Application of CeCHS57 gene in improving drought resistance of cyperus esculentus

Abstract

The invention discloses application of a CeCHS57 gene in improving drought resistance of cyperus esculentus, and relates to the technical field of biology. The nucleotide sequence of the CeCHS57 gene is as shown in SEQ ID NO. 1. According to the invention, the CeCHS57 gene is successfully cloned from cyperus esculentus, so that the blank of research on the drought-resistant functional gene of cyperus esculentus is filled. After the gene is over-expressed in cyperus esculentus and arabidopsis thaliana through genetic transformation, the activity of antioxidant enzymes such as SOD, CAT, POD and APX in plant leaves and root systems can be remarkably enhanced, active oxygen accumulated under drought stress is effectively eliminated, the content and conductivity of membrane lipid peroxidation products MDA are reduced, and cell membrane damage is reduced, so that the tolerance of plants to drought stress is improved. Phenotypic analysis shows that the growth state of transgenic arabidopsis thaliana under drought conditions is obviously superior to that of wild and mutant plants, and the drought resistance function of the CeCHS57 gene is proved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to... CeCHS57 Application of genes in improving drought resistance in tiger nuts. Background Technology

[0002] Drought, as one of the most widespread and severe abiotic stressors, poses a serious challenge to the sustainability of agricultural production. With the intensification of climate change and the increasing frequency of extreme droughts, the inhibitory effect on crop growth and development is becoming more pronounced, leading to significant yield fluctuations and even crop failure. Drought stress triggers a series of complex physiological, biochemical, and molecular-level response mechanisms in crops. The leaves, as the core organs of photosynthesis and transpiration, reduce water loss through thickening of the cuticle, densification of mesophyll cells, and stomatal regulation. The underground root system enhances water absorption capacity through adaptive changes such as elongating root length, increasing the number of root tips, and improving root vigor. However, the synergistic regulatory network, signal transduction pathways, and regulatory mechanisms of key functional genes in the aboveground and underground parts of crops under drought stress have not been fully elucidated, which greatly limits the breakthroughs and applications of stress-resistant crop breeding technologies.

[0003] Among numerous crops, tiger nuts, as an emerging oilseed crop with both ecological value and economic potential, have shown broad application prospects in oilseed production, saline-alkali land improvement, and ecological restoration due to their high oil content, rich nutritional components (including crude protein, starch, total sugar, and other nutrients), and strong environmental adaptability. Currently, research on tiger nuts mainly focuses on oil accumulation mechanisms, quality optimization, cultivation technology improvement, and comprehensive resource utilization. These technological achievements have laid the foundation for the large-scale cultivation of tiger nuts. However, in stark contrast, research on tiger nut stress tolerance, especially the elucidation of molecular mechanisms related to drought resistance, the discovery of functional genes, and genetic improvement research, is relatively lagging behind, becoming a key bottleneck restricting the development of its industry.

[0004] Although tiger nuts possess some drought tolerance potential under natural growing conditions, their drought resistance is still insufficient to withstand extreme drought environments. Under sustained drought stress, tiger nuts exhibit problems such as stunted growth, dwarfing, decreased photosynthetic efficiency, and hindered tuber development, ultimately leading to a significant reduction in yield and severely impacting their planting efficiency and promotion scope. With the continuous expansion of tiger nut cultivation areas, the risk of drought stress they face is becoming increasingly prominent, necessitating the use of molecular breeding techniques to enhance their drought resistance and ensure stable and high yields in different ecological regions. Furthermore, as a relatively new crop that has not yet been fully domesticated, tiger nuts have relatively limited genomic resources, making the discovery and identification of stress-related functional genes challenging. Current research has not yet clearly identified the core genes and key pathways regulating drought resistance, and effective molecular breeding targets and techniques are lacking.

[0005] Therefore, in-depth analysis of the molecular mechanisms by which tiger nuts respond to drought stress and systematic exploration of their drought-resistant functional genes can not only fill the gap in the research on tiger nut stress-resistance genes and improve the genetic regulatory network of drought resistance in oil crops, but also provide key gene resources and technical support for stress-resistance molecular breeding of tiger nuts. This has important theoretical significance and practical value for expanding the planting area of ​​tiger nuts, improving their stress resistance and stable yield capacity, and promoting the high-quality development of the tiger nut industry. Summary of the Invention

[0006] The purpose of this invention is to provide CeCHS57 The application of genes in improving the drought resistance of tiger nuts aims to address the problems existing in the aforementioned technologies. CeCHS57 Gene overexpression can effectively improve the drought resistance of plants, thus providing key gene resources and technical support for stress-resistant molecular breeding.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for improving plant drought resistance. CeCHS57 The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] The present invention also provides the above-mentioned CeCHS57 The application of genes in improving the drought resistance of plants.

[0009] The present invention also provides the application of a biomaterial in improving the drought resistance of plants, wherein the biomaterial is any one of (1)-(3): (1) Including the above CeCHS57 Gene expression cassette; (2) A recombinant expression vector containing the gene expression cassette; (3) A recombinant host cell containing the recombinant expression vector.

[0010] Furthermore, the plant is tiger nut or Arabidopsis thaliana.

[0011] The present invention also provides the above-mentioned CeCHS57 Application of genes in improving the heat stress resistance of microorganisms.

[0012] The present invention also provides the application of a biomaterial in improving the heat stress resistance of microorganisms, wherein the biomaterial is any one of (1)-(3): (1) Including the above CeCHS57 Gene expression cassette; (2) A recombinant expression vector containing the gene expression cassette; (3) A recombinant host cell containing the recombinant expression vector.

[0013] Furthermore, the microorganism is yeast.

[0014] The present invention also provides a method for improving the drought resistance of plants, comprising: taking the above-mentioned... CeCHS57 The gene was genetically transformed into a plant to construct an overexpression of the gene. CeCHS57 The steps involved in transgenic plants.

[0015] Furthermore, the plant is tiger nut or Arabidopsis thaliana.

[0016] The present invention also provides a method for improving the drought resistance of yeast, comprising: taking the above-mentioned... CeCHS57 Genetic transformation of the gene into yeast was used to construct an overexpression of the gene. CeCHS57 The steps involved in gene recombination in yeast.

[0017] The present invention discloses the following technical effects: This invention successfully cloned from tiger nuts. CeCHS57 This gene fills a gap in research on drought-resistant functional genes in tiger nuts. After overexpression in tiger nuts and Arabidopsis thaliana through genetic transformation, this gene significantly enhances the activity of antioxidant enzymes such as SOD, CAT, POD, and APX in the leaves and roots of the plants. It effectively scavenges reactive oxygen species accumulated under drought stress, reduces the content and conductivity of MDA (membrane lipid peroxidation products), and decreases cell membrane damage, thereby improving the plant's tolerance to drought stress. Phenotypic analysis showed that the transgenic Arabidopsis thaliana exhibited significantly better growth under drought conditions than wild-type and mutant plants, confirming that… CeCHS57 The drought-resistance function of genes. Furthermore, the transcriptomic and metabolomic combined analysis and screening strategy employed in this invention provides a feasible method for the efficient discovery of crop stress-resistance functional genes. The related genes and biological materials provide important tools for breeding stress-resistant crops, 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] Figure 1 The image shows the identification results of the target gene amplification product; where M is the DNA marker and 1 is the PCR amplification product. Figure 2 The figure shows the identification results of PCR products transformed into E. coli using the entry vector; where M is the DNA marker; and 1 is the PCR amplification product transformed into E. coli using the entry vector. Figure 3The figure shows the identification results of the PCR products of the expression vector transformed into Escherichia coli; where M is the DNA marker; and 1 is the PCR amplification product of the expression vector transformed into Escherichia coli. Figure 4 The figure shows the identification results of PCR products of Agrobacterium tumefaciens transformed with the expression vector; where M is the DNA marker; 1-3 are the PCR amplification products of Agrobacterium tumefaciens transformed with the expression vector. Figure 5 The image shows the identification results of transgenic yeast; where M is the DNA marker; 1-3 are the PCR amplification products of transgenic yeast. Figure 6 Phenotypic analysis diagram of transgenic yeast; Figure 7 The image shows the PCR identification results of Arabidopsis mutants; where M: molecular weight standard of 2000 marker; lanes 1-2: wild-type Arabidopsis Col-0; lanes 3-8: Arabidopsis mutants to be tested; lanes 1, 3, 5 and 7 are Lp+Rp amplification results; lanes 2, 4, 6 and 8 are Bp+Rp amplification results. Figure 8 The image shows the RT-PCR identification results of Arabidopsis mutants; where M represents the molecular weight standard of the 2000 marker; Col-0 represents wild-type Arabidopsis; and 2, 5, and 7 represent Arabidopsis mutants. Figure 9 Arabidopsis mutant SALK_076535C Statistical graph of relative expression levels; Figure 10 This is a Basta screening diagram for transgenic Arabidopsis thaliana. Figure 11 The image shows the identification results of transgenic Arabidopsis thaliana plants; where A represents... Bar Image A shows the test results of the test strip; Image B shows the PCR identification results of transgenic Arabidopsis thaliana. Figure 12 This is a diagram of RT-qPCR analysis of Arabidopsis thaliana plants; Figure 13 A diagram showing the analysis of chalcone synthase in the leaves and roots of transgenic Arabidopsis thaliana; Figure 14 This is a schematic diagram of the phenotypes of transgenic Arabidopsis thaliana under the control treatment (CK) and drought stress treatment (D); Figure 15 Statistical graphs showing the enzyme activities of antioxidant enzymes SOD(a), CAT(b), POD(c), and APX(d) in Arabidopsis leaves under different treatments; where CK represents the control treatment and D represents the drought stress treatment. Figure 16Statistical graphs showing the enzyme activities of antioxidant enzymes SOD (a), CAT (b), POD (c), and APX (d) in Arabidopsis roots under different treatments; where CK represents the control treatment and D represents the drought stress treatment. Figure 17 Membrane lipid peroxidation indices MDA (a), H2O2 (b), and O2 in Arabidopsis thaliana leaves under different treatments. - (c) and (d) are statistical graphs of electrical conductivity; where CK represents the control treatment and D represents the drought stress treatment. Figure 18 Membrane lipid peroxidation indicators (MDA, H2O2, O2) in Arabidopsis roots under different treatments - (c) and (d) are statistical graphs of electrical conductivity; where CK represents the control treatment and D represents the drought stress treatment. 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. 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.

[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 obvious 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] Example 1 1. Test materials The tiger nut variety "Jisha No. 2," a major variety promoted in Jilin Province, was used as the experimental material. This variety has a growth period of approximately 117 days, with bushy plants, narrow and long leaves, and yellow mature tubers. The plant height is 105-130 cm, with 75-95 tillers, a 100-seed weight of approximately 65 g, and round seed shape. Analysis by the Grain and Products Quality Supervision and Testing Center (Changchun) revealed the following main quality contents: crude protein 4.72%, crude fat 27.24%, crude starch 28.70%, and total sugar 16.10%.

[0026] 2. Drought stress experiment design After germinating tiger nut tubers, they were sown in a mixture of horticultural potting soil and vermiculite (volume ratio 1:1) and cultured in an artificial climate chamber until the 5-leaf stage. A control treatment (W) and a drought stress treatment (D) were then established. Control treatment: Maintain soil moisture content at field capacity; Drought stress treatment: Maintain soil moisture content at 50% of field capacity; Leaf and root samples were collected at 0, 3, 6, 9 and 12 days after treatment, and then flash-frozen in liquid nitrogen and stored at -80℃.

[0027] 3. Screening for drought-responsive genes (1) Phenotypic and physiological analysis of tiger nut seedlings under drought stress Tiger nut seedlings were subjected to drought stress. Phenotypic (biomass, leaf area, root system, and dry and fresh weight of leaves) and some physiological screening were used to determine that 6 days of stress was the optimal measurement time. Membrane lipid peroxidation index and physiological osmotic potential related index were used to prove that 6 days can be used as the sampling time for analysis of drought stress response.

[0028] (2) Analysis of the root and leaf response mechanism of tiger nut seedlings to drought Differentially expressed genes and differentially changed metabolites were observed in the leaves and roots of tiger nut seedlings under control and drought stress. The transcriptomic and metabolomic results were enriched in the phenylpropane biosynthesis (Map00940) and flavonoid biosynthesis (Map00941) pathways. The differentially expressed enzyme (chalcone synthase) that changes in common pathways was selected as the candidate enzyme encoding gene for further analysis.

[0029] (3) Identification and screening of CHS gene family members in tiger nuts A search of the tigernut reference genome in the CNGBdb database yielded a total of 66 genomes. CeCHS Family members, through analysis of conserved motifs, gene structure, and cisfunctional elements, revealed that members of five subfamilies of the CHS shared similar motif types, gene structures, and cisfunctional element types. GO and KEGG enrichment analyses showed... CeCHS Family members were enriched in pathways / entries related to flavonoid biosynthesis and chalcone synthase, and were screened based on expression levels. CeCHS57 Further analysis will be conducted on these genes as candidates for the CHS gene family.

[0030] CeCHS57 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0031]

[0032] Example 2 1. Cloning of the target gene according to CeCHS57 Cloning primers were designed based on the gene's CDS sequence, with the restriction enzyme sites being: Hind III and Bam HI. Using cDNA from the roots of Gisa 2 as a template, CeCHS57 The full-length sequence of the gene CDS after removing the stop codon was cloned to obtain the PCR product. Figure 1 The correctly amplified fragments were recovered using a gel extraction kit, and the recovered products were sequenced. The correctly sequenced PCR products were then stored for subsequent experiments.

[0033] 2. Construction of the introductory platform Using endonuclease Xho I and BamH II. The target fragment and the empty FU28 vector were double-digested, and the digestion products were ligated using ligase. The ligation products were then transformed into E. coli strains. DH5α In the middle; after the strain has grown well, use CeCHS57 Primers containing the full-length CDS sequences at both ends of the gene were used for PCR identification of the activated bacterial culture. The PCR products were detected by 3% agarose gel electrophoresis. The results are as follows: Figure 2 As shown, the target fragment appears at 1176 bp. After gel recovery of the target band and sequencing, the results were correctly aligned, indicating that the entry vector has been successfully transformed into E. coli, completing the entry vector transformation process. FU28-CeCHS57 The construction of ).

[0034] 3. Construction of expression carriers Using LR recombination reaction to transfer the target gene fragment on the entry vector ( CeCHS57 ) and related tags (GFP) were constructed into an empty expression vector pSOY1 Recombinant plasmids are formed on top ( pSOY1-CeCHS57 The recombinant plasmid was then transformed into an E. coli strain. DH5α In the process, after culturing the strain, a single colony is picked, and at the same time, the following methods are used: CeCHS57 Primers containing the full-length CDS sequences at both ends of the gene after activation DH5α The bacterial culture was identified by PCR, and the results were determined by 2% agarose gel electrophoresis. DH5α The bacterial culture PCR products were detected, and the results showed that the target band appeared at 1176 bp. Figure 3 The results of gel recovery of the target band and sequencing were correct, indicating that the expression vector was successfully transferred into E. coli.

[0035] 4. Transformation of Agrobacterium tumefaciens with expression vector Recombinant plasmids were extracted from the constructed expression vector *E. coli*. pSOY1 - CeCHS57 The recombinant plasmid was transformed into Agrobacterium tumefaciens strain using the freeze-thaw method. EHA105 In the process, after culturing the bacteria, a single colony is picked, and at the same time, the following methods are used: CeCHS57 Primers containing the full-length CDS sequences at both ends of the gene were used for PCR identification of the activated Agrobacterium tumefaciens culture. The PCR products of the Agrobacterium tumefaciens culture were detected by 2% agarose gel electrophoresis. The results showed that the target band appeared at 1176 bp. Figure 4 Furthermore, the results of gel extraction and sequencing of the target band were consistent, indicating that the expression vector was successfully transformed into Agrobacterium tumefaciens strain using the freeze-thaw method. EHA105 middle.

[0036] 5. Candidate genes of the CHS family in tiger nuts CeCHS57 Validation analysis of transgenic yeast 5.1 Construction of yeast recombinant expression vector choose pYES2 The vector is a yeast transformation vector, which utilizes homologous recombination to transfer the target gene fragment ( ) from the entry vector. CeCHS57 Constructing an empty expression carrier pYES2 Recombinant plasmids were formed and transformed into Escherichia coli strains. DH5α In the process, after culturing the strain, a single colony is picked, and at the same time, the following methods are used: CeCHS57 Primers containing the full-length CDS sequences at both ends of the gene after activation DH5α The bacterial culture was identified by PCR, and the results were determined by 2% agarose gel electrophoresis. DH5α The bacterial culture PCR products were detected. The results showed that the target band appeared at 1176 bp. Figure 5 The results of gel extraction and sequencing of the target band were correct, indicating that the expression vector was successfully transformed into E. coli, and the yeast recombinant expression vector was extracted. pYES2-CeCHS57, It can be used for subsequent yeast phenotypic verification experiments.

[0037] 5.2 CeCHS57 Construction and phenotypic analysis of transgenic yeast The constructed yeast recombinant expression vector pYES2-CeCHS57 With empty carrier pYES2 Converted to INVScI Recombinant yeast was obtained from competent yeast cells and inoculated onto drought stress medium and control medium for 7 days.

[0038] Drought stress culture medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L and 1 M mannitol; Control culture medium: yeast extract 10 g / L, peptone 20 g / L and glucose 20 g / L.

[0039] The results are as follows Figure 6 As shown, in the control culture medium, the expression vector pYES2-CeCHS57 Transforming yeast and empty vector pYES2 The transformed yeast cells showed basically consistent growth, indicating that no stress was exerted on the yeast cells in the control medium; in the drought-stressed medium, the expression vector... pYES2-CeCHS57 The transformed yeast grew much better than the empty vector. pYES2 The transformation of yeast indicates that the empty vector has been successfully adapted to drought-stressed culture medium. pYES2 Transformation of yeast caused stress, expression vector pYES2-CeCHS57 The drought tolerance of the transforming yeast is stronger than that of the empty carrier. pYES2 Transformed yeast, further explanation CeCHS57 It may have drought-resistant properties.

[0040] 6. CHS candidate gene for tiger nuts CeCHS57 Validation analysis of transgenic Arabidopsis thaliana 6.1 Analysis of Arabidopsis mutants The Arabidopsis homolog of the tigernut CHS gene was selected, and its mutants were purchased from Fuzhou Airosha Biotechnology Co., Ltd. (Fuzhou, Fujian, China). Information on the Arabidopsis mutants is shown in Table 2.

[0041] Arabidopsis mutant SALK_076535C Analysis revealed that the knockout gene in this mutant was... AT5G13930 (Table 1) is for CeCHS57 The homologous gene. The mutation type of this mutant is a T-DNA insertion mutation.

[0042] Table 1 Germplasm table of Arabidopsis mutants

[0043] 6.2 Molecular detection of Arabidopsis mutants 6.2.1 PCR identification of Arabidopsis mutants Using the Signal database (http: / / signal.salk.edu / tdnaprimers.2.html), Arabidopsis mutants purchased from the Arashare germplasm bank were analyzed. SALK_076535C PCR identification was performed on wild-type individuals. Col-0 Arabidopsis thaliana, heterozygous mutant Arabidopsis thaliana, and homozygous mutant were identified using the methods shown in Table 2. SALK_076535C- Lp, SALK_076535C- Rp and Bp primer pairs for Arabidopsis mutants SALK_076535C To conduct an identification, SALK_076535C- Lp+ SALK_076535C- The PCR amplification results of the Rp primers showed a negative band, i.e., Bp+. SALK_076535C- The Rp primer PCR amplification result showed a single target band, therefore it was identified as Arabidopsis thaliana. SALK_076535C The plant is a homozygous mutant. Figure 7 ).

[0044] Table 2. List of primers for PCR identification of Arabidopsis mutants

[0045] 6.2.2 RT-PCR and RT-qPCR identification of Arabidopsis mutants This invention aims to identify Arabidopsis thaliana mutants. SALK_076535C Whether transcription can be successfully performed at the mRNA level, and extraction of Arabidopsis mutants. SALK_076535C Total RNA from roots was reverse transcribed to obtain cDNA. This cDNA was then used as a template and... CeCHS57 RT-PCR amplification and RT-qPCR fluorescence detection were performed using CDS sequence-specific primers (Table 3). The RT-PCR products were detected by 2% agarose gel electrophoresis. The results showed that no transcription of the target gene was detected in the Arabidopsis mutant, confirming that this mutant was a homozygous Arabidopsis mutant. Figure 8 ).

[0046] Table 3. List of primers for RT-PCR identification of Arabidopsis mutants

[0047] Simultaneously, RT-qPCR fluorescence detection was used to... SALK_076535C Target genes in roots AT5G13930 Expression level analysis was performed, and the results of relative expression levels showed that in Arabidopsis mutants... AT5G13930 The expression level was sharply reduced compared to the wild-type Arabidopsis thaliana in each line, with a relative expression level less than 20% of that in the wild-type. Therefore, this data indicates that this mutant is a homozygous Arabidopsis thaliana mutant. Figure 9 ).

[0048] 6.3 Identification of Arabidopsis thaliana reintroduced and overexpressing plants 6.3.1 Basta screening of Arabidopsis thaliana replacement and overexpression plants Verification using Agrobacterium inflorescence staining method CeCHS57 The function of Arabidopsis thaliana wild-type Col-0 and mutants SALK_076535C As experimental material, the recombinant plasmid was used pSOY1 - CeCHS57 Transformation was performed on mutant and wild-type Arabidopsis thaliana materials to obtain Arabidopsis thaliana replenishment materials.35S : CeCHS57 / SALK_076535C ) and overexpression of Arabidopsis thaliana ( 35S : CeCHS57 / Col-0 Materials. Mature T0 generation Arabidopsis seeds were harvested after inoculation. The harvested T0 generation seeds were then mixed and sown in the soil. When the two young true leaves of the T1 generation plants were fully expanded, a Basta liquid solution (diluted 1:1000) was sprayed evenly. After 2-3 sprays, the Arabidopsis plants will be divided into two categories (…). Figure 10 One type of Arabidopsis thaliana will show yellowing leaves or gradually die (blue arrow pointing to the plant), indicating that the plant is a non-GMO Arabidopsis thaliana; the other type of Arabidopsis thaliana will remain green and grow normally (red arrow pointing to the plant), indicating that the plant is a GMO Arabidopsis thaliana positive plant. The positive Arabidopsis thaliana plants are transplanted into new small pots, and identification will be carried out after the seedlings have grown.

[0049] 6.3.2 Identification of Arabidopsis thaliana reintroduced and overexpressing plants Transgenic Arabidopsis thaliana (chassis material includes Arabidopsis thaliana mutants and wild-type) Col-0 ) to be carried out separately Bar Test strip detection and PCR identification: In Bar In the test strip test, the leaf is placed in a clean centrifuge tube, the extraction solution is added, the leaf is ground into powder using a small plastic pestle, and then inserted... Bar If two lines appear on the test strip, it means the gene has been transferred into the Arabidopsis plant. If only one line appears, it means the gene has not been introduced into the plant. Bar The test strip results show ( Figure 11 (A) Most Bar The appearance of two lines on the test strip indicates that the target gene has been transferred into the transgenic plant (the overexpressing Arabidopsis plant is...). 35S : CeCHS57 / Col-0 The replanted plants are 35S : CeCHS57 / SALK_076535C Total DNA was extracted from the leaves of transgenic Arabidopsis thaliana plants with two lines, and then used... CeCHS57 Primers for the full-length CDS sequences at both ends of the gene and Bar Primers for the gene sequence (Table 4) were used for PCR molecular identification of the transgenic plants. The PCR products were detected by 2% agarose gel electrophoresis. The results showed that no target band was present in the control plants, while it was present in the supplemented and overexpressing Arabidopsis plants (overexpressing Arabidopsis plants were...). 35S : CeCHS57 / Col-0 And replanting plants is 35S : CeCHS57 / SALK_ 076535CThe relevant target band was detected in ) Figure 11 (B)

[0050] Table 4. List of primers for PCR detection of transgenic Arabidopsis thaliana

[0051] Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 Under the same conditions that meet the growth requirements of Arabidopsis thaliana plants, total RNA was extracted from the roots of various Arabidopsis thaliana materials and cDNA was obtained through reverse transcription. CeCHS57 Specific primers for Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 qRT-PCR was performed for identification. The results showed that: CeCHS57 Expression levels in wild-type Arabidopsis thaliana Col-0 With mutant Arabidopsis thaliana SALK_076535C The expression level in the medium was very low, far lower than that in the replenished Arabidopsis plants ( 35S : CeCHS57 / SALK_ 076535C ) and overexpressing Arabidopsis thaliana plants ( 35S : CeCHS57 / Col-0 ),in CeCHS57 The expression level of [a substance] was higher in overexpressing plants than in replenished plants, which may be due to [a specific reason]. CeCHS57 With Arabidopsis AT5G13930 High gene homology may have an effect on CeCHS57 The expression plays a hindering role ( Figure 12 ).

[0052] 6.4 CeCHS57 CHS enzyme activity analysis of transgenic Arabidopsis thaliana Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0Under the same growth conditions that meet the requirements of Arabidopsis thaliana plants, the activities of chalcone synthase (CHS) in leaves and roots were measured, and the results are as follows: Figure 13 As shown: Mutants in leaves SALK_076535C The CHS enzyme activity was the lowest, indicating that homologous gene knockout affected the changes in CHS enzyme activity in Arabidopsis thaliana; while the replenishment material ( 35S : CeCHS57 / SALK_076535C The activity of CHS enzyme in the wild-type material was significantly increased, compared with that in the wild-type material. Col-0 The change is not significant compared to the previous one, indicating that... CeCHS57 The introduction of [a specific ingredient] can compensate for changes in CHS enzyme activity in Arabidopsis thaliana; the highest CHS activity was observed in Arabidopsis thaliana overexpression materials ([a specific ingredient]). 35S : CeCHS57 / Col-0 ),illustrate CeCHS57 Overexpression of the gene promotes increased CHS enzyme activity; in roots, CHS enzyme activity follows a similar pattern to that in leaves. SALK_076535C The CHS enzyme activity was the lowest, indicating that homologous gene knockout affected the changes in CHS enzyme activity in Arabidopsis thaliana; while the replenishment material ( 35S : CeCHS57 / SALK_076535C The activity of CHS enzyme in the wild-type material was significantly increased, compared with that in the wild-type material. Col-0 The change is not significant compared to the previous one, indicating that... CeCHS57 The introduction of [a specific ingredient] can compensate for changes in CHS enzyme activity in Arabidopsis thaliana; the highest CHS activity was observed in Arabidopsis thaliana overexpression materials ([a specific ingredient]). 35S : CeCHS57 / Col-0 ),illustrate CeCHS57 Overexpression of the gene can promote the enhancement of CHS enzyme activity.

[0053] All the above results demonstrate that the target gene has been successfully transferred into both mutant and wild-type Arabidopsis thaliana, and that Arabidopsis thaliana complementation and overexpression materials have been successfully obtained. Furthermore, the target gene has been functionally expressed in Arabidopsis thaliana.

[0054] 6.5 CeCHS57 Phenotypic verification of transgenic Arabidopsis Various Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The control treatment (W) and drought stress treatment (D) were performed according to the method in Example 1, respectively. Samples were taken after 6 days for phenotypic detection, and the results are shown in [Figure 1]. Figure 14 Under normal conditions, various Arabidopsis materials, such as wild-type Arabidopsis, exhibit... Col-0Mutant Arabidopsis thaliana SALK_ 076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The growth of Arabidopsis overexpression materials did not change significantly, but under drought stress (D) treatment, the growth of these materials increased. 35S : CeCHS57 / Col-0 Its growth was significantly better than that of wild-type Arabidopsis thaliana. Col-0 and Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C And the mutant Arabidopsis thaliana SALK_076535C Its growth is worse than that of wild-type Arabidopsis thaliana. Col-0 and Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_ 076535C This provides a preliminary explanation of the content in tiger nuts. CeCHS57 Genes are important candidate genes for responding to drought stress.

[0055] 6.6 CeCHS57 Physiological verification of transgenic Arabidopsis 6.6.1 Analysis of antioxidant enzyme activity in transgenic Arabidopsis thaliana Various Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The control treatment (W) and drought stress treatment (D) were performed according to the method in Example 1. After 6 days, samples were taken to measure the changes in antioxidant enzyme activity in the leaves. The results are as follows: Figure 15 As shown. Regarding superoxide dismutase (SOD) activity, Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The increase in activity was greatest in the leaves, at 57.63%, which was greater than that of Arabidopsis thaliana remediation materials. 35S : CeCHS57 / SALK_ 076535C (33.63%) and Arabidopsis wild-type materials Col-0 (31.76%), while the mutant Arabidopsis thaliana SALK_076535C The increase in activity was smallest in the leaves, at 10.46%; in peroxidase (POD) activity, the Arabidopsis overexpression material showed the highest increase. 35S : CeCHS57 / Col-0The increase in activity was greatest in the leaves, at 29.74%, which was greater than that in Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_076535C and wild-type Arabidopsis thaliana materials Col-0 And the mutant Arabidopsis thaliana SALK_076535C The increase in activity was the smallest in the leaves, at -2.50%; in catalase (CAT) activity, the Arabidopsis overexpression material showed the lowest increase. 35S : CeCHS57 / Col-0 The increase in activity was greatest in the leaves, at 56.02%, which was greater than that in Arabidopsis thaliana remediation materials. 35S : CeCHS57 / SALK_076535C (27.92%) and Arabidopsis wild-type materials Col-0 (23.66%), while the mutant Arabidopsis thaliana SALK_076535C The increase in activity was smallest in the leaves, at 11.27%; in ascorbate peroxidase (APX) activity, the Arabidopsis overexpression material showed the highest activity. 35S : CeCHS57 / Col-0 The increase in activity was greatest in the leaves, at 43.76%, which was greater than that in wild-type Arabidopsis materials. Col-0 (27.57%) and Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C (18.98%), while the mutant Arabidopsis thaliana SALK_ 076535C The increase in activity was smallest in the leaves, at 12.18%. These results indicate... CeCHS57 As a type of gene in secondary metabolism, genes can function by stimulating the activity of enzymes in the ROS enzyme system of leaves, thereby affecting the plant's ability to respond to drought.

[0056] Various Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The control treatment (W) and drought stress treatment (D) were performed according to the method in Example 1. After 6 days, samples were taken to measure the changes in the antioxidant enzyme activity of the roots. The results are as follows: Figure 16 As shown: Arabidopsis thaliana overexpression materials in SOD activity 35S : CeCHS57 / Col-0 The increase in activity was greatest in the root system, at 60.98%, which was greater than that of wild-type Arabidopsis materials. Col-0 (36.25%) and Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C(24.36%), while the mutant Arabidopsis thaliana SALK_076535C The increase in activity was smallest in the root system, at 19.30%; in POD activity, Arabidopsis overexpression materials showed the greatest increase. 35S : CeCHS57 / Col-0 The activity increase was greatest in the root system of Arabidopsis thaliana, at 26.59%, while in the replanted material of Arabidopsis thaliana... 35S : CeCHS57 / SALK_076535C Mutant Arabidopsis thaliana SALK_ 076535C Arabidopsis wild-type materials Col-0 The activity increases in the roots of Arabidopsis thaliana were 17.88%, 21.26%, and 18.85%, respectively; in CAT activity, Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The increase in activity was greatest in the root system, at 68.97%, which was greater than that of Arabidopsis thaliana remediation materials. 35S : CeCHS57 / SALK_076535C (44.44%) and Arabidopsis wild-type materials Col-0 (42.64%), while the mutant Arabidopsis thaliana SALK_076535C The increase in APX activity was smallest in the roots, at 12.24%; in APX activity, Arabidopsis overexpression materials showed the greatest increase. 35S : CeCHS57 / Col-0 The increase in activity was greatest in the root system, at 65.09%, which was greater than that of Arabidopsis thaliana remediation materials. 35S : CeCHS57 / SALK_076535C (45.02%) and Arabidopsis wild-type materials Col-0 (44.40%), while the mutant Arabidopsis thaliana SALK_076535C The increase in activity was the smallest in the root system, at 21.65%. These results indicate... CeCHS57 As a type of gene in secondary metabolism, genes can function by stimulating the activity of enzymes in the root ROS enzyme system, thereby affecting the plant's ability to respond to drought.

[0057] 6.6.2 Analysis of membrane lipid peroxidation index in tiger nut seedlings under drought stress Various Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C Arabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The control treatment (W) and drought stress treatment (D) were performed according to the method in Example 1. After 6 days, samples were taken to measure the membrane lipid peroxidation index in the leaves. The results are as follows: Figure 17As shown: In terms of malondialdehyde (MDA) content, the mutant Arabidopsis thaliana... SALK_076535C The content of [specific element] showed the largest variation, at 133.90%, which was greater than that of wild-type Arabidopsis thaliana. Col-0 (90.26%) and Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535C (86.27%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 The MDA content changed the least, at 57.71%; in O2 - In terms of content, mutant Arabidopsis thaliana SALK_076535C The content of [specific component] showed the largest variation, at 275.17%, which was greater than that of Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_076535C (112.12%) and Arabidopsis wild-type materials Col-0 (82.21%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 O2 - The content change was the smallest, at 48.53%; in terms of H2O2 content, the mutant Arabidopsis thaliana... SALK_076535C The content of [specific component] showed the largest variation, at 210.14%, which was greater than that of Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_076535C (155.32%) and Arabidopsis wild-type materials Col-0 (145.37%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 The H2O2 content showed the smallest change, at 96.22%; among the changes in electrolytic permeability, the mutant Arabidopsis thaliana... SALK_076535C The content of [specific component] showed the largest variation, at 136.93%, which was greater than that of Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_076535C (134.43%) and Arabidopsis wild-type materials Col-0 (125.44%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 The electrolytic permeability showed the smallest change, at 103.89%. These results indicate... CeCHS57 As a gene involved in secondary metabolism in tiger nuts, it can function to reduce membrane lipid peroxidation in leaves, thereby affecting the plant's ability to respond to drought.

[0058] Various Arabidopsis materials (including wild-type Arabidopsis) Col-0 Mutant Arabidopsis thaliana SALK_076535C Arabidopsis thaliana replenishment materials 35S : CeCHS57 / SALK_076535CArabidopsis thaliana overexpression materials 35S : CeCHS57 / Col-0 The control treatment (W) and drought stress treatment (D) were performed according to the method in Example 1. After 6 days, samples were taken to measure the membrane lipid peroxidation index in the roots. The results are as follows: Figure 18 As shown: In terms of MDA content, the mutant Arabidopsis thaliana SALK_076535C The content of [specific component] showed the largest variation, at 86.70%, which was greater than that of Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_076535C (68.86%) and Arabidopsis wild-type materials Col-0 (44.07%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 The MDA content changed the least, at 24.95%; in O2 - In terms of content, mutant Arabidopsis thaliana SALK_076535C The content of [specific component] showed the largest variation, at 96.38%, which was greater than that of Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_076535C (68.49%) and Arabidopsis wild-type materials Col-0 (54.90%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 O2 - The content change was the smallest, at 42.50%; in H2O2 content, the mutant Arabidopsis thaliana... SALK_076535C The content of [specific component] showed the largest variation, at 292.97%, which was greater than that of Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_076535C (196.87%) and Arabidopsis wild-type materials Col-0 (164.47%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 The H2O2 content showed the smallest change, at 126.25%; among the changes in electrolytic osmotic rate, the mutant Arabidopsis thaliana... SALK_ 076535C The content of [specific component] showed the largest variation, at 183.28%, which was greater than that of Arabidopsis thaliana replenishment materials. 35S : CeCHS57 / SALK_ 076535C (161.46%) and Arabidopsis wild-type materials Col-0 (138.16%), while Arabidopsis overexpression materials 35S : CeCHS57 / Col-0 The change in electrolytic permeability was the smallest, at 102.01%. These results indicate... CeCHS57 As a type of gene in secondary metabolism, genes can function to reduce membrane lipid peroxidation in the root system, thereby affecting the plant's ability to respond to drought.

[0059] 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 gene for improving drought tolerance in plants, characterized in that, CeCHS57 Its nucleotide sequence is shown in SEQ ID NO.

1. ​ 2. A method as claimed in claim 1 CeCHS57 Use of the gene for increasing drought tolerance in plants.

3. Use of a biomaterial for increasing drought resistance of a plant, characterized in that, The biomaterial is any one of (1)-(3): (1) a gene expression cassette comprising the gene of claim 1 CeCHS57 gene. (2) A recombinant expression vector containing the gene expression cassette; (3) A recombinant host cell containing the recombinant expression vector.

4. Use according to claim 2 or 3, characterized in that, The plant in question is tiger nut or Arabidopsis thaliana.

5. A device as described in claim 1 CeCHS57 Application of genes in improving the heat stress resistance of microorganisms.

6. The application of a biomaterial in improving the heat stress resistance of microorganisms, characterized in that, The biomaterial is any one of (1)-(3): (1) Containing the claims of claim 1 CeCHS57 Gene expression cassette; (2) A recombinant expression vector containing the gene expression cassette; (3) A recombinant host cell containing the recombinant expression vector.

7. The application according to claim 5 or 6, characterized in that, The microorganism is yeast.

8. A method for improving the drought resistance of plants, characterized in that, Including the one described in claim 1 CeCHS57 The gene was genetically transformed into a plant to construct an overexpression of the gene. CeCHS57 The steps involved in transgenic plants.

9. The method according to claim 8, characterized in that, The plant in question is tiger nut or Arabidopsis thaliana.

10. A method for improving the drought resistance of yeast, characterized in that, Including the one described in claim 1 CeCHS57 Genetic transformation of the gene into yeast was used to construct an overexpression of the gene. CeCHS57 The steps involved in gene recombination in yeast.

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