Application of potato StPAP12 gene in regulating potato leaf growth and resistance to potato late blight

Abstract

The application discloses application of a potato StPAP12 gene in regulation of potato leaf growth and resistance to potato late blight, and discloses for the first time a new function of the potato StPAP12 gene in regulation of leaf growth and development, breaks through the cognitive limitation that existing PAP gene family research is only focused on phosphorus nutrition metabolism and low-phosphorus stress response, and through construction of a transgenic strain with overexpression and RNAi interference, it is found for the first time that StPAP12 overexpression can significantly enhance the resistance of potato to the potato late blight caused by a pathogenic oomycete, and indoor potting inoculation experiments show that the overexpression strain has a significantly reduced disease area compared with the wild type, and the application fills the blank of the function research of PAP genes in resistance to oomycete diseases, and provides a new key gene resource for molecular breeding against the potato late blight.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the application of the potato StPAP12 gene in regulating potato leaf growth and resistance to late blight. Background Technology

[0002] Potatoes are a vital global food crop, ranking as the third largest staple food after rice and wheat, characterized by high yield, strong adaptability, and balanced nutrition. However, stable potato production has long been severely threatened by a devastating disease—late blight. Caused by the oomycete pathogen *Phytophthora infestans*, this disease is characterized by rapid spread and high destructive power, and remains the primary biological stress facing the global potato industry.

[0003] The occurrence and spread of late blight is a complex biological process. The pathogenic fungus *Phytophthora indicum* overwinters as mycelium, oospores, or sporangia in the soil or diseased plant debris, spreading rapidly with the help of wind, rain, and water flow. High humidity and cool conditions are particularly conducive to its infection. The pathogen invades through the epidermis of leaves or tubers, secreting effector proteins to suppress plant immunity and forming numerous mycelia that absorb nutrients, leading to water-soaked lesions on leaves and stems that quickly turn black and rot. On tubers, sunken brown lesions form, ultimately causing devastating loss of the entire plant or field of crop within a short period.

[0004] Faced with the challenge of late blight, control strategies have evolved from chemical dependence to integrated management. Early methods relied primarily on inorganic pesticides such as copper-based formulations, while modern methods widely utilize specific fungicides like metalaxyl. However, the pathogen readily develops resistance. Breeding resistant varieties through traditional hybridization and modern molecular breeding is a sustainable approach, but the continuous evolution of new pathogen races often leads to the "loss" of varietal resistance. Current research focuses on using biotechnology such as gene editing to cultivate broad-spectrum, durable resistant varieties, and combining this with a comprehensive management strategy that integrates precise prediction, resistant varieties, biological control, and scientific pesticide application to achieve sustainable control of this ancient disease. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a potato StPAP12 gene.

[0006] Another objective of this invention is to provide the application of the aforementioned potato StPAP12 gene in regulating potato leaf growth and resistance to late blight.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A potato StPAP12 protein, which is at least one of the following:

[0009] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0010] (b) Analogs of the amino acid sequence shown in SEQ ID NO: 1 that still enhance the resistance of potatoes to late blight by substitution, insertion or deletion of one or more amino acids.

[0011] The aforementioned potato StPAP12 protein can enhance the potato's resistance to late blight.

[0012] The nucleotide sequence of the gene encoding the aforementioned potato StPAP12 protein is obtained according to the codon coding rules; preferably, the nucleotide sequence is shown in SEQ ID NO.2.

[0013] A potato StPAP12 gene, the nucleotide sequence of which is at least one of the following:

[0014] (a) The nucleotide sequence shown in SEQ ID NO.2;

[0015] (b) Analogs of the nucleotide sequences in (a) above, obtained by base insertion, deletion, or substitution, that still enhance the resistance of potatoes to late blight.

[0016] An expression vector carrying the potato StPAP12 gene, comprising the nucleotide sequence shown in SEQ ID NO.2.

[0017] The vector backbone of the expression vector carrying the potato StPAP12 gene is pBWA(V)KS.

[0018] An Agrobacterium that expresses the potato StPAP12 gene in plants, including the aforementioned expression vector carrying the potato StPAP12 gene.

[0019] The above-mentioned potato StPAP12 protein, potato StPAP12 gene, expression vector carrying potato StPAP12 gene, and Agrobacterium expressing potato StPAP12 gene in plants are used to enhance the resistance of potatoes to late blight.

[0020] The above-mentioned potato StPAP12 protein, potato StPAP12 gene, expression vector carrying potato StPAP12 gene, and the application of Agrobacterium tumefaciens expressing potato StPAP12 gene in plants in potato breeding.

[0021] The above-mentioned potato StPAP12 protein, potato StPAP12 gene, expression vector carrying potato StPAP12 gene, and Agrobacterium expressing potato StPAP12 gene in plants are used to improve the ability of potatoes to resist low phosphorus stress.

[0022] The above-mentioned potato StPAP12 protein, potato StPAP12 gene, expression vector carrying potato StPAP12 gene, and the application of Agrobacterium expressing potato StPAP12 gene in plants in enhancing potato phosphorus absorption.

[0023] A method for enhancing the resistance of potatoes to late blight includes the following steps:

[0024] Agrobacterium, which expresses the StPAP12 gene that enhances potato resistance to late blight, was applied to potatoes to improve their resistance to late blight.

[0025] The application mentioned refers to the application to potato seedlings.

[0026] The potato in question is Yue Shu No. 1.

[0027] A biological agent to enhance potato resistance to late blight, comprising Agrobacterium that expresses the StPAP12 gene in plants to enhance potato resistance to late blight.

[0028] The Agrobacterium mentioned is Agrobacterium GV3101.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] This invention has significant advantages and effects compared to existing technologies: First, it reveals for the first time a novel function of the potato StPAP12 gene in regulating leaf growth and development, breaking through the limitations of existing PAP gene family research, which focuses solely on phosphorus nutrient metabolism and low phosphorus stress response. By constructing overexpression and RNAi-interference transgenic lines, morphological and cellular levels confirm that this gene positively regulates leaf cell expansion and negatively regulates cell division, determining the final size of organs by coordinating the balance between cell size and number. Overexpression lines show significantly increased leaf width, thickness, and leaf area, while interference lines show significantly reduced leaf area, providing a novel gene target and theoretical basis for crop plant architecture improvement and photosynthetic efficiency enhancement. Second, this invention is the first to discover that StPAP12 overexpression significantly enhances potato resistance to late blight caused by Phytophthora blight. Indoor pot inoculation experiments show that the diseased area of ​​overexpression lines is significantly reduced compared to wild types, filling a gap in research on the function of PAP genes in resisting oomycete diseases and providing a basis for research on resistance to oomycete diseases. Molecular breeding for late blight provides new key gene resources. Furthermore, this invention reveals for the first time that phosphate-solubilizing bacteria inoculation and StPAP12 overexpression have a synergistic effect in resisting late blight. Overexpressing lines showed a significantly lower incidence of late blight after inoculation with phosphate-solubilizing bacteria compared to lines overexpressing alone, providing an important theoretical basis for developing a new "microbial-genetic engineering" joint control strategy with both growth-promoting and biocontrol functions. In addition, subcellular localization analysis confirmed that StPAP12 is mainly located in the cell membrane, belonging to secretory PAPs, and possesses dual functions of phosphorus activation and utilization as well as immune defense, achieving synergistic regulation of efficient nutrient absorption and disease resistance, breaking through the limitations of the traditional "growth-defense" trade-off. Finally, this invention constructed overexpression and silencing vectors, established an Agrobacterium GV3101-mediated genetic transformation system, and developed a biological agent containing StPAP12-overexpressing Agrobacterium, which can be directly applied to potato disease-resistant breeding practices, showing good application prospects and industrialization value. Attached Figure Description

[0031] Figure 1 This is a graph showing the results of the StPAP12 gene expression level detection in Example 2.

[0032] Figure 2 This is a graph showing the results of yield and plant dry weight measurements in Example 3.

[0033] Figure 3 This is a graph showing the results of StPAP12 gene expression detection under inoculation conditions in Example 3.

[0034] Figure 4 This is a graph showing the effect of phosphate-solubilizing bacteria inoculation on phosphatase secretion in Example 3.

[0035] Figure 5 This is a graph showing the effect of phosphate-solubilizing bacteria inoculation on phosphorus absorption in Example 3.

[0036] Figure 6 This is a diagram showing the experimental results of the morphological characteristics of potato leaves in Example 4.

[0037] Figure 7 This is a diagram showing the experimental results of the cytological mechanism of potato leaf size in Example 4.

[0038] Figure 8 This is a diagram showing the experimental results of the effect of regulating the potato StPAP12 gene on the disease resistance of potatoes in Example 5. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0040] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0041] Example 1: Cloning of the potato StPAP12 gene

[0042] 1.1 Pretreatment of potato leaves

[0043] Using Yueshu No. 1 potato as the experimental material, potato microtubers were planted in a light-cultured chamber. Four-week-old large potato plants were taken, and mature leaves were quick-frozen in liquid nitrogen and stored in a -80 ℃ freezer for later use.

[0044] 1.2 Extraction of cDNA

[0045] RNA extraction was performed using the TRIZOL method. Potato leaf samples were freeze-ground into powder, then rapidly added to 1 ml of Trizol and vigorously mixed for 5 seconds. The mixture was incubated at room temperature for 20 min, then chloroform (200 μL) (1 / 5 volume of Trizol) was added and vigorously mixed for 1 min to form an emulsion. The emulsion was incubated on ice for separation, and then centrifuged at 12000 g at 4°C for 15 min. The supernatant was transferred to a 1.5 ml centrifuge tube, and an equal volume of isopropanol (pre-cooled to -20°C) was added. The mixture was inverted and incubated at -20°C for at least 1 h, then centrifuged at 12000 g at 4°C for 25 min. The isopropanol was removed, and the precipitate was retained. Add 1 ml of 75% ethanol (pre-cooled at -20 ℃), vortex to mix, then centrifuge at 12000 g, 4 ℃ for 5 min, remove the supernatant, dry in a clean bench for 5-15 min, add an appropriate amount of DEPC-treated ultrapure water to dissolve the RNA, and detect by OD value 260 / 280=1.9-2.1 or electrophoresis.

[0046] Using 1 μg of potato leaf RNA as a template, cDNA was synthesized using the Xinkailai All-in-one First-Strand Synthesis MasterMix (with dsDNase) reverse transcription kit. The cDNA synthesis was performed according to the kit instructions, and the reaction system and PCR conditions were as specified in the kit's manual.

[0047] 1.3 Sequence Amplification

[0048] Using the potato cDNA obtained in section 1.2 as a template, the full-length cDNA sequence of StPAP12 was amplified. First, the cDNA sequence of this gene was downloaded from the Potato Genome website (http: / / spuddb.uga.edu / index.shtml), and primers were designed using Primer 5.0 with reference to the transcriptome sequencing sequence.

[0049] StPAP12-F: ATGGTTATTGAAGAGGTTTATTCTGG;

[0050] StPAP12-R:CATTTCATGAAATAAGCTACAGAGGAT.

[0051] The full-length StPAP12 gene was obtained by PCR amplification using Takara PrimerSTAR Max DNA Polymerase. The PCR reaction system consisted of: 25 μL PrimeSTAR Max Premix (2×), 1 μL each of forward and reverse primers, 1 μL cDNA template, and 22 μL sterile water. The PCR amplification program was: 94℃ for 5 min, 98℃ for 10 sec, 55℃ for 15 sec, 72℃ for 1 min, for a total of 35 cycles, followed by 72℃ for 5 min and 4℃ for 15 min, to obtain the potato StPAP12 gene. The nucleotide and protein sequences of the potato StPAP12 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0052] Example 2: Construction of potato lines overexpressing and silencing the StPAP12 gene

[0053] 2.1 Construction of overexpression vectors Using the cloned StPAP12 cDNA as a template, the coding region was amplified using primers StPAP12-OE-F / R. The fragment and pBWA(V)KS overexpression vector were double-digested with BamHI and XbaI restriction endonucleases. Enzyme ligation was performed using DNA recombinase. The ligation product was added to DH5α competent cells, incubated on ice for 20 min, heat-shocked at 42℃ for 60 s, incubated on ice for 5 min, and then 700 μL of antibiotic-free LB liquid medium was added. The cells were incubated at 37℃ for 45 min using a shaker. 200 μL of the bacterial culture was evenly spread onto LB solid medium (LB + kana 50 mg / L) and incubated at 37℃ for 12 h. Single colonies were picked for PCR detection. Positive single-clone bacterial cultures were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing identification. The correct pBWA(V)KS-StPAP12-OE overexpression vector and bacterial culture were obtained through comparison and preserved.

[0054] StPAP12-OE-F:catcgtatgttgtcatgaaacggata;

[0055] StPAP12-OE-R: caaaataggcatgagttctgttcttg.

[0056] 2.2 Construction of gene silencing vector

[0057] Using the StPAP12 cDNA cloned in step (3) of Example 1 as a template, the following primers were used to clone the silent fragment.

[0058] Justice chain primer:

[0059] StPAP12-RNAi-SC-F (forward primer):

[0060] cagtGGTCTCacaacctggatttgtgtgtttacttataggcttagttttgag;

[0061] StPAP12-RNAi-SC-R (reverse primer):

[0062] cgatGGTCTCacaggaggactgcatttgagccaggttcatc;

[0063] Antisense primer:

[0064] StPAP12-RNAi-AS-F (forward primer):

[0065] cagtGGTCTCagggcaggactgcatttgagccaggttcatc;

[0066] StPAP12-RNAi-AS-F (reverse primer):

[0067] cagtGGTCTCatacactggatttgtgtgtttacttataggcttagttttgag.

[0068] The positive strand clone product was double-digested with AscI and SwaI, and then the RNAi vector pBWA(V)KS was double-digested with AscI and SwaI to construct the positive strand into the pBWA(V)KS vector. Following the method in 3.1, homologous recombination ligation reaction and bacterial single-clone identification were performed to obtain the pBWA(V)KS-StPAP12 (positive strand) vector. The antisense strand cloning product was double-digested with BamHI and SpeI. Then, the pBWA(V)KS-StPAP12 (positive strand) vector was double-digested with BamHI and SpeI. Following the method in 3.1, the antisense strand was constructed into the pBWA(V)KS-StPAP12 (positive strand) vector through enzyme-linked reaction and single-clone identification in bacterial culture. This formed the pBWA(V)KS-StPAP12 (positive strand-antisense strand) vector, which is the pBWA(V)KS-StPAP12-Ri gene silencing vector.

[0069] 2.3 Preparation of Agrobacterium

[0070] The pBWA(V)KS-StPAP12-OE overexpression vector constructed in 2.1 and the pBWA(V)KS-StPAP12-Ri gene silencing vector constructed in 2.2 were transformed into Agrobacterium GV3101, respectively. The specific steps are as follows: 100 μL of Agrobacterium competent cells were thawed on ice, and 1 μL of pBWA(V)KS-StPAP12-OE plasmid was added and mixed. The mixture was then incubated on ice for 20 min, frozen in liquid nitrogen for 5 min, incubated in a 30°C water bath for 5 min, and then placed on ice for 5 min. 1 mL of antibiotic-free LB liquid medium was added and cultured at 28°C and 175 rpm for 6 h. 200 μL of the bacterial culture was plated and placed in a biochemical incubator for 2-3 days at 28°C. Positive bacteria were screened by PCR, and after successful sequencing verification, Agrobacterium GV3101 containing the pBWA(V)KS-StPAP12-OE overexpression vector was obtained.

[0071] Following the steps described above, Agrobacterium GV3101 containing the pBWA(V)KS-StPAP12-Ri gene silencing vector was prepared.

[0072] 2.4 Transformation by Agrobacterium

[0073] Agrobacterium GV3101 containing the target gene plasmid was inoculated into LB medium (50 mg / L Kan) and activated twice. Single colonies were picked and cultured in LB liquid medium (20 ml, Rif 50 mg / L, Kan 50 mg / L) for 6 h at 28℃ and 240 rpm / min until OD600 = 0.5. After centrifugation at 5000 rpm for 6 min, the cultured cells were resuspended in 10 ml MS liquid medium (3% analytical sucrose) (OD600 = 1.5-2.0). Stem segments of robust tissue culture seedlings (Yueshu No. 1) grown for 3 weeks were taken and immersed in Agrobacterium inoculum for 15 min. The stem segments were removed and excess Agrobacterium was removed with sterilized filter paper. The infected stem segments were then spread out and transferred to co-culture medium and cultured in the dark at 26℃ for 2 days. Subsequently, they were transferred to bud differentiation medium containing 50 mg / ml hygromycin and 400 mg / L CEF until resistant buds grew to 0.5-1 cm. The buds were then cut off and transferred to MS medium + 50 mg / L Rooting medium containing hygromycin and 400 mg / L cephalosporin. When the transgenic seedlings reached a suitable size, they were potted, with 5 replicates per group.

[0074] Young leaves of transgenic potato plants were collected, and DNA was extracted as a template to amplify the overexpression vector GFP gene fragment and the gene silencing vector hygromycin (Hyg) gene fragment. Transgenic positive plants were obtained through screening. The amplification primers were:

[0075] GFP-F: 5'- GACCACATGAAGCAGCACGA-3';

[0076] GFP-R: 5'-CGCTTCTCGTTGGGGTCTTT-3';

[0077] Hyg-F: 5'-CGGTGTCGTCCATCACAGTT-3';

[0078] Hyg-R: 5'-TGACCTATTGCATCTCCCGC-3'.

[0079] After sequencing, positive plants with the highest StPAP12 expression were screened from the overexpression positive lines, and plants with the highest StPAP12 silencing efficiency were screened from the silenced positive lines for subsequent experiments.

[0080] 2.5 Determination of gene expression levels

[0081] Using cDNA from transgenic potato leaves as a template, qRT-PCR identification was performed according to the 2×SYBR Green qPCR Premix (Universal) (XKL0412, Kailai Enzyme, Guangzhou) kit. EF1α was used as an internal control gene (using primers StEF1α-qRT-F / R). Each sample was tested in quadruplicate. The reaction system and conditions followed the kit instructions. The primers used are as follows:

[0082] qRT-PCR primers for amplifying StPAP12:

[0083] StPAP12-qRT-F:GGTGGACGTTGTCTTTGCTGG;

[0084] StPAP12-qRT-R: TGCCCAATGCTTGCTTCTCG;

[0085] qRT-PCR primers for amplifying StEF1a:

[0086] StEF1α-qRT-F: 5'-GCCCATGGTTGTTGAGACCT-3';

[0087] StEF1α-qRT-R: 5'-TCTTGACAACACCGACAGCA-3'.

[0088] qRT-PCR results are as follows Figure 1 As shown in the figure, compared with the control, the expression level of the transgenic lines was significantly upregulated, while the expression level of the silent lines was significantly downregulated, indicating that the overexpression and gene silencing systems were successfully constructed. Subsequent experiments will use lines OX-12 and Ri-1 as overexpression and interference materials, respectively.

[0089] Example 3: Verification of the effect of regulating the potato StPAP12 gene on potato growth.

[0090] 3.1 Experimental Setup

[0091] The test materials were wild-type WT, transgenic line OX-12, and silent line Ri-1. A high-phosphorus group (LP) and a low-phosphorus group (LP) were set up. The high-phosphorus group was irrigated with 1 / 2 Hoagland nutrient solution containing 0.5 mM potassium dihydrogen phosphate, and the low-phosphorus group was irrigated with 1 / 2 Hoagland nutrient solution containing 0.01 mM phosphorus.

[0092] In addition, inoculation treatment (Bac) and non-inoculation treatment (CK) were set up. Two weeks after transplanting the inoculated tissue culture seedlings into the soil, inoculation began. Phosphate-solubilizing Bacillus cereus (GDMCC No: 67185), after activation, was cultured in liquid LB medium at 28°C until the OD600 reached approximately 0.8–1.0, and then inoculated near the roots of the potato seedlings, with 20 ml of culture medium per pot. The non-inoculated plants were not inoculated; all other culture conditions were the same.

[0093] 3.2 Determination of yield and dry weight of transgenic lines

[0094] After the experiment began, potato plants were cultured to maturity. Transgenic seedlings were transplanted and grown for 12 weeks before harvesting to determine the fresh and dry weight of the tubers.

[0095] Experimental results are as follows Figure 2 As shown, the yield and plant dry weight of the transgenic lines were measured. Compared with WT, the control treatment under LP conditions significantly increased potato yield and plant dry weight. The inoculation treatment significantly increased yield and dry weight compared with the control. This indicates that the gene is similar to the previously reported PAP gene, which has the function of increasing potato phosphorus uptake and utilization and promoting potato growth. In addition, the inoculation treatment also has the effect of promoting potato yield and increasing plant dry weight.

[0096] 3.3 Overexpression lines significantly enhanced StPAP12 gene expression

[0097] Take the potato root tissue obtained in 3.2, extract RNA and reverse transcribe cDNA according to the method in 1.2, and use the StPAP12 expression detection primers (StPAP12-qRT-F / R and StEF1α-qRT-F / R described in 2.5 of Example 2) to detect the StPAP12 expression level according to the reagent instructions of 2xQ5 SYBR qPCR Master Mix (TOLOBIO).

[0098] Experimental results are as follows Figure 3 As shown, the results of detecting gene expression levels in transgenic lines under phosphate-solubilizing bacteria inoculation conditions showed that under LP and CK treatments, overexpression significantly enhanced gene expression, while interference significantly downregulated it. Inoculation with phosphate-solubilizing bacteria significantly enhanced the expression level of overexpressing lines, while under high phosphorus conditions, the gene expression level was significantly reduced, indicating that the overexpression of this gene is regulated according to the phosphorus status of the growth environment.

[0099] 3.4 Overexpression of the StPAP12 gene increases the content of acid phosphatase in potato roots.

[0100] To determine the changes in acid phosphatase content in plants, approximately 0.2 g of fresh root tissue obtained in step 3.2 was taken and homogenized with 1.8 mL of 45 mM Hac-NaAc (pH=5). The homogenate was centrifuged at 12000 rpm for 30 min at 4 °C. The supernatant was transferred to a new centrifuge tube and stored at 4 °C for later use. X μL of the supernatant was taken (generally 10 μL for leaves and 150 μL for roots), and (500-X) μL of 45 mM Hac-NaAc (pH=5) and 2000 μL of 1 mM p-nitrophenyl phosphate cyclohexylamine (freshly prepared) were added. The reaction was carried out at room temperature for 15 min. The reaction was terminated by adding 1000 μL of 1 N NaOH, and the sample was measured at 405 nm within 30 min.

[0101] Experimental results are as follows Figure 4 As shown, the root tissue analysis revealed that, compared to WT, LP treatment increased phosphatase content through overexpression, while inoculation with phosphate-solubilizing bacteria significantly enhanced phosphatase secretion, indicating that inoculation with phosphate-solubilizing bacteria activates plant phosphatase secretion.

[0102] 3.5 Phosphate-solubilizing bacteria and StPAP12 gene expression enhance phosphorus uptake in plants.

[0103] The aboveground and root tissues obtained in step 3.2 were dried to constant weight and weighed. The tissues were then ground using a small sample grinder, and 0.1 g–0.3 g was weighed into the bottom of a digestion tube. 5 mL of H₂SO₄ was added and the mixture was digested overnight using the H₂SO₄-HClO₄ method until the liquid was clear. The volume was then adjusted to dilution with secondary water, and the total phosphorus content was determined using a flow analyzer. Phosphorus content (mg*P / plant) = phosphorus concentration (mg / g) × plant biomass (g / plant).

[0104] Experimental results are as follows Figure 5 As shown, the results of the total phosphorus content determination in the plants indicated that, compared with WT, overexpression of the StPAP12 gene increased phosphorus uptake in both phosphorus treatment and inoculation treatment, while the phosphorus content of the interference lines was not significantly different from that of WT. Furthermore, it was found that inoculation with phosphate-solubilizing bacteria significantly increased phosphorus uptake in the StPAP12 overexpressing lines, and overall, the high-phosphorus treatment resulted in greater phosphorus uptake than the low-phosphorus treatment, indicating that the phosphate-solubilizing bacterium Bac and the StPAP12 gene synergistically promote phosphorus uptake in potato plants.

[0105] Example 4: Effects of regulating the potato StPAP12 gene on potato leaves

[0106] 4.1 The gene StPAP12 regulates the morphological characteristics of potato leaves.

[0107] The test materials were wild-type WT, transgenic line OX-12, and silent line Ri-1. They were cultured according to the method of the low phosphorus group in Example 3.1. Four weeks after transplanting potato tissue culture seedlings, the leaf area, leaf length and leaf width were measured by taking the top leaf of the fourth pair of fully unfolded trifoliate leaves from the top of the plant.

[0108] Experimental results are as follows Figure 6 As shown, transgenic plants overexpressing and interfering with the StPAP12 gene were obtained through vector construction. Pot experiments revealed that, compared with the wild-type WT, overexpression of the StPAP12 gene significantly increased leaf width and leaf area, while the interfering lines significantly reduced leaf length, width, and leaf area. This indicates that the increase in cell number mainly stems from planar expansion.

[0109] 4.2 Cellular mechanism of StPAP12 gene regulating potato leaf size

[0110] Take the top leaf obtained in 4.1, use transparent tape to collect tissue cells from the underside of the leaf, stain with toluidine blue, observe and photograph under a microscope, and measure cell parameters using ImageJ software.

[0111] Experimental results are as follows Figure 7 As shown, observation of the epidermal cells of the apical leaves of transformed plants revealed that, compared with WT, overexpression of OX and interference with Ri significantly reduced cell area, cell length, and width, but significantly increased the number of cells per unit area. This indicates that the gene may have a dual regulatory function: on the one hand, promoting cell expansion (positively regulating cell size), and on the other hand, inhibiting cell division (negatively regulating cell proliferation). In the OX line, cell division inhibition was relieved, cell cycle activity was enhanced, and the cells exhibited a "small and numerous" characteristic; in the Ri line, cell expansion function was lost, possibly accompanied by disordered cell division, leading to a "small and few" developmental defect. This result is consistent with the "cell size-cell number tradeoff" theory of plant organ size regulation, providing cellular evidence for understanding the molecular mechanisms of leaf development. Figure 6 Morphological results revealed that this gene positively regulates cell expansion and negatively regulates cell division, thus determining the final size of the organ by coordinating the balance between cell size and number.

[0112] Example 5: Effect of regulating the potato StPAP12 gene on potato disease resistance.

[0113] The test materials were wild-type WT, transgenic line OX-12, and silent line Ri-1. Culture was performed according to the method described in Example 3.1 (low phosphorus group). After transplanting, the potato plants grew for 8 weeks until they reached the vegetative growth stage. Late blight resistance was then determined by inoculation with the pathogen. A strong strain "MZ" from our laboratory was selected as the resistance identification strain (the late blight strain is MZ15-30, which has been disclosed in the literature Zhou Y, Yang K, Yan Q ... Targeting of anti-microbial proteins to the hyphal surface amplifies protection of crop plants against Phytophthorapathogens Molecular Plant, 2021; 14, 1391-1403). The strain was sprayed on the potato plant leaves at a concentration of 300 sporangia / 10 μL. The inoculation temperature was set at 20°C, and a humidifier was used to maintain air humidity. The plants were cultured in the dark for 3 days. The disease incidence on all leaves was statistically analyzed according to the late blight disease severity level, and the incidence rate for each treatment was calculated.

[0114] Experimental results are as follows Figure 8 As shown, when potted plants were inoculated with late blight indoors, the growth of plants in the OX overexpression group was less affected, and the number of diseased leaves and the size of the infected area were significantly reduced. In contrast, the growth of plants in the WT and silent groups was significantly affected, and the number of diseased areas and infected leaves increased significantly. This demonstrates that overexpression of this gene can significantly improve the resistance of potato plants to late blight.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A potato StPAP12 protein, characterized in that it is At least one of the following: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) Analogs of the amino acid sequence shown in SEQ ID NO: 1 that still enhance the resistance of potatoes to late blight by substitution, insertion or deletion of one or more amino acids.

2. The potato StPAP12 protein according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the potato StPAP12 protein is shown in SEQ ID NO.

2.

3. A potato StPAP12 gene, characterized by... The nucleotide sequence is at least one of the following: (a) The nucleotide sequence shown in SEQ ID NO.2; (b) Analogs of the nucleotide sequences in (a) above, obtained by base insertion, deletion, or substitution, that still enhance the resistance of potatoes to late blight.

4. An expression vector carrying the potato StPAP12 gene, characterized in that: Includes the nucleotide sequence shown in SEQ ID NO.

2.

5. An Agrobacterium that expresses the potato StPAP12 gene in plants, characterized in that: Including the expression vector carrying the potato StPAP12 gene as described in claim 4.

6. The application of the potato StPAP12 protein as described in claim 1 or 2, the potato StPAP12 gene as described in claim 3, the expression vector carrying the potato StPAP12 gene as described in claim 4, and the Agrobacterium tumefaciens expressing the potato StPAP12 gene in plants as described in claim 5 in enhancing the resistance of potatoes to late blight.

7. The application of the potato StPAP12 protein according to claim 1 or 2, the potato StPAP12 gene according to claim 3, the expression vector carrying the potato StPAP12 gene according to claim 4, and the Agrobacterium tumefaciens expressing the potato StPAP12 gene in plants according to claim 5 in potato breeding.

8. A method for enhancing the resistance of potatoes to late blight, characterized in that... Includes the following steps: Agrobacterium, which expresses the StPAP12 gene that enhances potato resistance to late blight, was applied to potatoes to improve their resistance to late blight.

9. The method for enhancing potato resistance to late blight according to claim 8, characterized in that: The application refers to the application to potato seedlings; The potato in question is Yue Shu No.

1.

10. A biological agent that enhances the resistance of potatoes to late blight, characterized in that: Including Agrobacterium, as described in claim 5, which expresses the StPAP12 gene in plants to enhance resistance to late blight in potatoes.

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