP. Subsequently, the expression vectors were mixed with p19 in equal proportions and injected into leaves of Nicotiana benthamiana. After injection, the leaves were placed in dark culture at 28°C for 24 hours, and then cultured under light for 1-2 days. The injected leaves were taken and cut into discs with a diameter of 0.5 cm. They were soaked in DAPI (ratio of 1:1000) for 10 minutes, and then decolorized with PBS. The fluorescence signal was observed using a laser confocal microscope (TCS-SP8 Leica, Wetzlar, Germany). The fluorescence signal of GFP fluorescent protein was observed under excitation light of 488 nm, and the fluorescence signal of DAPI was observed under excitation light of 405 nm. The appearance of the cells when they were not fluorescent was observed under bright field, and finally an image after the bright field and dark field were fused together was generated.

[0049] Depend on Figure 3It can be seen that NtBES1S, NtBES1T, NtBZR1S, and NtBZR1T are located in the cell nucleus. Through the observation of GFP and DAPI signals, it was found that the number of detectable NtBES1 expressions was greater than that of NtBZR1, and the brightness was higher than that of NtBZR1. All four genes were mainly expressed in the cell nucleus. In addition, NtBZR1T was also found to be expressed on the cell membrane. This result not only shows that NtBES1S, NtBES1T, NtBZR1S, and NtBZR1T are mainly located in the cell nucleus, but also shows that the localization of the four genes is different, which means that there may be differences in function.

[0050] Example 3 Creation of functional loss materials and constitutive overexpression materials

[0051] Combined with the gene expression results in Example 2, we selected NtBES1S and NtBES1T to create tobacco genetic materials (function-deficient materials and constitutive overexpression materials) to analyze their functions in tobacco.

[0052] Overexpression vector construction

[0053] The overexpression vector pBI121 was double-digested (Xma I and Sac I) to linearize it, and then purified and recovered; the recombination primers were designed according to the restriction sites of the vector pBI121 and the sequence of the target gene, and the vector of the gene CDS sequence was used as a template to amplify the CDS fragment of the gene using PCR Mix (Vazyme, Nanjing) (primers are shown in Table 2) and purified and recovered. The PCR system was 25 μL, specifically as follows: Mix 12.5 μL, upstream primer 1 μL, downstream primer 1 μL, CDS plasmid 1 μL, ddH2O9.5 μL, and the amplification program was as follows: 95℃5min; 95℃15s, 60℃20s, 72℃1min 36 cycles, 72℃7min; 4℃ maintenance. The linearized vector and gene fragment were recombined using the Infusion recombinase of Takara Company, and the recombination system was as follows: Infusion 2μL, target fragment 4μL, linear vector 4μL. The recombinant plasmid was transformed into the DH5a strain by heat stimulation, and the colonies on the resistance plate were selected for PCR identification. The primer pbi121pJCF / R was used to detect the colonies with positive PCR and correct band size, and finally the colonies with positive PCR and correct band size were selected for sequencing. The sequencing results were compared by DNANAN, and the colonies with correct results were preserved and plasmids were extracted. The plasmids were transformed into Agrobacterium by heat stimulation, and finally the overexpressed Agrobacterium containing the target gene recombinant was obtained.

[0054] Gene editing vector construction

[0055] The NtBES1S and NtBES1T of common tobacco were compared and analyzed to find the sequence interval with 100% similarity and the PAM site near the start of the coding frame. The gRNA is marked by a red box ( Figure 4 A). Based on this, the common gRNAs for NtBES1S and NtBES1T were designed, and the target sequences that met the conditions were constructed into the pORE vector with AtU6-26 as the gRNA mediator and 2×35S promoter driving Cas9 to construct pORE NtBES1 Edit the vector.

[0056] Genetic transformation of tobacco by Agrobacterium tumefaciens-mediated leaf disc method

[0057] First, sterilize tobacco seeds as follows: soak in 75% ethanol for 90 seconds, turning the centrifuge tube over and over during this period; sterilize twice with 50% sodium hypochlorite + 1 drop of Tween 20, 15 minutes each time; finally, wash with sterile water at least 3 times. Then sow the seeds on a culture dish containing 1 / 2MS culture medium, and when the seedlings grow to about 0.5cm, transfer them to a culture bottle containing the same culture medium. When the tobacco seedlings grow to 5-8 leaves, select green leaves for Agrobacterium infection. Take the preserved Agrobacterium bacterial solution (-80℃) for activation, then take 100μL and add it to YEB liquid (generally use a triangular flask, 200ml / bottle), 28℃, 200 rpm, and culture under dark conditions until the bacterial solution OD = 0.6-0.8. The above bacterial solution was collected by centrifugation, resuspended and diluted to OD600 = 0.8 using MS0, and then 20 mg / L AS (acetosyringone) was added as the infection solution for standby use; the cultured sterile tobacco leaves were cut into 0.5*0.5 cm in size and soaked in the above infection solution for 8 minutes, during which the glass bottle was shaken continuously. The infected leaves were spread on the co-cultivation medium (G) with the veins facing up, and co-cultivated in the dark at 22°C or 23°C for 3 days. After S1, S2, S3 subculture and rooting culture, resistant plants were obtained. Plant genomic DNA was extracted from overexpression and gene-edited plants for PCR identification.

[0058] Total RNA of the overexpression PCR positive strains was extracted and fluorescence quantitative detection was performed. The fluorescence quantitative primers were BES1S-F / R, BES1T-F / R, BZR1S-F / R and BZRT-F / R, and the internal reference gene primer was NtActinK326F / R. The strains with higher expression levels in the two gene overexpression strains, NtBES1SOE53 and NtBES1TOE96, were selected and named OET and OES ( Figure 4E, F), used as overexpression materials for subsequent phenotypic analysis. PCR amplification of gene-edited resistant strains, amplification primers BES2SCJCF / BES2CJCR, BES2TCJCF / BES2CJCR, BZR1SCJ500F / BZR1CJ500R, BZR1TCJ500F / BZR1TJ500R.

[0059] Sequencing results showed that both NtBES1S and NtBES1T genes were edited, with NtBES1S missing one base and NtBES1T inserting one base ( Figure 4 B) Two corresponding editing lines were obtained, one of which was NtBES1S edited alone. CR (CR1), the other is NtBES1S edited by NtBES1S and NtBES1T at the same time CR BES1T CR (CR3). Further comparison of its encoded protein revealed that NtBES1T CR Only 66 amino acids, premature termination occurs, NtBES1S CR Only 48 amino acids, premature termination occurs ( Figure 4 C). Obtained NtBES1SOE, NtBES1TOE constitutive overexpression material, named CR3 ( Figure 4 D).

[0060] Example 4 NtBES1 affects the expression level of genes related to the T6P signaling pathway

[0061] By analyzing the transcriptional expression profiles of WT, CR3, OET, and OES, DEGs related to Inv, TPP, TPS, FK, and SnRK were screened from the differentially expressed genes in the transcriptome. Figure 5 As shown in the figure, the heat map results show that Inv-related genes are generally expressed at the highest level in CR3, and the expression levels of Nitab4.5_0002868g0010 and Nitab4.5_0000353g0100 in TPS are significantly lower in CR3 than in other materials, which is speculated to play a key role in the biosynthesis of T6P. Nitab4.5_0001992g0010 in TPP is expressed at the highest level in OET, and the expression level of SnRK in OET is high when the leaves are fully expanded, and the expression level decreases with leaf aging. The above analysis results indicate that NtBES1 is involved in the regulation of the expression of key genes in the T6P signaling pathway.

[0062] Example 5 NtBES1 affects the leaf T6P signaling pathway

[0063] Before the onset of leaf senescence (YL period), compared with the wild type WT control, NtBES1 gene deletion (CR3) significantly promoted the synthesis of T6P, and the T6P content increased ( Figure 6 A). The content of T6P in CR3 is 1.50 times that in WT, and 2.69 and 3.10 times that in OET and OES. The activities of TPS and TPP are increased ( Figure 6 C, D). When the leaves enter senescence (FEL, SL period); NtBES1 gene overexpression, especially NtBES1S (OES), increases the content of T6P, and at the same time increases the activities of TPS and TPP ( Figure 6 E, F). This indicates that NtBES1 has opposite regulatory patterns on T6P signaling before and after the onset of leaf senescence. In young (YL) leaves, NtBES1S and NtBES1T inhibit the activity of TPS and TPP and the synthesis of T6P. After the onset of senescence, NtBES1S and NtBES1T increase the activity of TPS and TPP and promote the synthesis of T6P. NtBES1T promotes the expression of NtTPS1 gene in fully expanded leaves, while NtBES1T and NtBES1S promote the expression of NtTPP gene in senescent leaves.

[0064] Example 6 Changes in T6P content after exogenous sucrose treatment of NtBES1 genetic material

[0065] Tobacco seedlings were grown in an artificial climate chamber (16h light: 8h dark), with a light intensity of 250±20μmol m-2s-1, and day and night temperatures of 26℃ and 24℃, respectively. When the tobacco seedlings grew to 60 days, 6 strong and uniform tobacco seedlings were selected, and the sixth leaf (fully expanded leaf, effective leaf from bottom to top) was selected for sucrose treatment. The beakers, punchers (1cm in diameter), tweezers and culture media required for the sucrose treatment experiment were sterilized in an autoclave in advance. The leaves were collected and washed in tap water for 15min, wiped dry with absorbent paper, and transferred to a clean bench, washed again with ddH2O for 15min, soaked in 70% ethanol for 30-60s, soaked in 3% sodium hypochlorite for 15min, and washed 2-3 times with ddH2O, and then punched holes in the middle of the leaves, avoiding the main veins and leaf edges. The leaf discs were floated in a 50 mL culture dish containing 1 / 2MS (Murasge and Skoog) medium containing 60 mM sucrose. Three culture dishes were set up for the sucrose treatment group and the control group, and about 50 leaf discs were placed in each culture dish. The culture medium was then sealed with a sealing film. The operation process required sterility to prevent the culture medium from being contaminated. The treated culture dishes were placed in the same conditions in the artificial climate chamber, and the experiment was treated for a total of 5 days. Sampling was performed in a clean bench, and the experiment was repeated three times, and three leaf discs were taken from each culture dish to reduce experimental errors.

[0066] Exogenous application of sucrose promotes the synthesis of T6P in mature tobacco leaves. Figure 7 As shown in the figure, in the control group, the content of T6P in CR3 (determined according to the instructions of the kit (provided by Suzhou Keming Biotechnology Co., Ltd.)) decreased continuously with the extension of treatment time, while the content of T6P in WT, OET and OES showed a trend of first increasing and then decreasing, among which the content of T6P in OET and OES reached the maximum on the fifth day (D5), and the content of T6P in WT was the highest on the first day (D1). After sucrose treatment, in WT, exogenous sucrose significantly promoted the increase of T6P content in leaves, and the content of T6P reached the maximum on the third day (D3) after treatment, which was 3.37 times that of the control group at the same time; before D3 treatment, the content of T6P in CR3 gradually decreased and was lower than that of the control group. At D5, the content of T6P increased significantly and was 2.17 times that of the control group at the same time. In OET, the content of T6P in leaves continued to increase under sucrose treatment, and at D5, the content of T6P was 4.37 times that of the control group at the same period; in OES, the content of T6P increased significantly at D1 of sucrose treatment, and was significantly higher than that in the control group, and then decreased. This result shows that NtBES1 significantly affects the content of T6P in leaves and promotes the accumulation of T6P in leaves after sucrose increase. In addition, in the process of exogenous sucrose-induced increase in T6P content, NtBES1T and NtBES1S have different effects. Specifically, the regulation of T6P signal by NtBES1T is induced by sucrose, and the regulation of T6P signal by NtBES1S is inhibited by sucrose.

[0067] Example 8 Effect of sucrose treatment on the TPP and TPS enzyme contents of NtBES1 genetic material

[0068] TPP (6-phosphate trehalose phosphatase) and TPS (6-phosphate trehalose synthase) assay: Enzyme-linked immunosorbent assay was used. The assay was performed according to the instructions of the kit (provided by Nanjing Camillo Bioengineering Co., Ltd.), and finally the fitting curve was calculated, and the concentrations corresponding to different samples were calculated based on the standard curve.

[0069] The trehalose 6-phosphate synthase (TPS) content was determined by enzyme-linked immunosorbent assay. Figure 8As shown. Before treatment, the TPS content in CR3 leaves was significantly higher than that in other materials, which was 1.78 times, 2.82 times and 2.23 times that in WT, OET and OES, respectively. In WT, with the extension of treatment time, the content of TPS in the control and sucrose treatments showed a trend of first increasing and then decreasing, with the highest content at D3, and sucrose treatment significantly promoted the increase of TPS content in leaves; in CR3, before treatment D3, the content of TPS gradually decreased under the control and sucrose treatments, and sucrose promoted the decrease of TPS in leaves, while at D5, sucrose significantly promoted the increase of TPS content, which was 2.12 times that of the control group at the same time; in OET, sucrose treatment significantly promoted the accumulation of TPS in leaves; in OES, exogenous sucrose promoted the increase of TPS content in leaves from D1.

[0070] The trehalose-6-phosphatase (TPP) content was determined by enzyme-linked immunosorbent assay. Fig. 9 As shown. The initial TPP content of CR3 was significantly higher than that of other materials, which was 1.35 times, 1.67 times and 1.81 times that of TPP in WT, OET and OES, respectively. In WT, the content of TPP in the leaves under the control and sucrose treatments showed a trend of first increasing and then decreasing, with the highest content on Day 1. Sucrose treatment significantly promoted the increase of TPP. In CR3, under the control treatment, the content of TPP in the leaves continued to decrease with the extension of treatment time, while under the sucrose treatment, the content of TPP did not change significantly before Day 3 of the treatment, and began to decrease significantly on Day 5, but was always higher than the control group. In OET, under the control treatment, the content of TPP in the leaves continued to increase with the extension of treatment time, which was completely opposite to the result in CR3. Under sucrose treatment, the TPP content in the leaves of OET continued to increase, and the contents on Day 3 and Day 5 were 3.46 times and 3.16 times that of the control group at the same period, respectively; the TPP content in OES was significantly promoted on Day 3 of sucrose treatment, which was 2.23 times that of the control group at the same period.

[0071] The above results indicate that NtBES1 affects the synthesis of T6P and plays a promoting role in the process of exogenous sucrose promoting the increase of TPS and TPP contents. The deficiency of NtBES1S and NtBES1T delays the decrease of TPS and TPP contents induced by sucrose, while overexpression, especially NtBES1T, significantly promotes the increase of TPS and TPP.

[0072] Example 9 Effect of sucrose treatment on the expression of NtTPS and NtTPP genes in NtBES1 genetic material

[0073] The kit for extracting Total-RNA was purchased from Acori Biotech. After obtaining RNA, the RNA concentration was measured and recorded using a spectrophotometer (TheImplen OD600 DilμPhotometerTM). The reverse transcription kit was purchased from Nanjing Novozyme Biotech. The specific steps are as follows:

[0074] Removal of genomic DNA: prepare a mixed solution in a 200μL centrifuge tube (RNase free), the system is TotalRNA: 1pg-1μg, 4×gDNAwiperMix 4μL, and use RNase-free ddH2O to make up the system to 16μL. Use a pipette to gently pipette to mix and centrifuge briefly, then set the PCR program to 42℃2min. Add 5×HiScriptIII qRT SuperMix 4μL to the reaction solution in step 1, and the reverse transcription reaction system. Use a pipette to gently pipette to mix and centrifuge briefly, and set the PCR program to 37℃, 15min; 85℃, 5sec. After the reaction, the cDNA obtained should be frozen in a -20℃ refrigerator if not used immediately. Use the AcoriTest Fluorescence Quantification Kit for fluorescence quantitative PCR amplification, using the Actin gene as the internal reference and 2 -ΔΔCT Gene expression analysis was performed.

[0075] The qPCR system was 2×SYBR GreenPro Taq HS 10μL, Primer-F 0.4μL, Primer-R 0.4μL, cDNA 1μL, ddH2O 8.2μL. The reaction program was 95℃3min for 1 cycle; 95℃5s, 60℃30s, 40 cycles; the melting curve program was 95℃15s, 60℃1min, 95℃15s, 1 cycle. The fluorescence quantitative primers were NtTPSF / R and NtTPPF / R.

[0076] The gene expression results of the gene encoding trehalose 6-phosphate synthase (TPS) NtTPS are as follows Fig.10 As shown in the figure, the expression level of NtTPS in each material was low. When the leaves entered senescence, the expression level of NtTPS first increased and then decreased. Compared with the control, NtTPS was significantly upregulated in WT at D5 under sucrose treatment. The NtTPS of OET and OES increased significantly in the sucrose-added group compared with the group without sucrose addition on Day 1 of sucrose treatment; while the expression of CR3 was not different from the control WT treatment on Day 1 and 5 after sucrose treatment, and was significantly decreased compared with the group with sucrose addition on D 3. This result indicates that NtBES1 affects the response of NtTPS gene to sucrose.

[0077] The gene expression results of the gene encoding trehalose-6-phosphatase (TPP) NtTPP are as follows Fig.11As shown in the figure, the expression level of NtTPP in each material was low in the control, and the expression level increased slowly with the extension of treatment time. Under sucrose treatment, the expression level of NtTPP in WT and CR3 materials increased significantly on Day 3, which was 3.66 times and 4.13 times of the control group at the same time, respectively, while the expression level of NtTPP in OET and OES increased significantly on Day 1 of sucrose treatment, which was 1.96 times and 1.73 times of the control group at the same time, respectively, indicating that when NtBES1 was overexpressed, sucrose treatment significantly promoted the increase of NtTPP gene expression level in leaves. Among them, the NtTPP of OET at D3 and D5 after sucrose treatment was 6.53 times and 10.06 times that of the control group at the same time, respectively, and the expression level of NtTPP in OES was 10.36 times that of the control group at D5. It shows that exogenous sucrose promotes the expression of NtTPP gene in leaves, and NtBES1 plays a promoting role in this process.

[0078] The primers used in the present invention are shown in Table 2. SPSS software (version 26.0) was used for data processing and one-way analysis of variance was performed using the LSD method (P<0.05). All data are expressed as mean ± standard error (SE). GraphPadPrism 9.0 and Origin2021 were used for drawing.

[0079] Table 2 Primers used in the present invention

[0080]

[0081]

Claims

1. A plant NtBES1 protein, characterized in that It includes NtBES1S protein and NtBES1T protein, the amino acid sequence of the former is shown in SEQ ID NO: 1, and the amino acid sequence of the latter is shown in SEQ ID NO:

2.

2. The gene encoding the plant NtBES1 protein according to claim 1.

3. The coding gene according to claim 2, characterized in that The encoding gene is selected from NtBES1S gene and / or NtBES1T gene, wherein the nucleotide sequence of NtBES1S gene is shown in SEQ ID NO:3, and the nucleotide sequence of NtBES1T gene is shown in SEQ ID NO:

4.

4. An overexpression material or a deletion material containing the coding gene according to claim 2 or 3.

5. Use of the plant NtBES1 protein according to claim 1 or the encoding gene according to claim 2 or 3 in regulating the level of 6-phosphate-trehalose T6P in tobacco leaves.

6. The use according to claim 5, characterized in that Before leaves mature, NtBES1S and NtBES1T genes negatively regulate and inhibit the synthesis of T6P; after leaves mature, NtBES1S and NtBES1T genes positively regulate and promote the synthesis of T6P.

7. The use according to claim 5, characterized in that Under the action of exogenous sucrose, the regulation of T6P by NtBES1T gene is induced by sucrose, while the regulation of T6P by NtBES1S gene is inhibited by sucrose.

8. A method for improving tobacco yield / quality, characterized in that: The yield / quality of tobacco is improved by introducing the coding gene described in claim 2 or 3 into tobacco.

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