Application of IbGATA8 protein in regulation and control of drought resistance of sweet potatoes

Abstract

The invention discloses application of IbGATA8 protein in regulation and control of drought resistance of sweet potatoes, and belongs to the field of genetic engineering. The IbGATA8 protein related to drought resistance is identified in sweet potatoes, the amino acid sequence of the IbGATA8 protein is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene IbGATA8 of the IbGATA8 protein is shown as SEQ ID NO.2. A sweet potato plant overexpressed with the IbGATA8 gene is obtained through an agrobacterium transfection method, and a drought stress experiment result shows that the IbGATA8 gene overexpressed plant is superior to a wild sweet potato in indexes such as growth state, fresh weight, stomatal opening, proline content, MDA content and hydrogen peroxide content; the overexpression of the IbGATA8 gene can significantly improve the drought resistance of sweet potatoes. The invention provides a new technical means for breeding the drought resistance of sweet potatoes, and has important application value in the aspect of improving the drought resistance of plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the application of IbGATA8 protein in regulating drought resistance in sweet potatoes. Background Technology

[0002] Drought is one of the most damaging abiotic stresses, severely impacting plant growth and development. It has become a major factor constraining global agricultural production, significantly affecting long-term crop yields and food security. Under drought conditions, plants face problems such as water shortage, oxidative stress, and metabolic disorders, leading to decreased yields and impaired quality. Taking sweet potato (Ipomoea batatas) as an example, drought stress significantly restricts the formation and development of its storage roots, posing a serious threat to sustainable agriculture in arid and semi-arid regions. Therefore, improving the drought resistance of sweet potato is of great significance.

[0003] The GATA transcription factor family plays a crucial role in regulating plant growth, development, and stress response. These proteins, characterized by conserved zinc finger domains, regulate various physiological processes, including light signaling, hormone responses, and abiotic stress tolerance. Currently, the evolutionary dynamics, structural diversity, and functional significance of GATA genes in sweet potato have not been reported. Therefore, cloning GATA transcription factor family-related genes in sweet potato and studying their functions is of great importance. There is an urgent need to discover new genes in the sweet potato GATA transcription factor family that regulate abiotic stress resistance and utilize them through bioengineering to improve plant stress resistance. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the IbGATA8 protein in regulating drought resistance in sweet potato, thereby addressing the problems existing in the prior art. This invention identifies an IbGATA8 protein with the amino acid sequence shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene, IbGATA8, is shown in SEQ ID NO.2. Overexpression of the IbGATA8 gene can significantly improve the drought resistance of sweet potato. This invention provides a new technical means for breeding sweet potato to improve drought resistance, has significant application value in enhancing plant drought resistance, and has broad application space and market prospects in the agricultural field.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an application of the IbGATA8 protein in any of the following:

[0007] (1) Regulating the drought resistance of sweet potatoes;

[0008] (2) Cultivating transgenic sweet potatoes with improved drought resistance;

[0009] (3) Prepare products that improve the drought resistance of sweet potatoes;

[0010] The amino acid sequence of the IbGATA8 protein is shown in SEQ ID NO.1.

[0011] The present invention also provides an application of the gene encoding the above-mentioned IbGATA8 protein in any of the following:

[0012] (1) Regulating the drought resistance of sweet potatoes;

[0013] (2) Cultivating transgenic sweet potatoes with improved drought resistance;

[0014] (3) Prepare products that improve the drought resistance of sweet potatoes;

[0015] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0016] Furthermore, the expression level of the coding gene was upregulated in sweet potato to improve the drought resistance of the sweet potato.

[0017] Furthermore, the method for upregulating the expression level of the coding gene includes overexpressing the coding gene in the sweet potato.

[0018] The present invention also provides an application of an overexpression vector, wherein the overexpression vector comprises the gene encoding the IbGATA8 protein;

[0019] The nucleotide sequence of the gene encoding the IbGATA8 protein is shown in SEQ ID NO.2;

[0020] The application is any one of the following:

[0021] (1) Regulating the drought resistance of sweet potatoes;

[0022] (2) Cultivating transgenic sweet potatoes with improved drought resistance;

[0023] (3) Prepare products that improve the drought resistance of sweet potatoes.

[0024] The present invention also provides the use of engineered bacteria comprising the above-described overexpression vector in any of the following:

[0025] (1) Regulating the drought resistance of sweet potatoes;

[0026] (2) Cultivating transgenic sweet potatoes with improved drought resistance;

[0027] (3) Prepare products that improve the drought resistance of sweet potatoes.

[0028] The present invention also provides a method for improving the drought resistance of sweet potatoes, including the step of upregulating the expression level of the gene encoding the IbGATA8 protein in sweet potato plants;

[0029] The nucleotide sequence of the gene encoding the IbGATA8 protein is shown in SEQ ID NO.2.

[0030] Furthermore, the upregulation of the expression level of the gene encoding the IbGATA8 protein includes the step of overexpressing the gene in the sweet potato plant.

[0031] This invention also provides a breeding method for sweet potatoes with improved drought resistance, comprising the following steps:

[0032] A vector encoding the IbGATA8 protein was constructed, and an engineered bacterium was made to infect sweet potato plants. The tubers of the sweet potato plants were harvested, and the tubers were used to regenerate sweet potato plants, thus obtaining the drought-resistant sweet potato.

[0033] The nucleotide sequence of the gene encoding the IbGATA8 protein is shown in SEQ ID NO.2.

[0034] Furthermore, the starting strain of the engineered bacteria is Agrobacterium K599.

[0035] The present invention discloses the following technical effects:

[0036] This invention identifies an IbGATA8 protein in sweet potato that is associated with drought resistance. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene, IbGATA8, is shown in SEQ ID NO.2. Sweet potato plants overexpressing the IbGATA8 gene were obtained using Agrobacterium-mediated transformation. Drought stress experiments showed that IbGATA8-overexpressing plants were superior to wild-type sweet potatoes in terms of growth status, fresh weight, stomatal aperture, proline content, MDA content, and hydrogen peroxide content. Overexpression of the IbGATA8 gene significantly improves the drought resistance of sweet potatoes. This invention provides a new technical means for breeding sweet potato to enhance drought resistance, has significant application value in improving plant drought resistance, and has broad application prospects and market potential in the agricultural field. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1The process diagram for obtaining transgenic sweet potato plants; where A is a sweet potato stem segment; B is the process of Agrobacterium infection of sweet potato stem segments; C is a transgenic sweet potato tuber; D is a transgenic sweet potato plant; E is an electrophoresis diagram of PCR detection of overexpressing sweet potato plants; F is an electrophoresis diagram of PCR detection of interfering plants;

[0039] Figure 2 The figure shows the results of the IbGATA8 gene expression characteristic identification experiment; where A is the expression pattern under PEG induction; B is the activation activity analysis of the IbGATA8 gene; and C is the subcellular localization result of the IbGATA8 gene, with a scale bar length of 5 μm.

[0040] Figure 3 Figure 1 shows the results of drought resistance identification experiments for IbGATA8 transgenic sweet potato plants and wild-type sweet potato plants; where A represents phenotypic identification results with a scale bar of 5 cm; B represents the statistical results of plant fresh weight; C represents the observation results of stomatal aperture with a scale bar of 100 μm; and D represents the measurement results of drought resistance-related indicators. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] The sweet potato variety Lushu 3 is described in the following literature: Wang Feibing. Obtaining and identifying the characteristics of sweet potato plants overexpressing IbMIPS1 and IbMVD genes [D]. China Agricultural University, 2015. The applicant promises to release the above-mentioned biological materials to the public within 20 years from the date of this application.

[0047] The vector pCAMBIA1300 is a product of UBO Biotechnology Co., Ltd., with product number VT1375.

[0048] The plant total RNA extraction kit was the Transzol Up Plant Total RNA Extraction Kit from TransGen Biotech (Beijing), product catalog number ET111.

[0049] The QuantScript RT Kit is a product of Tiangen Pharmaceuticals Co., Ltd. (Beijing), with product catalog number KR103.

[0050] Example 1: Obtaining the IbGATA8 gene

[0051] 1. Total RNA was extracted from the young leaves of sweet potato variety Lushu 3 using a plant total RNA extraction kit, and the total RNA was reverse transcribed into first-strand cDNA using the QuantScript RT Kit Quant cDNA Kit.

[0052] 2. Primers IbGATA8-F (SEQ ID NO.3) and IbGATA8-R (SEQ ID NO.4) were designed and synthesized. Using the cDNA obtained in step 1 as a template, PCR amplification was performed to obtain a PCR amplification product of approximately 975 bp, which was then sequenced.

[0053] Primer sequences:

[0054] IbGATA8-F: 5′-ATGGATTCAAATTTCGTCGATG-3′ (SEQ ID NO. 3);

[0055] IbGATA8-R: 5′-TCACTTGATCTCGGGTGGAG-3′ (SEQ ID NO. 4);

[0056] The results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID NO.2, with a length of 975 bp. The gene represented by this sequence was named the IbGATA8 gene, and the protein it encodes was named the IbGATA8 protein or protein IbGATA8. The amino acid sequence of the IbGATA8 protein was as shown in SEQ ID NO.1, with a length of 324 aa.

[0057] SEQ ID NO.1:

[0058] MDSNFVDEIDCGSFFDHIDELIDFPPENECAGVGSGDCANFPSIWDEPLPDSDPIFSAAHSHSASDLSAELSVPYEDIVQLEWLSAFVEESFSDGGLTLGKENYPIIPKPESDKKFQAASSPISVLESSSSSSSSNSSCSGGKVMPLSPSQRGAQRARSKRP RPVAFNPRRVMQLIETPQPESFADSPVKKVPRPPPAGAPEQKKKKKIKFSMPALPNPGGPDQDQPGAQPVRKCMHCEITKTPQWRAGPMGPKTLCNACGVRYKSGRLFPEYRPAASPTFVPALHSNSHKKVVEMRTKSVTSLEQSPVSSLVAPTVTAPPEIK.

[0059] SEQ ID NO.2:

[0060] ATGGATTCAAATTTCGTCGATGAAATAGACTGCGGCAGCTTCTTCGATCACATTGACGAACTGATCGATTTCCCCCCGGAAAATGAGTGCGCCGGTGTCGGCTCCGGCGACTGCGCCAATTTCCCCAGCATTTGGGACGAGCCGTTGCCGGATTCCGACCCCATTTTCTCCGCTGCCCACAGCCATTCCGCTTCCGACCTCTCCGCCGAGCTCTCAGTTCCGTACGAGGATATTGTGCAGCTGGAATGGCTTTCAGCCTTTGTGGAAGAATCATTCTCGGATGGAGGGCTAACTCTTGGAAAAGAGAACTATCCGATCATCCCGAAGCCGGAATCGGACAAGAAATTCCAGGCTGCTTCGAGCCCGATTTCGGTTCTTGAGAGCAGTAGTAGCAGCAGTAGCAGTAATTCGTCGTGCTCGGGGGGTAAGGTGATGCCGTTGAGCCCGAGCCAGCGTGGGGCGCAGCGTGCACGCAGCAAGCGCCCCCGCCCGGTGGCGTTTAACCCTAGGCGGGTGATGCAGCTGATTGAGACTCCGCAGCCCGAGAGCTTTGCGGATTCTCCCGTGAAGAAGGTTCCGAGGCCGCCACCAGCGGGAGCGCCCGAGCAGAAGAAGAAAAAGAAGATCAAGTTCTCAATGCCCGCCTTGCCTAATCCAGGCGGCCCGGATCAGGATCAGCCCGGGGCACAGCCAGTCAGGAAATGTATGCATTGCGAGATCACGAAGACGCCTCAGTGGAGGGCAGGTCCCATGGGGCCTAAAACGCTCTGCAACGCGTGTGGGGTTCGTTACAAATCGGGCAGGCTGTTCCCCGAGTACAGGCCTGCAGCCAGCCCTACATTCGTTCCGGCCCTTCACTCCAACTCCCACAAGAAGGTCGTCGAGATGAGAACCAAGTCGGTCACCAGCCTCGAGCAATCCCCCGTTTCTTCTCTCGTAGCTCCAACAGTCACTGCTCCACCCGAGATCAAGTGA。

[0061] Example 2: Application of IbGATA8 protein in improving drought resistance of sweet potatoes

[0062] 1. Construction of recombinant plasmid pCAMBIA1300-IbGATA8

[0063] 1.1 Fragment Amplification

[0064] A double-stranded DNA molecule with the nucleotide sequence shown in SEQ ID NO.2 was artificially synthesized. Using this double-stranded DNA molecule as a template, PCR amplification was performed using OE-F-KpnI and OE-R-XbaI as primers to obtain a double-stranded DNA fragment containing a restriction endonuclease KpnI recognition sequence at the 5' end and a restriction endonuclease XbaI recognition sequence at the 3' end.

[0065] OE-F-KpnI: 5′-acgggggacgagctc ggtacc ATGGATTCAAATTTCGTCGATGA-3′ (SEQ ID NO. 5);

[0066] Note: The underlined sequence is the recognition sequence of the restriction endonuclease KpnI;

[0067] OE-R-XbaI: 5′-aagatcttcgtcgac tctaga CTTGATCTCGGGTGGAGCAG-3′ (SEQ ID NO. 6);

[0068] Note: The underlined part is the recognition sequence of the restriction endonuclease XbaI.

[0069] 1.2 Double enzyme digestion

[0070] The vector pCAMBIA1300 was digested with restriction endonucleases KpnI and XbaI, and approximately 1 kp of the vector backbone was recovered.

[0071] 1.3 Homologous recombination

[0072] The double-stranded DNA fragment from 1.1 was ligated with the vector backbone recovered in 1.2 via homologous recombination to obtain the recombinant plasmid pCAMBIA1300-IbGATA8.

[0073] Based on the sequencing results, the recombinant plasmid pCAMBIA1300-IbGATA8 was modified as follows: the small fragment between the restriction endonuclease KpnI and XbaI recognition sequences of the recombinant plasmid pCAMBIA1300 was replaced with a DNA molecule with the nucleotide sequence shown in SEQ ID NO.2, expressing the IbGATA8 protein shown in SEQ ID NO.1.

[0074] 2. Construction of recombinant plasmid pFGC5941-IbGATA8

[0075] The pFGC5941 vector was double-digested with Xho I and Asc I. Using the CDS sequence of IbGATA8 as a substrate, the upstream fragment was amplified by PCR. The products were then homologously recombinated to construct the upstream vector pFGC5941-IbGATA8-Up. The primer sequences are as follows:

[0076] pFGC5941-IbGATA8-Up-F (Xho I):

[0077] 5'-tttggagaggacacgctcgagATGGATTCAAATTTCGTCGATGA-3' (SEQ ID NO. 7);

[0078] pFGC5941-IbGATA8-Up-R (Asc I):

[0079] 5'-ttaaatcatcgattgggcgcgccCGGAACTGAGAGCTCGGCG-3' (SEQ ID NO. 8).

[0080] After obtaining the upstream vector pFGC5941-IbGATA8-Up, double digestion with BamHI and XbaI was performed. Downstream fragment PCR amplification was then carried out using IbGATA8 CDS as a substrate. Homologous recombination of the two products yielded the pFGC5941-IbGATA8 vector. Primer sequences are as follows:

[0081] pFGC5941-IbGATA8-Down-F (BamH I):

[0082] 5'-aatttgcaggtatttggatccCGGAACTGAGAGCTCGGCG-3' (SEQ ID NO. 9);

[0083] pFGC5941-IbGATA8-Down-R (Xba I):

[0084] 5'-ggtcttaattaactctctagaATGGATTCAAATTTCGTCGATGA-3' (SEQ ID NO. 10).

[0085] 3. Obtaining IbGATA8 transgenic sweet potato plants

[0086] 3.1 Transplantation with Agrobacterium

[0087] The recombinant plasmid pCAMBIA1300-IbGATA8 was transformed into Agrobacterium rhizogenes K599 competent cells (purchased from Beijing Bairddi Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium, which was named K599 / pCAMBIA1300-IbGATA8.

[0088] The recombinant plasmid pFGC5941-IbGATA8-IbGATA8 was transformed into Agrobacterium rhizogenes K599 competent cells (purchased from Beijing Bairddi Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium, which was named K599 / pFGC5941-IbGATA8.

[0089] 3.2 Obtaining transgenic sweet potatoes

[0090] 3.2.1 Transformation

[0091] (1) Transformation of overexpression plants

[0092] The plasmid overexpressing IbGATA8 (pCAMBIA1300-IbGATA8) was introduced into Lushu No. 3 plants using an Agrobacterium-mediated transformation method, as follows:

[0093] 1) Stem segment pretreatment: Select healthy, fresh Lushu No. 3 stem segments for treatment. Each segment should contain a top stem tip and 5-8 nodes below it. Remove the leaves from the lower 2-3 nodes of the stem segment with scissors. Then, use a needle to make circular punctures around the nodes to create wounds. Make punctures evenly in the area between two nodes, with a spacing of approximately 0.5 cm. The treated sweet potato stem segments look like... Figure 1 As shown in Figure A.

[0094] 2) Infection: The infection process is as follows Figure 1 As shown in Figure B, the treated stem segments were immersed in the prepared K599 bacterial solution, ensuring that the wound area was completely immersed in the bacterial solution, and the infection was carried out at room temperature in the dark for about 12 hours.

[0095] 3) Obtaining transgenic sweet potato tubers: Infected stem segments are planted in soil, and transgenic tubers are harvested approximately 120 days later. Figure 1 As shown in C.

[0096] 4) Plant the genetically modified potato tubers to obtain genetically modified plants, such as... Figure 1 As shown in D.

[0097] (2) Interference with the transformation of expression plants

[0098] The recombinant plasmid (pFGC5941-IbGATA8) interfering with IbGATA8 expression was introduced into Lushu No. 3 plants using an Agrobacterium-mediated method, as follows:

[0099] 1) Stem segment pretreatment: Select healthy, fresh Lushu No. 3 stem segments for treatment. Each stem segment contains the top stem tip and 5-8 nodes below it. Remove the leaves from the lower 2-3 nodes of the stem segment with scissors, and then use a needle to make holes around the nodes to create wounds. Make holes evenly in the area of ​​two stem nodes, with a spacing of about 0.5 cm.

[0100] 2) Infection: Soak the treated stem segments in the prepared K599 bacterial solution, ensuring that the wound area is completely immersed in the bacterial solution, and infect at room temperature in the dark for about 12 hours.

[0101] 3) Obtaining genetically modified sweet potato tubers: The infected stem segments are planted in the soil, and the genetically modified tubers are harvested after about 120 days.

[0102] 4) Plant the genetically modified potato tubers to obtain genetically modified plants.

[0103] 3.2.2 Detection of transgenic plants

[0104] (1) PCR detection

[0105] Genomic DNA was extracted from transgenic and wild-type sweet potato plants using the CTAB method. PCR detection was performed using standard methods. The expected amplified fragment length for overexpression plants was approximately 1500 bp, and the expected amplified fragment length for interference expression plants was approximately 750 bp. The amplification primers used were:

[0106] 35S-F: 5'-TGACGCACAATCCCACTATCCT-3' (SEQ ID NO. 11);

[0107] IbGATA8-R: 5'-TCACTTGATCTCGGGTGGAG-3' (SEQ ID NO. 4);

[0108] int-F: 5'- CAACCACAAAAGTATCTATGAGCCT -3' (SEQ ID NO. 12);

[0109] int-R: 5'-TTCACATGTCAGAAACATTCTGATG-3' (SEQ ID NO. 13).

[0110] Add 2 μL of 10×PCR buffer, 1 μL of dNTP (10 mol / L), 1 μL each of primers (10 μmol / L), 2 μL of template DNA (50 ng / μL), and 1 μL of Taq DNA polymerase to a 0.2 mL Eppendorf centrifuge tube, and add H2O to a total volume of 20 μL.

[0111] The reaction program was 94℃ denaturation for 4 min, 57℃ annealing for 1.5 min, 72℃ extension for 1 min, 30 s, for a total of 35 cycles. Wild-type Lushu No. 3 sweet potato plants were used as negative controls, followed by electrophoresis detection.

[0112] See results Figure 1 In E and F, as can be seen from the electrophoresis diagram, both overexpressing and interfering transgenic plants can amplify the target band of the corresponding size, indicating that these sweet potato plants are transgenic sweet potato plants.

[0113] 3.3 Expression analysis of the IbGATA8 gene

[0114] PEGylated Lushu No. 3 plants 6000 RNA was extracted from whole plants using the TroZol kit after treatment for different time periods. The expression level of the IbGATA8 gene was detected by qRT-PCR using primers P1 and P2. The SYBR Premix Ex Taq kit was a product of TaKaRa (Takara Bio Inc., Dalian) (catalog number: RR420).

[0115] P1: 5′-GGGTAAGGTGATGCCGTTGA-3′ (SEQ ID NO. 14);

[0116] P2: 5′-TTCACGGGAGAATCCGCAAA-3′ (SEQ ID NO. 15).

[0117] The experimental results are shown in Figure 2 The results showed that drought stress induced upregulation of IbGATA8 gene expression.

[0118] 3.4 Subcellular localization of IbGATA8

[0119] The pCAMBIA1300-GFP vector was digested with Kpn I and Xba I, and the recombinant vector pCAMBIA1300-IbGATA8-GFP was constructed using homologous recombination. The primer sequences are as follows:

[0120] pCAMBIA1300-IbGATA8-GFP-F (Kpn I):

[0121] 5'- acgggggacgagctcggtaccATGGATTCAAATTTCGTCGATGA -3' (SEQ ID NO. 5);

[0122] pCAMBIA1300-IbGATA8-GFP-R (Xba I):

[0123] 5'-aagatcttcgtcgactctagaCTTGATCTCGGGTGGAGCAG-3' (SEQ ID NO. 6).

[0124] The constructed pCAMBIA1300-IbGATA8-GFP vector was transformed into rice protoplasts, and GFP and MCherry signals were observed.

[0125] 3.5 Analysis of the self-activation activity of IbGATA8 in yeast

[0126] Based on the IbGATA8 domain, the full-length (1-324 aa) and N-terminal (1-74 aa) and C-terminal (75-324 aa) fragments encoding the IbGATA8 protein were constructed into the pGBKT7 vector. Double digestion with Nde I and BamHI was performed. Amplification was performed using the IbERF109 CDS sequence as a substrate. The full-length IbGATA8 CDS sequence was amplified using IbGATA8-BD-F and IbGATA8-BD-R primers. N74 -BD-R primers were used to amplify the N-terminal (1-74 aa) sequence of IbGATA8, using IbGATA8 C249 -BD-F and IbGATA8-BD-R are primers used to amplify the C-terminal sequence (75-324 aa) of IbGATA8. The primer sequences are as follows.

[0127] pGBKT7-IbGATA8-BD-F (Nde I):

[0128] 5'-tcagaggaggacctgcatatgATGGATTCAAATTTCGTCGATGA-3' (SEQ ID NO. 16);

[0129] pGBKT7-IbGATA8 N74 -BD-R (BamH I):

[0130] 5'-ccgctgcaggtcgacggatccCGGCTCGAGAGTCAAGGC-3' (SEQ ID NO. 17);

[0131] pGBKT7-IbGATA8 C249 -BD-F (Nde I):

[0132] 5'-tcagaggaggacctgcatatgTACGAGGATATTGTGCAGCTG -3' (SEQ ID NO. 18);

[0133] pGBKT7-IbGATA8-BD-R (BamH I):

[0134] 5'-ccgctgcaggtcgacggatccTCACTTGATCTCGGGTGGAGC-3' (SEQ ID NO. 19).

[0135] The constructed vector, along with the positive control (pGBKT7-p53) and negative control (pGBKT7-Lam), were transformed into yeast strain Y2H. The cultured single clones were streaked onto SD / -Trp / -His / X-α-Gal medium and cultured at 30°C in the dark. The growth of yeast cells was observed after 3 days.

[0136] 4. Identification of drought resistance in transgenic sweet potato plants

[0137] 4.1 Experimental Methods

[0138] (1) Preparation of culture medium: The culture medium is a mixture of peat soil and vermiculite in a 1:1 ratio.

[0139] (2) Sweet potato plants with consistent growth status, including those overexpressing the IbGATA8 gene (OEG-1 / OEG-2), those with interfered IbGATA8 gene expression (RiG-1 / RiG-2), and wild-type sweet potato plants (WT), had stem segments of the same length cut and cultured in a culture medium to obtain sweet potato seedlings. The seedlings were then treated with Hogrange solution and a solution containing 20% ​​PEG, respectively. 6000 The Hogland solution was used for irrigation for 48 hours, corresponding to the normal water supply treatment (Control) and the simulated drought stress treatment (Drought).

[0140] (3) After 48 hours, observe and photograph the growth and development of sweet potato seedlings, weigh the fresh weight of the whole sweet potato plant, take leaves and observe the stomatal opening on the back of the leaves under a microscope, and measure the relevant indicators of stress.

[0141] Three independent biological replicates were set up for subsequent analysis.

[0142] 4.2 Experimental Results

[0143] like Figure 3 As shown in Figures A and B, under normal water supply conditions, the growth status of wild-type sweet potato plants, sweet potato plants overexpressing the IbGATA8 gene, or sweet potato plants with interference in IbGATA8 gene expression is basically the same, and there is no significant difference in the whole fresh weight of each sweet potato plant.

[0144] like Figure 3As shown in Figures A and B, under drought stress, wild-type sweet potato plants wilted significantly, while sweet potato plants overexpressing the IbGATA8 gene showed less wilting and exhibited significantly better growth than wild-type sweet potato plants. Sweet potato plants with impaired IbGATA8 gene expression wilted severely and showed yellowing. Compared with wild-type sweet potato plants, sweet potato plants overexpressing the IbGATA8 gene had significantly higher whole-plant fresh weight (P<0.01), while sweet potato plants with impaired IbGATA8 gene expression had significantly lower whole-plant fresh weight (P<0.01).

[0145] like Figure 3 As shown in Figure C, under drought stress, sweet potato plants overexpressing the IbGATA8 gene exhibited significantly smaller stomatal apertures compared to wild-type plants. This indicates that IbGATA8 gene expression can reduce stomatal aperture, effectively decreasing water loss and coping with drought stress. Conversely, sweet potato plants with impaired IbGATA8 gene expression showed significantly larger stomatal apertures, indicating that interference with IbGATA8 gene expression leads to a loss of its ability to regulate stomatal opening and closing, resulting in ineffective water loss control and difficulty in coping with drought stress. The stomatal aperture observation results are consistent with the results of sweet potato phenotypic observation and whole-plant fresh weight measurement.

[0146] 5. Determination of drought resistance-related indicators in IbGATA8 transgenic sweet potato plants

[0147] 5.1 Determination of proline content in IbGATA8 transgenic sweet potato plants

[0148] The proline content of wild-type sweet potato (WT) and two sweet potato plants, OEG-1 and OEG-2, that overexpress the IbGATA8 gene, and two plants, RiG-1 and RiG-2, that interfere with IbGATA8 gene expression, was determined using the PRO Assay kit (Comin, PRO 1-Y). The experiment was repeated three times, and the average result was taken.

[0149] The results are as follows Figure 3 As shown in Figure D, the proline content of OEG-1 and OEG-2 was significantly higher than that of WT, while the proline content of RiG-1 and RiG-2 was significantly lower than that of WT.

[0150] 5.2 Determination of malondialdehyde (MDA) content in IbGATA8 transgenic sweet potato plants

[0151] Malondialdehyde (MDA) content was determined in wild-type sweet potato (WT) and two sweet potato plants, OEG-1 and OEG-2, that overexpressed the IbGATA8 gene, and two sweet potato plants, RiG-1 and RiG-2, that interfered with IbGATA8 gene expression. The experiment was repeated three times, and the average result was taken.

[0152] The results are as follows Figure 3 As shown in Figure D, the malondialdehyde (MDA) content of OEG-1 and OEG-2 was significantly lower than that of WT, while the MDA content of RiG-1 and RiG-2 was significantly higher than that of WT.

[0153] 5.3 Determination of hydrogen peroxide content in IbGATA8 transgenic sweet potato plants

[0154] The hydrogen peroxide content was determined using the MDA Assay Kit (Suzhou Keming Biotechnology Co., Ltd., catalog number: H2O2-1-Y) for wild-type sweet potato (WT) and two sweet potato plants, OEG-1 and OEG-2, that overexpressed the IbGATA8 gene, and two plants, RiG-1 and RiG-2, that had IbGATA8 gene expression impaired. The experiment was repeated three times, and the results were averaged.

[0155] The results are as follows Figure 3 As shown in Figure D, the hydrogen peroxide content of OEG-1 and OEG-2 is significantly lower than that of WT, while the hydrogen peroxide content of RiG-1 and RiG-2 is significantly higher than that of WT.

[0156] The above results indicate that overexpression of the IbGATA8 gene can significantly increase the accumulation of proline in sweet potatoes and reduce the accumulation of malondialdehyde and hydrogen peroxide.

[0157] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an IbGATA8 protein in any of the following: (1) Regulating the drought resistance of sweet potatoes; (2) Cultivating transgenic sweet potatoes with improved drought resistance; (3) Prepare products that improve the drought resistance of sweet potatoes; The amino acid sequence of the IbGATA8 protein is shown in SEQ ID NO.

1.

2. The use of the gene encoding the IbGATA8 protein as described in claim 1 in any of the following: (1) Regulating the drought resistance of sweet potatoes; (2) Cultivating transgenic sweet potatoes with improved drought resistance; (3) Prepare products that improve the drought resistance of sweet potatoes; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. The application as described in claim 2, characterized in that, Upregulating the expression level of the coding gene in sweet potato improves the drought resistance of the sweet potato.

4. The application as described in claim 3, characterized in that, The method for upregulating the expression level of the coding gene includes overexpressing the coding gene in the sweet potato.

5. An application of an overexpression vector, characterized in that, The overexpression vector includes the gene encoding the IbGATA8 protein; The nucleotide sequence of the gene encoding the IbGATA8 protein is shown in SEQ ID NO.2; The application is any one of the following: (1) Regulating the drought resistance of sweet potatoes; (2) Cultivating transgenic sweet potatoes with improved drought resistance; (3) Prepare products that improve the drought resistance of sweet potatoes.

6. The use of an engineered bacterium comprising the overexpression vector of claim 5 in any of the following: (1) Regulating the drought resistance of sweet potatoes; (2) Cultivating transgenic sweet potatoes with improved drought resistance; (3) Prepare products that improve the drought resistance of sweet potatoes.

7. A method for improving the drought resistance of sweet potatoes, characterized in that, This includes steps to upregulate the expression of the gene encoding the IbGATA8 protein in sweet potato plants. The nucleotide sequence of the gene encoding the IbGATA8 protein is shown in SEQ ID NO.

2.

8. The method as described in claim 7, characterized in that, The upregulation of the expression level of the gene encoding the IbGATA8 protein includes the step of overexpressing the gene in the sweet potato plant.

9. A breeding method for sweet potatoes with improved drought resistance, characterized in that, Includes the following steps: A vector encoding the IbGATA8 protein was constructed, and an engineered bacterium was made to infect sweet potato plants. The tubers of the sweet potato plants were harvested, and the tubers were used to regenerate sweet potato plants, thus obtaining the drought-resistant sweet potato. The nucleotide sequence of the gene encoding the IbGATA8 protein is shown in SEQ ID NO.

2.

10. The method as described in claim 9, characterized in that, The starting strain of the engineered bacteria is Agrobacterium K599.

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