Application of GhSAD2 gene in regulating and controlling salt stress resistance of plants
By overexpressing or silencing the GhSAD2 gene in cotton and Arabidopsis, the plant's salt stress response was regulated, solving the growth and development problems of cotton in high-salt environments, improving the survival rate of Arabidopsis and reducing the salt stress tolerance of cotton.
Patent Information
- Application Number
- CN202510898965.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-01
- Publication Date
- 2025-10-17
AI Technical Summary
In the prior art, cotton growth and development are inhibited under high salt stress, fiber quality is reduced, the number of bolls is reduced, and yield is affected. The function of the SAD2 gene in cotton salt stress tolerance is not clear.
By overexpressing or silencing the cotton GhSAD2 gene in plants, the plant's salt stress tolerance response is regulated, and the recombinant plant expression vector is introduced into cotton or Arabidopsis to increase or decrease its tolerance to salt stress.
The survival rate of Arabidopsis under salt stress was significantly improved, and the tolerance of cotton under salt stress was reduced. The regulatory role of the GhSAD2 gene was verified by physiological and biochemical indicators.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of genetic engineering, in particular to the application of cotton GhSAD2 gene in regulating plant salt stress tolerance. BACKGROUND
[0002] Excessive soil salt content will adversely affect the growth and development of cotton, fiber quality and yield. Studies have shown that when the soil salt content is 0.2% to 0.3%, cotton emergence is inhibited; when the soil salt content is 0.4% to 0.5%, the seed germination potential is weakened and cannot emerge; when the soil salt content is greater than 0.65%, the seed germination rate is significantly reduced. Studies have found that salt stress can affect photosynthesis and nutrient uptake of cotton, resulting in leaf senescence and damage, dwarf plant type, delayed and shortened flowering and boll period, reduced fiber quality, and other adverse manifestations. High salt stress also causes cotton bolls to fall off, resulting in a decrease in the number of bolls, thereby greatly affecting cotton yield.
[0003] SAD2 (Sensitive to ABA and Drought 2) gene encodes a nuclear import protein with a beta domain and is sensitive to ABA and drought. Studies have shown that SAD2 plays an important role in the growth and development of Arabidopsis, such as mediating MYB4 transcription factor into the nucleus to participate in the UV-B response pathway of Arabidopsis; affecting the initial development of Arabidopsis trichomes by regulating AtGL1 and other nuclear genes; and participating in calcium ion and hydrogen peroxide-mediated apoptosis in Arabidopsis. However, the function of SAD2 gene in participating in cotton salt stress tolerance has not been clearly reported. SUMMARY
[0004] In view of the problems existing in the prior art, the present application provides the application of GhSAD2 gene in regulating plant salt stress tolerance, the ID of cotton GhSAD2 gene is GH_D02G0147, the full-length CDS sequence of the gene is 3105 bp, which expands the application range of cotton GhSAD2 gene.
[0005] The technical solution of the present application is: the application of GhSAD2 gene or expression cassette containing GhSAD2 gene or transformant or recombinant bacteria or recombinant plant expression vector in regulating plant salt stress tolerance, the CDS sequence of the GhSAD2 gene is shown as SEQ ID No. 1, and the plant is cotton or Arabidopsis.
[0006] Preferably, the application is overexpression of cotton gene GhSAD2 in plants to improve the tolerance of plants under salt stress, and the plant is cotton or Arabidopsis.
[0007] Preferably, the application is silencing cotton gene GhSAD2 in plants, reducing the tolerance of plants under salt stress, the plants being cotton or Arabidopsis.
[0008] The application also discloses a plant breeding method, and a mode of regulating the expression of a GhSAD2 gene in a target plant is overexpression or knockout of the GhSAD2 gene, a CDS sequence of the GhSAD2 gene is shown as SEQ ID No. 1, and the target plant is cotton or Arabidopsis.
[0009] The application also discloses a method for improving the tolerance of plants under salt stress, and a Gossypium hirsutum GHERF114 gene is introduced into plants by using a plant overexpression vector to obtain transgenic plants with improved tolerance under salt stress, and the plants are cotton or Arabidopsis.
[0010] The application has the following advantages and positive effects:
[0011] The application provides a cotton GhSAD2 gene, and a CDS sequence is shown as SEQ ID No. 1. Through a culture medium salt stress simulation experiment, it is proved that overexpression of the GhSAD2 gene significantly improves the survival rate of Arabidopsis under salt stress. Virus-induced gene silencing is used to interfere with the expression of the GhSAD2 gene, and the relative leaf water content and ion permeability of the gene-silenced plants are significantly reduced, the superoxide dismutase activity is significantly reduced, and the malondialdehyde content is significantly increased, which proves that the tolerance of cotton under salt stress is reduced after silencing the gene. The above results show that the cotton GhSAD2 gene plays an active regulatory role in plant salt stress tolerance. BRIEF DESCRIPTION OF DRAWINGS
[0012] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments, and it should be understood that the following drawings only show the embodiments of the application, and therefore should not be regarded as a limitation to the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.
[0013] Figure 1 is the gene expression amount of a GhSAD2 overexpression homozygote provided by the embodiments of the application, homozygote detection of a mutant, and survival rates of Col-0, mutant sad2 and GhSAD2 overexpression homozygotes OE#2 and OE#5 germinated in culture media with different NaCl concentrations for 8 days;
[0014] Figure 2 is a phenotype diagram of Col-0, mutant sad2 and GhSAD2 overexpression homozygotes OE#2 and OE#5 germinated in culture media with different NaCl concentrations for 8 days provided by the embodiments of the application;
[0015] Figure 3 TRV:GhSAD2 silencing cotton plants appear white phenotype, gene expression level and the results of the phenotype after salt stress;
[0016] Figure 4 TRV:GhSAD2 silencing plants after salt stress physiological and biochemical determination results. DETAILED DESCRIPTION
[0017] The specific embodiments of the present application are further described in detail below with reference to the accompanying drawings and examples.
[0018] The methods used in the following examples are conventional methods unless otherwise specified.
[0019] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.
[0020] Example 1 Cloning of cotton GhSAD2 gene
[0021] The total RNA of leaf tissue of Gossypium hirsutum wild line Malijialangte 85 (preserved by the wild cotton task group of the Cotton Research Institute, Chinese Academy of Agricultural Sciences) was extracted, and the first strand cDNA was reverse transcribed by reverse transcriptase after the gDNA of the total RNA was digested by DNase I, so as to obtain the cDNA of the leaf tissue of Malijialangte 85. The cDNA was used as a template, and the primer pair:
[0022] OE_GhSAD2_PF:
[0023] 5'-GAGAACACGGGGGACTCTAGAATGGATGTTCCAAGTCTTGTT GTTG 5'-3' (the underlined part is the recognition site of restriction enzyme XbaI)
[0024] OE_GhSAD2_PR:
[0025] 5'-TCTCCTTTACCCATGTTAATTAATGATGGTGGAGCGGCGGC-3' (the underlined part is the recognition site of restriction enzyme PacI)
[0026] PCR amplification of the full-length sequence of the GhSAD2 gene CDS obtained a PCR amplification product of about 3105 bp, and the fragment 1 was recovered; the plant expression vector pCAMBIA2300-35S was double digested by restriction endonucleases Xba I and Pac I, and a vector backbone 2 of about 11 k was recovered, and the fragment 1 and the vector backbone 2 were connected by using a homologous recombination enzyme to obtain a recombinant plasmid pCAMBIA2300-35S:GhSAD2; the recombinant plasmid was sequenced, and the sequencing result showed that the recombinant plasmid contained the DNA molecule shown in the sequence 1 in the sequence list (hereinafter named as the GhSAD2 gene).
[0027] Sequence 1:
[0028]
[0029] Example 2 Plant transformation and screening of Arabidopsis thaliana overexpressing the GhSAD2 gene
[0030] The recombinant vector pCAMBIA2300-35S:GhSAD2 obtained in Example 1 was transformed into Agrobacterium GV3101 competent cells and infected wild-type Arabidopsis thaliana (Col-0) by the floral dip method. The infiltration medium used for infection contained 4.3 g / L MS, 50 g / L (5%) sucrose, 0.5 g / L MES, 200 μL / L (0.02%) Silwet-77, pH 5.7. Arabidopsis inflorescences were placed in the transformation solution for 20-30 seconds. After infection, they were incubated in the dark for 24 hours and then transferred to normal conditions for cultivation. To improve transformation efficiency, the infection was repeated once after 1 week. T0 seeds were harvested after seed maturity.
[0031] After surface disinfection of the T0 generation seeds, resistant seedlings were screened on a 1 / 2MS medium containing 50 mg / L kanamycin. After vernalization for 3 days at 4°C, the seedlings were transferred to an artificial climate incubator with conditions set at 22°C, 16h light / 8h dark. After about 10 days of culture in the selection medium, the positive plants grew normally, while the leaves of the negative plants turned yellow and no longer grew. The positive seedlings were transplanted into nutrient pots, and the T1 generation seeds were harvested after the seeds matured. The T1 generation seeds were germinated on kanamycin selection medium, and the transgenic single copy strains were identified by counting the segregation ratio of seedling survival and death (approximately 3:1). The T2 generation seeds were harvested after the seeds matured. The T2 generation seeds were germinated again on kanamycin selection medium, and the transgenic homozygous strains were identified by counting the seedling survival rate. Leaf samples of the T2 generation seedlings were collected, and the primer pair:
[0032] qGhSAD2_PF: 5'-TCTAACTTGCAAGGAAACCGA-3'
[0033] qGhSAD2_PR: 5'-GTTGCAACAACTGGAACCAA-3'
[0034] GhSAD2 gene expression was measured by qRT-PCR to screen homozygous lines with high GhSAD2 expression. After the T2 seedlings matured, T3 seeds were harvested and two homozygous lines with high expression (OE-2 and OE-5) were selected for salt tolerance phenotype evaluation.
[0035] Example 3 Homozygous detection of Arabidopsis mutants
[0036] The mutant sad2 of Arabidopsis thaliana (No. N669077) was purchased from AraShare Arabidopsis Mutant Germplasm (https: / / www.arashare.cn / ), and the primer pair for detecting homozygosity of the mutant was obtained based on the sequence number of the T-DNA insertion mutant of Arabidopsis thaliana from the mutant website (http: / / signal.salk.edu / tdnaprimers.2.html):
[0037] LP: 5'-TGGACTGATTTGGTAGCCAAC-3'
[0038] RP: 5'-ATATAACGCTGGTGCTGTTGC-3'
[0039] The homozygosity of the mutant was identified by PCR amplification, and the homozygous mutant was obtained for phenotype identification.
[0040] Example 4: Analysis of the salt stress tolerance of the mutant and overexpression lines
[0041] In order to verify the sensitivity of seed germination of the mutant and overexpression lines to salt stress, the seeds of wild type material (Col-0), mutant (sad2) material and overexpression homozygous lines (OE-2 and OE-5) of Arabidopsis thaliana were surface sterilized and sown on 1 / 2MS medium containing different NaCl concentrations (100mM NaCl, 150mM NaCl and 200mM NaCl), vernalized at 4℃ for 3d, and then moved to a growth chamber with a photoperiod of 16h light / 8h darkness at 22℃. The survival rate was counted after 8d, and the growth of each line of Arabidopsis thaliana was recorded by taking pictures. The survival rate of the seed germination of the sad2 gene knockout line after 8 days was lower than that of the wild type, while the survival rate of the overexpression cotton GhSAD2 gene line was higher than that of the wild type. As shown in Figure 1 and Figure 2 .
[0042] Example 5: VIGS verification of the role of GhSAD2 in salt tolerance of cotton
[0043] The cDNA of Maligalant 85 was used as a template for PCR amplification using specific primers to construct a VIGS gene silencing vector. The primer pair is as follows:
[0044] VIGS_SAD2_PF:
[0045] 5'-GTGAGTAAGGTTACCGAATTCTTTGGACAACTACATATCCAGG GG-3' (the underlined part is the recognition site of restriction enzyme EcoRI)
[0046] VIGS_SAD2_PR:
[0047] 5'-TCCCCATGGAGGCCTTCTAGATACAAGAAGACATTTCAACCGT GG-3'(underlined is the recognition site of restriction enzyme Xbal)
[0048] The VIGS fragment of GhSAD2 gene was amplified, and the VIGS fragment was ligated to the TRV vector digested by EcoR I and Xba I by using homologous recombination enzyme. The correct ligation was verified by sequencing, and the TRV:GhSAD2 gene silencing vector was successfully constructed. The gene silencing vector was transformed into the competent cells of Agrobacterium GV3101. The TRV:00 Agrobacterium was used as a negative control, and the TRV:PDS Agrobacterium was used as a positive control. VIGS injection was performed.
[0049] The wild landrace of Gossypium hirsutum L. Marygallant 85 was used as the VIGS injection material. After the cotton seeds were germinated for 4 days, cotton seedlings with uniform size were selected and transferred to the water culture box and placed in the greenhouse under the condition of 28, ℃ 16h light / 8h darkness. When the two cotyledons of the seedlings were flat, VIGS injection was performed. The cotyledon back was lightly scratched with a syringe needle, and the bacterial solution was injected into the whole cotyledon. After the injection, the plants were cultured in the dark for 24h, and then transferred to normal conditions for culture. After the plants grew three true leaves, salt stress treatment was performed.
[0050] Example 6 Phenotypic identification of gene-silenced cotton under salt stress
[0051] The TRV:00 empty vector plants and the TRV:GhSAD2 gene-silenced plants at the three-leaf stage were subjected to salt stress (250mM NaCl) treatment. After 7d of salt treatment, the phenotype was observed. The results showed that, compared with the TRV:00 empty vector plants, the leaves of the TRV:GhSAD2 gene-silenced plants were more yellow, and the tolerance to salt stress was reduced, as shown in Figure 3 .
[0052] Example 7 Determination of physiological and biochemical indexes of gene-silenced cotton under salt stress
[0053] Leaf samples of TRV:00 empty vector plants and TRV:GhSAD2 gene silencing plants treated with salt for 0h and 48h were collected, and the relative leaf water content (RLWL) and ion permeability (IL) of the TRV:00 and TRV:GhSAD2 plants were determined. The superoxide dismutase (SOD) enzyme activity was determined by using an SOD kit (Solarbio), and the malondialdehyde (MDA) content was determined by using an MDA content determination kit (Solarbio), and the determination was performed according to the specific steps in the kit instructions. The experimental results show that the relative leaf water content of the TRV:GhSAD2 gene silencing plants is significantly reduced, and the ion permeability is significantly higher than that of the TRV:00 control plants; the SOD enzyme activity of the TRV:GhSAD2 gene silencing plants is significantly reduced, and the MDA content is significantly increased. As shown in Table 1. Figure 4
[0054] The wild germplasm GhSAD2 gene of Gossypium hirsutum is cloned from the wild germplasm of Gossypium hirsutum, and the cotton GhSAD2 gene is introduced into Arabidopsis thaliana, and the overexpression transgenic lines have enhanced salt tolerance. Specifically, the survival rate of the gene knockout lines after 8 days of seed germination is lower than that of the wild type, and the survival rate of the overexpression cotton GhSAD2 gene lines is higher than that of the wild type. The salt tolerance of the TRV:00 empty vector plants and the TRV:GhSAD2 gene silencing plants is compared, and the physiological and biochemical indexes are determined after salt stress treatment. The experimental results show that the relative leaf water content of the gene silencing plants is significantly reduced, and the ion permeability is significantly higher than that of the TRV:00 control plants; the SOD enzyme activity of the TRV:GhSAD2 gene silencing plants is significantly reduced, and the MDA content of the TRV:GhSAD2 gene silencing plants is significantly increased, which confirms that the salt tolerance of the gene silencing plants under salt stress is significantly reduced. It is shown that the GhSAD2 gene plays a role in enhancing the salt stress of plants.
[0055] The above examples are only preferred embodiments of the present application, and do not limit the other forms of the present application. Any person skilled in the art can modify or modify the above disclosed technical content as equivalent embodiments. However, any modification, equivalent change and modification made according to the technical essence of the present application to the above examples still belong to the protection scope of the technical scheme of the present application.
Claims
1. Use of the GhSAD2 gene or an expression cassette, transformant, recombinant bacterium, or recombinant plant expression vector containing the GhSAD2 gene in regulating plant salt stress tolerance, characterized in that: The CDS sequence of the GhSAD2 gene is shown in SEQ ID No.
1. The regulation is to overexpress the cotton gene GhSAD2 in the plant, thereby improving the plant's tolerance to salt stress, and to silence the cotton gene GhSAD2, thereby reducing the plant's tolerance to salt stress. The plant is cotton or Arabidopsis thaliana.
2. A plant breeding method, characterized in that: The expression of the GhSAD2 gene in the target plant is regulated by overexpressing or knocking out the GhSAD2 gene. The CDS sequence of the GhSAD2 gene is shown in SEQ ID No.
1. The target plant is cotton or Arabidopsis thaliana.
3. A method for improving plant tolerance to salt stress, characterized in that: The GhSAD2 gene is introduced into the plant using a plant overexpression vector. The CDS sequence of the GhSAD2 gene is shown in SEQ ID No. 1, thereby obtaining a transgenic plant with improved tolerance to salt stress. The plant is cotton or Arabidopsis thaliana.