SiPO gene for synergistically improving millet yield and drought resistance and application thereof

Abstract

The application discloses a kind of synergistically improving millet yield and drought resistance SiPO Gene and application thereof, and relates to the technical field of crop breeding genes.The nucleotide sequence as shown in SEQ ID NO.1 SiPO Gene is cloned from millet, and fluorescence quantitative expression analysis shows SiPO Participate in millet growth and development and drought stress response.In millet, after overexpression SiPO Compared with wild type, overexpression strain is significantly increased in plant height, stem diameter, ear length, ear width, grain number per ear, grain weight per ear, grain length, grain width and thousand-grain weight;Under strong drought conditions, the survival rate and chlorophyll content of overexpression strain are significantly increased;Under moderate drought conditions, the leaf color of overexpression strain is greener, the ear is larger, and the grain number per ear, thousand-grain weight and grain weight per ear are significantly better than those of wild type (WT);It is shown that SiPO Millet yield and drought resistance can be synergistically improved.

Description

Technical Field

[0001] This invention belongs to the field of crop breeding gene technology, and particularly relates to a method for synergistically improving millet yield and drought resistance. Sipo Genes and their applications. Background Technology

[0002] Discovering the genetic resources in millet that combine high yield and drought resistance can not only help increase millet production, but also provide new genetic resources and biological breeding technology support for stress-resistant millet molecular breeding, which has important practical significance for production.

[0003] Cytochrome P450 enzymes (CYPs) are a vital superfamily of redox enzymes in plants, widely involved in physiological processes such as the synthesis of secondary metabolites, regulation of growth and development, and stress responses. In the model plant Arabidopsis thaliana, numerous studies have revealed the core regulatory roles of CYPs in plant hormone metabolism pathways. Specifically, CYP94B3 and CYP94C1 regulate plant disease resistance by mediating the catabolism of jasmonic acid-isoleucine; CYP88A and CYP701A are key enzymes in the gibberellin biosynthesis pathway; and members of the CYP707A family play an important role in seed dormancy and drought response by catalyzing the 8′-hydroxylation of abscisic acid. Furthermore, CYPs are widely involved in the synthesis of diterpenoid bioactive substances. For example, CYP725A23-1, CYP725A22-1, and CYP725A55 are involved in the biosynthesis of the anticancer drug paclitaxel, while CYP728B70, CYP81AM1, CYP82D274, CYP71BE86, CYP71BE8, and CYP82D213 are closely related to the synthesis of triptolide, which has antitumor activity. These findings clearly demonstrate that the cytochrome P450 enzyme family plays an irreplaceable role in the regulation of plant secondary metabolism and the formation of stress-adaptive traits.

[0004] Premnaspirodiene oxygenase (PO) belongs to the cytochrome P450 family 71D subfamily (CYP71D) and is known to participate in the biosynthesis of phytoalexins, playing a role in disease resistance responses. However, current research on the biological functions of PO is still relatively limited, mainly focusing on its mechanism of action in phytoalexin synthesis pathways. There are no reports on whether this gene can synergistically improve crop yield and drought resistance. Summary of the Invention

[0005] To address the lack of breeding gene resources in existing technologies that can synergistically enhance crop yield and drought resistance, this invention provides a method for synergistically improving millet yield and drought resistance. Sipo Genes and their applications.

[0006] To achieve the above objectives, the present invention provides a method for synergistically improving millet yield and drought resistance. Sipo Gene, Sipo The cDNA sequence of the gene is shown in SEQ ID NO.1.

[0007] Preferred, Sipo The CDS sequence of the gene is shown in SEQ ID NO.2.

[0008] Preferred, Sipo The gene encodes the SiPO protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0009] Furthermore, the above-mentioned genes were overexpressed through genetic engineering. Sipo Gene synergy enhances millet yield and drought resistance; transgenic millet overexpression Sipo Positive yield and drought resistance phenotypes following gene expression include at least one of the following: increased plant height, increased stem diameter, increased ear length, increased ear width, increased number of grains per ear, increased grain weight per ear, increased grain length, increased grain width, increased thousand-grain weight, as well as improved survival rate, increased chlorophyll content, and increased yield under drought conditions.

[0010] On the other hand, the present invention also provides the above-mentioned Sipo Recombinant gene expression vectors.

[0011] Furthermore, the recombinant expression vector was constructed by cloning the CDS sequence shown in SEQ ID NO.2 into the UBi::pTCK303 vector.

[0012] On the other hand, the present invention also provides a method for synergistically improving millet yield and drought resistance, comprising: S1. Construct a CDS sequence containing the sequence shown in SEQ ID NO.2. Sipo Gene overexpression vectors; S2, will Sipo Gene overexpression vectors were transformed into Agrobacterium tumefaciens, and then millet was transformed using the inflorescence infection method; S3, after screening with hygromycin and identification by reverse transcription quantitative polymerase chain reaction, obtained Sipo Gene overexpression lines.

[0013] Furthermore, in S1, Sipo Gene overexpression vectors, through Sipo The gene was cloned into the UBi::pTCK303 vector to construct the gene.

[0014] Furthermore, cloning SipoThe upstream primer sequence of the gene is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.9.

[0015] On the other hand, the present invention also provides the above-mentioned Sipo Application of genes in the breeding of high-yield and / or drought-resistant varieties.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The present invention will Sipo The full-length cDNA of the gene was ligated into the pTCK303 expression vector started by UBi, and millet was transformed using the inflorescence infection method. Experiments confirmed that... Sipo Gene overexpression can cause millet to exhibit multiple positive phenotypes, such as increased plant height, thicker stems, larger ears of grain, larger kernels, increased yield, and enhanced drought resistance.

[0017] (2) Given Sipo The conservation of the gene in plants and the superior phenotype exhibited in transgenic millet suggest that the gene has broad application value. Sipo The gene provides new genetic resources for molecular breeding of drought-resistant, high-yield and high-quality millet, and will play an important role in the improvement of drought-resistant, high-yield and high-quality crop varieties, with broad application prospects. Attached Figure Description

[0018] 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.

[0019] Picture 1 for Sipo Results of quantitative real-time RT-qPCR analysis of gene expression levels in different tissues of millet; Picture 2 for Sipo A schematic diagram of a gene ligated into the pTCK303 expression vector; Picture 3 For different treatment groups Sipo Results of quantitative real-time RT-qPCR analysis of gene expression levels in millet stems, leaves, and roots; where A represents the expression levels after different treatment times with 3 μM ABA. Sipo Results of quantitative real-time RT-qPCR analysis of gene expression levels in millet stems and leaves; B represents the results of treatment with 3 μM ABA for different durations. Sipo Results of quantitative real-time RT-qPCR analysis of gene expression levels in millet roots; C represents the expression levels after different treatment times with 20% PEG. SipoResults of quantitative real-time RT-qPCR analysis of gene expression levels in millet stems and leaves; D represents the expression levels after different treatment times with 20% PEG. Sipo Results of quantitative real-time RT-qPCR analysis of gene expression levels in millet roots; Picture 4 For overexpression Sipo Results of quantitative real-time RT-qPCR analysis of target gene expression levels in strains; Picture 5 overexpression during seedling stage Sipo Observational and statistical results of plant height and stem diameter of transgenic lines and wild-type (WT) lines; where A represents overexpression at the seedling stage. Sipo Phenotypic diagrams of transgenic lines and wild-type (WT) lines; B represents overexpression at the seedling stage. Sipo Statistical results of plant height between transgenic lines and wild-type (WT) lines; C represents overexpression at the seedling stage. Sipo Statistical results of stem diameter of transgenic lines and wild-type (WT) lines; Picture 6 For synchronous overexpression Sipo Observation and statistical results of ear traits of transgenic lines and wild-type (WT) lines; where A represents synchronous overexpression. Sipo Phenotypic characteristics of immature panicles in transgenic and wild-type (WT) lines; B represents synchronous overexpression. Sipo Statistical results of panicle width of immature panicles of transgenic lines and wild-type (WT) lines; C represents synchronous overexpression. Sipo Statistical results of panicle length of immature panicles of transgenic lines and wild-type (WT) lines; D represents synchronous overexpression. Sipo Statistical results of the number of grains per mature panicle of transgenic lines and wild-type (WT) lines; E represents the number of grains per panicle at the same time point for overexpression. Sipo Statistical results of single ear grain weight of mature ears of transgenic lines and wild-type (WT) lines; Picture 7 For overexpression Sipo Seed size observation and statistical results of transgenic lines and wild-type (WT) lines; where A represents overexpression. Sipo Comparison of seed size between transgenic lines and wild-type (WT) lines; B represents overexpression. Sipo Statistical results of seed length between transgenic lines and wild-type (WT) lines; C represents overexpression. Sipo Statistical results of seed width between transgenic lines and wild-type (WT) lines; D represents overexpression. Sipo Statistical results of thousand-grain weight of transgenic lines and wild-type (WT) lines; Picture 8 For overexpression SipoThe results of drought resistance evaluation between transgenic lines and wild-type (WT) lines; where A shows the phenotypic diagrams under normal conditions, drought stress, and after rehydration; B shows the overexpression after drought treatment. Sipo Survival rates of transgenic lines compared to wild-type (WT); C represents overexpression after drought treatment. Sipo Results of chlorophyll content comparison between transgenic lines and wild-type (WT); Picture 9 Overexpression under long-term drought treatment Sipo Evaluation results of yield traits between transgenic and wild-type (WT) lines; where A represents overexpression under prolonged drought treatment. Sipo Phenotypic diagrams of transgenic lines and wild-type (WT) lines; B represents overexpression under prolonged drought treatment. Sipo A comparison of mature ear phenotypes between transgenic lines and wild-type (WT) lines; C is a statistical chart of the number of grains per ear after maturity; D is a statistical chart of the thousand-grain weight of the ear after maturity; E is a statistical chart of the single-ear grain weight of the ear after maturity. Detailed Implementation

[0020] 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.

[0021] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Unless otherwise specified, the term "parts" in this invention refers to parts by weight.

[0026] Obtained using the millet genome database website phytozome Sipo Gene sequence information was obtained by comparing the amino acid sequence encoded by the gene on the ClustalW website, which revealed that the SiPO protein is highly homologous in different species.

[0027] Sipo The full-length cDNA sequence of the gene is 2039 bp, as shown in SEQ ID NO.1, and the CDS sequence is 1530 bp, as shown in SEQ ID NO.2. The amino acid sequence of the SiPO protein it encodes is shown in SEQ ID NO.3.

[0028] SEQ ID NO.1: SEQ ID NO.2: SEQ ID NO.3: MDDYFYQSLLLSVAAVALLQLVKLALRPRPRLPPGPWKLPVIGSMHLVNVLPHRALRDLAAAHGPLMMLRLGETPLVVASSKETARAVLKTHDTNFATRPKLLAGEIVGYDWADILFSPSGDYWRK LRQLCAAEILSPKRVLSFRHIREDEVALKLAEIRAAGPSAPVNLSVMFHSLTNSIVSRAAFGKKRKNAPEFMAAIKAGVGLSSGFSIPDLFPTWTTVLAKITGMKRSLQDIHRTVDSILQEIIDERKA ILDEKVKSGAENAEENLVDVLIGLQEKGGFGFHLNNSRIKAIILDMFAGGTGTSASAMEWGMSELMRNPSVMKKLQGQIREAFHGKSVVTEADLQASNLRYLKLVIKEALRLHPPAPLLVPRESIEP CELDGYTIPAKSRVVINAFAIGRDPKYWDDAEEFKPERFEDGGVDFMGSSYEFLPFGSGRRMCPGFNYGLASMELALVGMLYHFDWSLPEGVAEVDMEEAPGLGVRRRSPLMLCATPFVPVVAVSTK. The Yugu No. 1 variety used in the examples was a gift from Professor Diao Xianmin in August 2017, and the wild-type millet (xiaomi) was a gift from Professor Wang Xingchun in December 2020.

[0029] Example 1 Roots, stems, leaves, spikes, and mature seeds of *Yugu No. 1* were collected under normal growth conditions, rapidly frozen in liquid nitrogen, and then ground into powder in liquid nitrogen. Total RNA was extracted from each tissue using the Biotech Universal Plant RNA Extraction Kit. Using the extracted RNA as templates, cDNA was synthesized via reverse transcription using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit.

[0030] Analysis using real-time quantitative RT-PCR technology Sipo Relative gene expression levels in various tissues. (Based on millet) SiACTIN The gene (Seita.5G393000) was used as an internal reference gene, and the primers for SiACTIN gene amplification were Actin_F and Actin_R; Sipo Gene amplification primers are Sipo _F and Sipo _R. Primer sequences are as follows: Actin_F (SEQ ID NO.4): 5′-TTGCTGACAGGATGAATGGC-3′; Actin_R (SEQ ID NO.5): 5′-CACATCTGCTGGAATGTGCT-3′; Sipo _F (SEQ ID NO.6): 5′-CGGGATGCTCTACCACTTCG-3′; Sipo _R (SEQ ID NO. 7): 5′-CTTCGTTGAGACAGCGACCA-3′.

[0031] The real-time quantitative RT-PCR reaction system is shown in Table 1.

[0032] Table 1. Real-time quantitative RT-PCR reaction system (15 μL)

[0033] The amplification program was as follows: 95.0℃ pre-denaturation for 60s; 95.0℃ denaturation for 10s; 60.0℃ annealing for 10s; 72.0℃ extension for 15s; acquisition of fluorescence signal (Plate Read); incubation at 65℃ for 20s; melting curve analysis: read every 0.5℃ from 65℃ to 95℃, hold for 1s; end the reaction.

[0034] The results are as follows Picture 1 As shown, Sipo The gene is mainly expressed in the roots during the seedling stage, followed by the spike, and expressed at a lower level in the stem, leaves and seeds.

[0035] Example 2 according to Sipo Primers were designed based on the CDS sequence of the gene for cloning. The specific steps are as follows: (1) Total RNA extraction: Total RNA was extracted from seedlings using the Biotech Universal Plant Total RNA Extraction Kit.

[0036] (2) Synthesis of the first strand of cDNA: The extracted total RNA was dissolved and its concentration was determined. Reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit. The reaction system was as follows: 5 μg of total RNA was added to 10 μL of 2× reaction buffer, 1 μL of oligo dT primer (0.5 μg / μL), 1 μL of reverse transcriptase, 1 μL of genomic DNA desminase, and sterile water was added to a total volume of 20 μL. The reaction program was as follows: incubation at 42℃ for 30 min, followed by heating at 85℃ for 5 min to inactivate the enzyme.

[0037] (3) Sipo Gene cloning: Upstream primer (SEQ ID NO.8): 5′- GATCC ATGGACGACTACTTCTACCAGTCGCTCC-3′; Downstream primer (SEQ ID NO.9): 5′- GAGCTC CTACTTCGTTGAGACAGCGACCACCG-3′.

[0038] The upstream primer introduces the BamHI restriction site (underlined), and the downstream primer introduces the SacI restriction site (underlined).

[0039] PCR amplification was performed using Novozymes high-fidelity enzyme (Vazyme#P505). The reaction system consisted of: 12.5 μL of 2× reaction buffer, 0.5 μL of deoxyribonucleic acid (dNTP), 1 μL of upstream primer, 1 μL of downstream primer, 0.5 μL of high-fidelity enzyme, 1 μL of cDNA template, and sterile water to a total volume of 25 μL.

[0040] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 35 cycles; final extension at 72℃ for 5 min; and incubation at 16℃.

[0041] After the reaction, agarose gel electrophoresis was performed. The gel containing the target band was excised and purified using a quick agarose gel DNA recovery kit (Cat#DP1722).

[0042] (4) Expression vector construction: Approximately 3.5 μL of the gel-recovered product was ligated to the pTCK303 expression vector driven by the UBi promoter (see schematic diagram of the vector). Picture 2(As shown), the operation steps were performed according to the EasyFusion Assembly Master Mix instructions. The ligation product was transformed into E. coli DH5α competent cells using the heat shock method, plated on LB agar plates containing kanamycin, and cultured overnight. White single colonies were picked for colony PCR identification, and positive clones were inoculated into LB liquid medium for overnight expansion.

[0043] (5) Extraction of plasmid DNA: Recombinant plasmid DNA was extracted using a high-purity plasmid mini extraction kit (CW0500A).

[0044] (6) Sequencing: The extracted recombinant plasmid DNA was sent to a sequencing company for sequence verification.

[0045] Example 3 Drought stress treatment Sipo Gene expression analysis: (1) Wild-type (WT) millet was used as material. After growing normally for 1 week, it was treated with an aqueous solution containing 3 μM ABA and 20% PEG at room temperature. Stems, leaves and roots were collected at 0, 3, 6 and 12 h and quickly placed in liquid nitrogen for freezing and preservation.

[0046] (2) Total RNA was extracted from the millet material at each of the above time points using a universal plant RNA extraction kit, and first-strand cDNA was synthesized according to the reverse transcription method described in Example 2.

[0047] (3) Analyzed by real-time quantitative RT-PCR Sipo The relative expression levels of genes at different treatment time points. Quantitative PCR was performed using a CFX96 Touch PCR instrument, following the instructions for the SYBR Premix Ex Taq II kit.

[0048] The results are as follows Picture 3 Middle A (stem and leaves) Picture 3 As shown in Figure B (root). In stems and leaves, 3 μM ABA treatment significantly induced... Sipo Gene expression was upregulated; in roots, treatment with 3 μM ABA for 6 h and 12 h resulted in upregulation. Sipo Gene expression was also significantly upregulated; while after 20% PEG treatment Sipo Gene expression is upregulated in stems and leaves, and significantly downregulated in roots. Picture 3 (C and D). The above results indicate that, Sipo The gene is mainly expressed in the roots of millet and participates in the regulation of millet growth and development as a drought stress response gene.

[0049] Example 4 Millet SipoGenetic transformation and screening of homozygous transgenic lines.

[0050] (1) Take 2 μL of the recombinant vector plasmid constructed in Example 2, transform it into Agrobacterium EHA105 competent cells, and plate culture until a single colony appears.

[0051] (2) Pick a single colony and inoculate it into 100 μL of LB liquid medium containing kanamycin and incubate for 4 h.

[0052] (3) Transfer the above 100 μL bacterial culture to 3 mL LB liquid medium containing kanamycin and incubate overnight for 7 h.

[0053] (4) Take 500 μL of bacterial culture and inoculate it into 50 mL of liquid pre-culture medium (pH 7.2) and incubate overnight for 14 h.

[0054] (5) Adjust the OD of the bacterial culture 600 The bacterial cells were collected by centrifugation at 2400×g for 5 min to a value of 1.0.

[0055] (6) Resuspend the bacterial cells in 40 mL of infection solution. The composition of the infection solution is as follows: 2.1 g K2HPO4, 0.9 g KH2PO4, 0.2 g (NH4)2SO4, 0.112 g citric acid, 0.66 mL glycerol, 0.0493 g MgSO4·7H2O, 3 g ascorbic acid, 0.4264 g MES, 7.2 g glucose, 200 mL distilled water, pH 5.8.

[0056] (7) Before infection, pre-treat the ears of grain (before flowering) with 1 mL of osmotic solution for 20 min. The osmotic solution formula is: 0.493 g MgSO4·7H2O, 0.3184 g MES, add distilled water to make up to 200 mL, and adjust the pH to 5.8.

[0057] (8) Infect the rice ears before flowering with pre-induced Agrobacterium for 20 min.

[0058] (9) Cover the infected ears of grain with an opaque plastic bag. Remove the plastic bag after 24 hours and continue normal cultivation until the seeds mature.

[0059] (10) Harvest the infected mature seeds and sow them on MS solid medium containing 90 mg / L hygromycin for screening. Select the lines with more green leaves and better growth as candidate transgenic materials (SiPO-OE).

[0060] (11) Total RNA was further extracted from the leaves of candidate transgenic seedlings, reverse transcribed into cDNA, and the expression level of the target gene was detected by qPCR. The results are as follows: Picture 4 As shown, the final result is Sipo -OE2、 Sipo-OE5、 Sipo -OE6 three stable overexpression lines.

[0061] Example 5 overexpression Sipo Transgenic lines and wild-type (WT) millet were planted under the same cultivation conditions, and phenotypic observations and statistical analyses were conducted.

[0062] (1) Overexpression during the seedling stage Sipo Plant height and stem diameter of transgenic lines and wild-type (WT) lines were observed and statistically analyzed, and the results are as follows: Picture 5 As shown. Compared with the wild type (WT), the transgenic lines showed significant increases in both plant height and stem diameter, with plant height increasing by approximately 32.2% and stem diameter increasing by approximately 18.1%.

[0063] (2) The characteristics of the panicle of immature panicles at the same time were observed, and the panicle length and width were measured and statistically analyzed. At the same time, the number of grains per panicle and the weight of grains per panicle after maturity were statistically analyzed. The results are as follows: Picture 6 As shown in the figure. Compared with the wild type (WT), the transgenic lines showed significant increases in panicle length, panicle width, number of grains per panicle, and grain weight per panicle, specifically an increase of approximately 33.3% in panicle length, approximately 5% in panicle width, 53.4% ​​in number of grains per panicle, and 61.4% in grain weight per panicle.

[0064] (3) Overexpression Sipo Seed size was observed and compared between the transgenic lines and wild-type (WT) lines, and seed length, width, and thousand-seed weight were statistically analyzed. The results are as follows: Picture 7 As shown in the figure. The results showed that, compared with the wild type (WT), the transgenic lines had a 5.6% increase in grain length, a 5.2% increase in grain width, and an 11.3% increase in thousand-grain weight.

[0065] Example 6 overexpression Sipo Identification of drought resistance in transgenic lines.

[0066] Mix vermiculite and potting soil in a 1:1 ratio, weigh out equal amounts, and place them in small pots. Allow water to seep through the drainage holes at the bottom of the pot until the soil is completely moistened. Then, allow the overexpressing agent to absorb water overnight. Sipo Transgenic and wild-type (WT) millet seeds were sown in small pots, 9 seeds per pot, covered with soil and lightly pressed down, and allowed to grow normally in a greenhouse for 30 days. Millet seedlings with uniform growth were selected, watering was stopped, and the plants were allowed to dry for 7 days. Afterward, they were rehydrated for 3-5 days. Survival rate and chlorophyll content were measured on the 10th day of the drought treatment.

[0067] overexpression Sipo Phenotypic differences between transgenic lines and wild-type (WT) under normal conditions, drought stress, and after rehydration are as follows: Picture 8As shown in Figure A, after drought treatment, the transgenic lines maintained good growth, with only a few leaves wilting and most leaves remaining green, while the wild-type (WT) plants wilted severely.

[0068] The survival rate and chlorophyll content statistics are as follows: Picture 8 B, Picture 8 As shown in Figure C. After drought treatment, overexpression... Sipo The survival rate and chlorophyll content of the transgenic lines were significantly higher than those of the wild-type (WT), indicating that overexpression... Sipo Genes can significantly enhance the drought resistance of millet.

[0069] Example 7 Overexpression under prolonged drought conditions Sipo Evaluation of yield traits in transgenic lines.

[0070] Mix vermiculite and potting soil in a 1:1 ratio, weigh out equal amounts, and fill flowerpots with the mixture. Sow an equal number of overexpressing seeds. Sipo Transgenic lines and wild-type (WT) millet. During normal growth until 15 days after flowering, both transgenic and wild-type (WT) materials were watered every 15 days and subjected to prolonged drought treatment throughout their growth period until seed maturity.

[0071] Overexpression under prolonged drought treatment Sipo Transgenic lines were observed and compared with wild-type (WT) immature ears at the same time, and ear length and width were measured and statistically analyzed. Simultaneously, the number of grains per ear, thousand-grain weight, and single-ear grain weight were statistically analyzed after maturity. The results are as follows: Picture 9 As shown.

[0072] The results showed that, compared with the wild type (WT), the transgenic lines under long-term drought conditions had significantly increased grains per ear, thousand-grain weight and single-ear grain weight, specifically an increase of 47.51% in grains per ear, an increase of 12.94% in thousand-grain weight and an increase of 61.38% in single-ear grain weight.

[0073] 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. A method for synergistically improving millet yield and drought resistance SiPO Genes are characterized by: SiPO The cDNA sequence of the gene is shown in SEQ ID NO.

1.

2. The method for synergistically improving millet yield and drought resistance according to claim 1 SiPO Genes are characterized by: SiPO The CDS sequence of the gene is shown in SEQ ID NO.

2.

3. The method for synergistically improving millet yield and drought resistance according to claim 1 SiPO Genes are characterized by: SiPO The gene encodes the SiPO protein, the amino acid sequence of which is shown in SEQ ID NO.

3.

4. The method for synergistically improving millet yield and drought resistance according to claim 1 SiPO Genes are characterized by: Overexpression via genetic engineering SiPO Gene synergy improves millet yield and drought resistance.

5. A method for synergistically improving millet yield and drought resistance as described in any one of claims 1-4 SiPO Recombinant gene expression vectors.

6. The recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector was constructed by cloning the CDS sequence shown in SEQ ID NO.2 into the UBi::pTCK303 vector.

7. A method for synergistically improving millet yield and drought resistance, characterized in that, Includes the following steps: S1. Construct a CDS sequence containing the sequence shown in SEQ ID NO.

2. SiPO Gene overexpression vectors; S2, will SiPO Gene overexpression vectors were transformed into Agrobacterium tumefaciens, and then millet was transformed using the inflorescence infection method; S3, after screening with hygromycin and identification by reverse transcription quantitative polymerase chain reaction, obtained SiPO Gene overexpression lines.

8. The method for synergistically improving millet yield and drought resistance according to claim 7, characterized in that: In S1, SiPO Gene overexpression vectors, through SiPO The gene was cloned into the UBi::pTCK303 vector to construct the gene.

9. The method for synergistically improving millet yield and drought resistance according to claim 8, characterized in that: clone SiPO The upstream primer sequence of the gene is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.

9.

10. A method for synergistically improving millet yield and drought resistance according to claim 7, characterized in that, SiPO Positive yield and drought resistance phenotypes in gene overexpression lines include at least one of the following: increased plant height, increased stem diameter, increased panicle length, increased panicle width, increased number of grains per panicle, increased grain weight per panicle, increased grain length, increased grain width, increased thousand-grain weight, as well as improved survival rate, increased chlorophyll content, and increased yield under drought conditions.

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