Application of StGDPD1 gene in improving salt tolerance of potato

Abstract

The application discloses application of StGDPD1 gene in improving salt tolerance of potatoes and belongs to the technical field of plant stress resistance. The application aims to improve the salt tolerance of potatoes and provides a method for molecular breeding of potato salt tolerance. The application provides application of StGDPD1 protein in improving salt tolerance of potatoes, and the amino acid sequence of the StGDPD1 protein is shown as SEQ ID NO. 2. The application has important significance for improving planting effect and yield of potatoes in saline areas.

Description

Technical Field

[0001] This invention belongs to the field of plant stress resistance technology, specifically involving the application of the StGDPD1 gene in improving the salt tolerance of potatoes. Background Technology

[0002] Potatoes are asexually propagated crops, and their growth and development process consists of the following stages: germination, seedling growth, tuber formation, tuber enlargement, starch accumulation, and maturity. In the early stages of potato growth, the nutrients stored in the tubers provide crucial support. Therefore, as the medium in direct contact with the tubers, a good soil environment is a key foundation for ensuring healthy potato growth. Loose, well-aerated soil with high organic matter content and a slightly acidic pH is most suitable for potato growth. Potatoes grown in such soil environments exhibit optimal growth performance, contributing to high yields and high-quality tuber production. Potatoes are moderately salt-tolerant but relatively sensitive to soil salinity. Soil electrical conductivity and salinity are positively correlated and can serve as important indicators of soil salinity, closely related to potato growth.

[0003] Studies have shown that when soil electrical conductivity exceeds a certain threshold, it significantly inhibits potato growth and development, causing salt stress. For example, when soil electrical conductivity exceeds 1.95 dSm⁻¹, seed potatoes will rot. The effects of salt stress on potatoes are particularly pronounced during the germination stage, leading not only to delayed emergence but also to impaired root formation. Roots are vital organs for absorbing water and nutrients, and salt stress reduces rooting rate, root length, and root vigor. Poor root development and weakened water absorption disrupt the water balance within potato cells, causing a decrease in cell turgor pressure, hindering cell elongation and division, and slowing potato growth. Changes in aboveground biomass during the seedling stage also directly reflect the impact of salt stress on potato plants. Under salt stress, soil salt is transported along with nutrients from the roots to the aboveground parts of the plant, inhibiting the growth of stems, leaves, and other organs, resulting in weakened growth vigor, reduced plant height and leaf area, and decreased biomass accumulation. During the tuber formation stage, salt stress inhibits tuber formation, reducing the number of tubers per plant. During tuber enlargement, the plant's nutrient demand reaches its maximum. At this time, salt stress weakens photosynthesis in the above-ground leaves and hinders nutrient absorption by the underground roots, both limiting tuber enlargement and reducing the accumulation of dry matter in the tubers. Ultimately, salt levels lead to a decrease in the number of tubers per plant and the average tuber weight, resulting in a decline in both total yield and quality. Salt stress has multiple harmful effects on potato growth, development, and internal physiology. These adverse effects collectively lead to reduced potato yield and lower tuber quality. In-depth research into the potato's response to salt stress and the identification of key salt stress-responsive genes are of great significance for improving potato cultivation effectiveness and yield in saline-alkali areas. Summary of the Invention

[0004] The purpose of this invention is to improve the salt tolerance of potatoes and to provide a method for molecular breeding of potatoes with salt tolerance.

[0005] This invention provides an application of overexpression of StGDPD1 protein in improving the salt tolerance of potatoes, wherein the amino acid sequence of the StGDPD1 protein is shown in SEQ ID NO.2.

[0006] This invention provides an application of overexpressing the StGDPD1 gene in improving the salt tolerance of potatoes, wherein the nucleic acid molecule of the StGDPD1 gene is shown in SEQ ID NO.2.

[0007] This invention provides the application of a recombinant vector containing an overexpression of the StGDPD1 gene in improving the salt tolerance of potatoes, wherein the nucleic acid molecule of the StGDPD1 gene is shown in SEQ ID NO.1.

[0008] This invention provides the application of recombinant microbial cells containing the StGDPD1 gene overexpressing in improving the salt tolerance of potatoes, wherein the nucleic acid molecule of the StGDPD1 gene is shown in SEQ ID NO.1.

[0009] This invention provides a breeding method for improving the salt tolerance of potatoes, the specific steps of which are as follows: Step 1: Ligate the gene shown in SEQ ID NO.1 with the overexpression vector to obtain the recombinant vector; Step 2: Transform the recombinant vector described in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: The recombinant Agrobacterium described in Step 2 was transferred into potato callus to induce transgenic potato plants, and positive transgenic potato plants were obtained after identification.

[0010] Further specifying, the overexpression vector in step 1 is pBWA(V)KS-eGFP.

[0011] This invention provides an application of overexpression of the StGDPD1 gene in promoting potato growth or antioxidant capacity, wherein the amino acid sequence of the StGDPD1 gene is shown in SEQ ID NO.2.

[0012] This invention provides the application of a recombinant vector or microbial recombinant cell containing an overexpression of the StGDPD1 gene in promoting potato growth or antioxidant capacity, wherein the amino acid sequence of the StGDPD1 gene is shown in SEQ ID NO.2.

[0013] Further, promoting potato growth is defined as increasing potato plant height, root length, fresh weight, and dry weight.

[0014] This invention provides an application of a recombinant vector or microbial recombinant cell containing an overexpression of the StGDPD1 gene in increasing the content of glycerol-3-phosphate (G3P) in potatoes, wherein the amino acid sequence of the StGDPD1 gene is shown in SEQ ID NO.2.

[0015] Beneficial Effects: This study identified the salt tolerance of 10 potato varieties (lines), screening out Dongnong 303, which exhibits strong salt tolerance, and Atlantic, which exhibits poor salt tolerance. Growth indicators (such as plant height and root length) and physiological indicators (such as osmotic regulators and antioxidant enzyme activity) of Dongnong 303 under salt stress were analyzed. Subsequently, transcriptomic and metabolomic sequencing of the Dongnong 303 root system was performed to screen for DEGs and DEMs. Through joint analysis of the transcriptomic and metabolomic sequencing results, key metabolic pathways co-enriched by DEGs and DEMs were identified, leading to the discovery of an important gene in salt stress response: the glycerol phosphate diesterase gene (StGDPD1). Overexpression and knockout vectors were constructed. Using Agrobacterium-mediated transformation, the StGDPD1 overexpression vector was used for transient genetic transformation of Nicotiana benthamiana and stable genetic transformation of the potato variety Atlantic; the StGDPD1 knockout vector was used for stable genetic transformation of the potato variety Atlantic. The specific location of the StGDPD1 protein in cells was determined by transient expression in tobacco. Transgenic plants were subjected to salt stress via hydroponics. Phenotypic, growth, and physiological indicators of StGDPD1-overexpressing potatoes and stgdpd1 mutant potatoes were measured and analyzed to verify the salt tolerance function of the StGDPD1 gene. The G3P content in the roots, stems, and leaves of StGDPD1-overexpressing potatoes and stgdpd1 mutant potatoes after salt treatment was measured and analyzed to verify the regulatory role of StGDPD1 in G3P content. Attached Figure Description

[0016] Figure 1Figure 1 shows the construction results of the pNK2-KRSN-stgdpd1 knockout vector; Note: a) Agarose gel electrophoresis of StGDPD1 clone; M: Marker lane 1: PCR product of StGDPD1 clone b) Agarose gel electrophoresis of E. coli transformation; M: Marker lanes 1-10: PCR product of bacterial culture c) Agarose gel electrophoresis of Agrobacterium transformation; M: Marker lanes 1-10: PCR product of bacterial culture; Figure 2 Image showing the results of PCR detection of DNA from StGDPD1 transgenic potatoes; Figure 3 Figure 1 shows the construction results of the overexpression vector pBWA(V)KS-StGDPD1-eGFP; Note: a) Agarose gel electrophoresis of clones T1-T4; M: Marker lanes 1-2: PCR products of clones T1-T4 b) Agarose gel electrophoresis of E. coli transformation; M: Marker lanes 1-10: PCR products of bacterial culture c) Agarose gel electrophoresis of Agrobacterium transformation; M: Marker lanes 1-10: PCR products of bacterial culture; Figure 4 Figure showing the identification results of stgdpd1 mutant potatoes; Note: a) DNA PCR detection of stgdpd1 transgenic potatoes; M: Marker; Lane 1: ddH2O; Lane 2: pNK2-KRSN-stgdpd1 Agrobacterium plasmid; Lane 3: Non-transgenic material; Lanes 4-14: Transgenic plants used for identification; b) Detection of stgdpd1 mutant potatoes; Figure 5 Figure 1 shows the qRT-PCR results of transgenic plants; Note: a) StGDPD1 gene expression level in WT and StGDPD1 overexpressing potatoes; ***: P < 0.001 b) StGDPD1 gene expression level in WT and stgdpd1 mutant potatoes; ***: P < 0.001; Figure 6 Phenotypic results of potatoes overexpressing StGDPD1 under salt stress; Note: a) Phenotyps of the hydroponic treatment CK group; b) Phenotyps of the hydroponic treatment 5 days with NaCl; c) Phenotyps of the hydroponic treatment 8 days with NaCl. Figure 7 Figure 1 shows the results of morphological index analysis of StGDPD1 overexpressing potatoes under salt stress; Note: a) plant height b) root length c) fresh weight d) dry weight; ns: no significant difference, ***: P < 0.001, **: P < 0.01, *: P < 0.05; Figure 8Figure 1 shows the results of physiological index analysis of StGDPD1 overexpressing potatoes under salt stress; Note: a) CAT activity b) POD activity c) SOD activity d) APX activity e) Pro content f) SP content g) MDA content h) O2- content i) Root activity; ns: no significant difference, ***: P < 0.001, **: P < 0.01, *: P < 0.05; Figure 9 Phenotypic results of stgdpd1 mutant potatoes under salt stress; Note: a) Phenotyps of the hydroponic treatment CK group; b) Phenotyps of the hydroponic treatment 3 days with NaCl; c) Phenotyps of the hydroponic treatment 5 days with NaCl. Figure 10 Figure 1 shows the results of morphological analysis of stgdpd1 mutant potatoes under salt stress; Note: a) plant height b) root length c) fresh weight d) dry weight; ns: no significant difference, ***: P < 0.001, **: P < 0.01, *: P < 0.05; Figure 11 Figure 1 shows the results of physiological index analysis of stgdpd1 mutant potato under salt stress; Note: a) CAT activity b) POD activity c) SOD activity d) APX activity e) Pro content f) SP content g) MDA content h) O2- content i) Root activity; ns: no significant difference, ***: P < 0.001, **: P < 0.01, *: P < 0.05; Figure 12 Figure 1 shows the results of the analysis of the effect of StGDPD1 on G3P content under salt stress; Note: a) G3P content in WT / StGDPD1 overexpressing lines under salt stress; ns: no significant difference, ***: P < 0.001, **: P < 0.01, *: P < 0.05 b) G3P content in WT / stgdpd1 mutant lines under salt stress; ns: no significant difference, ***: P < 0.001, **: P < 0.01, *: P < 0.05; Figure 13 This describes the genetic transformation process for potatoes. Detailed Implementation

[0017] The vector used for potato knockout was pNK2-KRSN-ccdb, purchased from Wuhan Boyuan Biotechnology Co., Ltd., and the Dicotyledonous Gene Editing Vector Kit (G418), catalog number: #REC52D. The vector used for potato overexpression was pBWA(V)KS-eGFP, purchased from Wuhan Boyuan Biotechnology Co., Ltd., Dicotyledonous Subcellular Localization Vector Kit (G418), catalog number: #REC11D.

[0018] GP3 is glyceraldehyde-3-phosphate.

[0019] Example 1. Obtaining overexpression recombinant vectors by cloning genes 1. PCR amplification of the StGDPD1 CDS sequence Download potatoes from the Phytozome database StGDPD1 Gene( PGSC0003DMG403020240 CDS sequence. The expression vector pBWA(V)KS-eGFP and its... were analyzed using the software Snapgene. StGDPD1 CDS sequence, using the homologous arm of expression vector pBWA(V)KS-eGFP and StGDPD1 CDS design seamless cloning primers.

[0020] StGDPD1 CDS sequence: (SEQ ID NO.1) StGDPD1 protein sequence: (SEQ ID NO.2) MALTAAVHISDVPNLDQVPDKYATRFSSQGIEIGRTSKFLVVGHRGNGMNLLQSADRRMKAVKENSILSFNAAANYPIDFIEFDVQVTKDDCPVIFHDDFILTQHNGTVYERRITELSLAEFLSYGPQREEGVAGKPLMRKTKDGKIVSWTVETDDSACTLKEAFEKVNPSIGFNIELKFDDHI VYQQDYLIHVLKAVLHVVLEYAKDRPIIFSSFQPDAALLVKNLQTCYPVFFLTNGGTEIYYDVRRNSLEEATKLCLEGGLEGIVSEVKGIFRNPGLVNKIKESKLSLLTYGKLNNVPEAVYMQHLMGIDGVIVDFVEQVTEAVCKLVKKPDEVLLEGEEKVQNRPQFSQRELSFLLKLIPELIQ.

[0021] Primer sequences are shown in Table 1.

[0022] Table 1 StGDPD1 PCR amplification primers for genes

[0023] Total RNA was extracted from the roots of virus-free tissue culture seedlings of Dongnong 303, and cDNA was obtained by reverse transcription. High-fidelity polymerase (KOD One) from Toyobo (Shanghai) Biotechnology Co., Ltd. was used. TM PCR Master Mix), using cDNA as a template, StGDPD1 -F、 StGDPD1 -R is the primer, and the target gene is amplified using PCR technology. 10 μL of KOD One is used. TM PCR MasterMix was prepared using 0.6 μL each of F and R primers, 1 μL of sample cDNA, and 7.8 μL of ddH2O. StGDPD1 The PCR products were recovered by gel electrophoresis. The PCR products were identified by 1% agarose gel electrophoresis, and the target band (1107 bp) was cut from the gel and recovered by gel electrophoresis using an agarose gel recovery kit. The recovered product was labeled as rDNAG1.

[0024] Results: The first strand of cDNA obtained by reverse transcription of total RNA extracted from the roots of Dongnong 303 was used as a template. StGDPD1 -F、 StGDPD1 -R is a primer used for cloning the target fragment. For example... Figure 1 As shown in a, the PCR product and StGDPD1 The gene fragment lengths (1107 bp) are basically the same.

[0025] 2. Obtaining overexpression vectors The expression vector pBWA(V)KS-eGFP was digested with Eco31I to obtain the linear vector pBWA(V)KS-eGFP. The gene gel recovery product rDNAG1 was ligated with the vector digestion and purification product pBWA(V)KS-eGFP to obtain the ligation product. The ligation product was transformed into E. coli by heat shock. The bacterial culture corresponding to the positive single colony was taken, amplified and shaken, and the plasmid was extracted using a plasmid miniprep kit (purchased from TIANGEN) for subsequent transformation into Agrobacterium. The plasmid transformed into E. coli was identified by PCR primers of 35S+StGDPD1-F and 35S+StGDPD1-R. The PCR product was transformed into Agrobacterium by liquid nitrogen freeze-thaw method, and the target band (1056 bp) was detected by 1% agarose gel electrophoresis.

[0026] Table 2 StGDPD1 PCR amplification primers for overexpression vector

[0027] Results: The target fragment was recovered from the gel and ligated with the purified vector pBWA(V)KS-eGFP digestion product, then transformed into E. coli DH5α using 35S+. StGDPD1 -F 、 35S+ StGDPD1 -R is a primer used for E. coli culture PCR. For example... Figure 1 As shown in b, all PCR products amplified bands of approximately 1056 bp, indicating they were all positive clones. Sequencing of the bacterial culture confirmed the presence of pBWA(V)KS- StGDPD1 The -eGFP plasmid has been successfully transformed into E. coli. The overexpression vector pBWA(V)KS -StGDPD1 -eGFP was transformed into Agrobacterium GV3101, with 35S+ StGDPD1 -F、35S+ StGDPD1 -R is a primer used for Agrobacterium-mediated PCR. For example... Figure 1 As shown in c, the PCR products all amplified bands of approximately 1056 bp, indicating that pBWA(V)KS -StGDPD1- The eGFP plasmid has been successfully transferred into Agrobacterium.

[0028] Transgenic potato plants were created using potato stem segment genetic transformation technology. Transgenic potato plants containing the overexpression vector pBWA(V)KS were used. -StGDPD1- A resuspension of Agrobacterium tumefaciens eGFP was prepared and used to infect pre-cultured Atlantic virus-free tissue culture seedling stem segments in a clean bench. Figure 13 The process involves cultivation, with dedifferentiation medium used in the pre-culture and co-culture stages, and redifferentiation medium used in the callus induction and shoot induction stages. Once the shoots reach approximately 2 cm in length, they are cultured in rooting medium, ultimately yielding regenerated transgenic potato plants. StGDPD1 Genetically modified plants.

[0029] 3. Identification of genetically modified potatoes: Extracted non-GMO Atlantic and 11 strains StGDPD1 Using the genomic DNA of the transgenic potato plant as a template, primer 35S+ was used. StGDPD1 -F、35S+ StGDPD1 -R, perform PCR, then electrophoresis on a 1% agarose gel to check if the target band (1056 bp) is correct. If correct, it is considered a valid target band. StGDPD1 Overexpression positive plants.

[0030] Results: 11 plants were extracted. StGDPD1 Genomic DNA from transgenic regenerated plants was analyzed by PCR. For example... Figure 2 As shown, the overexpression vector pBWA(V)KS -StGDPD1- eGFP Agrobacterium plasmid and 10 StGDPD1 All positive lines of transgenic potatoes showed amplification bands of approximately 1056 bp, while no amplification bands were observed in non-transgenic materials and ddH2O.

[0031] Example 2. Obtaining the knockout vector 1.4 target sites, and the target site sequences are shown in Table 3.

[0032] Table 3 sgRNA target sequences

[0033] The four designed sgRNA target sequences were tandemly linked with the pNK2-KRSN-ccdb vector sequence and sent to Shanghai Sangon Biotech Co., Ltd. for full-gene synthesis of the sgRNA target and vector tandem sequences T1-T4. Primers containing BsaI restriction sites were designed. stgdpd1 -F、 stgdpd1 -R, using KOD One TM PCR Master Mix, designed stgdpd1 -F、 stgdpd1 -R represents the primers used to amplify the target fragment using PCR technology. The primer sequences are shown in Table 4.

[0034] Table 4 Primers for PCR amplification from T1 to T4

[0035] The PCR product was recovered by gel extraction and labeled as rDNAG2.

[0036] 2. T1-T4 was ligated into the pNK2-KRSN-ccdb vector to obtain a ligation vector. The ligation product was then transformed into *E. coli* via heat shock. JP- stgdpd1 -F、JP- stgdpd1 -R is the primer for bacterial culture PCR. Electrophoresis was performed on a 1% agarose gel to check if the target band (1243 bp) was correct. The plasmid transformed into *E. coli* was then transformed into *Agrobacterium* using the liquid nitrogen freeze-thaw method, and JP- stgdpd1 -F、JP- stgdpd1 -R is the primer for bacterial PCR. Electrophoresis was performed on a 1% agarose gel to check if the target band (1243 bp) was correct. Table 5 StGDPD1 PCR amplification primers for knockout vector

[0037] Results: Using the synthesized T1-T4 sequences as templates, stgdpd1 -F、 stgdpd1 -R is the primer used for T1-T4 target fragment cloning, such as Figure 3 As shown in a, the PCR product is basically consistent with the length of the target fragment (569 bp).

[0038] After gel recovery of the target fragment, it was digested and ligated with the vector pNK2-KRSN-ccdb, then transformed into E. coli DH5α, and ligated with JP- stgdpd1 -F 、 JP- stgdpd1 -R is a primer used for E. coli culture PCR. For example... Figure 3 As shown in b, all PCR products amplified bands of approximately 1243 bp, indicating they were all positive clones. The bacterial culture sequencing was accurate, confirming that pNK2-KRSN- stgdpd1 The plasmid has been successfully transferred into E. coli.

[0039] Knockout vector pNK2-KRSN- stgdpd1 Transfected with Agrobacterium GV3101, using JP- stgdpd1 -F 、 JP- stgdpd1 -R is a primer used for Agrobacterium-mediated PCR. For example... Figure 3 As shown in c, the PCR products all amplified bands of approximately 1243 bp, indicating that pNK2-KRSN- stgdpd1 The plasmid has been successfully transferred into Agrobacterium.

[0040] 3. Genetic transformation of transgenic potatoes: Transgenic potato plants were created using potato stem segment genetic transformation technology. The knockout vector pNK2-KRSN was used. -stgdpd1Agrobacterium bacterial suspension was prepared into a resuspension, which was then used to infect pre-cultured Atlantic virus-free tissue culture seedling stem segments in a clean bench. Figure 13 The process shown is as follows: dedifferentiation medium is used in the pre-culture and co-culture stages, and redifferentiation medium is used in the callus induction and shoot induction stages. Once the shoots reach approximately 2 cm in length, they are cultured in rooting medium to ultimately obtain transgenic potato regenerated plants. stgdpd1 Genetically modified potato plants.

[0041] Identification of genetically modified potatoes Extracted non-GMO Atlantic and 11 strains stgdpd1 Using the genomic DNA of the transgenic potato plant as a template, primers Cas9- stgdpd1 -F、Cas9- stgdpd1 -R, perform PCR, then electrophoresis on a 1% agarose gel to check if the target band (755 bp) is correct. If correct, it is considered a valid target band. stgdpd1 Transgenic positive plants. Primer sequences are shown in Table 6.

[0042] Table 6 stgdpd1 Primers for verifying transgenic positive plants

[0043] Extracted non-GMO Atlantic and 10 strains stgdpd1 Using genomic DNA from transgenic positive potato plants as a template, primer BJ- stgdpd1 -F、BJ- stgdpd1 -R, send for sequencing. The sequencing results should be correct. stgdpd1 Mutant plants. Sequencing primer sequences are shown in Table 7.

[0044] Table 7 stgdpd1 Validation primers for mutant plants

[0045] Results: 11 plants were extracted. stgdpd1 Genomic DNA from transgenic regenerated plants was analyzed by PCR. For example... Figure 4 As shown in a, the knockout vector pNK2-KRSN- stgdpd1 plasmids and 10 stgdpd1 All positive transgenic potato lines showed amplified bands of approximately 755 bp, while non-transgenic materials and ddH2O showed no amplified bands. Sequencing of the positive lines was performed, such as... Figure 4 As shown in b, stgdpd1-1 A 39 bp deletion occurred between target sites one and four. This mutation type terminated translation at the 15th amino acid, reducing 354 amino acids and resulting in a 6-amino acid mutation. stgdpd1-2A 25 bp deletion occurred between target sites 3 and 4. This mutation type terminates translation at amino acid 19, reducing 350 amino acids and resulting in a 1-amino acid mutation. stgdpd1-3 A 46 bp deletion occurred between target sites three and four. This mutation type terminates translation at amino acid 34, reducing translation by 335 amino acids, resulting in a 14-amino acid mutation. Therefore, sequencing results indicate a total of 3... stgdpd1 Editing of positive lines of transgenic potatoes.

[0046] 4. qRT-PCR detection of relative expression levels Take 10 whole, non-GMO Atlantic plants respectively StGDPD1 Overexpression of potato plants and 3 plants stgdpd1 Mutant potato plants were ground into powder in a pre-cooled mortar under liquid nitrogen. RNA was extracted using the Trizol method and reverse transcribed. StGDPD1 -F、q StGDPD1 -R is the primer used for qRT-PCR detection.

[0047] Table 8 StGDPD1 qRT-PCR primers for genes

[0048] like Figure 5 As shown in 'a', potato plants overexpressing the gene were selected. StGDPD1 The line with the highest relative gene expression was named StGDPD1-1 , StGDPD1-2 and StGDPD1-3 . StGDPD1-1 , StGDPD1-2 , StGDPD1-3 in strains StGDPD1 The relative expression levels of the gene in the whole plant were significantly higher than those in non-transgenic potatoes, being 23.44, 21.96, and 15.03 times higher than those in WT, respectively, indicating that... StGDPD1 Genes in StGDPD1-1 , StGDPD1-2 , StGDPD1-3 Overexpression in transgenic lines.

[0049] like Figure 5 As shown in b, mutant potato plants were selected. StGDPD1 The strain with the lowest relative gene expression level was named stgdpd1-1 , stgdpd1-2 and stgdpd1-3 . stgdpd1-1 , stgdpd1-2 , stgdpd1-3 in strains StGDPD1 The relative expression levels of the gene in the whole plant were significantly lower than those in non-transgenic potatoes, being 0.02, 0.07, and 0.22 times that of WT, respectively, indicating that... StGDPD1exist stgdpd1-1 , stgdpd1-2 , stgdpd1-3 Expression was reduced in mutant lines.

[0050] Example 3. StGDPD1 Verification of salt tolerance function of genes 1. Compare WT with the three genes with the highest relative expression levels. StGDPD1 Overexpression lines StGDPD1-1 , StGDPD1-2 , StGDPD1-3 3 stgdpd1 mutant lines stgdpd1-1 , stgdpd1-2 , stgdpd1-3 Propagation was carried out by cuttings on MS solid medium. After 14 days of culture in a tissue culture room, the seedlings were hardened off and then propagated using hydroponics. StGDPD1 Gene function.

[0051] After 7 days of cultivation and once the plants had adapted, two treatment groups were set up. The CK group continued to be watered with 1 / 2 MS liquid medium in the hydroponic box, while the NaCl group had its hydroponic box replaced with 1 / 2 MS liquid medium containing 220 mM NaCl. Each strain had 3 replicates in each treatment group, arranged randomly, and the liquid medium was updated every 3 days.

[0052] After significant differences were observed, photos were taken on the same day. Photos were taken of the hydroponic containers and the root systems. Four growth indicators—plant height, root length, fresh weight, and dry weight—were measured for the WT group and the three overexpression lines / WT and the three mutant lines (CK and NaCl groups). Nine physiological indicators were measured in the roots, stems, and leaves of the WT group and the three overexpression lines / WT and the three mutant lines (CK and NaCl groups). All indicators were measured in triplicate. In addition to the above photography and measurements, the remaining hydroponic containers were continued to be treated until the plants began to show signs of death.

[0053] 2. Phenotypic observation and index determination of Dongnong 303 under salt stress: On day 10 of treatment, after a significant phenotypic difference was observed between the NaCl group and the CK group, photographs were taken on that day. The flowerpots were photographed. The tissue culture seedlings were then carefully separated from the soil, and the roots were washed with distilled water and photographed. Four growth indicators—plant height, root length, fresh weight, and dry weight—were measured, and nine physiological indicators were measured using a kit.

[0054] Table 9. Reagent kits used for measuring physiological indicators involved in this experiment.

[0055] 3. Under salt stress StGDPD1 Phenotypic analysis of overexpressed potatoes: After 5 days of hydroponic treatment, WT and StGDPD1The three overexpression lines showed significant phenotypic differences under CK and NaCl conditions. Figure 6 ).like Figure 6 As shown in a, under control conditions, StGDPD1 The three overexpression lines were significantly taller and had more vigorous root systems than the WT line. Figure 6 As shown in b, under 220 mM NaCl stress, WT began to show typical salt damage symptoms, including leaf yellowing and stem lodging; in contrast, StGDPD1 The three overexpression lines still maintained normal growth with bright green leaves and upright stems. These results indicate that... StGDPD1 Overexpression of genes can not only promote root development in plants, but also delay the onset of salt stress symptoms and significantly improve the salt tolerance of plants.

[0056] After 8 days of hydroponic treatment, if Figure 6 As shown in c, the WT plant's leaves were completely wilted, indicating severe water loss; the stems collapsed, losing physiological activity, and the plant as a whole was completely dead. StGDPD1 Although the three overexpression lines showed partial yellowing and mild wilting of leaves, they were not completely dehydrated; the stems remained upright and retained physiological activity. These results indicate that... StGDPD1 Overexpression of this substance can effectively delay the onset of salt damage symptoms and improve the salt tolerance of potatoes.

[0057] In summary, StGDPD1 Overexpression of this substance can not only directly promote root development to enhance the plant's resistance, but also significantly delay the appearance of salt damage symptoms, thus positively regulating potato salt tolerance.

[0058] 4. Salt stress StGDPD1 Analysis of potato morphological indicators by overexpression: like Figure 7 As shown, the morphological indicators measured after 5 days of hydroponic treatment indicate that... StGDPD1 Overexpression of the gene significantly affected the growth characteristics of Atlantic potato plants. In the CK group, compared with WT, StGDPD1 The three overexpression lines showed significant improvements in multiple morphological parameters. Specifically, plant height increased by 14.23%–26.97%; root length increased by 13.61%–41.36%; and fresh weight increased by 38.76%–60.29%. While dry weight showed an increasing trend (1.82%–39.39%), it did not reach a statistically significant level. Under 220 mM NaCl stress conditions, StGDPD1The three overexpression lines exhibited stronger salt tolerance. Compared with WT, the plant height of the three overexpression lines increased significantly by 20.13%–33.56%; root length increased significantly by 10.56%–26.71%; fresh weight increased significantly by 1.30–1.55 times; and dry weight increased significantly by 75.00%–90.00%.

[0059] In conclusion, under normal growth conditions, StGDPD1 Overexpression of the gene can promote plant growth, especially in terms of plant height, root length, and fresh weight; under salt stress conditions, StGDPD1 The three overexpression lines showed stronger salt tolerance, with significantly better performance than the control (WT) lines in four morphological parameters: plant height, root length, fresh weight, and dry weight. Compared to the control (CK), under salt stress conditions... StGDPD1 The three overexpression lines showed a more pronounced growth advantage, exhibiting a greater increase in biomass accumulation (fresh weight and dry weight).

[0060] 5. Salt stress StGDPD1 Analysis of overexpression in potato physiological indicators: like Figure 8 As shown, physiological indicators measured after 5 days of hydroponic treatment indicated that, in the CK group, compared with WT, StGDPD1 The activities of antioxidant enzymes (CAT, POD, SOD, APX), the content of osmotic regulators (Pro, SP), and the oxidative damage indicators (MDA, O2) of the three overexpression lines were compared. - The content of NaCl did not show a significant difference with root activity. Under 220 mM NaCl stress treatment, StGDPD1 The three overexpression lines exhibited significantly enhanced antioxidant enzyme activity. Compared to WT, StGDPD1 The three overexpression lines showed significant increases in the activities of CAT, POD, SOD, and APX in root, stem, and leaf tissues. CAT activity increased by 22.62%–120.49%, 33.71%–65.95%, and 18.76%–25.64% in roots, stems, and leaves, respectively; POD activity increased by 97.39%–128.50%, 2.36–3.22 times, and 1.00–1.96 times, respectively; and SOD activity increased by 37.51%–44.93%, 47.83%–53.58%, and 14.33%–26.16%, respectively. APX activity showed the most significant increase, rising by 1.87–2.65 times, 1.45–2.03 times, and 1.89–2.39 times in roots, stems, and leaves, respectively. StGDPD1 The three overexpression lines showed significantly enhanced osmotic regulation capabilities in root, stem, and leaf tissues compared to WT. Among them, StGDPD1The Pro content in the three overexpression lines increased by 18.31%–36.29%, 33.20%–49.14%, and 19.13%–24.21% in roots, stems, and leaves, respectively; the SP content increased by 6.05%–8.69%, 63.49%–88.48%, and 55.40%–60.41% in roots, stems, and leaves, respectively. Compared with WT, StGDPD1 The three overexpression lines showed MDA and O2 levels in root, stem, and leaf tissues. - The content was significantly reduced. Specifically, the MDA content decreased by 39.68%–43.92% in roots, 42.40%–44.80% in stems, and 18.75%–46.50% in leaves; O2 - The content decreased by 22.08%~22.89%, 27.62%~60.77%, and 48.90%~53.69% in roots, stems, and leaves, respectively. Compared with WT, StGDPD1 The root activity of the three overexpression lines was significantly increased by 22.92% to 33.75%.

[0061] In summary, overall, under the CK group conditions, StGDPD1 The three overexpression lines showed no significant differences in physiological parameters compared to WT. However, under 220 mM NaCl treatment, compared to WT, StGDPD1 The three overexpression lines showed significantly increased activities of CAT, POD, SOD, and APX, significantly increased contents of Pro and SP, and significantly increased contents of MDA and O2. - The content decreased significantly, while root activity increased significantly. This indicates... StGDPD1 The three overexpression lines enhanced salt tolerance by significantly increasing the activity of antioxidant enzyme systems, promoting the accumulation of osmotic regulatory substances, effectively reducing membrane lipid peroxidation damage, and maintaining high root vigor.

[0062] 6. Salt stress stgdpd1 Phenotypic analysis of mutant potatoes: After 3 days of hydroponic treatment, WT and 3 stgdpd1 The mutant lines showed significant phenotypic differences under CK and NaCl conditions. Figure 9 ).like Figure 9 As shown in a, under CK conditions, 3 stgdpd1 The mutant lines were significantly shorter than the WT lines, and their root systems were also sparser and shorter. For example... Figure 9 As shown in b, under 220 mM NaCl stress, three stgdpd1 The mutant lines began to show symptoms of leaf dehydration, with a 100% rate of stem lodging; in contrast, the WT lines maintained upright stem growth. These results indicate that... StGDPD1 Gene mutations not only impair root development in plants but also make them more sensitive to salt stress.

[0063] After 5 days of hydroponic treatment, if Figure 9 As shown in c, there are 3 stgdpd1 After lodging, most leaves of the mutant line completely wilted, indicating severe dehydration and loss of physiological activity, ultimately leading to complete death. In contrast, the WT line, initially lodging, while showing partial yellowing and mild wilting of leaves, had not yet completely lost water and retained physiological activity. These results indicate that... StGDPD1 The mutation can accelerate the wilting of plants under salt stress and significantly reduce the plant's tolerance to salt stress.

[0064] In summary, StGDPD1 Mutations in this substance can inhibit normal root development, accelerate the appearance of salt damage symptoms in plants, make plants more sensitive to salt stress, and reduce their salt tolerance.

[0065] 7. Salt stress stgdpd1 Morphological analysis of mutant potatoes: like Figure 10 As shown, the morphological indicators measured after 3 days of hydroponic treatment indicate that... StGDPD1 Gene mutations significantly affected the growth characteristics of Atlantic potato plants. In the CK group, compared with WT, three... stgdpd1 Several morphological parameters of the mutant lines were significantly reduced. Specifically, plant height decreased by 14.29%–21.74%; root length decreased by 22.01%–35.22%; and fresh weight decreased by 28.05%–36.99%. Although dry weight showed a decreasing trend (0.75%–28.36%), the difference was not statistically significant. Under 220 mM NaCl stress, three... stgdpd1 The mutant lines exhibited lower salt stress tolerance. Compared to WT, 3 stgdpd1 The mutant lines showed a 2.84%–11.37% reduction in plant height, but this was not statistically significant; a 30.88%–42.46% reduction in root length; a 30.58%–59.50% reduction in fresh weight; and a 27.03%–40.54% reduction in dry weight. In summary, StGDPD1 Gene mutations significantly negatively impacted the growth, development, and salt tolerance of potato plants. Under normal growth conditions, the mutant exhibited obvious developmental defects, with significantly reduced plant height, root length, and fresh weight, and a decreasing trend in dry weight. Under salt stress, the mutant's salt tolerance was significantly weakened, with all morphological indicators (especially root length, fresh weight, and dry weight) significantly lower than the mean wt. This indicates... StGDPD1 Genes play a dual role in regulating normal plant growth and development as well as salt tolerance. The loss of their function can lead to hindered plant development and reduced stress resistance.

[0066] 8. Salt stress stgdpd1Analysis of physiological indicators of mutant potatoes: like Figure 11 As shown, physiological indicators measured after 3 days of hydroponic treatment indicated that, in the CK group, compared with the WT group, the three... stgdpd1 The activities of antioxidant enzymes (CAT, POD, SOD, APX), the content of osmotic regulators (Pro, SP), and the oxidative damage indicators (MDA, O2) of mutant strains. - The content of the substance did not show a significant difference with root activity.

[0067] Under 220 mM NaCl stress treatment conditions ( Figure 11 ), 3 stgdpd1 The mutant lines showed a significant decrease in the activity of most antioxidant enzymes in root, stem, and leaf tissues. Compared with WT, the activity of all three... stgdpd1 The mutant lines showed CAT activity decreased by 21.43%–24.42%, 47.94%–54.92%, and 11.73%–18.18% in roots, stems, and leaves, respectively; POD activity decreased by 8.13%–12.20%, 13.03%–15.50%, and 25.98%–28.36% in roots, stems, and leaves, respectively, although the decrease in POD activity in roots was not statistically significant; SOD activity decreased by 22.77%–28.08%, 23.51%–25.02%, and 15.65%–25.41% in roots, stems, and leaves, respectively; APX activity decreased by 59.75%–67.27%, 16.47%–25.33%, and 38.96%–51.10% in roots, stems, and leaves, respectively. (3 lines) stgdpd1 The mutant lines showed a significantly reduced ability to regulate osmosis in root, stem, and leaf tissues compared to the WT lines. Among them, three... stgdpd1 The Pro content in the mutant lines was reduced by 23.31%–59.63%, 53.79%–67.51%, and 22.97%–28.04% in roots, stems, and leaves, respectively; the SP content was reduced by 51.20%–61.80%, 17.52%–20.31%, and 9.72%–23.37% in roots, stems, and leaves, respectively. Compared with WT, the three... stgdpd1 The mutant lines showed MDA and O2 in root, stem, and leaf tissues. - The content increased significantly. Specifically, the MDA content increased by 69.23%–95.60% in roots, 71.43%–108.33% in stems, and 85.77%–150% in leaves; O2... - The content increased by 49.19%~59.87% in roots, 31.31%~38.67% in stems, and 40.82%~69.97% in leaves. Compared with WT, the three... stgdpd1 The root activity of the mutant lines was significantly reduced by 27.59% to 29.42%.

[0068] In summary, under the conditions of group CK, 3 stgdpd1 The mutant lines showed no significant differences in physiological parameters compared to WT. However, under 220 mM NaCl treatment, compared to WT, the three mutant lines... stgdpd1 The mutant strain showed significantly reduced overall activities of CAT, POD, SOD, and APX, significantly reduced contents of Pro and SP, and significantly reduced contents of MDA and O2. - The content increased significantly, and root activity decreased significantly. This indicates that salt stress caused three... stgdpd1 The mutant strain exhibits disordered antioxidant enzyme system, cellular osmotic imbalance, aggravated membrane lipid peroxidation damage, and root function decline, leading to its high sensitivity to salt stress.

[0069] 9. StGDPD1 Analysis of the regulatory effect on G3P content: WT and the three with the highest relative expression levels StGDPD1 Overexpression lines StGDPD-1 , StGDPD-2 , StGDPD-3 3 stgdpd1 mutant lines stgdpd1-1 , stgdpd1-2 , stgdpd1-3 The cultivation and treatment methods were the same as in 2.2.11. After differences were observed, root, stem, and leaf samples were taken, and the G3P content in the roots, stems, and leaves of WT and three overexpression lines / WT and three mutant lines was determined using a kit. The kit model was G0920W, and the supplier was Gree.

[0070] StGDPD1 The gene encodes GDPD, a key enzyme that catalyzes the formation of G3P from glycerophosphate diesters. In plant salt stress, G3P plays a crucial role in membrane lipid remodeling and osmotic regulation.

[0071] like Figure 12 As shown in a, WT / StGDPD1 In the overexpression lines, compared with WT in the CK group, StGDPD1 The G3P content in the three overexpression lines was significantly increased in roots, stems, and leaves, increasing by 1.57–1.86 times, 2.00–2.63 times, and 75.00%–105.00%, respectively. Under 220 mM NaCl treatment, compared with WT, StGDPD1 The G3P content in the three overexpression lines increased significantly in the roots, stems and leaves, by 1.88–2.84 times, 95.45%–150.00% and 1.65–2.39 times, respectively.

[0072] like Figure 12 As shown in b, WT / stgdpd1 In the mutant lines, in the CK group, compared with WT, 3 stgdpd1The G3P content in the mutant lines was significantly decreased in roots and leaves, by 79.29%–82.42% and 85.81%–86.88%, respectively, and by 25.00%–50.00% in stems, but the difference was not statistically significant. Under 220 mM NaCl treatment, compared with WT, StGDPD1 The G3P content in the three mutant lines was significantly reduced in the roots, stems and leaves, decreasing by 50.53%~54.83%, 61.28%~75.00% and 85.76%~87.90%, respectively.

[0073] In summary, StGDPD1 Overexpression of this compound increased G3P content under both normal growth and salt stress conditions. StGDPD1 The mutation led to a significant decrease in G3P content.

Claims

1. The application of overexpression of the StGDPD1 gene in promoting potato growth or antioxidant capacity, characterized in that, The amino acid sequence of the StGDPD1 gene is shown in SEQ ID NO.

2.

2. The application of recombinant vectors or recombinant microbial cells containing the StGDPD1 gene in promoting potato growth or antioxidant capacity, characterized in that, The amino acid sequence of the StGDPD1 gene is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, Promoting potato growth aims to increase potato plant height, root length, fresh weight, and dry weight.

4. The application of recombinant vectors or microbial recombinant cells containing the StGDPD1 gene in increasing the content of glycerol-3-phosphate (G3P) in potatoes, characterized in that, The amino acid sequence of the StGDPD1 gene is shown in SEQ ID NO.2.

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