Grape vvi hkt1;1 promoter and application

Abstract

This invention provides a promoter for VviHKT1;1 in grapes and its application, belonging to the field of plant genetic engineering technology. Specifically, it provides a promoter PS that responds to salt stress, the nucleotide sequence of which is shown in SEQ ID NO.1; a recombinant vector, expression cassette, recombinant cell, or recombinant bacteria containing the PS promoter with the nucleotide sequence shown in SEQ ID NO.1; a promoter PL that responds to salt stress, the nucleotide sequence of which is shown in SEQ ID NO.2; a recombinant vector, expression cassette, recombinant cell, or recombinant bacteria containing the PL promoter with the nucleotide sequence shown in SEQ ID NO.2; promoters PL and PS have the effect of responding to salt stress. When the salt concentration increases, promoters PS and PL can enhance the expression of the VviHKT1;1 gene. The effect has a certain tissue specificity at different growth stages of plants and can effectively improve the salt tolerance of transgenic plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the grape VviHKT1;1 promoter and its applications. Background Technology

[0002] Recent studies have found that the HKT1 gene family has Na + Transport function, which can be achieved by participating in the transport of Na within plants. + Long-distance transportation and Na + The distribution of salt in various tissues and organs affects plant salt tolerance and even determines the plant's salt tolerance strategy.

[0003] Grapes are perennial fruit trees with a known complete genome sequence. Previous studies have used quantitative trait loci (QTLs) to locate the HKT1 gene cluster on chromosome 11, which plays a decisive role in grape salt tolerance. This gene cluster includes 6 HKT1 genes, and preliminary studies have found that VviHKT1;1 may play a key role. IS,Gilliham M,Jha D,et al(2009).Shoot Na + In the study "Exclusion and Increased Salinity Tolerance Engineered by Cell Type-Specific Alteration of Na+ Transport in Arabidopsis. The Plant Cell, 21(7):2163–2178", the AtHKT1 gene of Arabidopsis was overexpressed using a constitutive promoter and a root-stem cell-specific promoter, respectively. The results showed that overexpression of AtHKT1 in the whole plant using a constitutive promoter caused transgenic plants to become hypersensitive to salt, while the salt accumulation in the aboveground parts of plants specifically overexpressed in root-stem cells was greatly reduced, and the plants were more salt-tolerant.

[0004] There are many studies on promoters that affect plant resistance in the existing technology, but there are no reports on the VviHKT1;1 promoter, and it is unclear whether it will affect the salt tolerance effect of VviHKT1;1 in plants.

[0005] Chinese patent document CN109266649A (application number: 201811183462.6) discloses the CDM1 promoter, an inducible promoter that responds to salt stress. The CDM1 promoter was cloned from the promoter region of the Arabidopsis thaliana CDM1 gene. When transgenic plants were treated with a salt-containing culture medium, it was found that the CDM1 promoter could enhance the expression of the exogenous gene GUS, resulting in darker staining of the transgenic plants, proving that the CDM1 promoter is significantly induced by salt. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides the grape VviHKT1;1 promoter and its applications.

[0007] This invention cloned VviHKT1;1 promoters of different lengths, approximately 1700 bp and 2300 bp, from the grape 'Syrah' genome using the same set of primers, naming them PS (short promoter) and PL (long promoter), respectively. This invention discovered that promoters PS and PL respond to salt stress; in culture media with high salt concentrations, promoters PS and PL can enhance gene expression, increase the salt tolerance of transgenic plants, and their effects exhibit tissue specificity at different growth stages of the plant.

[0008] The technical solution of the present invention is as follows:

[0009] A promoter PS that responds to salt stress, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] Recombinant vectors, expression cassettes, recombinant cells, or recombinant bacteria containing the PS promoter of the nucleotide sequence shown in SEQ ID NO.1.

[0011] The application of the promoter PS in improving the salt tolerance of transgenic plants.

[0012] Application of the promoter PS in salt-tolerant plant breeding.

[0013] According to a preferred embodiment of the present invention, the promoter PS acts on the gene VviHKT1;1 in improving the salt tolerance of transgenic plants.

[0014] According to a preferred embodiment of the present invention, the plant is grape or Arabidopsis thaliana.

[0015] According to a preferred embodiment of the present invention, the promoter PS is used in the expression of the gene VviHKT1;1.

[0016] A promoter PL that responds to salt stress, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0017] Recombinant vectors, expression cassettes, recombinant cells, or recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO.2 of the PL promoter.

[0018] The application of the promoter PL in improving the salt tolerance of transgenic plants.

[0019] The application of the promoter PL in salt-tolerant plant breeding.

[0020] According to a preferred embodiment of the present invention, the promoter PL acts on the gene VviHKT1;1 in improving the salt tolerance of transgenic plants.

[0021] According to a preferred embodiment of the present invention, the plant is grape or Arabidopsis thaliana.

[0022] According to a preferred embodiment of the present invention, the promoter PL is used in the expression of the gene VviHKT1;1.

[0023] Beneficial effects

[0024] 1. The promoters PL and PS provided by this invention have a salt stress response function. Increased salt concentration enhances gene expression, especially the expression of the gene VviHKT1;1. Their function exhibits tissue specificity at different growth stages of the plant, effectively improving the salt tolerance of transgenic plants.

[0025] 2. In this invention, the transgenic plants driven by the promoter PL not only exhibit better growth characteristics under long-term salt stress, but also produce more seeds to reproduce offspring, and their reproductive capacity is much higher than that of PS-VviHKT1;1 overexpressing plants and wild type. Attached Figure Description

[0026] Figure 1 This is a PCR amplification diagram of the promoter of the grape VviHKT1;1 gene.

[0027] In the figure: (a) amplified fragment PS; (b) fragment PL;

[0028] M1: DL2000 DNA Marker; M2: DL5000 DNA Marker; Lane 1: VviHKT1; 1 promoter PS amplification fragment; Lane 2: VviHKT1; 1 promoter PL amplification fragment.

[0029] Figure 2 This is a diagram showing the alignment of the Shiraz VviHKT1;1 gene promoter PL,PS with the grape VviHKT1;1 reference genome sequence.

[0030] Figure 3 A schematic diagram of the construction process of the plant expression vector 1300GN::P-GUS.

[0031] Figure 4 The detection diagram shows the GUS fusion vector for promoter digestion identification.

[0032] In the figure: (a) Enzyme digestion verification of 1300GN::PS-GUS; (b) Enzyme digestion verification of 1300GN::PL-GUS;

[0033] M: DL2000 DNA Marker; Lanes 1-2: Recombinant plasmid 1300GN::PS-GUS; Lanes 3-4: Recombinant plasmid 1300GN::PL-GUS.

[0034] Figure 5 A schematic diagram of the GUS fusion expression vector;

[0035] In the figure: (a) Schematic diagram of recombinant vector 1300GN::PS-GUS; (b) Schematic diagram of recombinant vector 1300GN::PL-GUS.

[0036] Figure 6 This is a PCR detection image of Agrobacterium colonies;

[0037] In the figure: (a) PCR identification of 1300GN::PS-GUS colonies; (b) PCR identification of 1300GN::PL-GUS colonies; M: DL2000 DNA Marker; Lanes 1-4: PCR amplification products of 1300GN::PS-GUS colonies; Lanes 8-11: PCR amplification products of 1300GN::PL-GUS colonies.

[0038] Figure 7 Screening diagram of transgenic Arabidopsis thaliana on 1 / 2 MS medium with hygromycin;

[0039] In the figure: (a) 1300GN::PS-GUS transgenic plant; (b) 1300GN::PL-GUS transgenic plant.

[0040] Figure 8 This is an RT-PCR identification diagram of transgenic Arabidopsis thaliana;

[0041] In the figure: (a) 1300GN::PS-GUS transgenic plant; (b) 1300GN::PL-GUS transgenic plant; M: DL2000 DNA Marker; lanes 1 and 8: wild-type Arabidopsis thaliana; lanes 2-7: PCR products of 1300GN::PS-GUS transgenic plant; lanes 9-14: PCR products of 1300GN::PL-GUS transgenic plant.

[0042] Figure 9 GUS staining analysis of 7-day-old transgenic Arabidopsis seedlings;

[0043] In the figure: ac: WT (wild-type Arabidopsis thaliana), PS-GUS-overexpressing Arabidopsis thaliana, and PL-GUS-overexpressing Arabidopsis thaliana under 1 / 2 MS conditions; df: 100 mmol·L⁻¹ -1 WT (wild-type Arabidopsis thaliana), PS-GUS-overexpressing Arabidopsis thaliana, and PL-GUS-overexpressing Arabidopsis thaliana after 24 hours of NaCl stress.

[0044] Figure 10 Image showing GUS staining analysis of transgenic Arabidopsis thaliana after 20 days of growth;

[0045] In the figure: a, i, q and e, m, u are the rosette leaves of the control and salt treatment, respectively; b, j, r and f, n, v are the flowers of the control and salt treatment, respectively; c, k, s and g, o, w are the stems of the control and salt treatment, respectively; d, l, t and h, p, x are the roots of the control and salt treatment, respectively.

[0046] Figure 11 GUS staining analysis of transgenic Arabidopsis thaliana after 30 days of growth;

[0047] In the figure: a, i, q and e, m, u are the stem leaves of the control and salt treatment, respectively; b, j, r and f, n, v are the stems of the control and salt treatment, respectively; c, k, s and g, o, w are the inflorescences of the control and salt treatment, respectively; d, l, t and h, p, x are the seed pods of the control and salt treatment, respectively.

[0048] Figure 12 This is a diagram illustrating the construction process of the plant expression vector pCLB1301NH-P-VviHKT1;1.

[0049] Figure 13 A graph showing the germination rate of Arabidopsis thaliana seeds under salt stress;

[0050] In the figure: (a) Germination rate phenotype of control group plants; (b) Germination rate phenotype of plants after salt treatment; (c) Germination rate data of plants; Different lowercase letters in the same treatment conditions in the figure indicate that the differences between samples reached the significance level (P<0.05).

[0051] Figure 14 This is a graph showing the growth analysis of wild-type and transgenic Arabidopsis thaliana after salt stress.

[0052] Figure: (a) Phenotype of plants before and after salt treatment; (b) Root length data of plants; (c) Fresh weight data of plants;

[0053] In the figure, different lowercase letters under the same treatment conditions indicate that the differences between samples reached the significance level (P<0.05).

[0054] Figure 15Figure showing the effect of VviHKT1;1 overexpression on salt stress tolerance in transgenic Arabidopsis thaliana;

[0055] In the figure: (a) Phenotypes of WT and VviHKT1;1 overexpression lines under normal conditions; (b) Phenotypes of WT and VviHKT1;1 overexpression lines after salt treatment; (c) Proline content in plant leaves before and after salt treatment; (d) Malondialdehyde (MDA) content in plant leaves before and after salt treatment; Different lowercase letters in the figure under the same treatment conditions indicate that the differences between samples reached the statistical significance level (P<0.05).

[0056] Figure 16 Figure 1 shows the sodium and potassium ion content in WT and transgenic plants under salt treatment conditions.

[0057] In the figure: (a) Na+ content in leaves; (b) Na+ content in roots. + Content; (c) K in leaves + Content; (d) K in roots + Content; (e) Na in leaves + / K + ; (f) Na in the root + / K + In the figure, different lowercase letters under the same treatment conditions indicate that the differences between samples reached a significant level (P<0.05).

[0058] Figure 17 Figure showing the effect of VviHKT1;1 gene overexpression on reproductive growth in Arabidopsis thaliana;

[0059] In the figure: (c) Effect of salt stress on the expression level of VviHKT1;1 in the leaves of transgenic lines; (d) Effect of salt stress on the expression level of VviHKT1;1 in the roots of transgenic lines; (e) Effect of salt stress on the expression level of VviHKT1;1 in the stems of transgenic lines; Different lowercase letters in the figure indicate that the differences between different treatments reached the statistical significance level (P<0.05). Detailed Implementation

[0060] The technical solution of the present invention will be further explained and described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0061] Unless otherwise specified, the drugs and reagents used in the examples are common market products. Any content not described in detail in the examples is based on the prior art.

[0062] Experimental Example 1

[0063] Cloning and sequence alignment analysis of the grape VviHKT1;1 promoter

[0064] Using genomic DNA from the 'Shiraz' grape as a template, amplification was performed using specific primers F244-S / R2648-B. Electrophoresis results showed that two amplification products of different lengths were obtained in this variety, approximately 1700bp and 2300bp in size. Figure 1 (a) and (b) are denoted as PS and PL, respectively. Sequencing and sequence analysis using biological software revealed that the PS and PL sequences, compared to the GeneBank VviHKT1;1 reference genome sequence, exhibited approximately 270-300 bp deletions in the -192bp to -511bp and -1811bp to -2081bp regions upstream of the start codon ATG, respectively. Figure 2 The nucleotide sequence of promoter PS is shown in SEQ ID NO.1, and the nucleotide sequence of promoter PL is shown in SEQ ID NO.2.

[0065] Primer sequences:

[0066] F244-S:5'-CCTTTGCTCATATGTTCACTGCTT-3'SEQ ID NO.4;

[0067] R2648-B: 5'-GAGGAATGGAATGAAATAACGAGG-3' SEQ ID NO.5.

[0068] Experiment Example 2

[0069] Construction and identification of the grape VviHKT1;1 promoter fusion expression vector with GUS

[0070] To further verify the activity and expression patterns of the two VviHKT1;1 gene promoters cloned in Experiment 1, this invention selected a GUS reporter gene fusion vector (1300GN) suitable for promoter expression analysis. The construction process of the expression vector is as follows: Figure 3 As shown, based on the characteristics of the multiple cloning site on 1300GN, Bam HI restriction sites were introduced into the 5' end of the VviHKT1;1 gene promoters PS and PL, respectively, and Sal I restriction sites were introduced into the 3' end during primer design. Protective bases were introduced at both ends of the two restriction sites. The two VviHKT1;1 gene promoters obtained were then modified at the end by PCR reaction.

[0071] pET30a was selected as the intermediate cloning vector. The promoter and pET30a were ligated via homologous recombination. Subsequently, pET30a-PS / PL and the 1300GN vector carrying the GUS gene were double-digested. The promoters PS and PL were ligated into the 1300GN vector using T4 DNA ligase and transformed into E. coli Fast T1 competent cells. The cells were then treated with 50 μg / mL... -1 Positive clones were screened on Kanamycin LB agar plates. Single colonies were picked from the plates and inoculated onto plates supplemented with 50 μg / mL. -1 The culture was carried out overnight at 37°C with shaking in Kan's LB liquid medium. The cultured bacterial solution was used for plasmid extraction and enzyme digestion identification. Two recombinant vectors were verified by double digestion with BamHI / SalI.

[0072] The result showed two bands, such as Figure 4 As shown in Figure a, one band is approximately 10 kb in length, consistent with the size of the vector fragment; the other band is approximately 1.7 kb in size, consistent with the size of the promoter PS. This indicates that the VviHKT1;1 promoter PS was successfully ligated to the 1300 GN vector. Figure 4 As shown in Figure b, the length of the vector fragment is the same as... Figure 4 The expression vectors 'a' and 'a' are consistent, but the target gene fragment size is significantly larger than 2000 bp, proving that the promoter PL has also been successfully ligated into the 1300GN vector. The correctly identified recombinant plant expression vectors were named 1300GN::PS-GUS and 1300GN::PL-GUS, respectively. Their corresponding schematic diagrams are shown below. Figure 5 As shown.

[0073] The recombinant expression vector was transformed into Agrobacterium GV3101 chemically competent cells. Single colonies with normal growth status were randomly selected from selection plates for PCR detection. Figure 6 Electrophoresis results showed that bacteria with a distinct band at a size of 300 bp were positive. The positive bacteria were inoculated into a solution containing 50 μg / mL... -1 Kan and 25 μg·mL -1 Agrobacterium GV3101 was cultured in LB liquid medium containing rifampicin and then sent to the company for sequencing for further validation. Ultimately, Agrobacterium GV3101 cells carrying the recombinant expression vectors 1300GN::PS-GUS and 1300GN::PL-GUS were obtained.

[0074] Experimental Example 3

[0075] Agrobacterium GV3101, carrying recombinant expression vectors 1300GN::PS-GUS and 1300GN::PL-GUS respectively, obtained in Experiment 2, was used to infect Arabidopsis thaliana. The seeds collected after infection were then inoculated using sterile toothpicks onto a solution containing 25 μg / mL of [agent name missing]. -1Screening was performed on hygromycin on 1 / 2 MS plates, such as... Figure 7 As shown. Leaves of the obtained T1 generation transgenic plants were cut, and total RNA was extracted from the transgenic plants. The transgenic Arabidopsis thaliana was identified at the transcriptional level using semi-quantitative RT-PCR. Figure 8 Agarose gel electrophoresis results showed that, compared with wild-type Arabidopsis, transgenic seedlings were those with a band at the 100bp position matching the expected fragment length. Ultimately, six lines were obtained from the 1300GN::PS-GUS transgenic Arabidopsis, and five lines were obtained from the 1300GN::PL-GUS transgenic Arabidopsis.

[0076] Experiment Example 4

[0077] GUS staining analysis of transgenic Arabidopsis at different developmental stages

[0078] Plants transformed into the 1300GN::PS-GUS and 1300GN::PL-GUS plant expression vectors were named PS-GUS and PL-GUS, respectively. On day 6 of growth on 1 / 2 MS solid medium, five plants with similar growth were selected from each vector and transferred to 1 / 2 MS and 100 mmol·L⁻¹ solid medium, respectively. -1 Incubate on NaCl+ 1 / 2 MS solid medium for 24 h, then perform GUS staining analysis ( Figure 9 The staining was observed under a microscope. Results showed that wild-type Arabidopsis plants (WT), used as a negative control, did not show a blue color; in transgenic Arabidopsis, all tissues of both PS-GUS and PL-GUS showed a blue color, but the blue in the leaves of PL-GUS was clearly deeper than that of PS-GUS in the control group, while there was no significant difference in color in the roots; 100 mmol·L⁻¹ -1 After 24 hours of NaCl treatment, the blue color in PS-GUS leaves deepened, while the change in roots was not obvious. However, the blue color in PL-GUS leaves and roots did not change significantly before and after salt treatment.

[0079] Next, wild-type Arabidopsis thaliana and transgenic plants grown in 1 / 2 MS medium for 7 days were transplanted into soil and continued to grow until 20 days later, when the plants began to bolt and flower. 100 mmol·L⁻¹ of 100 mmol·L⁻¹ of 100 mmol·L⁻¹ was used as the treatment. -1 After 24 hours of NaCl treatment, GUS staining analysis was performed on the rosette leaves, inflorescences, reproductive stems (referred to as stems), and roots. Figure 10 (Among them, the staining of rosette leaves and roots under control conditions was different from that under control conditions.) Figure 9The results were consistent: none of the WT tissues showed color change. The rosette leaves of PL-GUS were significantly darker blue than those of PS-GUS, while there was no significant difference in color between the two in the roots. After salt treatment, the blue color of the PS-GUS rosette leaves deepened slightly, but remained lighter than that of PL-GUS, although there was no significant difference in the blue color of the PL-GUS rosette leaves after salt treatment. In the flowers and stems, PS-GUS showed a light blue color, while PL-GUS showed a dark blue color. Salt treatment deepened the blue color in the stems and inflorescences of both transgenic plants to varying degrees, but overall, the blue color of PS-GUS in the stems and flowers was lighter than that of PL-GUS. When the plants continued to grow for 30 days, seed pods appeared, and the same 100 mmol·L⁻¹ solution was used. -1 After 24 hours of NaCl treatment, various tissues of the plant were stained, such as... Figure 11 As shown: Under control conditions, PL-GUS exhibited a blue hue in its stems, leaves, stems, inflorescences, and seed pods, and this blue hue was significantly deeper than that of PS-GUS. However, the different tissues of PL-GUS before and after salt treatment were incomparable due to the intense blue color; PS-GUS stems, leaves, stems, and inflorescences all showed a light blue hue under control conditions. After salt treatment, the blue color in PS-GUS stems, leaves, stems, and inflorescences deepened, but remained lighter than that of PL-GUS. Furthermore, PS-GUS seed pods did not show any coloring before or after salt treatment.

[0080] The experimental results above show that GUS is distributed in the aboveground parts and roots of Arabidopsis thaliana, including rosette leaves, stems, stem leaves, and flowers, at different developmental stages of PS-GUS and PL-GUS overexpression lines. In particular, GUS is distributed along the vascular bundles of the roots, stems, and leaves, which is beneficial for unloading Na+ from the xylem sap of the roots and stems. + This allows the gene to enter the surrounding parenchyma cells. Regardless of salt stress, the GUS activity of the PL-GUS overexpressing lines in rosette leaves, stems, stem leaves, and flowers was much higher than that of the PS-GUS lines. In the aboveground parts, the expression level of the PL-driven gene was much higher than that of the PS. In the roots, there was little difference in GUS activity between the two lines, and neither showed a significant response to salt.

[0081] Experimental Example 5

[0082] Using cDNA from the roots of 'Crimson Seedless' grape seedlings as a template, a 1635 bp VviHKT1;1 sequence was cloned, and its nucleotide sequence is shown in SEQ ID NO.3. Amino acid sequence analysis revealed that this sequence belongs to the highly active VviHKT1;1.

[0083] The cDNA sequence with KpnⅠ and Bam HI restriction sites at both ends was ligated to pET30a via homologous recombination to obtain pET30a-VviHKT1;1, and positive clones were screened. The promoter sequences PS and PL with SalⅠ and Bam HI restriction sites at both ends were ligated to the pCLB1301NH vector to obtain two recombinant plasmids, pCLB1301NH-PS and pCLB1301NH-PL, which were transformed into Fast T1 competent cells, and positive clones were screened by colony PCR. pET30a-VviHKT1;1 was double-digested with KpnⅠ and Bam HI, ligated to the similarly double-digested plant expression vector pCLB1301NH-PS / PL, and transformed into Fast T1 chemocompetent cells. Positive clones pCLB1301NH-PS-VviHKT1;1 and pCLB1301NH-PL-VviHKT1;1 were screened. The successfully validated plasmid was sent to Qingdao Paiseno Biotechnology Co., Ltd. for sequencing. The construction process of pCLB1301NH-PS / PL-VviHKT1;1 is detailed below. Figure 12 The successfully constructed plant expression vector was transformed into Agrobacterium competent cells, which were then used to infect Arabidopsis thaliana. The resulting transgenic plants were named PS-VviHKT1;1 and PL-VviHKT1;1, respectively.

[0084] Experimental Example 6

[0085] Effects of VviHKT1;1 gene overexpression on seed germination rate under salt stress

[0086] Three PL-VviHKT1;1 (PL.L1, PL.L2, PL.L3) transgenic lines and three PS-VviHKT1;1 (PS.L1, PS.L2, PS.L3) transgenic lines were simultaneously sown with WT, for a total of seven lines, in an environment containing 0 mmol·L⁻¹. -1 NaCl and 100 mmol·L -1 In 1 / 2 MS medium containing NaCl. Phenotypic analysis of the plants showed that seeds from the control group (WT) and both overexpressing plants germinated normally, while those from the 100 mmol·L⁻¹ medium... -1 Wild-type seed germination was significantly inhibited on NaCl medium. Figure 13 (a, b). Statistical analysis of germination rates over 7 consecutive days showed that, under control conditions, there was no significant difference in germination rates between wild-type Arabidopsis and all transgenic Arabidopsis, indicating that the introduction of VviHKT1;1 did not affect normal seed germination. After salt treatment, the germination rates of seeds from both wild-type and transgenic plants were lower than under control conditions, and the decrease in WT seed germination rate was much greater than that of the two overexpression lines (a, b). Figure 13(c) However, there was still no significant difference in seed germination rate between PS-VviHKT1;1 and PL-VviHKT1;1 transgenic plants. This indicates that under salt stress conditions, these two VviHKT1;1 overexpression lines showed good salt tolerance during seed germination, but at this stage, there was no significant difference in salt tolerance between them.

[0087] Experimental Example 7

[0088] Effects of VviHKT1;1 gene overexpression on seedling growth under salt stress

[0089] After WT, PS-VviHKT1;1, and PL-VviHKT1;1 transgenic Arabidopsis thaliana germinated and grew for one week on 1 / 2 MS medium, Arabidopsis thaliana seedlings with relatively uniform root length were selected and transferred to a medium containing 0 mmol·L⁻¹. -1 NaCl and 100 mmol·L -1 Plant phenotypes were observed and root length and fresh weight were measured on 1 / 2 MS medium containing NaCl after 8 days. Results showed that under control conditions, WT and all transgenic plants exhibited similar growth, and there were no significant differences in root length and whole-plant fresh weight among the different lines. Figure 14 (a, b, c) After salt treatment, the root length of both wild-type and transgenic plants was significantly reduced compared to the control condition. However, the root length of the PL-VviHKT1;1 and PS-VviHKT1;1 transgenic lines was significantly longer than that of the WT line, and their fresh weight was also significantly greater than that of the WT line. This indicates that the growth of both WT and transgenic plants was inhibited under salt stress, but the degree of inhibition was significantly higher in the WT line than in these two transgenic lines. However, at the seedling stage, there was no significant difference in root length between the PL-VviHKT1;1 and PS-VviHKT1;1 transgenic lines, but the fresh weight of the PS-VviHKT1;1 transgenic line was slightly smaller than that of the PL-VviHKT1;1 transgenic line, although this difference was not statistically significant except for PS.L2.

[0090] Experimental Example 8

[0091] Effects of VviHKT1;1 gene overexpression on salt tolerance of seedlings under salt stress

[0092] Salt tolerance of various transgenic lines was assessed using Arabidopsis thaliana grown in nutrient soil for 25 days. Figure 15 Phenotypic changes in plants before and after salt treatment could be observed. In the control group, WT and the two overexpressing plants showed similar growth with no significant difference. Using 100 mmol·L⁻¹ salt... -1 After 10 days of irrigation with NaCl nutrient solution, the leaves of all strains began to turn yellow, showing varying degrees of salt damage. However, compared with the PS-VviHKT1;1 and PL-VviHKT1;1 transgenic lines, the WT leaves were significantly smaller. Figure 15 (b). However, there was no significant difference between the PS-VviHKT1;1 and PL-VviHKT1;1 transgenic lines.

[0093] Proline, as an osmotic regulator, shows a positive correlation between its accumulation and resistance to osmotic stress. In the control group, there was no significant difference in proline content in the leaves of the WT, PS-VviHKT1;1, and PL-VviHKT1;1 transgenic lines. (Using 100 mmol·L⁻¹) -1 NaCl stress treatment resulted in an increase in proline content in the leaves of all these plants, indicating that both WT and transgenic plants can rapidly accumulate proline to reduce cell osmotic potential and prevent cell dehydration under salt stress. However, the proline accumulation in the leaves of PS-VviHKT1;1 and PL-VviHKT1;1 transgenic lines was significantly higher than that in WT. Figure 15 (c) Similarly, there was no significant difference in leaf proline content between the two transgenic plants, PL-VviHKT1;1 and PS-VviHKT1;1, after salt stress. Figure 15 (c)

[0094] Malondialdehyde (MDA) is the final breakdown product of unsaturated fatty acids in biological membranes. It accumulates in large quantities under stress conditions, and its content is positively correlated with the damage to plant cell membranes. For example... Figure 15 As shown in Figure d: Under normal conditions, there was no significant difference in MDA content in the leaves of WT and various transgenic lines; after salt stress, the MDA content in WT leaves increased significantly, while it remained basically unchanged in the PL-VviHKT1;1 and PS-VviHKT1;1 transgenic lines, indicating that the cell membranes of WT plants suffered more severe salt stress damage.

[0095] Experimental Example 9

[0096] VviHKT1;1 gene overexpression inhibits Na+ in seedlings under salt stress + and K + Content effect

[0097] Salt stress can disrupt the plant's ion absorption, by altering the Na+ content within plant cells. + / K + and Na + This concentration, in turn, causes ion toxicity to plants. Maintaining low Na+ concentrations in the cytoplasm... + Concentration and high K + Concentration is a necessary prerequisite for various physiological processes. Arabidopsis thaliana plants cultured in soil for 25 days were subjected to a 100 mmol / L concentration. -1 Ten days after irrigating with NaCl, leaf and root tissues were taken for NaCl analysis. + and K +Content determination and analysis. From Figure 16 Figures a and b show that in the control group, the leaves and roots of the WT and PS-VviHKT1;1 and PL-VviHKT1;1 transgenic lines showed Na + The content was very low, and there were no significant differences among the samples; after salt treatment, the Na content in the leaves and roots of all strains was very low. + The content increased significantly, and the increase was greater in WT than in PS-VviHKT1;1 transgenic line than in PL-VviHKT1;1 transgenic line; while before and after salt treatment, K + The K content varied little across the leaves and roots of different strains, and the K content in the leaves and roots of different strains was relatively high. + The content difference is also very small. Figure 16 (c, d). Na+ in plant salt tolerance + / K + It plays a crucial role in cell stability, from Figure 16 In sections e and f, we can see that the leaves and roots of each strain in the control group contain Na... + / K + There were no significant differences and the ratios were much lower than those of the corresponding salt treatment groups; Na content in leaves and roots of each plant under salt stress was significantly lower. + / K + The Na content in roots was significantly elevated, with the increase in leaves (WT) > PS-VviHKT1;1 transgenic line > PL-VviHKT1;1 transgenic line, while the increase in roots (WT) > all transgenic plants. For the difference in root Na content between PL-VviHKT1;1 and PS-VviHKT1;1 transgenic plants... + / K + The overall difference was smaller in the leaves, especially in the roots of PS.L2 plants, where Na + / K + There was no significant difference compared to PL.L2 and PL.L3. The results above indicate that under salt stress, compared to WT, VviHKT1;1 overexpressing plants showed better Na... + The content was significantly reduced, maintaining a low level of Na within plant cells. + / K + Therefore, overexpression of VviHKT1;1 in Arabidopsis enhances the salt tolerance of the plants. Additionally, salt treatment of PL-VviHKT1;1 transgenic plants showed increased Na... + / K + The salt tolerance was significantly lower than that of PS-VviHKT1;1 transgenic plants, suggesting that PL-VviHKT1;1 overexpression plants have stronger salt tolerance than PS-VviHKT1;1 plants.

[0098] Experimental Example 10

[0099] Seed yield is a key indicator for evaluating the economic efficiency of crops and also an important indicator for assessing plant evolutionary adaptability. Using a solution containing 100 mmol·L⁻¹...-1 NaCl nutrient solution was used to irrigate Arabidopsis thaliana plants grown in soil for 25 days until they flowered and produced seeds, and their phenotypes were recorded by photograph. Figure 17 As shown in Figures a and b: In the control group, the WT, PS-VviHKT1;1 and PL-VviHKT1;1 transgenic lines showed no phenotypic differences during reproductive growth; unsurprisingly, after salt treatment, the PS-VviHKT1;1 and PL-VviHKT1;1 overexpressing plants grew much better than WT, and the bolting height and the number of seed pods produced by PL-VviHKT1;1 plants also far exceeded those of PS-VviHKT1;1 plants.

[0100] In summary, overexpression of the VviHKT1;1 gene in Arabidopsis thaliana enhances the salt tolerance of the plants, resulting in transgenic plants harvesting more seeds after salt treatment compared to wild-type plants. Among them, PL-VviHKT1;1 overexpressing plants exhibit the strongest salt tolerance and have a much higher reproductive capacity than PS-VviHKT1;1 overexpressing plants and wild-type plants.

[0101] To further clarify the response characteristics of the transgenic Arabidopsis VviHKT1;1 gene under salt stress, RNA was extracted from the roots, leaves, and stems of WT, PS-VviHKT1;1, and PL-VviHKT1;1 transgenic plants, respectively. The expression profiles of the VviHKT1;1 gene in different tissues before and after salt stress were detected using qPCR. Figure 17 (c, d, e).

[0102] The results are as follows:

[0103] ① Under normal conditions, the VviHKT1;1 gene can be expressed in the roots, stems and leaves of Arabidopsis thaliana overexpressing PS-VviHKT1;1 and PL-VviHKT1;1, and the expression level in the leaves is much higher than that in the roots.

[0104] ② After salt treatment, the expression level of VviHKT1;1 gene in the leaves of PS-VviHKT1;1 transgenic lines was significantly increased, while the expression level in the leaves of PL-VviHKT1;1 transgenic lines was significantly decreased, resulting in a significantly higher VviHKT1;1 expression level in the leaves of PS-VviHKT1;1 transgenic lines than that in PL-VviHKT1;1 transgenic lines.

[0105] ③ The expression levels of VviHKT1;1 in the roots of these two transgenic plants were similar, and compared with the control, the expression levels of VviHKT1;1 in both plants did not change significantly under salt stress.

[0106] ④ After bolting, the expression levels of VviHKT1;1 in the stems of the two transgenic plants differed significantly. Under control conditions, the expression level of VviHKT1;1 in the stems of PL-VviHKT1;1 overexpressing Arabidopsis thaliana was higher than that in PS-VviHKT1;1 overexpressing Arabidopsis thaliana. After salt treatment, the expression level of VviHKT1;1 did not change significantly in the stems of PS-VviHKT1;1 transgenic Arabidopsis thaliana, but it increased significantly in the stems of PL-VviHKT1;1 transgenic lines PL.L1 and PL.L2, which were 1.62 and 3.94 times that of the control group, respectively.

[0107] ⑤ Based on the determination of germination rate, root length, and fresh weight, this invention found that there was no significant difference in salt tolerance regulation between PS and PL promoters during seed germination and seedling growth. Similarly, in mature Arabidopsis thaliana, no difference in salt tolerance could be observed between PS-VviHKT1;1 and PL-VviHKT1;1 overexpressing Arabidopsis thaliana in terms of growth performance, proline content, and MDA content. However, after salt treatment, the Na+ content in the leaves of PL-VviHKT1;1 transgenic plants increased significantly. + / K + The salt tolerance of PL-VviHKT1;1 overexpressing plants was significantly lower than that of PS-VviHKT1;1 transgenic plants, suggesting that the salt tolerance of PL-VviHKT1;1 overexpressing plants may be stronger than that of PS-VviHKT1;1.

[0108] ⑥ After long-term treatment with NaCl until flowering and seed production, the growth of both VviHKT1;1 overexpressing plants was significantly better than that of WT plants. Furthermore, the bolting height and the number of seed pods produced by PL-VviHKT1;1 overexpressing plants were also significantly higher than those of PS-VviHKT1;1 overexpressing plants. Analysis of VviHKT1;1 expression levels in the roots, stems, and leaves of both PS-VviHKT1;1 and PL-VviHKT1;1 overexpressing Arabidopsis thaliana before and after salt treatment suggests that the NaCl unloaded in the stems of PL-VviHKT1;1 transgenic plants... + There will be more, including Na + Thin-walled cells confined to the xylem of the stem. On the one hand, they can restrict Na+. + Transporting sodium to more important reproductive organs, flowers and seeds, will, on the other hand, reduce sodium levels. + The amount accumulated in the leaves will increase the amount of Na. + Distributed to the stem.

[0109] The promoters PL and PS provided by this invention have a salt stress response. Increased salt concentration enhances gene expression, particularly the expression of the gene VviHKT1;1. At different growth stages of plants, the effects of promoters PL and PS exhibit tissue specificity, both enhancing plant salt tolerance. The PL-driven VviHKT1;1 tissue expression pattern is significantly superior to that of PS.

[0110] The PL-VviHKT1;1 transgenic plants not only exhibited better growth characteristics under long-term salt stress, but also produced more seeds to reproduce offspring, and their reproductive capacity was much higher than that of PS-VviHKT1;1 overexpressing plants and wild type.

[0111] sequence list

[0112] PS Starter

[0113] SEQ ID NO.1

[0114]

[0115] PL starter

[0116] SEQ ID NO.2

[0117]

[0118] HKT1.1 cDNA

[0119] SEQ ID NO.3

[0120]

Claims

1. A promoter PS responsive to salt stress, characterized in that, The nucleotide sequence of the promoter PS is shown in SEQ ID NO.

1.

2. A recombinant vector, characterized in that, A PS promoter containing the nucleotide sequence shown in SEQ ID NO.

1.

3. An expression cassette comprising, A PS promoter containing the nucleotide sequence shown in SEQ ID NO.

1.

4. A recombinant cell, characterized in that, A PS promoter containing the nucleotide sequence shown in SEQ ID NO.

1.

5. A recombinant bacterium, characterized in that, A PS promoter containing the nucleotide sequence shown in SEQ ID NO.

1.

6. Use of the promoter PS of claim 1 for the expression of a gene VviHKT1;1 for increasing the salt tolerance of transgenic plants. The plants mentioned are grapes and Arabidopsis thaliana.

7. The use according to claim 6, characterized in that, Application of the PS promoter in salt-tolerant plant breeding.

8. A promoter PL responsive to salt stress, characterized in that, The nucleotide sequence of promoter PL is shown in SEQ ID NO.

2.

9. A recombinant vector, characterized in that, The PL promoter contains the nucleotide sequence shown in SEQ ID NO.

2.

10. An expression cassette comprising, The PL promoter contains the nucleotide sequence shown in SEQ ID NO.

2.

11. A recombinant cell, wherein, The PL promoter contains the nucleotide sequence shown in SEQ ID NO.

2.

12. A recombinant bacterium, characterized in that, The PL promoter contains the nucleotide sequence shown in SEQ ID NO.

2.

13. The use of the promoter PL of claim 8 to operate a gene VviHKT1;1 for increasing salt tolerance in transgenic plants. The plants mentioned are grapes and Arabidopsis thaliana.

14. The application as described in claim 13, characterized in that, Application of the promoter PL in salt-tolerant plant breeding.

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